Advances in Parasitology Volume 34

Advances in PARASIT0LOGY

VOLUME 34

Editorial Board

C. Bryant Department of Zoology, Australian National University, G.P.O. Box 4, Canberra, A.C.T. 2601, Australia C. Combes Laboratoire de Biologie Animale, Universite de Perpignan, Avenue de Villeneuve, 66860 Perpignan Cedex, France J.P. Kreier Department of Microbiology, College of Biological Sciences, Ohio State University, 484 West 12th Avenue, Columbus, Ohio 43210-1292. USA

W.H.R. Lumsden 17A Merchiston Crescent, Edinburgh EHlO 5AX, UK E.J.L. Soulsby Department of Clinical Veterinary Medicine, University of Cambridge, Madingley Road, Cambridge CB3 OES, UK K.S. Warren Comprehensive Medical Systems, Inc., 461 Fifth Avenue, New York, N.Y. 10017, USA P. Wenk Tropenmedizinisches Institut , Universitat Tiibingen, D7400 Tiibingen 1, Wilhelmstrasse 31, Federal Republic of Gemany

M. Yokogawa Department of Parasitology, School of Medicine, Chiba University, Chiba, Japan

Advances in PARASITOLOGY Edited by

J.R. BAKER The Royal Society of Tropical Medicine and Hygiene, London, England

R. MULLER International Institute of Parasitology, St Albans, England and

D. ROLLINSON The Natural History Museum, London, England VOLUME 34

ACADEMIC PRESS Harcourt Brace & Company, Publishers London San Diego New York Boston Sydney Tokyo Toronto

ACADEMIC PRESS LIMITED 24/28 Oval Road LONDON NWl 7DX United States Edition published by ACADEMIC PRESS INC. San Diego CA92101

Copyright 0 1994, by ACADEMIC PRESS LIMITED This book is printed on acid-free paper All Rights Reserved No part of this book may be reproduced in any form by photostat, microfilm, or any other means, without written permission from the publishers

A catalogue record for this book is available from the British Library ISBN 0-12-0317344

Typeset by J&L Composition Ltd, Filey, North Yorkshire Printed in Great Britain by T.J. Press (Padstow) Ltd, Padstow, Cornwall

CONTRIBUTORS TO VOLUME 34 P.J. BRINDLEY,Molecular Parasitology Unit, and Tropical Health Program, Queensland Institute of Medical Research, The Bancroft Centre, 300 Herston Road, Brisbane, Queensland 4029, Australia J.M. CRAMPTON, Wolfson Unit of Molecular Genetics, Liverpool School of Tropical Medicine, Liverpool L 3 5 Q A , U K C.H. GREEN, Department of Veterinary Medicine (Tsetse Research Group), Bristol University, Langford, Bristol BS18 7DU, UK

R. HALL,Department of Biology, University of York, York Y O 1 5 D D , UK D.W. HALTON, Comparative Neuroendocrinology Research Group, School of Biology and Biochemistry, The Queen’s University of Belfast, Belfast BT7 I N N , Northern Ireland, UK A. G . MAULE,Comparative Neuroendocrinology Research Group, School of Clinical Medicine, The Queen’s University of Belfast, Belfast BT7 I N N , Northern Ireland, U K

C . SHAW,Comparative Neuroendocrinology Research Group, School of Clinical Medicine, The Queen’s University of Belfast, Belfast BT7 1N N , Northern Ireland, U K

D . SMART,Comparative Neuroendocrinology Research Group, School of Clinical Medicine, The Queen’s University of Belfast BT7 I N N , Northern Ireland, U K A.P . WATERS,Department of Parasitology, Postbus 9605, 2300 R C Leiden, The Netherlands

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PREFACE As readers will already have noticed, Advances in Parasitology has had a “face lift” - a redesigned cover and some typographical changes which will, we hope, serve to make the series more attractive and a little easier to read. Coupled with these changes we intend to increase the frequency of publication from annual to roughly biannual. By so doing we hope to be able to give better coverage to the many recent and exciting advances brought about by the application of new approaches in molecular parasitology, immunology and epidemiology of parasitic diseases. In order to help with this new initiative the series has also acquired a third editor, Dr David Rollinson of the Department of Zoology at the Natural History Museum in London. David Rollinson will be known to most, if not all, readers of Advances in Parasitology both as a highly respected parasitologist and as an editor of various books on the subject. We, the “old timers” (John Baker and Ralph Muller) warmly welcome him as a long-time friend and new colleague, and we feel sure that, with these changes, the series is established on a firm base for continued expansion into the 21st century. The volume starts with two reviews concerned with molecular studies in relation to malaria, one concerned primarily with the insect vectors, the other with the parasites. Malaria is an increasingly important health problem in the world today. Effective control has been hampered by the development of insecticide resistance by the mosquito vectors and of drug resistance by the malaria parasite. Julian Crampton of the Liverpool School of Tropical Medicine reviews two important areas of research, both of which concern vector biology and malaria control. The first is the use of DNA probe technology to improve vector identification techniques. Many mosquito vectors are members of sibling species complexes which cannot be easily distinguished by morphological characters. There is a need to identify species in control programmes as the different species within a complex may exhibit differences in ecology, vectorial capacity and response to control measures, and new methods of DNA analysis can provide much needed alternatives for mosquito identification. The second area of research is quite novel and concerns the genetic manipulation of anophelines so as to disrupt the transmission cycle. The advent of vii

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recombinant DNA technology and transgenic techniques now provides the means for the controlled genetic manipulation of vector genomes by the direct introduction of DNA into the insect germ line. A fascinating insight into the development of transgenic techniques for mosquitoes is provided, and the potential value and use of this remarkable technology are detailed. Ribosomal RNA (rRNA) genes have been the subject of a number of detailed studies in recent years. Andrew Waters from the University of Leiden reviews the impact of such studies as they relate to the genus Plasmodium by considering three research areas: molecular biology, molecular phylogeny and diagnostics. This work clearly shows how unsuspected and interesting biological findings can emerge from detailed molecular research. Plasmodium species appear to have evolved an apparently unique response to the problem of living in mosquitoes and vertebrate hosts, and that is the switching ribosome. Two different types of rRNA gene, each containing unit specific sequence elements, are expressed in a stage specific manner. Nucleotide sequences of rRNA genes can be useful for constructing phylogenies. One of the intriguing findings, confirming earlier suspicions, is that the major human pathogen P . fulciparum has a unique and distant relationship to other human and primate malarias. Sequence variation within the rRNA gene has also been exploited in polymerase chain reaction based diagnostic tests for the detection and identification of the malaria parasite - all this from one gene family! A question that has taxed many parasitological minds is the extent and nature of molecular mimicry between parasite and host. Roger Hall from the University of York provides a refreshing and detailed overview of this topic and concludes that the existence of molecular mimicry as an adaptation of parasites to their hosts is an undoubted reality. He explores this fascinating area by first considering what is meant by molecular mimicry, how it might be studied, the consequences to host and parasite, and how widespread is it. Numerous examples of mimicry, some of them speculative, are considered and placed in four main classes: cytoadhesive proteins and their receptors; effectors of the immune system; hormones, serum proteins and their receptors; and cytoskeletal and muscle proteins. There are difficulties in recognizing and providing clear evidence for mimicry but in a few cases compelling explanations are available with some of the best examples being associated with microbial infections and autoimmune syndromes. Traditionally, research on the chemotherapy and the immunology of parasitic infections has been carried out independently and the interaction and possible synergy between the effects of drugs and the immune response of the host has been little studied. Recently, enhancement or even dependence of effective chemotherapeutic action on host immune defences has been reported in various parasitic infections and Paul

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Brindley from the Queensland Institute of Medical Research reviews new and important studies linking the efficacy of antischistosomal drugs to host immunological responses to schistosomes. He concludes that achieving a semi-immune state through repeated infection, or perhaps through vaccination, may allow lower curative doses of antischistosomal drugs to be used than in non-immune patients. While neuroendocrine secretion in vertebrates has been recognized for several decades, many new regulatory peptides have been identified and isolated in the last twenty years. More recently, research on the origins of peptidergic signalling in invertebrate neurons has been made possible by advances in the techniques of radioimmunoassay and immunocytochemistry. David Halton and colleagues at the Queen’s University in Belfast have been in the forefront of research in helminth neuropeptides, and here review this exciting and fruitful field of study. Many newer anthelmintics appear to act against parasite neuromusculature and it is likely that, when helminth regulatory peptides and their receptors have been fully characterized and a physiological function assigned to them, they will prove valuable as targets for drug action. The very elegant results which can be obtained demonstrating neuropeptide immunoreactivity in whole mounts of platyhelminths are illustrated by the cover photograph. Finally, Chris Green (University of Bristol) has contributed an excellent review of the possibility of tsetse control by trapping. Although not a new topic, having been tried since at least 1910 with varying degrees of success (see Glasgow, J.P. and Potts, W.H., 1970, in “The African Trypanosomiases” edited by H .W. Mulligan; London: George Allen and Unwin, for a history of the early work), considerable advances have been made in recent years -largely as a result of greater understanding of what attracts Glossina to traps and the work of Einar Bursell, Chris Green and others on the use of olfactory attractants based originally on chemicals in the breath of cattle. With increasing fears about the development of insecticide resistance by insects, and the recognition of the adverse ecological effects of wide-scale application of insecticides, the “ecologically friendly” method of trapping has once again come to the fore amongst methods of controlling tsetse and, hence, both human and animal African trypanosomiasis. JOHN BAKER RALPH MULLER DAVID ROLLINSON

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CONTENTS CONTRIBUTORS TO VOLUME 34 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . PREFACE. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Molecular Studies of Insect Vectors of Malaria J.M. Crampton 1. General Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 2. DNA Probes for the Identification of Malaria Vectors . . . . . . . . . . . . . . . . . . 3 3. Genetic Manipulation of Malaria Vectors . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25

The Ribosomal RNA Genes of Plasmodium A.P. Waters

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1. Introduction . . . . . . . . . . . . . .

3. Inferences of Phylogeny Based 4. Species Identification Based on Ribosomal RNA .......... ..................................

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Molecular Mimicry R. Hall Introduction . . . . . . . . . . . . . . . .......................... Adaptive Mimicry ...................... Consequential Mimi : Autoimmunity . . . . . . . . . . . . . . . . . . . . . . . . . . How Does Molecula Considerations . . . . . . 5. Conclusions . . . . . . ........................ Acknowledgements References . . . . . . .

1. 2. 3. 4.

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Relationships Between Chemotherapy and Immunity in Schistosomiasis P.J. Brindley 1 . Introduction . . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . . 134

134 . . . . . . . . . . . . . . . . . . . 135 136 5. Antimonials . . . ............................. 137 . . . . . . . . . 139 ....................................... 143 ............ . . . . . . . . . . . . . . . . . . . 155 ...................................... 156 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . 156

3. Schistosomicidal Chemotherapy

Regulatory Peptides in Helminth Parasites D.W. Halton, C. Shaw, A.G. Maule and D. Smart 1. Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Occurrence and Distribution of Regulatory Peptides in Helminths . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ....... ... 3. Quantification and Characterization of Regulat es in Helminths . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .............. 4. Isolation and Structure of Helminth Regulatory Peptides . . . . . . . . . . . . . . . 5. Evolutionary Aspects of Helminth Regulatory Peptides . . . . . . . . . . . . . . . . 6. Functional Aspects of Helminth Regulatory Peptides . . . . 7. Future Developments . . . . . . . . ................... Note Added in Proof . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . .............................

164 168

184 195 201 214 217

Bait Methods for Tsetse Fly Control C.H. Green 1. 2. 3. 4. 5.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Attractants for Tsetse Flies . . . . . . . . . . . . . . . Bait Systems for Tsetse Control . . . . . . . . . . . . Programmes of Tsetse Control Using Bait Systems .................... Conclusions . . . .

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Molecular Studies of Insect Vectors of Malaria Julian M. Crampton Wolfson Unit of Molecular Genetics, Liverpool School of Tropical Medicine, Liverpool, L3 5QA, UK

1. General Introduction

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2. DNA Probes for the Identification of Malaria Vectors . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Current methodsfor the identification of malaria vectors . . . . . . . . . . . . . . . . 2.2. Approaches to developing DNA-based methods for identification purposes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3. Development of a DNA probe method for the simple identification of malariavectorspecimens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4. DNA probes for vector identification: the future . . . . . . . . . . . . . . . . . . . . . . . .

1 3 3 5 7 8

3. Genetic Manipulation of Malaria Vectors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 3.1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 3.2. The requirements for genetic manipulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 3.3. The potential application of transgenic technology to malaria vectors . . . . . . 21 3.4. Transgenic mosquitoes: the future in relation to the control of malaria . . . . . 25 Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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1. GENERAL INTRODUCTION

Despite enormous efforts over many years, malaria is an increasingly important health problem in the world today. Some 500 million people are estimated to be affected each year, with approximately 3 million deaths (Sturchler, 1989). The incidence of this disease is increasing due largely to the development of insecticide resistance by the mosquito vectors and by the appearance of drug resistance in the malaria parasite. These factors are exacerbated by migration of increasing numbers of people from non-endemic areas to regions where malaria is prevalent. ADVANCES IN PARASITOLOGY VOL 34

ISBN 0-12431734-6

Copyright0 1994 Academic P m s I.rmifed A / / ngho ofrepruducrion in m y furm reserved

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Research over the last century has increased our understanding of the complex relationship between insect vectors, the parasite causing the disease and the human host. One way of interrupting this cycle is to suppress the insect which acts as the vector and a number of techniques have been tried. These include chemical control through insecticides and larvicides, biological control through natural predators or toxins, such as that produced by Bacillus thuringiensis, environmental control through the removal of breeding sites and the raising of public awareness and genetic control through the mass release of sterile males. Another genetical approach, which may become a possibility through the advent of recombinant DNA technology, would be to compromise the vectorial capacity of the insect. Despite the current trend towards integrated pest management, in which several approaches are used in combination to suppress insect populations, the main emphasis for mosquito control has been the elimination of breeding sites and the application of chemical agents. There are now considerable problems associated with the use of synthetic insecticides. The most important of these is the seemingly inevitable evolution of resistance and there are now populations which are multiply resistant to all four classes of insecticidal compound (organophosphates, organochlorines, carbamates and pyrethroids). Coupled with this is the high cost of developing and registering new insecticidal variants, increasing legislation over their use and growing environmental awareness over their toxic residues. Genetic control, through the mass release of sterile male mosquitoes, has also been attempted but with little success (Grover, 1985). This is now thought to be due to the poor competitive mating ability of treated males following chemical or radiological sterilization. Less traumatic sterilization treatments may increase the effectiveness of such a strategy, as demonstrated by the successful eradication of the screw-worm fly from the Southern USA (Krafsur etal., 1987). However, such autocidal strategies of population control often prove prohibitively expensive since they involve the repeated mass release of treated males (McDonald et al., 1977). The problems associated with the widespread and continuing reliance on chemical insecticides has stimulated interest in alternative methods for malaria control. What is required is the development and evaluation of a new generation of methodologies which will have a profound and longlasting effect on malaria transmission. Clearly, biotechnology and molecular biology can play a central role in the search for, and production of, such new tools. The development of potential antimalarial vaccines and biotechnologically produced larvicidal compounds are obvious examples of the power of the approach. Perhaps surprisingly, this technology has not been applied to the anopheline vectors of malaria until very recently.

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Advances in the molecular analysis of vector-parasite relationships and vector molecular biology now make such an approach very attractive especially as past experience has shown that vector control is an effective way of disrupting malaria transmission. Two applications of molecular biology will be considered in relation to vector biology and malaria control. The first is the utilization of DNA probe technology to improve vector identification techniques. The aim here is to enhance the efficiency of existing control measures by providing accurate data about vector populations. The second is a completely revolutionary approach to malaria control through genetic manipulation of anophelines so as to disrupt the parasite transmission cycle. There are many other areas where molecular biology will have a profound impact on our understanding of the insect vectors of malaria. However, these two examples serve to emphasize the power of this technology and they are reviewed below.

2. DNA PROBES FOR THE IDENTIFICATION OF MALARIA VECTORS 2.1. Current Methods for the Identification of Malaria Vectors

Many mosquito vectors are members of sibling species complexes. Sibling species are reproductively distinct but cannot be distinguished by morphological features alone and require alternative methods for identification. It is important to distinguish these species in control programmes as the different species within a complex may exhibit differences in ecology, vectorial capacity and response to control measures (White, 1982). There are a number of techniques available for the identification of vector specimens and these are discussed below. The definitive method for species identification of mosquito malaria vectors such as the Anopheles gambiae complex, which includes the major vectors of malaria in Africa, is to perform crossing experiments of the offspring of the unknown specimen against laboratory colonies of known species (Davidson, 1964, 1966; Davidson and Hunt, 1973). Although this method has been of great value in the elucidation of species within a number of anopheline complexes, it is far too laborious and time consuming to use for routine identification of individuals. Many external morphological characteristics of anopheline species have been examined in an attempt to identify species-specific features. In the Anopheles gambiae complex, some characteristics of Anopheles melas are fairly reliable for separation of this species from freshwater Anopheles

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gambiae (Ribbands, 1944; Muirhead-Thompson, 1948). However, no reliable features have been found which allow the identification of the other species in this complex. The most reliable and widely used method for species identification is that of the polytene banding technique (Coluzzi and Sabatini, 1967, 1968, 1969; Green, 1972; Davidson and Hunt, 1973; Coluzzi et al., 1979). For species of the Anopheles gambiae complex, this technique allows the separation of the three freshwater species, Anopheles gambiae s.s., Anopheles arabiensis and Anopheles quadriannulatus by diagnostic X chromosome inversions, and identification of the other species using more subtle autosomal features. The main disadvantages of this method are that identification is limited to fourth instar larvae or semigravid blood-fed females and that the technique requires a good deal of skill to perform. The number of specimens which may be processed in a given time is also limited. Isoenzyme typing (Mahon el al., 1976; Miles, 1976, 1978) is also routinely used in some laboratories for species identification. This method relies on different species having different electromorphs of a number of enzymes. Starch gel electrophoresis is usually used and, in some cases, the gel is split to allow the assay of two different enzymes. This technique is time consuming and relatively expensive in terms of equipment and materials. The number of specimens which may be processed individually is also limited and some specimens give anomalous results. Specimens must be stored frozen in liquid nitrogen prior to identification due t o the lability of the enzymes on which the technique relies. The use of cuticular and internal hydrocarbons to identify Anopheles gambiae species has been investigated (Carlson and Service, 1979, 1980; Hamilton and Service, 1983). In this technique hydrocarbons are extracted using an organic solvent and analysed by gas chromatography. This method has not been fully evaluated, but the requirement for sophisticated equipment to obtain and handle data, together with the time-consuming nature of identification by this equipment makes this an unlikely technique for routine species identification. Some success in differentiating Anopheles gambiae sibling species using the fluorescent stain, Hoechst 33258, has been described (Gatti et al., 1977; Bonaccorsi et al., 1980). This reagent preferentially stains A-T rich regions in DNA and so allows the position of heterochromatic blocks of DNA to be visualized on mitotic chromosomes. Unfortunately, in addition to major differences in banding patterns between species, a significant level of intraspecies variation is also evident. This geatly reduces the potential of this method for routine species identification.

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2.2. Approaches to Developing DNA-based Methods for Identification Purposes

The problems discussed above have led to the investigation of DNA probe technology which is cheap, accurate and easy to use. There are essentially three approaches which may be taken when developing identification techniques based on DNA technology and these are discussed below. 2.2.1. The Directed Approach The first, which may be termed an orientated or directed approach, involves the cloning of a DNA sequence which has a high probability of displaying sequence variation between the species within a complex. An example of such a sequence is the ribosomal DNA. There are multiple copies of these genes in all organisms; each copy of the gene cluster is separated by spacer regions (Gale and Crampton, 1989). It is the DNA sequence of these spacer regions which commonly varies significantly between closely related species. Probe systems can, therefore, be generated which take advantage of this variation to identify each specimen. A number of researchers have utilized this approach to generate speciesspecific probes. One good example is the work of Collins et al. (1987, 1988) who defined probes which could be used to identify mosquitoes of the Anopheles gambiae complex. The method involved extracting the DNA from each specimen, digesting the DNA with restriction enzymes and fractionating the DNA by agarose gel electrophoresis. Subsequently, the digested DNA was transferred to nitrocellulose by Southern blotting following denaturation and hybridized to the 32P-labelled DNA probe. Specimens could then be identified by the size of the resulting band following autoradiography of the hybridized Southern blot. More recently, the same group have modified this procedure to utilize the polymerase chain reaction (PCR) for vector identification (Paskewitz and Collins, 1990). A set of three DNA primers has been derived from the ribosomal gene region of species in the Anopheles gambiae complex. Purified DNA isolated from each mosquito specimen is mixed with the primers and a PCR reaction carried out. Depending on the species of the specimen, an amplification product characteristic of that species is generated during the reaction and this can be visualized by agarose gel electrophoresis. This technique has great potential for species identification, although the reagents for PCR amplification are not cheap. 2.2.2. The “Shotgun” Approach The second method for generating DNA probes may be termed a “shotgun” approach. This involves the selection of any piece of DNA from

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the genome of the insect which is present in one of the species of interest but not in the others. Such a probe lends itself to being used in a very simple assay as it gives a yes-no answer, i.e. the target DNA sequence is either absent or present and thus gives a simple and unambiguous answer. Thus, in the ideal situation, a series of such probes, one for each of the species within a complex, will allow each specimen to be identified. A drawback to the method is, however, that a series of probes may be required to identify fully each mosquito specimen. The progress which has been made towards the development of this type of DNA probe system is described more fully in section 2.3. 2.2.3. Random AmpliJied Polymorphic DNA (RAPDs) The RAPD technique is PCR based and permits scores of markers to be assayed on DNA extracts from a single mosquito (Williams et al., 1990, 1993). In order to achieve this, the RAPD PCR reaction uses a single, short primer (usually about 10 bases in length) of a randomly chosen sequence. For a RAPD band to be produced during the PCR reaction, the primer needs to anneal to a binding site which is within 2-3 kilobase pairs of another oppositely orientated binding site. Where this occurs, the single oligonucleotide primer can prime amplification in both the forward and reverse direction resulting in the production of a PCR product. A typical RAPD reaction produces multiple amplification products, each representing a discrete genetic locus, and these can be readily fractionated and analysed by agarose gel electrophoresis. RAPD bands display a high degree of polymorphism so that screening taxa of interest with a number of different primers has proved to be a very rapid method for generating species-specific markers (Arnold et al., 1991). Another advantage of the RAPD technique is that RAPD markers derive from multiple loci and thus have the potential to provide information on the genetic structure of a population. Recently, Wilkerson et al. (1993) have begun to apply the RAPD technique to species of the Anopheles gambiae complex of mosquitoes. They have made a preliminary survey of a large number of RAPD primers in order to identify differences in the PCR products between Anopheles gambiae S . S . and Anopheles arabiensis. Seven primers produced diagnostic bands for these two species. The approach clearly has potential although the technique has to be used with caution. For example, the reproducibility of the PCR reactions is not reliable so that even fairly strong RAPD bands will occasionally fail to be produced in a particular amplification reaction; the absence of bands should not be used to draw inferences without confirmation from a number of duplicate reactions; in some cases there has also been difficulty in

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reproducing the same RAPD product profile using the same primers, template and reaction conditions between different laboratories.

2.3. Development of a DNA Probe Method for the Simple Identification of Malaria Vector Specimens

By using the “shotgun” approach we have developed DNA probes for the identification of species in the Anopheles gambiae complex which includes the major vectors of malaria in Africa. Six species within this complex have been identified on the basis of mating incompatibility (Davidson et al., 1967) - Anopheles gambiae s . s . , Anopheles arabiensis, Anopheles quadriannulatus, Anopheles melas, Anopheles merus and Anopheles bwambe. The first two are the most important vectors and are widely distributed throughout tropical Africa. Differential screening of clone banks generated from the DNA of each of those species has allowed the isolation of a number of cloned DNA probes which may be used for the identification of five of the six species in the complex (Gale and Crampton 1987a, b, 1988). In a similar way, DNA probes have also been developed to distinguish species in the Anopheles dirus complex (Panyim et al., 1988a, b) which are major vectors in Thailand and species of the Anopheles farauti complex of Australia (Cooper et al., 1991; Booth et al., 1991). Subsequent work has been directed towards refining these DNA probes and simplifying the methods for using them so that they become available for entomologists in the field. One of the major refinements has involved simplification of the sample preparation technique so that now all that is required is to squash a portion of the mosquito specimen, usually the head, directly onto a nylon filter membrane (Hill et al., 1991a). A further refinement has been to derive short oligonucleotide DNA probes from the original cloned sequences for both the Anopheles gambiae probes (Hill et a l . , 1991b) and the Anopheles furauti probes (Hartas et al., 1992). Such oligo-probes have a number of advantages for vector identification. First, they are cheap and easy to synthesize chemically; second, they may be tagged during synthesis so that they can be used with a variety of reporter molecules other than radiochemicals; and third, the kinetics of hybridization using singlestranded oligo-probes are such that the time taken for the identification process can be greatly shortened (Crampton et a l . , 1989). In addition to the use of oligo-probes, the conditions for hybridizing the probes have been greatly modified. The aim here has been to reduce both the reliance on laboratory equipment and the complexity of the hybridization solutions so that they are both cheap and very stable. It is now possible to carry out the whole process at room temperature and with a very simple

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hybridization solution which can be pre-mixed as a dry powder and stored for long periods at room temperature (Hill et al., 1992). A major drawback of DNA probe systems is that they have previously relied on the use of radiochemical labelling as a means for detecting hybridization to a target DNA sequence. Major advances in the utilization of nonradioactive reporter molecules have now made it possible to incorporate these techniques into a simplified vector identification protocol. A comparison of the available non-radioactive labelling and detection techniques has shown that the E-link plusTMsystem produced by ICI does not suffer from background problems, is the most sensitive and the cheapest ( 2 4 centslspecimen) of the current alternative systems (Hill et al., 1991a). In addition, it may be used with squash-blotted specimens (i.e. the DNA does not have to be isolated or purified from each specimen) and the result can be detected either as a colour change on the filter or by photographic detection of chemiluminescence. Similar results using the non-radioactive system have been reported by Cooper et al. (1991) using cloned probes to identify species of the Anopheles farauti complex. Further refinements of the detection system are underway to shorten and simplify this aspect of the process. The systems which have evolved for the use of DNA probes for mosquito identification are easy and quick to use and, above all, are cheap enough to allow many specimens to be identified at minimal cost. Indeed, the methodologies in their present form are now being assessed by a number of laboratories by comparing them with existing identification techniques (Collins et al., 1988). 2.4. DNA Probes for Vector Identification: The Future

An important aspect of this technology is that the method can be used with any probes which may be developed in the future. In addition, probes which would provide other useful epidemiological data, such as those for Plasmodium falciparum detection (Delves et al., 1989), blood meal analysis, or insecticide resistance determination could also be incorporated into this methodology. Hopefully, with further improvements and simplification of the technique and the development of probes for a range of mosquito species complexes, DNA probes technology may shortly realize its full potential in the field of mosquito identification. 3. GENETIC MANIPULATION OF MALARIA VECTORS 3.1. Introduction

Control of insect populations by genetic means is not new. A number of vector control programmes have utilized the mass release of sterile

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9

males, the generation of cytologically induced sterility through translocation and the use of mating behaviour to transfer lethal or sterilizing agents between the members of insect populations. The advent of recombinant DNA technology and transgenic techniques, however, now provides the means for the controlled genetic manipulation of vector genomes by the direct introduction of DNA into the insect germ line. Such manipulation of medically important insects would enable desirable modifications to be inherited by subsequent generations, potentially removing the need for frequent mass releases. Two particular advantages of using transgenic technology over classical genetics for future manipulation are evident and should be emphasized. One is the potential to exploit genes and gene constructs across species barriers, and the other the ability to introduce particular, defined sequences without the genome disruption of a conventional cross. The ability to introduce and express foreign genes and/or disrupt existing gene functions may lead to the development of a variety of control strategies. It may be possible, for example, to produce pathogen refractory strains, to reduce reproductive potential or vector competence, or to increase vector susceptibility to existing control measures. Clearly, the usefulness of genetic manipulation as an approach to the control of insect-borne disease has first to be assessed and there will be many practical, and perhaps ethical, difficulties to overcome. Nevertheless, transgenic technology clearly has considerable potential both for the furtherance of our understanding of mosquito molecular biology and for the development of novel genetic control strategies. 3.2. The Requirements for Genetic Manipulation

Despite the dramatic advances made recently with respect to genome manipulation in Drosophilu melunoguster there is an urgent need for a much greater understanding of the molecular biology of mosquito disease carriers. This must include an analysis of the complexity and organization of their genomes and an understanding of the distribution of coding and repetitive sequences. These details form an integral part of the design and interpretation of cloning and hybridization experiments. In addition, the exploitation of transgenic technology requires methods for the introduction of DNA both into living mosquitoes and into cultured cells. Ideally, this will involve a transformation vector which is capable of directing efficient and stable integration into the chromosomes of the recipient. The introduced DNA has not only to be expressed but should also carry a selectable marker for the identification of transformed individuals or cells. Ultimately, there will be a need to study alternative promoter and enhancer sequences so that the spatial and temporal expression of the

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introduced DNA may be controlled. Finally, none of this will be of any relevance unless appropriate genetic target systems can be identified, cloned and characterized at the molecular level. 3.2.1. Genome Organization and Complexity

If the genomes of insect disease vectors are to be manipulated in a controlled and directed fashion, it is important to determine the size of the genomes involved. Genome organization, that is the nature and dispersion pattern of repetitive sequences and how they are organized in relation to the coding sequences, is also important. This is because it will have a profound influence on the types of manipulation which can be envisaged and the approaches to be adopted in order to identify and to clone sequences of interest. Until recently, very little was known about the size and organization of mosquito genomes. However, the DNA of all higher eukaryotes is conveniently subdivided into three components, namely highly repetitive, moderately repetitive and unique or single copy sequences and the organization of these components can be determined through experiments which involve denaturing the DNA and measuring the reassociation of complementary strands over time. Generally, highly repetitive sequences will reassociate most quickly since there are so many copies, whereas unique sequences will reassociate more slowly. The complexities of these components and of the total genome are determined by comparing the reassociation values with that of a single copy sequence of known complexity, namely E. coli DNA. Of equal importance, however, is the way in which the different components are distributed throughout the genome. Black and Rai (1988) suggest that the existence of two basic organization patterns throughout all higher eukaryotes is indicative of a set of rules governing the establishment and spread of repetitive elements. The first pattern is known as short period interspersion (SPI), in which 1-2 kb segments of single copy sequence alternate regularly with short (0.2-0.6 kb) or moderately long ( 1 4 kb) repetitive sequences. This pattern is characteristic of the majority of animal species. The second pattern, long period interspersion (LPI), is characterized by long (5-6 kb) repetitive sequences alternating with very long (> 13 kb) uninterrupted stretches of unique sequence DNA. Clearly, the evolution of such organization is an interesting phenomenon in itself and the family Culicidae (to which the mosquitoes belong) may be of particular interest. According to Black and Rai (1988), this is the only family so far shown to contain species exhibiting both patterns of organization and it may, therefore, explain how the transition from one to the other occurs. Black and Rai (1988) have analysed the genomic DNA from four species of mosquito, namely Anopheles quadrimaculatus, Culex pipiens, Aedes

11

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albopictus and Aedes triseriatus. More recently, Cockburn and Mitchell (1989) have shown that the genomes of anopheline mosquitoes are generally relatively small and exhibit the LPI pattern of repetitive sequences. For example, the genome of Anopheles gambiae, the major mosquito vector of malaria in Africa, has a genome size of 2.7 x 10' bp (Besansky and Powell, 1992). This is in marked contrast to Aedes aegypti which has the more complex SPI pattern (Warren and Crampton, 1991). The Aedes aegypti genome is also relatively large (8.3 x 10' bp), being five times the size of the Drosophila genome or, to place this in context, one third the size of the human genome. Thus, almost every clone isolated from Aedes aegypti genomic libraries will contain repetitive DNA and this may well mask the hybridization characteristics of sequences of interest (particularly single copy sequences) when using complex genomic probes. This is in direct contrast to organisms like Drosophila or Anopheles, which display the LPI pattern, where cloned sequences are more likely to consist entirely of either repetitive or unique DNA. In insect studies to date, Anopheles quadrimacufatus, Anopheles gambiae and Drosophila melanogaster have been shown to exhibit the LPI pattern (Cockburn and Mitchell, 1989). Conversely, Aedes aegypti, Culex pipiens, Aedes triseriatus and Aedes albopictus exhibit the SPI pattern (Black and Rai, 1988; Crampton et al., 1990b). Clearly, there appears to be no simple relationship between genome size and organization.

3.2.2. Codon Usage in Mosquitoes The number of mosquito genes which have been cloned and sequenced is still very limited. However, sufficient sequences are now available for Aedes aegypti, Aedes albopictus and Anopheles gambiae to be able to begin the analysis of codon usage for these species of mosquito. In the case of the two species of Aedes mosquitoes, there is some preference for C G at the third base position, but this is not a general rule (Argentine and James, 1993). A comparison of codon usage for three homologous genes, an amylase, maltase and trypsin, of Aedes aegypti and Drosophila melanogaster showed that Drosophifa has a much stronger bias for C G at the third base when compared with Aedes. These conclusions are, however, limited by the very small sample size for the construction of the codon usage tables. In the case of Anopheles gambiae, rather more sequences have been determined, including 14 chromosomal genes (Besansky, 1993). There is variation in the degree of codon bias among the chromosomal genes, with a general bias in favour of C G at the third base position. Thus, although there are some general similarities in codon usage between Aedes, Anopheles and Drosophila in the preference for G and C-ending codons, it would

+

+

+

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appear that each has evolved a distinct pattern of codon usage. Such codon preferences will clearly be of relevance when it comes to the preparation of oligonucleotide probes and PCR primers for the identification and cloning of mosquito homologues of genes previously described in Drosophila. 3.2.3. Genome mapping

The molecular mapping of a number of insect genomes other than Drosophila has now been initiated. Foremost amongst these, is the programme which aims to map the genome of the mosquito, Anopheles gambiae, and uses the approach initially developed for the molecular mapping of the Drosophila genome (Saunders et al., 1989; Siden-Kiamos et al., 1990). A preliminary, low-resolution map for part of this genome has been generated using PCR-amplified, microdissected segments from polytene chromosomes as segment-specific probes and markers (Zheng et al., 1991). It is hoped that a complete molecular map will be generated which can then be correlated with the genetic map and the very detailed cytological map which is available for this insect. Such maps will be of considerable benefit when the mobility of potential DNA vector systems are to be tested, and to explore the nature of transgene integration events and their long-term stability in insect genomes. 3.2.4. Methods for Introducing DNA into Mosquito Embryos and Adults

(a) Microinjection of embryos. Here, we describe the system which we have developed in our own laboratory for transformation of the mosquito Aedes aegypti (Morris et al., 1989). However, similar techniques have been used elsewhere both for Anopheles and for other Aedes species, and all are based in general on the methods developed for Drosophila melanogaster. Unlike that of Drosophila, the rigid, opaque endochorion of the mosquito embryo cannot be removed, and the embryos are extremely sensitive to desiccation. However, glass capillaries with tips of 100-300 pm X 4-10 pm can be used to puncture the rigid endochorion without tearing it and deliver the DNA solution without damage to the embryo. The slightly viscous DNA solution cannot be expelled manually from such a fine needle and is therefore injected by means of a two-phase nitrogen supply. The lower pressure prevents backflow and the higher pressure delivers 160-800 pl of DNA solution (corresponding to 1-5% of the embryo volume) into the posterior pole of the embryo at the syncytial blastoderm stage, before cell partitioning occurs. This is where the pole cells, which are the germ line primordia, develop. Experience from Drosophila suggests that injection of DNA close to the site of pole cell formation is not critical to germ line incorporation, but the timing is clearly important if the DNA

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13

is to be taken up by the developing germ line cells. All of our injections are normally completed within 2 h of oviposition. Diving and after injection, the embryos are covered with a water-saturated halocarbon oil, which permits the normal uptake of water until they are returned to standard insectary conditions. In this way DNA has been introduced into mosquito embryos (Miller et a f . , 1987; McGrane et al., 1988; Morris et al., 1989), with survival rates comparable to those obtained with Drosophila (Spradling and Rubin, 1982). (b) The biolistic technique. The biolistic technique, which uses high velocity DNA-coated, tungsten microprojectiles to deliver DNA directly into the nuclei (Sandford et al., 1993), has been successfully applied to introduce DNA into large numbers of dechorionated Drosophifa embryos (Baldarelli and Lengyel, 1991). Transient expression from the P160 construct, a plasmid containing the actin 5C promoter fused to the E. cofi P-galactosidase gene, was detected. In addition, one germline transformation event was obtained when embryos were bombarded with the pn25.7wc helper and the P element vector 2pCasPeR in a ratio of 1:1.7. Once this method has been optimized it will have the advantage that a very large number of embryos (between 3000-5000) may be bombarded in one experiment, thus overcoming the time-consuming microinjection process. This would be particularly advantageous in a situation where microinjection survival rates are low and/or the microinjection process is difficult. These considerations have led Mialhe et al. (1992) to test the biolistic method for gene transfer in Anopheles gambiae mosquitoes. In these experiments a construct made up of the luciferase gene under the control of the Drosophifa heat shock promoter, hsp70, was used as the reporter gene system. Transient expression of the reporter gene was detected, and experiments are currently underway to select for stable transformants using the biolistic method and a construct which incorporates a selectable marker. (c) Sperm-mediated gene transfer. Considering reports of spermmediated gene transfer in rabbits, sea urchins, mice and chickens, some efforts have been directed towards determining whether or not insect sperm can transfer genes. The honey bee, Apis meffifera,has been used to examine this transformation technique, and was investigated by inseminating honey bee queens with sperm that had been incubated with a 1 kb linear DNA fragment, and assaying the DNA of Go progeny by the polymerase chain reaction (Milne, 1992). Preliminary evidence indicates that DNA incubated with bee sperm was present in about 30% of the progeny, appeared to be in the genome, only occurred in a fraction of the cells, and was probably not transferred by incorporation into the sperm genome. These preliminary results indicate that honey bee sperm can transfer genes into the genome. This method may represent an attractive

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alternative to microinjection in gene transfer technology in terms of its ease, success rate and broad applicability to other insects, but to date has not been attempted with insects other than the honey bee. This technique may work in other insects for which instrumental insemination with sperm has not been perfected by placing a concentrated DNA-containing solution in the reproductive tracts of females before natural mating to treat the sperm in vivo. However, it is clear that there is still a considerable amount of work to be done before sperm-mediated transformation can be considered for the genetic manipulation of mosquito malaria vectors. (d) Transformation of symbionts as a means of introducing genes into mosquitoes. Microinjection of embryos has been the system which has received most attention to date for producing transgenic insects. Another very interesting possibility is the idea of transforming symbionts isolated from the insect gut or ovary with gene constructs and reintroducing them back into the insect vector. This approach has received particular attention in tsetse flies where the gut symbionts have been isolated, cultured and transformed and shown to express the introduced DNA (Beard et al., 1993). The ability to introduce gene constructs into the insect via symbionts may be a very attractive alternative to direct genome manipulation, particularly in the case of gut symbionts, where they would be ideally placed for the expression of antiparasitic agents. Clearly, however, this form of manipulation would not be stable unless the symbionts were inherited in some way. Although this may be true for the Rickettsia-like microorganisms (RLMOs) which are present in the ovaries of tsetse flies and Culex mosquitoes, symbionts such as these would be difficult to reintroduce into the adult fly. In addition, RLMOs have not been detected in anophelines, but some consideration is now being given to the idea of introducing RLMOs isolated from other mosquito species into anophelines. If this is successful and the RLMOs survive and are passed through subsequent generations, this approach may provide a potentially powerful means for introducing specific gene constructs into anopheline mosquitoes. Gut symbionts, although easy to isolate, transform and reintroduce into the insect midgut, nevertheless may prove unsatisfactory as a means to introduce specific genes into large populations of insects as they are unlikely to be passed on to subsequent generations. Thus, although gut symbionts would seem to be ideally placed in terms of the expression of antimalarial factors, they do not seem to provide a realistic mechanism for the creation of mosquitoes which are refractory to the malaria parasite. 3.2.5. D N A Vector Systems Used in Mosquito Transformation Germ line transformation of insects and transfection of cultured cells requires the establishment of appropriate techniques for the introduction

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of a eukaryotic D N A transformation vector. This should be capable of directing the efficient and stable integration of D N A sequences into the chromosomes of the recipient, as well as providing some means for the identification of transformants. The transposable genetic P element from Drosophila melanogaster has been used in a variety of insect species in an attempt to create transgenic individuals and appropriate transformation vector constructs are now used as a routine research tool in Drosophila. P elements (Engels, 1988) are a distinct class of transposable element discovered when a syndrome of correlated genetic traits (hybrid dysgenesis) occurred if male flies from P strains (normally from wild populations) were mated with M strain females (normally from laboratory populations). A series of abnormalities resulted, which included high rates of mutation, male recombination, chromosomal rearrangements, sterility, abnormal germline development and meiotic drive. Genetic observations showed that a number of these mutations were unstable and had high reversion frequencies, all of which led to the hypothesis that P strains carry a family of transposable sequences called P elements. The abnormalities only arise in hybrids because of the absence of a repressive factor which, although, not fully understood, is normally found in P strains. It is now thought that P factors have arisen in wild Drosophila only within the past 30 years. The P element family consists of intact elements (2.9 kb) and a heterogeneous group of smaller elements (0.5-2.5 kb) which are derived from the intact element by internal deletion. The intact element encodes a transposase enzyme which catalyses excision and transposition. The smaller elements lack this function and are incapable of transposing themselves. They can, however, transpose when supplied with functional transposase in trans by an intact element. All P elements carry perfect 31 bp inverted repeat sequences at their termini that are absolutely required for excision and transposition. This process results in an 8 bp duplication at the target site. P elements are capable of precise excision which leaves behind a single copy of the 8 bp sequence. More frequently they will excise imprecisely leaving part of the element behind or removing flanking sequences. The pUChsneo transformation vector (Pirrotta, 1988) carries only the inverted terminal repeats of the P element. Between these termini it has been engineered to carry a neomycin resistance gene driven by the Drosophila heat shock promoter hsp70, which serves as a marker for the identification of transformed individuals, and a site where foreign D N A can be ligated. The inverted repeats are joined by 500 bp from the white locus of Drosophila which may function as a hybridization probe to distinguish random integration events from precise P-mediated transposition (Miller et al., 1987). The latter would involve only those sequences within, and including, the inverted terminal repeats of the P element, but excluding the white locus DNA. In our experience, however, this diagnostic feature

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is limited, perhaps because of a low frequency of precise excisions. The site of integration of foreign DNA within the genome, rather than the precision of the mechanism, is likely to be of greater importance to normal gene expression. Transposition of vector sequences and integration into the genome will only be mediated by the terminal repeats if they are supplied with a functional transposase. A helper plasmid, pUChsn(A2-3), which carries the entire P element transposase coding region is therefore introduced with the transformation vector. Although the helper carries most of the P element sequences, it is unable to transpose and integrate into the genome because of a specific deletion in one of its terminal repeats (Steller and Pirrotta, 1985). The sole function of the helper is therefore to supply transposase and, in Drosophila, further injections of helper have been used to switch transposition on in those generations following the initial transformation. Normally, P element transposition occurs only in the germ line of Drosophila because the intron between open reading frames 2 and 3 of the coding sequence is not removed in somatic tissue. However, transposase expression from constructs which have been modified in vitro to remove this intron does allow transposition in somatic tissue. There is now good evidence that similar processing problems prevent normal P element transposition in non-drosophilids (O’Brochta and Handler, 1988) although excision has been observed in mammalian and yeast cells (Rio et al., 1988). If there is a requirement for non-P element encoded proteins to achieve transposition in Drosophila, then the distribution of homologous genes in related genera may explain the divergent results obtained in other insects. The basis of the experimental design for the creation of transgenic mosquitoes is based on that developed for Drosophila melanogaster. Go individuals which survive microinjection with the P element vector/helper DNA, pUChsneolpUChsx(A2-3), are mated inter-se and allowed to produce progeny. These GI individuals are the first which might be expected to express antibiotic resistance throughout all tissues and larvae are therefore subjected to selection with the neomycin derivative, G418. The molecular nature of any transformation events is determined by DNA analysis using radioactively labelled transformation vector DNA to probe Southern blots of genomic DNA extracted from the putative transformants and their progeny (Miller et al., 1987; Morris et al., 1989). Intact vector P elements have been detected in 5 1 0 % of adults that have developed from injected mosquito embryos (Go), confirming that the introduced DNA is not immediately broken down by the mosquito. Furthermore, we have detected the chromosomal integration of vector DNA in several Go individuals. This probably reflects direct incorporation into a proportion of the somatic cell nuclei since it is only in the following generation (GI)

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that we might expect a germ line integration event to have been transmitted to every nucleus. More promisingly, vector DNA has been identified in the chromosomes of the G I and G2 progeny of injected embryos, suggesting that integration has occurred in the germ line of the mosquito and that this DNA shows normal Mendelian inheritance. Some of these events, however, appear to be unstable from one generation to the next and this phenomenon, together with the molecular basis of the transformation events, awaits further investigation. As indicated above, chromosomal integration of the introduced P element DNA has been observed in both Anopheles and Aedes mosquitoes and the integration events appear, in some cases, to be heritable and clearly involve the germ line of the transgenic mosquitoes (Miller et al., 1987; McGrane et al., 1988; Monis et al., 1989). Although these events did not result from normal P element transposition, some functional role of the P sequences can not be excluded. This is particularly true since similar experiments in Lucilia cuprina (Atkinson et al., 1992; T. Howells, personal communication) and Ceratitis capitata (Malacrida et al., 1992; M. Ashburner, personal communication) have failed to produce any integration of vector sequences. Research in other laboratories is now being directed towards the identification of these accessory Drosophila proteins, and the cloning of the genes involved may facilitate high efficiency P transposition in non-drosophilids. However, it is clear from this work and from other experiments involving the transfection of the same DNA into cultured mosquito cells (Lycett et al., 1989) that the P element system in its present form is not suitable for routine use in the mosquito. Thus, whereas the means are currently available for introducing DNA into both mosquito embryonic germ lines and cultured cells, a major stumbling block is the lack of an appropriate, high efficiency, DNA transformation vector system for manipulating the mosquito genome. In addition to the use of the P element-derived DNA vector systems, experiments to assess the mobility of a number of other transposable elements in mosquitoes have been attempted. These experiments have included the use of fully processed cDNA copies of both the Ac and Spm elements from Zea mays (Comley et al., 1992) which have been shown to transpose actively in a number of evolutionarily disparate organisms and may prove to act autonomously in mosquitoes (Kunze and Starlinger, 1989). In addition, experiments involving the introduction of the transposable elements hobo, gypsy and mariner into Anopheles gambiae embryos are currently underway (K. Vernick, personal communication). 3.2.6. The Search for a Mosquito Transformation System It is clear that the germ line integration events so far observed in mosquitoes do not involve normal P element transposition. The absence

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of this controlled mobility poses certain limitations, for example with respect to transposon tagging for functional cloning (see Section 3.3.1). Research elsewhere is concentrating on the precise mechanism of P transportation and attempts are being made to modify the P element system for more general use (O’Brochta, 1990). Such research may yet lead to the “universal vectors” originally envisaged. At the same time, there is the possibility that P elements may never function as efficient transposition-mediated transformation vectors in mosquitoes. This concern has stimulated the search for alternative systems for the transformation of mosquito vectors of malaria. (a) The FLPIFRT recombinase system in mosquitoes. Low frequency illegitimate recombination events such as those isolated from previous attempts to transform mosquitoes could be utilized effectively if the integrated sequence served as a target for a heterologous high frequency recombination system. Morris et al. (1991) have reported the activity in mosquito embryos of a yeast recombinase, FLP, acting on a specific target DNA sequence, FRT, isolated from the yeast 2 pm plasmid. In a series of experiments, plasmids containing the FLP recombinase under the control of the Drosophila melanogaster hsp70 heat shock promoter were coinjected with target plasmids containing F R T sites. FLP-mediated recombination was detected between: (i) target sites located on separate plasmids resulting in the formation of dimers or higher order multimers and (ii) target sites located on the dimers reformed in (i), leading to resolution of the dimers to their original monomeric forms. Synthetic FRT sites were also used and gave rise to similar results to those obtained using the FRT sites originally isolated from the yeast 2 pm plasmid. This successful demonstration of yeast FLP recombinase activity within the mosquito embryo suggests a possible future application of this system in establishing transformed lines of mosquitoes. (b) Transposable genetic elements in the mosquito genome. The isolation of endogenous transposable genetic elements may ultimately prove central to the development of efficient transformation and transposon tagging systems in mosquitoes. A number of approaches have been taken to identify such mobile elements and one of these was to analyse specific gene systems, such as the ribosomal DNA of mosquitoes, in an attempt to isolate variants of these genes which may have arisen from the insertion of transposons. No such insertions have, as yet, been identified in Aedes aegypti DNA (Gale and Crampton, 1989) but insertion events have been detected in the rDNA of Anopheles gambiae and these elements are being fully defined (Paskewitz and Collins, 1989). The elements appear to resemble a particular class of mobile element known as non-viral retroposons. It is unlikely, however, that these elements will prove useful as transformation vectors because of the ill-defined nature of their mode of

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transposition. We have recently adopted an alternative strategy to identify directly a specific class of mobile elements, known as retrotransposons, in the mosquito DNA. The approach relies on utilizing the characteristic biochemical and structural properties of these elements. This work has led to the successful isolation of several retrotransposon-like elements from the Aedes aegypti genome (Crampton et al., 1990a, b). More recently, we have used the polymerase chain reaction (PCR) to develop a particularly rapid methodology for identifying endogenous retrotransposon-like elements in mosquito DNA (Warren and Crampton, 1991). A similar approach has also been employed to identify sequences related to the mariner transposable element from Drosophila mauritiana in the genomes of a wide range of insect vector species, including Anopheles gambiae (Robertson, 1993). However, all the mariner-like elements which have been identified in Anopheles gambiae appear to be defective and thus not capable of transposition or mobilization. Attempts are underway to identify a functional element, and to construct a functional element from the available mosquito-derived mariner sequences. Once such elements have been isolated, fully characterized, and their ability to transpose autonomously established, they may be engineered to form the core of a transformation vector system. (c) Markers for the selectionlidentijication of transformed individuals. A very important feature to be incorporated into a DNA transformation vector for use in mosquitoes is an improved selectable marker system for the identification of transformed individuals. A number of research laboratories have now reported problems with the existing neomycin resistance technique. In our experience, the main problem is the low activity of the neomycin phosphotransferase produced by the Tn5 neo gene in the transformation vector. The system is also less than satisfactory in that mosquitoes exhibit a spectrum of sensitivities to G418 and the survival of transformed mosquitoes relies on high levels of expression of the resistance gene. In addition, Aedes aegypti cells appear to have a tendency to retain intact vector plasmids which do not integrate into the recipient chromosomes but which transiently express antibiotic resistance. Alternative selectable markers, including the Tn903 neomycin resistance gene, which has been reported to show much higher phosphotransferase levels in yeast cells (Langhinrichs et al., 1989), and hygromycin resistance, together with alternative promoters to drive their expression, are now being investigated. A considerable body of work now suggests that the Drosophila heat shock promoter currently incorporated in the P element transformation vector constructs, is not entirely satisfactory for driving the expression of selectable marker gene systems in mosquitoes (Lycett et al., 1989). Ultimately, therefore, cloned phenotypic markers available for use in transgenic mosquitoes, such as the eye colour mutations used routinely

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in Drosophila, would appear to offer the most efficient selection system for incorporation into a mosquito DNA transformation vector. Eye colour mutations exist in both Aedes and Anopheles, and recently the gene coding for white has been isolated and characterized from Anopheles gambiae (F.H. Collins, personal communication). (d) Stage- and tissue-spec@ promoterlenhancer sequences. At some stage it will be desirable to express defined genes in mosquitoes and other insects in a tissue- or stage-specific fashion. For this to be envisaged, stageand tissue-specific promoters have to be defined. None are, as yet, available but attempts to characterize the DNA sequences responsible for expressing certain genes, particularly in mosquito systems, are underway by identifying genes which are expressed in a tissue specific fashion and then defining the upstream, putative tissue-specific promoter sequence. One example of this approach, has been the identification of an Aedes aegypti sequence which is only expressed in the female salivary gland (James et al., 1989). The expectation is, therefore, that this will allow the definition of a salivary gland-specific promoter sequence which may eventually allow the controlled expression of an introduced gene sequence in this tissue. More recently, trypsin genes have been cloned and characterized from Aedes aegypti (Barillas-Mury et al., 1991), the blackfly, Simulium vittatum (Ramos et al., 1993) and Anopheles gambiae (Muller et al., 1993). In each case, the expression of one or more of these trypsin genes has been shown to be induced in the insect midgut by a blood meal. It is therefore likely that gut-specific, blood meal-inducible promoters will shortly be available for each of these insects. Such promoters are clearly of interest as they will allow the expression of antiparasitic agents in the insect gut when it takes a blood meal, that is, when the insect first comes in contact with the organisms which it can transmit to the human population. However, it will be necessary to develop methods for establishing the functionality of putative promoter sequences. To this end, we and others have begun to develop the methodologies for transfecting mosquito cells in culture. The simple, controlled environment of cultured cells allows one to follow the expression of cloned genes, and so delineate promoter and enhancer sequences. Genetically manipulated cell cultures are also able to over-produce specific proteins which facilitates their isolation and purification. Cultured mosquito cells have been used to examine different transfection techniques and vectors and to help establish a suitable system for germ line transformation of Aedes aegypti (Lycett, 1990). Initially, experiments involved introduction of the P element vector and helper constructs into several cell lines by a variety of techniques devised to generate transient cell membrane pores, including calcium phosphate precipitation (Wigler et al., 1977), dextran sulphate (Lopata et al., 1984), polybrene (Durbin and

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Fallon, 1985), electroporation (Chu et al., 1987) and lipofection (Felgner et al., 1987). Much of this work has concentrated on the immortal Mos20 fibroblast cell line which was derived from minced, trypsinized, neonate larvae of the Aedes aegypti London strain in 1969. Polybrene and electroporation mediated transfection have proved to be most successful for these cells, producing approximately 30 and 4000 transformants per lo6 cells, respectively. Subsequently, constructs incorporating the chloramphenicol acetyl transferase (CAT) reporter gene system have been utilized to optimize expression of the CAT gene under the control of the Drosophila heat shock promoter, hsp70, in the Mos20 mosquito cultured cells (Lycett et al., 1989, 1992; Lycett and Crampton, 1993). This type of approach, in addition to experiments utilizing a range of reporter genes and microinjection of embryos, will eventually allow fully functional constitutive, stage- or tissue-specific mosquito promoters to be defined. 3.3. The Potential Application of Transgenic Technology to Malaria Vectors

Once the systems necessary to create transgenic mosquitoes have been developed, how may this technology be applied? Two aspects will be discussed in order to illustrate the potential of the technology. The first deals with the use of the technique for analytical purposes and the second with applying transgenic technology to medically significant mosquito populations. 3.3.1. Transgenic Technology as an Analytical Tool

The introduction and insertion of a mobile genetic element at or near a particular locus can cause that allele to mutate producing a structural or developmental effect. In Drosophila in particular, transposable genetic elements (TGEs) have been used as mutagens in order to clone genes or gene clusters of interest via transposon tagging (Bingham et al., 1981; Searles et al., 1982). In essence, the T G E is introduced into the germ line of the insect by microinjection of the embryo, and the progeny scored for mutants in the phenotype of interest. Subsequently, cloned T G E probes are used for in situ hybridization to chromosomes of mutant and wild-type individuals. This identifies a TGE “newly” integrated at or near the genetic locus of interest. DNA clones are then retrieved from a genomic library prepared from the mutant stock using the TGE DNA as a probe. DNA sequences adjacent to the TGE in such clones represent the gene for the locus of interest. This approach is an extremely powerful application of the technology, as it allows the cloning of genes purely on the basis of their function.

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3.3.2. Genetic Manipulation of Malaria Vectors In the long term, perhaps the most exciting applications of transgenic technology will be in malaria vector populations in the field. Clearly, the type of manipulation to be envisaged will depend on the target insect and the scale of its medical impact. For example, transgenic technology may eventually have a role to play in controlling malaria by providing the means to suppress vector populations by rendering them vulnerable to subsequent control measures, such as insecticide susceptibility, temperature sensitivity o r ability to survive diapause. A second possibility and, perhaps, a more exciting approach, would be to alter the ability of the insect to transmit the disease.

3.3.3. Potential Target Genes for Manipulation Several types of useful target gene can be envisaged. There are those that render populations vulnerable to subsequent control measures (such as insecticide susceptibility, temperature sensitivity or inability to survive diapause); those that interrupt disease transmission by replacing vector with non-vector forms, and those that disrupt normal fertility, development or behaviour. In this respect there are a number of obvious targets for manipulation including the genes involved in the mosquito immune system, developmental control genes, genes influencing mosquito behaviour and insecticide resistance genes. Changes in esterase activity are correlated with insecticide resistance in a number of insects and the esterases which are involved in organophosphate resistance in Culex quinquefasciatus have been cloned and characterized (Mouche et al., 1986; Merryweather et al., 1990). In addition, the Aedes aegypti cytochrome P-450genes which are involved in more general detoxification mechanisms have now been isolated (Bonet et al., 1990). A number of genes are of particular interest because they are directly implicated in the ability of the insect to transmit parasites. Examples include the filarial susceptibility (p") and Plasmodium susceptibility (pls) loci of Aedes aegypti. The p" locus is genetically well defined and we have good data on its linkage relationships. Refractoriness to infection is due to a partially sex-linked, dominant gene (Macdonald and Ramachandran, 1965). There is marked variation in the susceptibility of this mosquito to different filarial worms, although all of the alleles concerned map at about the same place on the sex chromosome. Also of particular interest is a strain of Anopheles gambiae which has been selected for refractoriness to the malaria parasite and characterized genetically (Collins et al., 1986). However, no gene or gene product has yet been defined or identified at the molecular level which controls susceptibility of the vector to a parasite. Attempts are currently underway to clone these genes but it is difficult to

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undertake such a cloning excercise in the absence of any knowledge of the gene product. Clearly, the use of transgenic technology through transposon tagging will assist in the characterization of refractory genes and their products. 3.3.4. Creating an “Incompetent” Mosquito

An important genotypic characteristic not met by the majority of genes encoding refractoriness is that any such gene introduced into the mosquito would have to be capable of altering phenotype through the expression of a single gene copy. Unfortunately, at present, there is no gene or gene product defined at the molecular level which is known directly to affect phenotype in relation to parasite development in, or transmission by, mosquitoes. In contrast, a number of molecules are known to affect the transmission of malaria by anophelines. Foremost among these are the socalled transmission blocking vaccines, which can achieve a total transmission blockade (Winger et al., 1987). These vaccines attack antigens present on the gametes and ookinetes of the malaria parasite and antibodies which recognize these antigens are able to block the development of the parasite in the mosquito midgut. A very exciting possibility, therefore, is to introduce the genes coding for such antibodies into the mosquito genome thus directly conferring the transmission blocking phenotype to the insect. In this case, a transgenic mosquito would be created incorporating an antibody gene expressed in the insect midgut in response to a blood meal, and which therefore blocks transmission of malaria. This type of approach is attractive for a number of reasons. It eliminates the need for the detailed molecular analysis of refractory mechanisms in mosquitoes and it would be a “dominant” gene system (i.e. one gene copy only would be needed in each cell of the mosquito). The antigen targets on the stage of the malaria parasite present in the mosquito are highly conserved, suggesting that the parasite may be less able to avoid this type of transmission control mechanism. Finally, the use of transgenic insects incorporating an antibody gene could be applied to any vector transmitted pathogen (parasite or viral) where a target antigen can be identified as being inhibited by the expressed molecule. To date, mouse antibody genes have been cloned and introduced into mosquito cells in culture and mouse Fab molecules have been expressed and detected using immunohistochemical staining techniques. Efforts are currently underway to clone the transmission blocking antibody genes themselves and to assess their functionality in vivo. The trypsin genes from the mosquito Anopheles gambiae have recently been cloned (Muller et al., 1993) and the expression of two of the genes have been found to be induced in the female mosquito midgut when it takes a blood meal. It may

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therefore be possible in the near future to create a mosquito expressing the transmission blocking antibody genes in such a way as to block or disrupt the transmission of malaria. If successful, transgenic mosquitoes expressing antimalarial antibodies may represent a potential strategy for controlling malaria and may establish a precedent for a wide range of new antidisease strategies. 3.3.5. Transgenic Mosquitoes in Natural Populations

Now that the technology for the introduction of artificial genes is becoming established, it is necessary to consider the problems likely to be faced in experimental and natural populations. It may well be that such a situation would disrupt the normal adaptive process and therefore be opposed by natural selection. If this is so then some form of drive mechanism may be needed to force the desired gene through the population. This is not an alien concept to those who have worked on the genetic control of insect populations. However, the testing of such mechanisms has been limited since, in reality, they have awaited the advent of recombinant DNA technology to provide the necessary raw material. Two types of drive mechanism have been suggested. One is meiotic drive, where a given chromosome is transmitted to more than the expected 50% of offspring. Any desirable genes linked to the driven chromosome would eventually approach fixation even with the release of relatively few individuals. There is experimental evidence to support the use of meiotic drive in Aedes aegypti. This mechanism, driven by the MD locus has been used to force the marker gene re (red eye) into a laboratory cage population (Wood et al. , 1977). Interestingly, meiotic drive also occurs during hybrid dysgenesis and it might, therefore, also be possible to exploit this phenomenon by using P elements in Aedes aegypti. The second type of drive mechanism is the exploitation of genetic traits that reduce heterozygote fitness (Curtis and Graves, 1988). For example, the gene to be driven could be introduced into a translocation chromosome such that viable and fertile homozygotes were formed, whereas heterozygotes would display reduced fertility or viability. In this way, translocations, pericentric inversions, inter-racial hybrid sterility, cytoplasmic incompatibility and compound chromosomes all have potential since, in each case, hybrids have reduced fitness. Such mechanisms require larger release numbers since there is no exponential increase in the frequency of the driven chromosome as with meiotic drive. However, fixation of desirable genes would occur more quickly than with meiotic drive because of the reduced fitness of heterozygous combinations. Efficiency could be improved by providing the released individuals with some form of temporary advantage. For example, insecticide resistance could be incorporated into the genome

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and then insecticide applied (Whitten, 1970). Ideally, the insecticide resistance gene would be fused to the desirable gene and introduced as a unit to prevent disruption of useful combinations by meiotic recombination. The most useful end result of such programmes would be the progressive replacement rather than the eradication of disease-transmitting populations since an emptied ecological niche might be colonized rapidly by migration of wild types. 3.4. Transgenic Mosquitoes: The Future in Relation to the Control of Malaria

Eventually, embryo transformation will provide the raw material to test the proposed drive mechanisms in laboratory and natural populations. The questions posed by considering the release of transgenic insects emphasizes the need to assess the biological consequences of such a release. It is, however, difficult to gauge the possible hazards of such a release in the absence of experimental evidence and these ethical and safety considerations need to be faced at an early stage. In order to undertake an informed appraisal where the possible net benefits may be balanced against the potential hazards, considerable effect will have to be devoted to utilizing caged populations and the controlled release of molecularly tagged individuals together with mathematical modelling of these populations. There is clearly some way to go before any release of transgenic insects can be considered. The power of the technology is, however, so enormous that it must be explored and there is every indication that over the next few years the potential of transgenic technology in insects will be fully exploited. ACKNOWLEDGEMENTS

This investigation received financial support from the UNDPNirorld BanM WHO Special Programme for Research and Training in Tropical Diseases, the Wolfson Foundation, Wellcome Trust, Medical Research Council and Liverpool University. The author is a Wellcome Trust Senior Research Fellow in Basic Biomedical Sciences. REFERENCES Argentine, J.A. and James, A.A. (1993). Codon preference of Aedes aegypti and Aedes albopictus. Insect Molecular Biology 1, 189-194. Arnold, M.L., Buckner, C.M. and Robinson, J.J. (1991). Pollen-mediated introgression and hybrid separation in Louisiana irises. Proceedings of the National Academy of Sciences, USA 88, 1398-1402.

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Saunders, R.D.C., Glover, D.M., Ashburner, M., Siden-Kiamos, I., Louis, C., Monastirioti, M., Savakis, C. and Kafatos, F.C. (1989). PCR amplification of DNA microdissected from a single polytene chromosome band: a comparison with conventional microcloning. Nucleic Acids Research 17, 9027-9037. Searles, L.L., Jokerst, R.S., Bingham, P.M., Voelker, R.A. and Greenleaf, A.L. (1982). Molecular cloning of sequences from a Drosophila RNA polymerase XI locus by P element transposon tagging. Cell 31, 585-592. Siden-Kiamos, I., Saunders, R.D.C., Spanos, L., Majerus, T., Trenear, J., Savakis, C., Louis, C., Glover, D.M., Ashburner, M. and Kafatos, F.C. (1990). Towards a physical map of the Drosophila melanogaster genome: mapping of cosmid clones within defined genomic divisions. Nucleic Acids Research 18, 626145270. Spradling, A.C. and Rubin, G.M. (1982). Transposition of cloned P elements into Drosophila germ line chromosomes. Science 218, 341-347. Steller, H. and Pirrotta, V. (1985). Transposable P vector that confers selectable G418 resistance to Drosophila larvae. EMBO Journal 4, 167-171. Sturchler, D. (1989). How much malaria is there Worldwide? Parasitology Today 5, 39. Warren, A.M. and Crampton, J.M. (1991). The Aedes aegypti genome: complexity and organisation. Genetical Research (Cambridge) 58, 225-232. White, G.B. (1982). Malaria vector ecology and genetics. British Medical Bulletin 38, 207-212. Whitten, M.J. (1970). Use of chromosome rearrangements for mosquito control. In “The Sterility Principle for Insect Control or Eradication”, pp. 399-410. International Atomic Agency Symposium, Athens. Wigler, M., Siverstein, S., Lee, L.S., Pellicer, A,, Cheng, Y. and Axel, R. (1977). Transfer of purified Herpes virus thymidine kinase gene to cultured mouse cells. Cell 11, 223-232. Wilkerson, R.C., Parsons, T.J., Albright, D.G., Klein, T.A. and Braun, M.J. (1993). Random amplified polymorphic DNA (RAPD) markers readily distinguish cryptic mosquito species (Diptera: Culicidae: Anopheles). Znsect Molecular Biology 1, 205-21 1. Williams, J.G.K., Kubelik, A.R., Livak, K.J., Rafalski, J.A. and Tingey, S.V. (1990). DNA polymorphisms amplified by arbitrary primers are useful as genetic markers. Nucleic Acids Research 18, 65314535, Williams, J.G.K., Rafalski, J.A. and Tingey, S.V. (1993). Genetic analysis using random amplified polymorphic markers. Methods in Enzymology 218, 704-740. Winger, L., Smith, J.E., Nicholas, J., Carter, E.H., Tirawanchai, N. and Sinden, R.E. (1987). Ookinete antigens of Plasmodium berghei: the appearance of a 21 kD transmission blocking determinant on the developing ookinete. Parasite Immunology 10, 193-207. Wood, R.J., Cook, L.M., Hamilton, A. and Whitelaw, A. (1977). Transporting the marker gene re (red eye) into a laboratory cage opulation of Aedes aegypti (Diptera: Culicidae), using meiotic drive at the M locus. Journal of Medical Entomology 14, 461-464. Zheng, L., Saunders, R.D.C., Fortini, D., della Torre, A., Coluzzi, M., Glover, D.M. and Kafatos, F.C. (1991). Low resolution map of the malaria mosquito, Anopheles gambiae. Proceedings of the National Academy of Science USA 88, 11187-11 191.

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The Ribosomal RNA Genes of Plasmodium Andrew P.Waters

Department voor Parasitologie. Rijksuniversiteit te Leiden. Leiden. The Netherlands

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.1. Molecular biology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.2. Molecular phylogeny . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.3. Diagnostics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

34 34 34 34

2. Molecular Biology of rRNA Genes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Life cycle considerations . . . . . . . . . . . . .............. 2.2. Organization of nuclear rRNAgenes . . 2.3. Sequence and expression of the rDNA units of Plas 2.4. Transcriptional control of rRNA gene expression in 2.5. Genetic control of expression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.6. Transcription factors and rDNA . . . . . . ........................... 2.7. 5 s RNA genes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.8. Organellar rRNA genes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.9. The production of mature rRNA: processingof the primary transcript . . . . . 2.10. Ribosomes . . . ............................................ 2.11. Plasmodium ribosomal RNAsequences . . . .......................

35 35 37 40 42 45 46

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3 . Inferences of Phylogeny Based on Ribosomal RNAAnalyses . . . . . . . . . . . . . . . . . 58 . . . . . . . . . . . . 58 3.1. Earlier classifications . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. Molecular-based estimations . . . . . . . . . . . . . . . . . . 3.3. Evolution of the rDNA units within the genome . . . . . . . . . . . . . . . . . . . . . . . . 64 4 . Species Identification Based on Ribosomal RNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1. The principle of direct detection of rRNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2. Ribosomal RNAgene-based PCR . . . . . . . . . . . . . . . . . . . . . . . ... ... 4.3. Drug testing and rRNA diagnosis . . . . . . . . . . . . . . . . . . . . . . .

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5. Perspective . . . . . . . . . . . .

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References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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ADVANCES IN PARASITOLOGYVOL 34 ISBN (L12431734-5

CopyrrghtO lW4 Academic Press Limited A / / rights of reproduchon in sny form reserved

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1. INTRODUCTION

The study of ribosomal RNA (rRNA) genes offers the biologist at least three possible research areas: (1) molecular biology; (2) molecular inference of phylogeny based upon molecular sequence data; and (3) rRNA-based specific detection of organisms which can be used diagnostically. 1.1. Molecular Biology

This first area concerns the function of the rRNA genes and that is to provide the major portion of the RNA components of the ribosome which is the protein synthetic apparatus of the cell. The ribosome consists of a highly ordered and conserved proteidRNA complex which acts as the catalytic centre for the many ordered chemical reactions necessary for protein formation. Thus the processes of ribosomal RNA (rRNA) production from the genes that encode it, the post-transcriptional modifications, its export from the nucleus and its assembly into active ribosomes is a window into the molecular biology of any particular cell type. Much has been learned about these processes from the study of a great number of systems and the current knowledge of the molecular biology of rRNA genes in Plasmodium will be highlighted against that from other chosen systems. 1.2. Molecular Phylogeny

The ribosome is responsible for protein synthesis in all cells and some organelles with the general exception of viri. The constancy of this role in the cell has resulted in a generally conserved structure/function relationship between all equivalent rRNA molecules. This is especially true as the perhaps unexpected centrally catalytic nature of the rRNA molecules within the ribosomal processes emerges (Dahlberg, 1989). This constancy, which covers both prokaryotes and eukaryotes, is in turn exploited by those with an interest in phylogeny. The small subunit (SSU) rRNA molecule provides a dataset of which a large part is structurally conserved in all organisms. By comparing the structurally conserved regions of the SSU rRNA genes of different organisms it is possible to draw inferences of phylogeny. 1.3. Diagnostics

The RNA product of the ribosomal RNA genes is generally the most abundant macromolecule in any given cell. If it is possible specifically to

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detect the rRNA of a particular organism then it is possible to use that detection in a diagnostic manner. The abundance of the rRNA in the cell also provides the potential for great sensitivity of detection. It has proved possible to exploit all of these avenues in the study of Plasmodium and this review will provide a summary of progress of research and attempt to place the findings in the context of the biology and known natural history of the genus.

2. MOLECULAR BIOLOGY OF rRNA GENES 2.1. Life Cycle Considerations

Normally it can be taken for granted that readers of a focused review such as this are sufficiently familiar with the organism under discussion as to render consideration of its life cycle redundant. In the case of Plasmodium, the molecular biology of the rRNA genes is apparently so linked to the biological progession of the life cycle that it is illuminating to preface discussion of the genes with a short description of the life cycle of this genus. The members of the unicellular apicomplexan parasitic genus Plasmodium employ a dichotomous life style existing in association with an invertebrate (usually Anopheline) mosquito vector and a vertebrate host (Figure 1). The individual species within this genus are responsible for the various forms of the disease malaria. The range of vertebrate host species affected by different malaria species is enormous and worldwide; primates and lower mammals, birds and reptiles are all susceptible to different species of the parasite. A general description of the life cycle can be considered as follows: vertebrate infection is initiated by mosquito bite (Figure 1, I). A fully differentiated form of Plasmodium, the sporozoite, is released into the vertebrate blood stream from the store in the salivary glands of the mosquito. In mammalian malaria the sporozites clear rapidly to the liver entering hepatocytes (Figure 1, 11). Within the hepatocyte the parasite undergoes a period of differentiation and multiplication giving rise to a mature form, the extra-erythrocytic schizont, which contains within it thousands of individual parasite forms, merozoites. These are released into the bloodstream, initiating the second blood-borne phase of the infection (Figure 1, 111). The merozoites invade bloodstream erythrocytes and also undergo a characteristically periodic phase of intracellular differentiation and asexual multiplication that involves progression through distinct morphologies culminating in the production of an erythrocytic schizont which again contains merozoites but usually only 10-32. Upon reinvasion, the merozoite

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ANDREW P. WATERS

I

SPoRoZo'TES

3

IN MOSQUITO

0 UT

Figure 1 Life cycle of Plasmodium spp. The life cycle of Plasmodium spp. is illustrated as its passage through the human host. The roman numerals (I-VI) indicate the reference points in the life cycle mentioned in the text. The symbols A and C represent expression of the different forms of the small subunit ribosomal RNA genes through the life cycle. A and C are capitalized where they represent the major expressed form and are given in lower case when they are the minority form. [C] indicates the precursor form of the C SSU rRNA gene observed in gametocytes of P . falciparum (Waters et al., 1989).

THE RIBOSOMAL RNA GENES OF PLASMODlUM

37

can either reinitiate the asexual erythrocytic cycle or embark upon sexual differentiation and the process of gametocytogenesis. Gametocytogenesis involves the production of gametocytes (male and female) which are dormant gamete precursors (Figure 1, TV). Once formed, these forms are relatively stable and remain dormant until ingested by the mosquito as part of the blood meal during feeding. Once in the acidic environment of the insect midgut the gametocytes are triggered into final sexual differentiation forming the gametes which then fertilize to give the zygote and initiate the insect phase (Figure 1, V). The zygote develops into an ookinete which is motile and penetrates the midgut wall entering the lumen forming the oocyst (Figure 1 , VI). Here, reminiscent of schizogony, the oocyst again undergoes differentiation and multiplication resulting in the formation of a cell which upon rupture releases thousands of sporozoites which make their way to the salivary gland thus completing the cycle. From this it is clear that the progression of the parasite through its life cycle is a tremendous feat of biology involving the invasion of multiple tissues and structures of both hosts which is reflected in the various distinct and complex physical forms that the parasite displays. These forms and progressions result from the coordinated stage-specific expression of genes which is itself superimposed on a background expression of housekeeping genes. What might be anticipated about the biology of the parasite housekeeping from its life cycle? It has to perform the fundamental processes common to all cells in a variety of environments. The clearest distinction between these environments is at the simple level of host and vector., The host, usually warm blooded (or sun basking), offers a completely different set of environmental influences and selective pressures to the cold-blooded anopheline mosquito. It has become apparent through study of the ribosomal RNA (rRNA) genes of malaria parasites that this fluctuating environment may have provided the stimulus for a number of peculiar and unique phenomena. 2.2. Organization of Nuclear rRNA genes

2.2.1 General There are a number of structural ribosomal RNA molecules which are involved in the formation of an active eukaryotic ribosome. These are usually identified by their size and sedimentation properties as 28S, 18S, 5.8s and 5 s . The genes encoding these structural RNA species have a typical eukaryotic arrangement that is found in both lower and higher organisms. The major rDNA unit is polygenic and encodes three structural rRNA molecules (28, 18 and 5 . 8 s ) and will be discussed first. The genes

38

ANDREW P. WATERS

Typlcal Eukaryotlc rDNA Unit Arrangement

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THE RIBOSOMAL RNA GENES OF PLASMODlUM

39

within the unit are always identically disposed, separated and flanked by spacer regions (Figure 2A). The gene order within the unit is thus External Transcribed Spacer (ETS), 18S, Internal Transcribed Spacer 1 (ITSl), 5.8S, ITS2, 28s and ETS. The unit is transcribed as a single polygenic transcript. The 18s rRNA molecule is also called the small subunit (SSU) rRNA and the 28s the large subunit (LSU) rRNA according to the particular ribosomal subunit they are assembled into. There is also a typical higher order organization of the rDNA units themselves as they are dispersed usually at a single genomic locus in large tandem head to tail arrays. The copy number of the individual units is high and can vary from 100s to 1000s. Each unit usually has intrinsic promoter and terminator information although adjacent units may affect transcription of their neighbours (e.g. Xenopus luevis, De Winter and Moss, 1986; Labhart and Reeder, 1986). The genomic environment of each rDNA unit is actively homogenized such that hybridization of an rRNA probe to Southern blots of restricted DNA usually gives a single very intense band of hybridization (Dover, 1982; Flavell, 1986). 2.2.2. Plasmodium The overall organization of the rDNA units has been studied in most detail in Plasmodium berghei and data will be presented mainly from this system. Figure 2 Genomic organization of the nuclear and 35 kb circle encoded rRNA genes. A. General organization of eukaryotic rDNA units. These are shown as a tandem array. The expanded region shows the typical arrangement of the structural genes within an individual unit. This detail of the unit organization is given for the P. berghei A unit which is typically eukaryotic. It must be emphasized that this is for illustration purposes only, the unit does not exist as part of a tandem array in Plasmodium but as disparate units arranged in the genome as shown in B. Restriction endonuclease cut sites are indicated as follows: E = Eco RI, N = Nde I, Ns = Nsi I, H3 = Hind 111, RV = Eco RV. ITS indicates internal transcribed spacer and ETS, external transcribed spacer. B. Organization of the rDNA units of P. berghei. The genomic location of the four rDNA units on the 14 chromosomes of a reference clone of P. berghei. The same organization has been found in all clones and lines of P. berghei with one exception (Janse er al., 1992). Note the location of the 5s RNA genes on chromosome 12. The crude organization of the individual units is given to the right of the chromosome map. Restriction enzyme sites are indicated as before. Additional sites are: K = Kpn I, Pv = Pvu I, P = Pst I. Gene abbreviations are: CSP circumsporozoite protein; DHFR dihydrofolate reductase; PbS21 ookinete specific 21 kDa protein of P berghei (Paton et al., 1993). C. Organization of the rRNA genes on the 35 kb circle of Plasmodium. The organization is illustrated for P. fakiparum and is modified from Gardner et al. (1993), with permission. The relative position and direction of transcription of the LSU and SSU genes is given. The positions of tRNA genes (t) between units is indicated and the fragments given by Hind I11 digestion of the circle are numbered at the top. The asterisk marks the region of unique sequence which is flanked by the invert repeat regions which contain the rRNA genes.

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ANDREW P. WATERS

Other species of Plasmodium will be referred to at appropriate points in the text. Dame and McCutchan (1983a, b) demonstrated the overall organization of the rDNA units of the NYU strain of P. berghei and showed that the organization of the units differed from the normal in two remarkable ways. Although the gene order within each individual unit was that of a typical eukaryote as described above, both the copy number and the higher order organization of the units relative to one another were unusual. Copy number analysis demonstrated that there were only four units per haploid genome and that they were unlinked (no closer than 150 kbp) to one another. Each of the four units (termed A , B, C and D) had a related but unique array of restriction enzyme sites and were subsequently shown to be on separate chromosomes (A.P. Waters and C. Janse, unpublished Figure 2B). Other species of Plasmodium all seem to have a greater copy number than the rodent malarias (all the rodent malarias appear to have a total of four units each one on a separate chromosome in the main homologous to those in P. berghei, A.P. Waters and C. Janse). The typical estimation appears to be between 8 and 10 copies (Langsley et al., 1983; Unnasch and Wirth, 1983). The restriction maps of the four units suggested a further subdivision could be made recognizing conserved features between the A and the B units (A-type) and a similar pairing of the C and D units (C-type). The terminology of the genes for P. fafciparum is somewhat confusing since they were also referred to in their order of characterization. The A SSU rRNA gene of P. fakiparum is equivalent in its expression pattern to the A gene from P. berghei but the B gene of P. fafciparum is equivalent to the C gene of P. berghei (McCutchan et al., 1988). The terms A and C will be used throughout this review (precedented in Waters et al., 1989). 2.3. Sequence and Expression of the rDNA Units of Plasmodium

2.3.1. Sequence of the SSU Genes Two fragments of the P. berghei rDNA were sequenced, each representing an SSU rRNA gene, one A and the other C. When the compiled sequences were compared with each other the results of the restriction mapping were borne out but gave more detail. The sequences of the genes were 96.5% identical; however, the sequences representing the 3.5% difference between the two genes were clustered rather than randomly dispersed throughout the genes (Gunderson et al., 1986, 1987). These unit specific regions lay in expansion regions of the SSU rRNA molecule, that is regions whose sequence complexity is not necessarily conserved between any pair of organisms (Clark et al., 1984). These regions lie outside the conserved

THE RIBOSOMAL RNA GENES OF PLASMODIUM

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core sequence and have unknown functions. Secondary structures could be predicted, however, which were more or less equivalent (Gunderson et al., 1987; Waters et al., 1991), structures that are supported by compensatory mutations a criterion that is applied as a test of validity (Hancock et a/., 1988). The clustering of the sequence differences between the two units in non-conserved regions of the SSU molecule had two effects; one was that both units contained all the known information to form active subunits that could be assembled into ribosomes and the second was that it was possible to synthesize oligonucleotide probes complementary to the regionalized differences as tools to dissect the pattern of expression of the two units. Two probes in particular, TM3 (C specific) and TM4 (A specific), were used to study the pattern of expression of the C- and A-type units. Both probes detect both rDNA units of their subclass (i.e. C- or A-type). When hybridized to preparations of total RNA from the various stages of the life cycle of the parasite it was clearly seen that the A-type gene was preferentially expressed in asexual parasites at a ratio of 20:l of A:C and that the ratio was more than reversed in oocyst RNA purified from infected mosquitoes (it is actually very difficult to observe A gene expression in sporozoites) (Gunderson et al., 1987). Direct RNA sequencing from the appropriate parasite forms confirmed the hybridization data. The pattern of rRNA gene expression is summarized in Figure 1 superimposed on the life cycle. All Plasmodium species so far investigated have been shown to have two distinct types of rRNA gene which display varying degrees of regionalized sequence similarity (range 83-97%). Oligoprobes directed at these differences have also always shown a similar pattern of differential gene expression. Currently this holds true for rodent, avian, primate and human malarias. 2.3.2. Sequence of the LSU Genes Two large subunit rRNA genes have been sequenced from P. falciparum and show the same pattern of regionalized differences (A.P. Waters, J.-D. Li and T.F. McCutchan, unpublished) and represent members of the C- and A-type units. The asexually expressed molecule is predicted to be -3800 nt long and again contains all the conserved regions expected of a functional molecule and folds into an appropriate secondary structure. Confirmation that this unit was expressed in asexual bloodstage forms was obtained by comparison with cDNA clones. The asexually expressed gene corresponds in its restriction pattern to the previously identified pPFrib2 unit (Langsley et al., 1983). The second clone that has been sequenced (A.P. Waters, J.-D. Li and T. McCutchan, unpublished) does not contain the complete LSU rRNA gene but does resemble pPFribl and has every

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ANDREW P. WATERS

restriction site reported by Langsley et al. (1983). However, it also has additional sites for the same enzymes. Nucleotides which are involved in recognition of the assembled small subunit to give the functional ribosome are also conserved. The 5’ region of the asexually expressed LSU molecule has appropriate complementarity with the 5.8s RNA molecule forming a functional hybrid that is typically eukaryotic (Gutell and Fox, 1988). The sequence of the LSU genes from P. berghei is largely uncharacterized, however it has been shown that the mature LSU rRNA species in asexual parasites is naturally nicked to give two species of 3200 and 800 nt respectively (Miller and Ilan, 1978; Dame and McCutchan, 1983b). 2.3.3. Sequence of the 5.8s Genes 5.8s RNA is a eukaryotic specific feature of ribosomes. The prokaryotic equivalent is contiguous with the LSU and 5.8s base pairs at the 5’ end of the eukaryotic LSU (Jacq, 1981). The 5.8s rRNA genes have been divided into five classes on the restriction fragment length polymorphism defined by AT-rich cutting restriction enzymes. Four gene types have been sequenced and show variation at the 3‘ end of the structural molecule (Shippen-Lentz et al., 1987,1990). One gene type was shown to be present in the genome 30-fold more abundantly than the other four. A 5.8s gene was also sequenced from the same clone containing the 28s gene (A.P. Waters and T. McCutchan, unpublished) which was most similar (yet not identical) to one of the minor units (unit IV) described by Shippen-Lentz et al. (1990) yet this clone was equivalent to pPFrib2. Since the rDNA unit copy number has been estimated as only 8 (Langsley et al., 1983), there are data which cannot be reconciled at present. The class I1 and IV molecules are capable of hybridizing to the 5’ end of the asexually expressed LSU molecule (A.P. Waters and T. McCutchan, unpublished). 2.4. Transcriptional Control of rRNA Gene Expression in Plasmodium

The demonstration of the ability of the parasite to express different distinct rRNA genes at different points in its life cycle has a number of implications which must be considered in the light of the circumstances of the life cycle. The switch in rRNA expression occurs at points of proliferation in the life cycle resulting in different forms of the parasite using different stagespecific ribosomes. Is there a mechanism that removes one form of the ribosome allowing its replacement with the alternative? Both the ookinete and the newly invaded exoerythrocyctic cell undergo multiple rounds of cell division giving rise to thousands of daughter forms which are either

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sporozoites (C unit expression) or merozoites (A expression), respectively. Therefore, replacement of the ribosome might be more effectively regarded as dilution of the existing ribosome population reducing it to an undetectable level. The C and A genes encoding rRNA must also be differentially expressed and the logical level at which this might be controlled is through the promotion of transcription. These rDNA units might therefore be thought of as switchable, expressed at different stages of the life cycle. So when might the switch in expression occur? As previously discussed the progression of the life cycle involves exposure to two very different environments provided by host and vector. The simplest expectation, but not the only possibility, would be that the switch would take place upon transfer of the appropriate parasite form from vector to host and vice versa. In other words during occupancy of the vertebrate liver and in the insect midgut. It used to be impossible to observe these phenomena in a single Plasmodium species and many of the early studies were performed in a variety of systems using different species. The majority of the data that are now available come from the study of two systems, the P. berghei model in rodents and the human parasite, P. falciparum in culture. 2.4.1. The A to C Switch Using P. falciparum it has been possible to demonstrate that this onset of C gene transcription does indeed take place in a manner linked to the developmental pathway of the parasite. In the gametocytes of this species, a C gene precursor product accumulated which contained the SSU gene. The precursor was some lo00 nt longer than the mature SSU. Oligonucleotide mapping suggested that the extra sequence material was contiguous with the 5’ end of the SSU region, therefore including all or part of the ETS region. The precursor was apparently stable in P. falciparum gametocytes and only minimal processing of the precursor to the mature SSU form was apparent. After activation and fertilization of the gametocytes, however, two events were stimulated; increased transcription of the C gene and processing of the precursor form to give the mature SSU transcript. Culturing of the zygotes allows their continued differentiation and 16 h after fertilization the precursor was still visible but large amounts of the mature transcript were also accumulating. The caveats associated with this work are that it relies on cultured material and that ookinete culture is not a wholly successful undertaking with P. falciparum. Zygote formation is efficient but only a small percentage develop into truly mature ookinetes which can be used to infect mosquitoes. Recently we have been able to repeat these observations using the now highly developed rodent model of P. berghei (Waters et al., 1993~).In this model it is now possible

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to obtain large amounts of pure gametocytes and to activate, fertilise and culture these through to ookinetes which are highly infective to Anopheles stephensi. In this model we see a different pattern of C gene expression during sexual development that occurs in two phases. Increased expression is not visible until 14 h after fertilization in ookinetes and is due to the upregulation of transcription. There is no apparent accumulation of precursor There is a small earlier peak of expression molecules (Waters et al., 1993~). at 20 h after merozoite invasion of erythrocytes when both asexual and sexual cells are trophozoites and morphologically indistinguishable. This may be the equivalent of the C gene expression seen in the developing gametocytes of P. falciparum. Can we reconcile the observed differences in C gene expression during gametocytogenesis in P. berghei and P. falciparum? The two species are quite distinct biologically and phylogenetically. Whereas one would expect the overall features of gametocyte development to be very similar, the duration of development, which takes 26 h in P. berghei and 8 days in P. fulciparum, is quite different. Thereafter the timing of the progression of the life cycle through the vector is quite similar. The appearances of the gametocytes are also quite distinct, those of P. falciparum produce avianlike scythe shaped forms whereas those of P. berghei do not deform the host erythrocyte. The appearance of the nucleus during gametocytogenesis in the two species may also be quite significant. An electron-dense area that has been interpreted as being the usual site of rRNA transcription, the nucleolus, is plainly visible in mature female gametocytes of P. fulciparum (Sinden, 1982), the species which produces the precursor, whereas none is visible in the equivalent forms of P. berghei (Sinden, 1978). The early burst of C-type transcription in P. berghei occurs before gametocytes and trophozoites can be morphologically distinguished. One may question, however, if a nucleolus is to be expected at all given the low copy number of the rDNA units and their disparate location in the genome. All of these differences may result in species specific requirement or preparation for the introduction of C unit transcripts. Both models may simply need more detailed investigation to demonstrate equivalent transcriptional events. 2.4.2. The C to A Switch This has been demonstrated using P. berghei sporozoites isolated from infected mosquitoes which are then used to infect in vitro cultures of human hepatoma cells. Full and efficient development of the liver stage forms occurs in a manner that mimics the development in the liver of the vertebrate host. Using the specific oligonucleotide probes the appearance of the A gene was monitored and was detectable 8 hours after infection

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45

of the liver cells. Some of the parameters of liver stage development were also investigated. Invasion of sporozoites was necessary but not sufficient for switching which required some intracellular development. Irradiated, attenuated sporozoites were also capable of switching to A gene expression demonstrating that attenuated sporozoites develop through at least one observable developmental change (Zhu et al., 1990). This work also demonstrated that simple transition of sporozoites from 18°C to 37°C is not sufficient to trigger the switch in rRNA expression. Interaction of the sporozoite with the hepatocyte and development is required. The evolution of the dual rRNA system is part of a more complex response to the differences in environment provided by host and vector. 2.5. Genetic Control of Expression 2.5.1. General

Promoter elements and enhancers which control the onset and degree of transcription of rRNA genes vary widely in their positioning relative to the SSU. This is due to variation in length of the ETS region which effectively separates the promoter from the SSU. In yeast the 5’ ETS is 696 nt long (Klootwijk and Planta, 1989); in mouse, 4 kb (Miller and Sollner-Webb, 1981). In Saccharomyces cerevisiae the promoter is contained within the 212 nt adjacent to the transcription start site with a bidirectional 190 bp enhancer sequence located a further 2 kbp upstream (Elion and Warner, 1984, 1986; Johnson and Warner, 1989) that also has terminator activities (Kempers-Veenstra et al., 1986; Mestel et al., 1989) and demonstrable regulatory activity (Morrow et al., 1993; Schultz et al., 1993). Linker scanning analysis of the yeast promoter divided it into three domains; 111 (-146 to -76), I1 (-70 to -50) and I(-28 to +8) where 0 is the start of transcription (Kempers-Veenstra et al., 1985; Musters et al., 1989). Disruption of domains I and I1 almost abolished transcription and phase spacing of these two domains was also important implying that a transcription factor(s) might bind simultaneously to both elements (Musters et al., 1989).

2.5.2. Plasmodium Little is known at present about transcriptional start sites or promoter structures of any genes still less about functional characterization of such elements. Research in this area is hampered by a lack of basic tools such as transcription lysates of nuclei from Plasmodium for in vitro analysis of

46

ANDREW P. WATERS

transcription and stable transfection systems of Plasmodium for in vivo analysis. The recent description of transient transfection of zygotes of P. gallinaceum (Goonewaradene et al., 1993) will allow preliminary in vivo analyses to be made. A further complication is the demonstrable sequence conservation in the upstream regions of both A gene units in P. berghei and similar conservation between both C-type units (A.P. Waters and C. Janse, unpublished) which will defeat attempts to analyse unmodified units in vivo. Nuclear run-on analyses of rRNA transcription have been performed with nuclei isolated from P. falciparum and transcription of the A-type units has been shown to be resistant to high levels of a-amanitin (Rudenko and van der Ploeg, 1989). This indicates that transcription of the genes is carried out by a polymerase molecule with a drug resistance profile similar to that of a typical RNA polymerase I. Using PCR probes it has been possible to demonstrate that the transcription of the A-type genes of P . berghei initiates less than 1 kb upstream and that a processing site lies within 0.5 kb of the 5' end of the SSU gene (A.P. Waters and C. Janse, unpublished). Further mapping will reveal the exact location of the start of transcription and upstream processing sites. The predicted length of the A-type unit ETS is similar to that found in yeast. The sequence of the ETS of both the A and C genes of P . berghei has been determined and compared and shows little sequence homology to each other or other ETS regions. The actual figure of the aligned sequences is over 60% homologous but this is merely a reflection of the difficulties faced by alignment programmes trying to align two sequences of -80% AT content. 2.6. Transcription Factors and rDNA

2.6.1. General Transcription factors are proteins which interact either with the transcriptional enzymes, RNA polymerases I, I1 and 111, with specific DNA sequences that are important in the promotion of transcription or interact with both enzyme and DNA. These factors are responsible for both the enhancement of transcription and the accuracy of its initiation. It has been possible to characterize some of the transcription factors associated with the promotion of transcription of the complex rRNA gene unit in several systems. What has emerged from biochemical and genetic studies is a picture which demonstrates both unique and universal features associated with the promotion of transcription in general. Two factors, termed promoter selectivity factor (SL1) and upsteam binding factor (UBF), have been biochemically characterized that act as

THE RIBOSOMAL RNA GENES

OF

PLASMODIUM

47

transcription factors for rRNA genes in humans (Learned et af., 1986).UBF and RNA pol I are interchangeable between closely related species (Bell et al., 1990), however SL1 is species specific (Learned et al., 1985; Bell et al., 1989, 1990), responsible for recognition of the cognate template and therefore site specific. This is somewhat similar to the situation with RNA pol I1 with its requirement for a combination of general transcription factors and site-specific enhancers (Reinberg and Roeder, 1987). The similarity between the interactions of these two polymerases is further emphasized with the demonstration that they both are influenced by the general transcription factor known as the TATA-binding protein (TBP). TBP, which is known to be integral to the general activity of RNA pol I1 and bind to the TATA box DNA element in the promoter region of all pol I1 transcribed genes, also forms part of the SL1 complex (along with three other Tightly Associated Factors (TAFs) ). TBP is, therefore, also involved in the promotion of RNA pol I activity and rRNA transcription (Comai et al., 1992). Further genetic evidence showed the central role of TBP as a cofactor of transcription mediated by all three polymerases in yeast (Cormack and Struhl, 1992; Schultz et af., 1992). It is not clear how the product of a single gene (in yeast) is capable of differential interaction with the three types of RNA polymerase molecule. Perhaps multiple forms of TBP exist, generated by forms of post-transcriptional processing. Evidence in support of this latter point shows that different temperaturesensitive mutations within TBP can differentially affect the ability of the mutant protein to act as a co-factor to specific RNA polymerases (Schultz et a f . , 1992). Proteins binding to enhancer elements have also been demonstrated for example Reblp (Morrow et al., 1989) and Abflp (Morrow et al., 1990) both bind to different regions of the 190 bp rDNA enhancer element of S. cerevisiae. The combination of enhancer and transcription factors is thought to regulate transcription in one of two ways (Reeder, 1989), either through alteration of the receptivity of the gene for active polymerase or increased loading onto pre-assembled activation complexes (Conconi el a f . , 1989). 2.6.2. Plasmodium TBP has been cloned from the human parasite, P. falciparum (McAndrew et al., 1993). It is a widely diverged protein compared with those characterized from other species. In general it is highly conserved protein which shows > 80% identity in the -180aa C-terminal core domain (Greenblatt, 1991). However, TBP from P. falciparum shows only 3844% identity and is conserved primarily in length. A significant number of the basic amino acid residues arginine and lysine are positionally conserved (seven of nine

48

ANDREW P. WATERS

universally conserved positions (McAndrew et al., 1993)) which may influence binding to other protein factors (Buratowski and Zhou, 1992). The protein is so different that one is forced to consider if this gene is indeed a diverged member of a multigene family and that the true homologue of TBP is yet to be discovered. For instance, the otherwise conserved proline at position 65 of S . cerevisiae TBP (position 50 in P. falciparum) that determines ability to influence RNA pol I (Schultz et al., 1992) is not conserved in the TBP of P. falciparum (McAndrew et al., 1993). It is speculated that the unusual AT-rich constitution of the P. falciparum genome has resulted in the widely divergent form of TBP in this parasite (McAndrew et al., 1993). The gene encoding this form of TBP is single copy and located on chromosome 5 (McAndrew et al., 1993). 2.7. 5s RNA Genes

2.7.1. General The 5s RNA molecule consists of an approximately 120 nt transcript produced by RNA polymerase I11 which is associated with the large ribosomal subunit and the peptidyltransferase activity (Garrett et al., 1981). Like their complex counterpart (the 18, 5.8, 28s rRNA encoding unit), the genes are usually present in multiple copies that cluster in the genome but at a locus which is separate from that of the complex unit. The copy number of the 5s genes is usually in proportion with the copy number of the complex unit, reflecting the molar requirement for the respective transcripts. Yeast has an unusual organization of its rDNA where the complex unit and the 5s units are interdigitated in an equimolar tail-totail configuration. The two units are transcribed independently of each other (Gudenus et al., 1988). The promoter for transcription of 5s genes is internally disposed in the structural sequence of the gene (Ciliberto et al., 1983). A species-specific family of transcription factors (termed TFIIIA, B and C) promote transcription. The gene regulates its transcriptional binding through feedback inhibition (Brow and Geidushek, 1987), the 5s RNA molecule competing with the gene for binding to TFIIIA (Geidushek and Tocchini-Valentini, 1988). The general transcription factor TBP also forms part of the active transcription complex for this class of genes (Cormack and Struhl, 1992; Schultz er al., 1992; White er al., 1992). 2.7.2. Plasmodium The genes encoding the 5s RNA species are typical of most eukaryotes except that, in P. falciparum, they have been shown to be of low copy

THE RIBOSOMAL RNA GENES OF PLASMODIUM

49

number, matching that of the complex unit (Shippen-Lentz and Vezza, 1988). The three 5s RNA genes are physically linked to one another and contained within 1.5 kbp of DNA at a locus that is not linked to a complex unit of rRNA. Recent evidence demonstrated that in P. berghei the genes are located on chromosome 12, the same chromosome as the complex unit A but the relative positions of the two loci are unknown at present (A.P. Waters and C. Janse, unpublished). Earlier mapping work suggested that they are at least 5 kbp distant from any of the complex rDNA units (Dame and McCutchan, 1984). The three genes in P. falciparum code for identical transcripts with the exception of one or two uridine residues at the 3' end. Each of the units has a typical oligo(dT) pol I11 termination sequence at its 3' end. The three units are all expressed in asexual parasites although at different ratios (Shippen-Lentz and Vezza, 1988). It is not known if this ratio alters during the progression of the life cycle and neither is the significance of the 3' terminal polymorphisms. Each gene contains elements homologous with consensus internal elements involved in promotion of transcription and the gene can be transcribed in an heterologous, transformed human cell transcription extract (Shippen-Lentz and Vezza, 1988). This sugests that the promoter elements are sufficiently conserved to be functional. 2.8. Organellar rRNA Genes

Plasmodium parasites possess two extrachromosomal DNA elements that appear to replicate independently of nuclear DNA. One is a circularly disposed 35 kbp element whereas the second is a -6.0 kbp element which is highly repeated and can be found as a tandem array. It is unclear at the moment which of the various organellar structures contain either of these elements. Both elements contain distinct rDNA moieties which are transcribed and will be described separately.

2.8.1. The 35 kbp Circular DNA Element The 35 kbp circular DNA element was discovered as an aberrantly migrating band of P. knowlesi parasite DNA during buoyant density gradient separation (Williamson et al., 1985) and has subsequently been demonstrated in other parasites including P. falciparum. It has a number of genes that have been identified and include RNA polymerase subunit rpoB and rpoC genes and a palindromic element which contains both large and small rRNA genes and separated by tRNA genes. The rRNA genes are therefore present in two copies per circle arranged as mirror images of each other centred around the 3' end of the SSU. The relative arrangement

50

ANDREW P. WATERS

of the SSU and the LSU units within each palindromic unit is unusual since they are head t o head with each other (Figure 2C). Thus the transcription of the molecules is on opposite strands and independent unless a bidirectional promoter is invoked. The two copies of the LSU rRNA gene are identical for 300 nt at their 3’ end and may well be completely identical (Gardner et al., 1993) and thus as yet there is no evidence for their stagespecific transcription; the genes are transcribed throughout asexual growth. Evolutionary implications drawn from the sequence analysis of these molecules will be described in a later section. Both the SSU and the LSU genes contain the appropriate structural information to form active organellar ribosomes (Gardner et al., 1991, 1993).

2.8.2. The 6 kbp element The 6 kbp DNA element is a mitochondrial DN A-like element originally demonstrated in the rodent parasite, P. yoelii (Vaidya and Arasu, 1987) 5.8 kbp in length. The element is tandemly repeated in a head to tail fashion and the repeat unit cross-hybridized with Plasmodium DNA from other rodent species as well as primate, human and avian parasites. In P. yoelii and the avian parasite, P. gallinaceum, transcription of the fragment was demonstrated in vertebrate bloodstream, asexual parasite forms and the transcripts included fragments of rRNA-like molecules as well as cytochrome b and cytochrome oxidase subunit I (Aldritt et al., 1989; Vaidya et al., 1989). Complete sequence characterization of the element and database searching revealed the presence of rDNA-like fragments typical of a mitochondrial arrangement, transcription taking place on both strands of the DNA (Aldritt et al., 1989; Suplick et a l . , 1990; Feagin et al., 1992). There is sufficient sequence complexity in the rRNA fragments to cover the majority of the mitochondrial core sequences of both the large and small subunits suggesting functionality (Feagin et al., 1992). 2.9. The production of Mature rRNA: Processing of the Primary Transcript

2.9.1. General Processing is the series of cleavage events by which the primary multigene transcript of the complex rDNA unit is cut to yield the three mature structural rRNA molecules, the 5.8S, the 18s and the 28s. A minimum of six cuts will be required at exact points in the transcript to achieve the appropriate products although there are usually more. This is a highly ordered process which is mediated by both RNA and protein moieties

51

THE RIBOSOMAL RNA GENES OF PUSMODKJM m I

"

I

1

Lm

I1

,

I

I

1

Gene

Transcrlptlon

Tc

.

*

35s

-,C

Primary Transcript

32s

27SA A3

27SB

18s

-

28s

Mature Species

Figure 3 Processing of ribosomal RNA. The A unit of P. berghei is illustrated and detailed as in Figure 2A. Processing of the transcript is illustrated by showing the pathway used in S. cerevisiae and is modified from Mattaj et al. (1993). Asterisks indicate the processing events which are affected by either defects in or depletion of the indicated snoRNA species or nuclear proteins and are referred to in the text; f indicates cuts inhibited during gametocytogenesis leading to accumulation of the C gene precursor rRNA in P. fakiparum. The sizes of the precursors are indicated and the cuts are denoted in order, A referring to the production of the 18s species and B refemng to production of the 28s species. Tc indicates the theoretical start of transcription in the A unit.

many of which are very conserved. In yeast, the number and order of the cleavage events is known and many of the components involved in processing have been defined genetically. The state of knowledge of these events is summarized in Figure 3 showing the order and number of the cleavage events and the RNA and protein components known to mediate those events. Several (12 in yeast) small nucleolar RNA molecules (snoRNAs) play a role in rRNA maturation, e.g. snoU3 is involved in the

52

ANDREW P. WATERS

A0 cut in the 5’ ETSl region (Hughes et al., 1987; Hughes and Ares, 1991) and snoRNA U14 (Zagorski et al., 1988; Li et al., 1990); snoRNA30 (Morrissey and Tollervey, 1993) and snoRNAlO (Tollervey, 1987) are all involved in the production of mature 18s rRNA. The mechanism of action is not entirely clear at present for any of the snoRNA species. In the case of U3 snoRNA, it is thought to involve base pairing between a conserved region in the U3 molecule and a region of the ETS1, in a manner analogous to the snoRNA molecule involvement in intron splicing from pre-mRNA (Mattaj et al., 1993). A role for proteins in these events has been defined genetically. The protein fibrillarin is associated with all 12 known yeast snoRNAs (Schimmang et al., 1989) and is involved in all facets of post-transcriptional modification of the primary precursor since every cleavage event as well as methylation and ribosome assembly can be affected by different defects in this conserved nucleolar protein (Tollervey et al., 1991, 1993). The protein encoded by GAR1 in yeast is associated with snoR10, 30 and 31 (Girard et al., 1992; Morrissey and Tollervey, 1993) and again defects or depletion of this molecule leads to failure to accumulate 18s rRNA. Not surprisingly, all of the protein and RNA mediators mentioned above are essential for viability in yeast. 2.9.2. Processing of the r R N A Transcripts of Plasmodium The stage-specific predominance in expression of different forms of rRNA molecules raises the question of the processing of these molecules. Three possibilities present themselves: they may be (1) entirely stage specific having no shared mediators or sequence elements; (2) entirely conserved, using identical processes; (3) a mixture of 1 and 2. The evidence is far from complete and is currently being addressed preliminary work in this laboratory would suggest that option 3 is possibly correct. As stated earlier, sequence analysis of the ETS regions of C and A units of P. berghei shows that there is very little homology between the sequences. However, the 10 nucleotides immediately adjacent to the mature 5‘ SSU terminus are identical in both C and A units and the first five are conserved in other Plasmodium species (Goman et al., 1991; A.P. Waters, unpublished), S. cerevisiae (Yeh and Lee, 1992) and Tetrahymena thermophila (Miiller and Eckert, 1989). This implies that the factors and mechanisms which are responsible for the formation of the mature 5 ’ terminus of the SSU molecule are conserved (see below) but that other cut sites and promoters may well be stage and unit specific. Indeed the sizes of the precursors which predominate in either asexual bloodstage forms or in dividing oocyst in the mosquito show distinct size differences indicating that even if similar mechanisms are employed, sequence divergence

THE RIBOSOMAL RNA GENES OF PLASMODlUM

53

between the C- and A-type units has minimally involved insertions and/or deletions. For instance, there is demonstrable size variation in the ITS 1 of the C and A units of P. berghei (Dame and McCutchan, 1984). Furthermore, our own sequence data show there is immediate divergence of the sequence of ITS1 at its junction with the 3' end of the SSU (A.P. Waters, T. McCutchan and C. Janse). This would imply that either distinct mechanisms are involved in the processing events or that the sequence of the 18s rRNA alone determines the position of the cut. Uniquely in P. berghei there is also polymorphism at the very 3' end of the structural 18s unit (Gunderson et a f . , 1987) reinforcing the thought that unit specific mechanisms exist. None of the factors which have been described in the general section detailing processing of the preribosomal RNA have as yet been identified in Plasmodium although elements involved in splicing of mRNA have been described (Francoeur ef al., 1985). Of course, the interest will lie in the existence of multiple forms of such factors each produced in a stage-specific manner and associating with a particular transcript from a C- or A-type gene. Post-translational modifications may well alter the transcript specificity of these factors. If multiple genes exist for these factors they may have the ability to act on both transcripts but with reduced efficiency on the non-cognate form reflecting the divergence. Future research should answer these questions. 2.10. Ribosomes

2.10.1. General Ribosomes consist of a complex of the rRNA species already described and proteins. Two distinct subunits, the large and the small, associate around mRNA to form the active ribosome. Transient association with amino acid-charged tRNA molecules translates the mRNA and forms the covalent bonds between the amino acid residues building the protein. For thorough reviews about ribosome structure, function and activities, see Stern et al. (1989), Atkins et al. (1990), Ramagopal et al. (1992) and Lake (1985). 2.10.2. Plasmodium The ribosomes of Plasmodium have not been investigated in much detail chiefly due to problems in obtaining sufficient biomass and purity. In earlier research ribosomes were isolated from asexual bloodstage parasites of a variety of species, e.g. P. knowlesi (Sherman et a f . ,1975), P. lophurae

54

ANDREW P. WATERS

(Sherman and Jones, 1976,1977;Wallach and Boeke, 1983) and P. berghei (Miller and Ilan, 1978). The normal 30s and 50s sizes of the subunits were demonstrated as well as the presence of the rRNA species. In the case of P. berghei it was shown that the large rRNA molecule is nicked to give two species of approximately 800 nt and 3000 nt (Miller and Ilan, 1978). Cell-free translation lysates have been produced and shown to be translationally competent (Wallach and Boeke, 1983). Miller and Ilan (1984) compared the ribosomal protein spectrum of P. berghei and host rat liver and showed that numerous distinctions could be made. Beyond this little has been done to further our knowledge of ribosomes at the molecular level. Recently a random genome sequencing project has isolated the genes for several ribosomal proteins (J. Dame, Genbank Submissions) and a protein encoding elongation factor la which is ribosome associated (J. Dame; D. Williamson, both Genbank Submissions). Clearly one of the most interesting questions will be how many of the ribosomal components are stage specific? This addresses the level of duplication of the ribosomal proteins, the extent to which such components are interchangeable in C and A ribosomes and the possible role of stagespecific post-translational modification that may influence the function of specific ribosome components (this happens in Dictyostelium discoideum (Ramagopal, 1992)). Although it is possible to obtain sufficient material from asexual bloodstages to address these questions the difficulty will lie with obtaining sufficient amounts of material from the mosquito stages of the parasite. The potential of the in vitro culture systems for the oocyst stages of the parasite may provide this (Warburg and Miller, 1992). 2.1 1. Plasmodium Ribosomal RNA Sequence

2.11.1. Function All nuclear SSU rRNA genes which have been sequenced from any Plasmodium species contain all of the conserved sequence and structural elements which will allow the formation of an active 30s ribosomal subunit capable of interacting with the 50s subunit. Much of the sequence, and therefore structure, is conserved between the various characterized species of Plasmodium. Similarly in the one completely sequenced nuclear LSU gene, an A type of P. fakiparum all conserved known functional sequence motifs are typically eukaryotic in composition. Sequence data from a Ctype LSU gene from P. fakiparum and an A LSU gene from P. berghei support this. The sequence of extrachromosomal rDNA elements also contain all of these conserved elements (or most in the case of the 6.3 kbp

THE RIBOSOMAL RNA GENES OF PLASMODlUM

55

mitochondria1 element). Readers are referred to the reviews mentioned above for detailed analysis of structure and function. 2.1 1.2. Drug Resistance Within the Ribosome It has become apparent that the rRNA molecules are not merely scaffold for the ribosome serving to maintain the appropriate steric relationships of the active ribosomal proteins. Instead the catalytic nature of the rRNA itself has been steadily revealed over recent years (Dahlberg, 1989). Many antibiotics inhibit the ribosomal activities as their mechanism of action and subsequently have been shown to interact directly with rRNA (De Stasio et al., 1988). A consequence of this is that point mutation changes in the sequence of both the SSU and LSU can generate resistance to many antibiotics either specifically or generically. Again with the parasite possessing three types of ribosome in different compartments at any one point in its life cycle there are numerous potential targets for appropriate antibiotics. The sequence of rRNA genes, once elucidated, allows a search for the conserved active structures and also predictions about the natural antibiotic resistance spectrum of an organism. This latter approach was successfully applied to Giardia where the predicted resistance and sensitivity spectrum was demonstrated in cultures (Edlind, 1989). Jenson et al. have extensively studied the drug sensitivity of Plasmodium parasites and this affords the opportunity to correlate their observations with predictions that might be made from the sequences. The presence of stagespecific ribosomes allows the possibility of the display of a different drug sensitivity spectrum throughout the life cycle (Geary and Jensen, 1983; Divo et al., 1985; Geary et al., 1988, 1989). A review of the positions within the rRNA units where variation is associated with resistance to antibiotics reveals that none of these are variant between stage-specificunits (Table 1). Indeed there are no known antibiotic-interactive positions which vary between species of Plasmodium. However, sporozoites, asexual bloodstage forms and gametocytes have been reported as having different sensitivities to various antibiotics (Geary et al., 1989). This may be due to the distinctions that can be made between nuclear and extachromosomally encoded rRNAs. Stage-specific susceptibility to particular drugs can also be explained either by differences in uptake or by pharmacokinetic distinctions of the vertebrate and invertebrate hosts and not due to known differences in the rRNA target. It remains possible that novel changes might occur in the ribosomes of Plasmodium that may facilitate resistance. Only a few naturally occurring sequence forms predict specific antibiotic resistance in Plasmodium, for example, streptomycin. Resistance in bacteria to this drug is known to be due to mutations at positions ASz3+G and q l p U (E. coli 16s numbering)

56 Table I

ANDREW

P. WATERS

Summary of ribosomal RNA sequence and antibiotic drug resistance.

Antibiotic

Streptomycin Spectinomycin Hygrom ycin Paromomycin Kanamycin and gentamycin Kanamycin and apramycin Aminoglycosides

Ribosomal target

Resistance changea

ssu ssu ssu ssu ssu ssu ssu ssu

LSU LSU' Erythromycin and LSU MLSg Lincomycin LSU Clindamycin and LSU chloramphenicol Chloramphenicol LSU LSU LSU LSU LSU Thiostrepton LSU

Plasmodium Prediction Observed

equivalent Nu. Ci. Mi. U C C Resistant A A G Resistant C' C C Resistant? U U U Sensitive C C C Sensitive Cd C C Sensitive?

Resistant ?

Sensitive Resistant

Gd A A Sensitive? Resistant G

G G Sensitive

Ue A C Resistant? Ae G G Resistant? Sensitive G A U Sensitive

Erythromycin

AIm+G,U,C

G G

G G Sensitive G G Sensitive

A U A G A G

A U A G A A

Sensitive Sensitive

A Sensitive U Sensitive A Sensitive G Sensitive Sensitive A Sensitive G Resistant? ND

Nucleotide numbering is given for E. coli. Changes given are those reported in literature. Spectrum of resistance is seen with all possible replacements of C in order of strength they are G > A > U. ' Range of associated changes at other positions in SSU confer either temperature sensitivity to the resistance or abolish it. Plasmodium has sensitive genotype at all reported positions of this type. Methylation status of any residue of Plasmodium rRNA is not known. These changes confer resistance singly possibly due to disruption of mutual base pair formation. Natural changes in Plasmodium form base-paired combination. Resistance might not be expected and is not seen. Also confers resistance to chloramphenicol. g MLS = Macrolide, linocomycin and streptogramin B. ND = no published data. a

in the 16s rRNA molecule (Montandon et af., 1986). All nuclear 18s rRNA genes characterized so far in Plasmodium normally have a U residue at the equivalent position to CgI2(in P. berghei) and an altered sequence surrounding the A523 (Gunderson et al., 1986, 1987). Additionally the 6 kb rDNA fragment of P. fakiparum has a A,,,-G replacement (Feagin et

THE RIBOSOMAL RNA GENES OF PLASMODlUM

57

al., 1992). Consistent with this, all species of Plasmodium tested so far with this antibiotic are also resistant to streptomycin (Geary and Jensen, 1983; Puri and Dutta, 1982) although there is also evidence to suggest that streptomycin is not taken up well by infected erythrocytes (Geary et a f . , 1988). A combinatorial effect may well explain the lack of activity. Conversely all of the positions in the LSU where change is associated with resistance in bacteria are conserved in the sensitive form in P . falciparum (A.P. Waters and T. McCutchan, unpublished). Thus the parasite is expected to be sensitive to erythromycin which confirms the observed effect of the drug in culture (Geary and Jensen, 1983). Kanamycin is an example where an antibiotic is not active against malaria parasites when activity would be predicted from the 18s rRNA sequence (Table 1) (Divo et af., 1985). This can possibly be explained by lack of uptake of the drug since when erythrocytes are pre-loaded with kanamycin inhibitory effects are seen (Krugliak et al., 1987). Table 1 summarizes some of the sites of action in the ribosome of antibiotics and the predicted and actual effect on Plasmodium. It is anticipated that the nuclear encoded ribosomes would be sensitive to the peptide antibiotics, ricin and a-sarcin. Both the nuclear and the mitochondria1 LSU fragment have an altered thiostrepton site A I o 6 p G (E. cofi 23s rRNA numbering) (Feagin et al., 1992; A.P. Waters and T. McCutchan, unpublished. 2.11.3. The Function of Stage-Spec#c Sequence Differences All of the regionalized sequence differences between stage-specific SSU rRNA molecules of Plasmodium lie in what are termed expansion regions of the molecule. Expansion regions are located between the highly conserved “core” regions. Each expansion region is predictably either species specific showing no variation between C and A genes or stage specific and to date has no associated specific function. However, specific secondary structures can be predicted (Gunderson et a f . , 1987) in which compensatory base changes appear indicating requirement for a particular structure (Hancock et a f . ,1988). Equivalent structures between distantly related members of Plasmodium are not easy to define. A species specific region that forms a roughly equivalent structure in the 18s RNA has been reported (Waters et a f . , 1991) but there are no stage-specific equivalents. One of the attractive possibilities was that there might have been sequence motifs in the stage specific regions which facilitated mRNA association with the appropriate ribosome form in a manner equivalent to the bacterial Shine-Delgarno sequence. Such a motif might be expected to be conserved in equivalent regions between species and found at the 3’ end of the 18s molecule. No such motif is seen; however, a survey of stage specific genes similarly failed to reveal sequence homologies in the mRNA species that

58

ANDREW P. WATERS

may have implicated a different region of the rRNA molecules. A number of stage-specific pairs of SSU genes have now been sequenced (A.P. Waters, D. Higgins and T. McCutchan, submitted) revealing that closely related parasites (such as vivax-like primate malaria) show close similarity in the stage-specific motifs. Conserved stage-specific nucleotides and motifs are easily identified to the extent that the cognate stage-specific genes are more similar to one another than to the gene in the same species expressed at the other part of the life cycle. This will be more fully addressed in the following section. It provides a strong argument for the necessity of the identity of stage-specific rRNA genes although precise function cannot be attached to any particular domain or motif. A further stage-specific activity of the ribosomes is participation in the specific removal of the active ribosome population during replacement and introduction of the ribosome population that accompanies the stagespecific switching. This phenomenon has been initially addressed in cultured P . falciparum sexual stage parasites where the specific degradation of A gene SSU sequences was observed (Waters et al., 1989). Curiously the initial cleavage site was mapped to within a few nucleotides (nt 1641-1689 in P . falciparum) in a highly conserved region of the molecule shared not only by Plasmodium SSU rRNA genes but also all those of the various hosts. It has been postulated that the incoming ribosome population is protected from the activity of the putative ribonuclease either by compartmentalization or possibly by specific folding patterns induced by the stage-specific regions which obscure the cut site (Waters et al., 1989). The possibility that dilution of the current ribosome complement by the introduced form is the major apparent mechanism in the apparent removal of the existing ribosome population has already been discussed. Such a mechanism would allow for the conserved nature of the observed cut point in SSU rRNA. However, the pattern of degradation points to some level of temporal control of ribosome removal which perhaps serves to protect the incoming ribosome population (Waters et al., 1989).

3. INFERENCES OF PHYLOGENY BASED O N RIBOSOMAL RNA ANALYSES 3.1. Earlier Classifications

3.1.1. The Phylum, Apicomplexa and the Class, Sporozoa The genus Plasmodium is contained within the phylum Apicomplexa (Levine et al., 1980) which is defined by the possession of all or part of a

THE RIBOSOMAL RNA GENES OF PLASMODlUM

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characteristic set of structures that together form what is termed the apical complex. It is a wide-ranging phylum and debate continues about membership and even if the emphasis placed on the apical complex as the defining characteristic for intraphyletic relationships was appropriate (Gajadhar et al., 1991). On the basis of biological criteria and light microscopy, all parasitic protozoa lacking contractile vacuoles, locomotor organelles and which utilize sexual reproduction as well as asexual fission were included in the class Sporozoa. All members produce encysted sporozoites (Kudo, 1966). More recently, Barta (1989) selected a group of 26 “biologically valid” characters of this class to produce a tree which demonstrated among other features, that the heteroxenous (use of more than one host during the life cycle) life cycle has been independently evolved on more than one occasion within the phylum; that blood infectious parasites did not cluster together and the definitive host is also the ancestral host. In the case of Plasmodium this would mean that the anopheline mosquito was ancestral. As Gajadhar et al. (1991) point out such assessments are qualitative and without the safeguard of a more quantitative approach. 3.1.2. The Genus Plasmodium Inferring the phylogeny within a genus may be no more simple. Although one is dealing with a group of organisms that are closely related and share a large number of features of their biology and life cycle, one is still left with the problem of deciding which are the significant characteristics upon which to make a valid estimation of phylogeny. Garnham (1966) produced the most comprehensive review of the phylogeny of Plasmodium based on his own choice of morphological and biological considerations. The criteria selected by Garnham were: 1. The vertebrate host; 2. The size of the erythrocytic schizont; 3. The shape of the gametocyte; 4. The site and shape of the exoerythrocytic schizont; 5 . The number of merozoites produced by the exoerythrocytic schizont. The host was considered to be by far the most important of these criteria. These criteria enabled Garnham to erect nine subgenera - three mammalian, four avian and two saurian - emphasizing host importance. Primate and human malaria was distributed in two subgenera, Plasmodium plasmodium and Plasmodium laverania. The latter contains only two species recognizing the unique aspects of the biology and morphology of P. falciparum the malignant tertian disease of humans, and its chimpanzee equivalent P. reichenowi. The rest of mammalian malaria is contained in

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Plasmodium vinckeia an artifice purely of convenience containing malaria species infective to rodents, lemurs and water buffalo. However, the construction of the P. vinckeia subgenus emphasizes the limitation of this type of classification. Without better datasets to compare the species it was not possible to generate anything other than groups of similar parasites with no means of looking at the relationships within and between these groups. This limitation was emphasized when McCutchan et al. (1984) studied the nucleotide composition of the genomic DNA of a number of malaria parasites, a property successfully used to compare bacteria. This study confirmed the distinctive nature of P. falciparum grouping it apart from other human and primate malarias and placing it in a group with avian and rodent malarias all of which had an AT-rich genome (> 80%). Killick-Kendrick (1978) also commented on the unusual distribution of rodent malaria, its restriction not only to a particular geographical region (the high altitude forests of East Africa), but also to such a small number of species of susceptible rodent (Gramnomys and Thamnomys, thicket rats), the closest vertebrate host sharing a susceptibility to malaria being brush-tailed porcupines. This led to his suggestion that rodent malarias were a “gift” rather than an “heirloom” suggesting an evolutionary history that had not followed strict lines of the natural law of co-evolution of host and parasite.

3.2. Molecular-based Estimations Woese and co-workers have pioneered the use of SSU rRNA sequences as a dataset for the comparative inference of phylogeny (Woese, 1987; Olsen and Woese, 1993). The SSU can be used in such studies because of the degree of conservation associated with the molecule. A constant structure/function relationship exists between all SSU sequences from any organism. This is especially true if one examines “core” sequence where the structure is even more highly conserved. The molecule provides a dataset of -1400 nucleotides which can be assigned to core and is sufficient for statistical analysis. A brief review of the stastistical methods which can be used has recently been published (Waters et a f . , 1993b; for more detail consult the references therein). The molecule is considered to be an excellent model for the inference of deep branches within phylogenetic trees. If the goal is the evaluation of a group of more closely related organisms then one has two choices one can use as much of the molecule that can be unambiguously aligned on a structural basis or choose a different molecule. As molecular data accumulate it will be possible to make composite trees based upon data from a number of molecular and biological sources. Such a wealth of data is not yet available, however

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much complete SSU data is and this has been exploited to produce a series of phylogenetic analyses which address the phylogenetic groupings of the species within Plasmodium and to analyse the relative position of the genus within the Apicomplexa. 3.2.1. Apicomplexa and Sporozoa The most recent study based on SSU sequence regarding the relationships with Apicomplexa implied that Plasmodium has a common ancestry with dinoflagellates as represented by the free-living dinoflagellate, Crypthecodinium cohnii. These two classes form a separate grouping with ciliates distinct from other protists. The study also revealed that within Apicomplexa, Plasmodium was relatively diverged more distant than Theiferiaannufuta,a tick-borne piroplasmic parasite of cattle, or Sarcocystis murk, a coccidial parasite that transfers between cat and mouse, were to each other (Gajadhar et al., 1991). This was surprising given the similarities in biology of Theiferia and Plasmodium and accelerated rates of evolutionary change within Plasmodium was postulated as a possible explanation (Gajadhar et af., 1991). A further puzzle concerning the origins of Plasmodium was demonstrated by the phylogenetic analysis of the rRNA genes contained on the 35 kb circular DNA element described earlier. Most unusually when the analyses were performed using the LSU rRNA gene of P . fafcipurum (Gardner el al., 1993) the most similar sequences were found to be the 23s rRNAs of chloroplast origin implying that the source of the circle was photosynthetic and possibly pointing towards the overall origin of the genus. This was substantiated by an analysis and comparison of a second gene on the 35 kb circle, the rpoC gene (Howe, 1992). Interestingly, when the SSU gene of this element was subject to the same analysis it grouped with the mitochondria1 rRNA genes of fungi, ciliates and Chfamydomonas reinhardtii (Wilson et af.,1991) which may be more in agreement with the analyses of the nuclear encoded genes. The authors were concerned, however, about bias that may be due to AT richness within the genes. 3.2.2. Plasmodium The analyses based on the nuclear-encoded SSU rRNA genes had a different purpose and that was to answer questions about the relationships between the different Pfasmodium species. Published work utilizes analyses of the SSU A genes, i.e. those expressed in the asexual bloodstage forms found in the vertebrate host. There were two reasons for this: (1) to avoid the possibility that the results would be biased by using a mixture of genes expressed in different stages which may evolve at different rates; (2) the

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ANDREW P. WATERS t--

-~ - ' 1% dislanca

rodent P. berghei

9

A

Tgx ,

P. malariae 65.6%

vlvax 'tertlan'

'avian'

P.

VlVaX

P. lophurae

A / , A 65.2% 65 2%

P. tragile tragle 62 2% 62.2%

P. gallinamurn

c

-

*'

A 64.4%

P. talcipamtn

C

Figure 4 Phylogeny of Plasmodium based upon SSU rRNA gene sequences. A phylogenetic tree is illustrated modified, with permission, from Waters et al. (1993a). Except where indicated the analysis is of A genes of the included species. The tree is crudely subdivided to indicate the statistically significant phylogenetic groupings inferred from previous analyses. The percentage figures indicate the AT content of the gene included in the analysis.

main interest in malaria is its effect upon its vertebrate host making the gene expressed in those hosts a more appropriate basis for the analysis. The results of the analyses have again been summarized in the same review (Waters et al., 1993b) and deal with the results of two reports (Waters et al., 1991, 1993a). An unrooted tree is given in Figure 4 and shows the inferred relationship between three human, two avian, three primate and one rodent species. In general the groupings favoured by Garnham (1966) proved to be accurate and confirm that the vertebrate host can be viewed as the single most important phylogenetic characteristic of malaria parasites. The most surprising finding of the studies was the confirmation and clarification of the earlier genome composition analysis (McCutchan et af.,

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1984) and of Garnham’s suspicions that the major human pathogen P. falciparum has a unique but distant relationship to other human and primate malarias. In fact, it is most closely and statistically significantly associated with avian malaria but not P. berghei rodent malaria, the other parasite with an AT-rich genome. The overall branching order and topology of the tree combined with the statistical analysis was not compatible with Farenholz’s rule of parasitehost co-evolution implying that a lateral transfer effectively between vertebrate hosts had to be invoked (Waters et al., 1991). The lateral transfer would involve birds and humans or some closely related primate from which the parasite was then transferred into man. A number of possible scenarios for the origins and natural history of P. falciparum have been suggested and summarized (Waters et al., 1993b). These include: 1 . The parasite has a long association with man but was originally a chronic and benign infection that has evolved towards virulence as the host population has increased; 2. Man’s agricultural activities created the conditions for the introduction of a novel pathogen into the population, with the virulence that is often (and sometimes mistakenly (Read and Schrag, 1991)) thought to signal a new host-pathogen combination; 3. The possible role of the chimpanzee as an intermediate host facilitating the introduction of malignant tertian malaria should not be overlooked; 4. Both avian parasites and P. falciparum are related to an as-yetundescribed parasite; 5. The humadprimate form of the ancestor passed from primates to birds and gave rise to the highly successful avian parasites. Clearly this list is not exhaustive and some of the above are not mutually exclusive. One of the interesting features of the latest survey is that it suggests distinct lines of descent for the three human malaria species included in the analysis. P . vivax clusters strongly with the monkey tertian malarias of Southeast Asia as well as with the only quotidian primate malaria, P. knowlesi. This points strongly to an Asian origin of vivax malaria as suggested by Coatney et al. (1971), subsequently distributing from there as man migrated (Bruce-Chwatt, 1965). Quartan malaria also has its own distinct lineage, clustering neither with the vivax-like parasites nor with the aviadfalciparum cluster. This would imply that quartan parasites have a deep lineage and that the separation of quartan from tertian is an ancient event in the history of primate malaria. The same is clearly not true of the quotidian primate malaria which is shown to be a relatively recent experiment within the vivax branch. Due to an uncertainty about the relative position of the rodent lineage and

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P. malariae and the position of the root for the tree, the provenance of human quartan malaria may be completely distinct from that of tertian. If so, the generation of the lineage which resulted in modern P. malariae could have arisen before the rodent lineage. We have suggested that the subgenus Plasmodium plasmodium now be subdivided and that all quartan parasites within that genus be included in a new subgenus Plasmodium quartans to recognize the deep division in the tree (Waters et al., 1993b). Future analysis will demonstrate if all the quartan parasites truly have the same lineage. It is worth also commenting on the phenomenon of AT-rich genomes. Plasmodium parasites have some of the most AT-rich genomes reported. The distribution of the composition is not uniform, structural genes having a higher CG content although still below normal values. The AT content of the SSU genes is similarly lower than the overall A T content of the genome for each species, although it has been noted that for other species (e.g. Drosophila melanogaster) there can be a different bias in base composition that is gene dependent (Hancock et al., 1988). The interesting feature of the AT content of the various SSU genes represented in the tree is that there is not a gradient of AT content across the tree. Thus, whatever the influence that overall genome composition has on the composition of the individual SSU genes it seems to have little influence on the topology of the tree. In this light it will be very interesting to see a similar comparison of the SSU genes of the 35 kb circles where the suspicion is that A T content of the genes compared influenced the analysis (Wilson et al., 1991). 3.3. Evolution of the rDNA Units Within the Genome

How are the forces of divergence, concerted evolution and selection reflected in the status of the nuclear rDNA units in Plasmodium? P. berghei has four nuclear rDNA units organized into two classes C and A and although the two classes are homogeneous they are still maintained as distinct forms in the genome. The level of sequence identity between the two A-type units extends outside the structural regions of the unit (the 18S, 5.8s and 28s) at least into the 5 ’ ETS and ITS1. The same is true for the C-type units yet there is very little sequence similarity between the C and A ETS regions. All four units exist on separate chromosomes. There must be a mechanism(s) which facilitates the active tandem evolution of both unit pairs. Is there any significance in the position of stage-specific genes from the same species on a phylogenetic tree which might reflect these forces and mechanisms? The placement of the C gene units from P. berghei, P. gallinaceum and P. falciparum illustrates the situation. In

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each case the stage-specific genes group with the second type from the same species (Figure 4, A.P. Waters, D. Higgins and T. McCutchan, unpublished). This implies that overall homogenization of the rDNA unit complement within a species occurs at the same time and to a greater extent than the maintenance of stage-specific regions. Thus in P. berghei, the four rDNA units homogenize with sufficient frequency to maintain the species-specific component as the most significant characteristic of all units. Therefore, the sequence of a Plasmodium SSU gene is characteristic first of a member of Plasmodium, second of the species and finally of the stagespecific unit. Homogenization therefore acts at two levels, species and stage specifically; the former involves regions within the SSU unit whereas the latter demonstrably affects each of the ETS, SSU and ITS1. These arguments are made on the basis of the knowledge about the SSU region and the flanking ETS and ITS1 and will be clarified as the remainder of the units from multiple species are characterized. It can also be seen from the phylogenetic tree (Figure 4) that the C units have the longest branch lengths compared with the equivalent A gene from the same species. This might imply that there is a different rate of evolution in the stage-specific pairs effected either by accelerated accumulation of mutations or reduced correction. A mechanism for the latter can be envisaged since the process of homogenization of two loci necessitates mutual hybridization and, effectively, exchange of sequence content. The rDNA units are maintained on separate chromosomes and the rate of effective accumulation of mutation (drift) can essentially be viewed as a function of the frequency with which homogenization can occur relying on the three-dimensional distribution of the rDNA units in the nucleus. Thus the prediction would be that the C units are so disposed that homogenization is less frequent than between A units. Thus the organization of the rDNA units within Plasmodium provides a fascinating insight into the organization, evolution and maintenance of the genome of malaria parasites.

4. SPECIES IDENTIFICATION BASED ON RIBOSOMAL RNA 4.1. The Principle of Direct Detection of rRNA

Classical detection of microorganisms for medical diagnosis has relied upon culture of those organisms on defining media. This is a labourintensive and time-consuming process requiring highly skilled personnel and a sophisticated laboratory setting. More recent technologies have sought to simplify the process using, initially, antibody-based techniques and lately nucleic acid-based techniques. The most well-known application

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is based on the polymerase chain reaction (PCR) technology, however, it is also possible to base detection systems on the direct detection of rRNA and its genes. PCR uses thermostable DNA polymerases to amplify a DNA target almost limitlessly. By judicious choice of the target DNA (defined by the oligonucleotides which determine both ends of the amplified target) the appearance of a specific amplified DNA band can be diagnostic. The potential of the PCR technology is huge and ingenious applications are revolutionizing molecular biology, medical science, forensic science and many other fields. The rRNA-based alternative for the simple detection of a microorganism is also polymerase amplified but in this case the amplification is the natural amplification of transcription carried out by the RNA polymerase I responsible for rRNA synthesis within the cell. In Plasmodium it has been estimated that in a schizont there are lo6 ribosomes and therefore the same number of 18 and 28s molecules.

Figure 5 Secondary structure of a small ribosomal subunit of Plasmodium berghei. The secondary structure of the A type SSU ribosomal RNA of P . berghei is reproduced from Waters and McCutchan (1990) with permission. This structure is generally highly conserved for all SSU rRNA molecules. Shaded areas depict the expansion regions which serve as the source of diagnostic oligonucleotides.

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Therefore if specificity and sensitivity of detection of these targets can be achieved the rRNA of any organism can serve as a diagnostic target. Detection is achieved through the hybridization of the target RNA to a diagnostic antisense oligonucleotide which is modified to facilitate detection of hybridization. At present, the target RNA is immobilized and the hybridization is driven by the presence of excess oligonucleotide. The presence of species-specific regions within the SSU has already been mentioned in the previous section. Figure 5 demonstrates the regional organization of the molecule; the shaded areas represent the expansion regions which contain genus, species and, in Plasmodium, stage-specific information. Alignments of the SSU sequence will provide the information for the design of oligonucleotides which will hybridize specifically to the SSU of a single species thereby permitting the specific detection and identification of that species. The principle of the method for the specific detection of malaria was initially shown for the human malarias (Waters and McCutchan, 1989). The characterization of a single species-specific region of the SSU rRNA molecule provided sufficient information to allow the synthesis and use of oligonucleotides to discriminate unambiguously between nucleic acid preparations from each species. The sensitivity was also shown to be potentially sufficiently sensitive for use in field applications (20 parasites detectable) and relatively simple methods of preparation of the samples were applicable to the whole process (La1 et al., 1989; Waters and McCutchan, 1989). For the simple discrimination between parasite and host about 65% of the parasite SSU sequence is available as a target for detection so the simultaneous use of multiple oligonucleotides is possible (Waters and McCutchan, 1990). Characterization of the sequence of the LSU molecules will simply expand the database for the construction of diagnostic oligonucleotides. Figure 6 illustrates the potential of the methodology to discriminate between the human malaria species. The limitations of the current methodology relate to its application to the field situation. Methodologies need to be developed and refined such that the principle of the process can be carried out in rudimentary field laboratories with a mimim of equipment and training. Ideally the process should be adapted to dipstick technologies, however cost considerations may prove limiting. Why is there a perceived requirement for molecular diagnostic technologies in tropical disease and malaria in particular? Current methodologies in use are based on the identification of the parasite itself. The, by now, almost traditional method is the microscopic observation of the parasite on Giemsa-stained blood smears. The process is cheap, sensitive and the different human malarias can be discriminated on the basis of appearance. The major disadvantage lies in the fact that it is a labour-intensive method. Highly trained personnel spend 5-10 minutes

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Figure 6 Demonstration of the specificity of ribosomal RNA-based detection of human Plasmodium species. Radiolabelled oligonucleotide probes specific for the SSU rRNA gene of each of the human malarias indicated were hybridized to strips of nitrocellulose to which parasite RNA had been immobilized. Specific detection of each species was possible. (From Waters and McCutchan (1990), with permission.)

examining each slide. In endemic areas the burden on the microscopists is simply too great and that individual soon ceases to be reliable. Therefore, a method that offers simple, sensitive, rapid and unambiguous detection and identification that requires little or no judgemental input from the user is very attractive. An alternative holistic method is the quantitative buffy coat (QBC@)process where parasites are detected through nuclear uptake of a fluorescent dye and discriminated from other nucleated cells on the basis of their unique sedimentation properties during centrifugation (Spielman et al., 1988). The method is easy, fast and sensitive and, with a small amount of training, the user can distinguish P. fulciparum and P . vivax and it may be used in the assessment of drug resistance in the field (Garin et al., 1992). However, it is not cheap and requires a centrifuge. Models are being developed which can be powered by car batteries.

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4.2. Ribosomal RNA Gene-Based PCR

As stated above the oligonucleotides which are input into a PCR define the amplified DNA fragment. Thus knowledge of the sequence of the target DNA is essential in order to design appropriate oligonucleotides. For the detection of human malaria parasites by PCR the SSU gene sequences are the only dataset for all four parasites from which oligonucleotides could be designed such that the equivalent target molecule in each species is amplified. Furthermore due to the configuration of the SSU molecule, as described above, a number of options are available in the design of PCR experiments. It is possible of course to use unique pairs of primers for each species, however it is also possible to use a single common primer, the species specificity being supplied by the second oligonucleotide. In such experiments single tube detection of a mixture of human parasites can be achieved with just five primers, the single common primer and four species specific. With four possible products a method for discrimination must also exist, with careful choice of oligonucleotides this could be on the basis of size. Another possibility is to label each species specific primer with a different fluorescent tag which would then allow specific detection of the polymer. The potential that the SSU genes offer for the design of diagnostic PCR has been exploited by Snounou et al. (1993) who show that specific-sized DNA fragments can be amplified in a species-specific manner from field samples and that mixed infections can be identified. The value of PCR for mass field diagnosis has been questioned on the basis that it is too technical and too easy to contaminate the working environment such that completely erroneous results may be obtained (Waters and McCutchan, 1990). However, for epidemiological surveys where the samples are handled in a carefully controlled laboratory environment PCR should prove a valuable approach. Some misgivings may still apply. The enormous sensitivity of the technique means that detection of a single parasite is feasable. In endemic areas it has been suspected that individuals may well have a constant low-grade parasitaemia which, although not normally detectable upon microscopic examination of blood smears, would routinely show positive in PCR analysis. If true, then PCR is unlikely to give an indication of the dynamics of an epidemic. Competitive PCR can give accurate quantitation, but this is not feasible for epidemiological surveys. PCR may well prove to be very useful for the diagnosis of malaria infections in tourists. These are by and large naive subjects unlikely to be harbouring low-grade parasitaemias and the diagnosis would be carried out in a well-equipped laboratory by personnel well trained in various applications of the PCR process.

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4.3. Drug Testing and rRNA Diagnosis

One novel approach which combines an assay for the efficacy of novel drug compounds and rRNA of Plasmodium lies in a screen for drugs which affect the liver stages of the parasite. As discussed in an earlier section the switch from C to A rRNA unit expression occurs within 8 hours of the sporozoite invading the liver. This then provides a means to assess the development of the exoerythrocytic stages and the ability of drugs to prevent either the switch or the subsequent development and amplification of the A gene signal. This method has been explored recently in the P. berghei in vitro culture system in the HepG2 human hepatoma cell line (Li et al., 1991). The recent development of the in vitro culture system for the mosquito stages of Plasmodium will allow similar assays to be developed for this part of the life cycle (Warburg and Miller, 1992).

5. PERSPECTIVE

The study of rRNA genes in Plasmodium has provided valuable information about the molecular biology, biology and evolutionary history of members of the genus. These members have evolved an apparently unique response to the problem of a heteroxenous life style in mosquitoes and vertebrate hosts and that is the switching ribosome. Two different types of rRNA gene containing unit-specific sequence elements are expressed in a stage-specific manner. Is this organization and expression of any biological significance? We can only answer probably; the regionalized organization of the stage-specific elements within the structural units of the two types of rDNA unit can be viewed in two ways: 1. The regions where the observed sequence differences accumulate are the only portions of the molecule where such changes can be tolerated without any deleterious effects on the functionality of the molecule. 2. There is a functional consequence to the accumulation of the stagespecific differences which allows the stage-specific ribosome to operate with maximum efficiency in its particular environment. There is no direct biological evidence which supports the strict requirement for the two types of ribosomes, however, indirect arguments can be used to address the question. The core sequence that is absolutely conserved in structure is restricted to about 1200 nt of the 2100 nt of the Plasmodium SSU unit. A further 600 nt form genus-specific sequence elements and structures, the remainder form either species-specific elements or stage specific. There is no consensus structure or sequence element for either C

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or A genes that can be found in all species (Enea and Corredor, 1991; A.P. Waters, D. Higgins and T. McCutchan, unpublished), although the relative position of the stage-specific domains within the molecule is conserved within the genus. Despite the lack of consensus, the sequence distinctions between the two classes are maintained in the genome which implies either an inability to homogenize the sequences or a biological requirement. Furthermore, according to the phylogenetic analysis, the multicopy C- and A-type genes evolve at different rates demonstrating the existence of mechanisms which discriminate between the rDNA units in the extent and possibly frequency of homogenization. The most important piece of evidence regarding the requirement for the maintenance of the different forms of the rRNA remains the fact that they are expressed in a stage-specific manner. Thus the parasite has in place mechanisms which allow for the more or less complete replacement of the ribosome population and performs this replacement at points in the life cycle where it parasitizes a new host or vector. This is a complex process requiring events some of which will be described in more detail below but a less than comprehensive list would include: maintenance of different units in the genome; their differential transcription; processing, transport and assembly into the ribosomes. Furthermore, during the replacement process, the existing ribosome population must be dissembled and removed. Only when stable transfection of Plasmodium becomes available will it become possible to address directly the question of the essential nature of the switching ribosomes. Has the switching ribosome evolved on more than one occasion? Developmentally distinct ribosomes have been described in Dictyostelium discoideum where the ribosomal protein content varies in a stage-specific manner on a constant rRNA component (Ramagopal, 1992). Clustered heterogeneity in SSU rRNA genes of a single species have also been reported for the halophilic Archebacterium Haloarcula marismortui which has two rRNA operons (Mylvaganam and Dennis, 1992). The sequence heterogeneity forms identical predicted secondary structures with compensatory base changes. The two genes are co-transcribed and equally represented. There is no evidence for differential expression of the two operons. Thus so far the stage-specific expression of heterogenous rRNA genes is unique to Plasmodium, to date there are no reports of this phenomenon in other heteroxenous organisms. Barta (1989) has suggested that the heteroxenous nature of apicomplexan life cycles has evolved independently, which would imply that the spectrum of adaptations would be species specific and not necessarily include stage-specific ribosomes. Clearly the study of rRNA genes has given insight into the biology of Plasmodium and continues to have much to offer and many challenges for the future.

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ACKNOWLEDGEMENTS

The author would like to acknowledge his colleagues in the Department of Parasitology of Rijksuniversiteit te Leiden, The Netherlands, especially Chris Janse for dicussions and critical review of the manuscript. I would also like to acknowledge Tom McCutchan who established the investigation of ribosomal RNA in Plasmodium and is at the heart of the discovery of the unique features of the system and with whom I was fortunate enough to train. REFERENCES Aldritt, S.M., Joseph, J.T. and Wirth, D.F. (1989). Sequence identification of cytochrome b in Plasmodium gallinaceum. Molecular and Cellular Biology 9 , 3614-3620. Atkins, J.F., Weiss, R.B. and Gesteland, R.F. (1990). Ribosome gymnastics degree of difficulty 9.5, style 10.0. Cell 62, 413-423. Barta, J.R. (1989). Phylogenetic analysis of the class Sporozoea (phylum Apicomplexa, 1970): evidence for the independent evolution of heteroxenous life cycles. Journal of Parasitology 7 5 , 195-206. Bell, S.P., Pikaard, C.S., Reeder, R.H. and Tijan, R. (1989). Molecular mechanisms governing species-specific transcription of ribosomal RNA. Cell 59, 489497. Bell, S.P., Jantzen, H.-M. and Tijan, R. (1990). Assembly of alternative multiprotein complexes directs rRNA promoter selectivity. Genes and Development 4, 943-954. Brow, D.A. and Geidushek, E.P. (1987). Modulation of yeast 5s RNA synthesis in vitro by ribosomal protein YL3. Journal of Biological Chemistry 262, 13953-13958. Bruce-Chwatt, L.J. (1965). Paleogenesis and paleoepidemiology of primate malaria. Bulletin of the World Health Organisation 32, 363-387. Buratowski, S. and Zhou, H. (1992). Transcription factor IID mutants defective for interaction with transcription factor IIA. Science 255, 1130-1132. Ciliberto, G., Raugei, G., Costanzo, F., Dente, L. and Cortese, R. (1983). Common and interchangeable elements in the promotors of genes transcribed by RNA polymerase 111. Cell 32, 725-733. Clark, C.G., Tague, B.W., Ware, V.C. and Gerbi, S.A. (1984). Xenopus laevis 28s ribosomal RNA: a secondary structure model and its evolutionary and functional implications. Nucleic Acids Research 12, 6197-6220. Coatney, G.R., Collins, W.E., Warren, McW. and Contacos, P.G. (1971). “The Primate Malarias”. US Dept of Health, Bethesda, USA. Comai, L., Tanese, N. and Tijan, R. (1992). The TATA-binding protein and associated factors are integral parts of the RNA polymerase I transcription factor, SLI. Cell 68, 965-976. Conconi, A., Widmer, R.M., Koller, T. and Sogo, J.M. (1989). Two different chromatin structures coexist in ribosomal RNA throughout the cell cycle. Cell 57, 753-761.

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Molecular Mimicry Roger Hall

Department of Biology, University of York, York, YO1 5DD, U K

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81 ....................... 81 1.1. Definition . . . . . . . . . . . . . . 1.2. How is molecular mimicry 1.3. What are the potential consequences 1.4. How widespread is molecular mimicry? . . . . . . . . . . 84 2. AdaptiveMimicry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85 2.1. Mimicry of cytoadhesive proteins and their receptors 85 94 2.2. Mimicry of effectors of the immune system ............................ 2.3. Hormones, hormone receptors and serum protein receptors . . . . . . . . . . . 100 2.4. Cytoskeletal and muscle proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Consequential Mimicry: Autoimmunity 3.1. Bacterial mimicry . 3.2. Protozoal mimicry 3.3. Viral mimicry . . . . .

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4. How Does Molecular Mimicry Cause Disease?Autoimmune Considerations 5. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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1. INTRODUCTION 1.I.Definition

Molecular mimicry has been defined in several different ways. The first formal definition, provided by Damian (1964), states that molecular ADVANCES IN PARASITOLOGY VOL 34 ISBN % I 2 4 3 1 7 3 4 4

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mimicry is the sharing of antigenic determinants between a parasite and its host. An alternative, provided by Oldstone (1989a) states, that molecular mimicry is the possession of similar structures between molecules produced by dissimilar genes. He qualifies this statement by pointing out that this sharing can be at the level of linear sequence a n d o r conformation. There are problems with both definitions in the opinion of the present author. Damian’s is hampered by relying solely on an immunological criterion. Functional homologies (either of a linear or conformational nature) may be immunologically inert and, although biologically meaningful, would be disallowed under Damian’s definition but be accepted by Oldstone’s criteria. Conversely, important immunological cross reactions between highly conserved molecules, e.g. heat-shock proteins (with serious autoimmune consequences), would fit the Damian concept but are not acceptable under Oldstone senw stricto. A further complication is that Oldstone’s definition is restricted to proteins and thus excludes carbohydrate and lipid structures whereas these could be accommodated under Damian’s definition. I should, at this point state that I am not trying to imply that there is a rift between these two scientists. Rather, I am merely using their definitions as a means to discuss and highlight a central dilemma I have encountered whilst writing this review. Namely, what is molecular mimicry? The problem, at least in part, is due to the fact that molecular mimicry is quite simply not a single concept. At the very least, it is a term that embodies two essentially different notions which I shall term “adaptive mimicry” and “consequential mimicry”. I shall try to explain what I mean by these terms. The term “adaptive mimicry” is essentially self-explanatory and means that a parasite molecule mimics a host molecule for a biological reason. That reason may be immune evasion (Bloom, 1979) or subversion of some host-function for its own physiological or biochemical purposes, e.g. mimicry of a host ligand in order to recognize and enter a target cell. I would say that this definition would be best served by stating that true adaptive mimicry is where the homologous functional structure resides in a parasite molecule which is otherwise dissimilar to the host molecule. Such a state of affairs of course could arise by convergent evolution or, most intriguingly, by horizontal transfer of genetic material from host to parasite. It is arguable that functional mimicry may not be reflected at all in structural similarity but is still able to satisfy the definition of adaptive mimicry I am trying to espouse. The term “consequential mimicry” does not imply (but equally does not exclude) any adaptive force behind the observations of shared structures. Equally it does not impose the stricture that I put upon my definition of adaptive mimicry, namely that the homology need reside in an otherwise dissimilar molecule. The main point is that the result of the mimicry has

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direct consequences for the host; this can be achieved by sharing of phylogenetically conserved molecules or by small regions of similarity in evolutionarily distinct molecules. It is largely identical to Damian’s definition and I have introduced it really to allow a discussion, in this review, of the large and fascinating field of literature claiming that many autoimmune diseases are a result of shared, cross-reacting epitopes between parasites and their hosts. For the purposes of this review I have also taken the liberty of broadening the term parasite to include any invasive microbe including bacteria, fungi and viruses as there appears to be no good intellectual reason for making a distinction between these and the “traditional” parasites (helminths and protozoa). 1.2. How is Molecular Mimicry Studied?

There are three basic approaches to uncovering mimicked structures.

1. A search for immunologically shared determinants between parasite and host molecules using antibodies and less often cellular-based assays. Such an approach may uncover both adaptive and consequential mimicry although proving either is by no means a simple process. It will also undoubtedly reveal a proportion of fortuitously shared structures that are both biologically meaningless to the parasite and have no consequences for the host. One advantage of this approach is that there is, in principle, no chemical bias and shared lipid, carbohydrate and protein epitopes can be revealed. 2. The direct demonstration of molecules in the parasite which mimic defined host functions. Such demonstrations, by functional assays, are highly likely to be biologically meaningful. 3. Direct sequence comparisons between host molecules and parasite molecules by searching protein databases. This is obviously restricted to protein molecules and a degree of “noise” is inevitable especially if one allows short matches of say four amino acids. This approach, however, does allow for rapid comparisons to be made and some functional motifs may be predicted as well as potential immunological cross reactions suggested. All three approaches have been employed and examples of each are available and will be discussed at appropriate points. 1.3. What are the Potential Consequences of Molecular Mimicry?

The consequences of molecular mimicry are discussed by Damian (1989) and can be broken down into those that bear on the parasite and those

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that affect the host. In the context of the parasite the direct consequence will be enhanced fitness either by immune evasion (Sprent, 1962; Dineen, 1963; Damian, 1964) or by mimicry of functional structures facilitating physiological interaction with the host. From the point of view of the host the consequences are thought to be essentially two fold. 1. The evolution of extreme polymorphism of the mimicked structure in an attempt to become less susceptible to the parasite at the level of the population. Much speculation that polymorphism of blood group antigens, immune response and immune suppression genes is due to parasite pressure has been advanced (Snell, 1968; Mitchison and Oliveira, 1986), and mimicry may be the major underlying factor. As with many evolutionary questions the evidence for this is at once compelling but a’t the same time merely circumstantial. 2. The breakdown of self-tolerance, the development of an autoimmune response eventually leading to pathology. Essentially this is a mechanistic explanation for many autoimmune diseases the aetiology of which had until recently remained mysterious. These ideas are discussed extensively in recent reviews (Oldstone, 1987, 1989a, b; Barnett and Fujinami 1992; Tsuchiya and Williams, 1992; Horsfall, 1992; Baum et af., 1993) and will be expanded later in this article.

1.4. How Widespread is Molecular Mimicry?

Examples of molecular mimicry are now manifold and found in viruses, bacteria, protozoa and helminths (see Oldstone, 1989b). Perhaps the scale of the phenomenon is best illustrated in a study by Srinivasappa (Srinivasappa et al., 1986; Oldstone, 1989a) the results of which are reported in Table 1. The cross reactivity on uninfected host tissues of a panel of 708 monoclonal antibodies raised against 13 different viruses was assessed; 34 antibodies cross reacted which gives a figure of 4.8%. In another study, using the database sequence comparison approach, McLaughlin et af. (1987) compared six of the repeated antigen sequence motifs of plasmodia1 origin with human proteins; 29 matches of at least four amino acids were found. The biological relevance, if any, of these statistics is not clear. As stated earlier some of them are bound to be fortuitous “noise”. However, there are now many examples where the homology is so strong or the link between cross reactivity and autoimmunity so great that they cannot be dismissed lightly. In the remainder of this review I hope to demonstrate that molecular mimicry is a real phenomenon with definite consequences for host and parasite.

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Table I The extent of molecular mimicry as determined by monoclonal antibody cross reactivity between viruses and their host. Virus Coxsackie B4 Japanese encephalitis Lymphocytic choriomeningitis Theiler’s virus Measles Rabies Vesicular stomatitis Herpes Simplex (I) Vaccinia Dengue Cytomegalovirus Human Mouse HIV-1 Total

Number of monoclonals tested 66 34 174 64 39 80 37 20 16 132

Number reacting with uninfected tissues 1 6 3 9 5 2 2 1 1 0

24 14 8

1 0 1

708

34

Based on a study of Srinivasappa et al. (1986) and reproduced from Oldstone (1989a) with permission of the author and Springer-Verlag.

2. ADAPTIVE MIMICRY 2.1. Mimicry of Cytoadhesive Proteins and Their Receptors

2.1.1. Thrornbospondin E Region and Proteins of Malaria The circumsporozoite protein (CSP) of malaria provides possibly the bestdefined example of molecular mimicry which can be shown to have a direct functional role. The CSP is the major surface protein of malaria sporozoites and it has been much characterized as a vaccine candidate (Nussensweig and Nussensweig, 1989). The structure of the protein, which is essentially the same in all species of malaria examined, is shown diagrammatically in Figure 1A (reviewed in Kemp et al., 1987). It consists of three clearly defined motifs known as regions I and I1 plus the central repetitive domain (Dame et a l . , 1984). The repeats which form about one-third to one-half of the molecule vary between species in both unit length and sequence although the total length is fairly well conserved. Species with small repeat units have more motifs than species with large repeat units, e.g. approximately 40 repeats of NANP in P . fulciparurn versus 12 repeats of GQPQAQGDGANA in P . knowlesi. Regions I and I1 on the other hand are highly conserved across the species boundary. The high level of

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Generalised Structure o f CSP Slgnal Repeats

sequence

Charged area

Charged area REGION I

A

REGION I1

amino acid no. Pf Pv

Pk Pc Pb Py

345-372 312-329 297-314 312-329 274-298 302-319

E E E E E E

W W W W W W

S T T S S S

P P P P Q Q

C C C C C C

S S S S N S

V V V V V V

T T T T T T

C C C C C C

G G G G G G

N V N K S S

G G G G G G

I V V V I V

Q R R R R R

V V I M V V

R R R R R R

I S R R K K

K R K K R R

THROMBOSPONDIN amino acid no. 369-386 425-442 482.499

0

Figure 1 Structural features of the circumsporozoite protein (CSP) of Plasmodium spp. (A) Schematic of the overall architecture of the CSP. (B) Sequence comparison of region I1 of the CSP from various Plasmodium spp. to the three type I repeats of the cytoadhesive E region of thrombospondin. Homologous residues between thrombospondin and the various CSP sequences are enclosed in the main box. The Plasmodium spp. are abbreviated as follows: Pf, P . falciparum; Pv, P . vivax; Pk, P . knowlesi; Pc, P . chabaudi; Pb, P . berghei; Py, P . yoelii. Modified from Robson et al. (1988). with permission.

conservation of region I1 is shown in Figure 1B where the relevant sequences of six mammalian malaria species are compared. It is region I1 with which we are concerned in this review. The region I1 sequence of Plasmodium fafciparum (and other species of malaria) bears a striking homology to the type I repeats of the E regions of the cytoadhesive molecule thrombospondin (TS) (Frazier, 1987). The type I repeats are one of four regions in TS that mediate cytoadhesion (Prater et af., 1991). Furthermore, a study by Rich et af. (1990) demonstrated that the highly conserved VTCG motif (Figure 1B) was critical for adhesion to a number of human haematopoetic cell lines. Evidence has been published that this domain recognizes sulphated glycoconjugates (Holt et af., 1990). The sequence match, shown in Figure l B , extends over 18 amino acids with the P . fafciparum sequence being identical with one of the three E region sequences at 15 of the 18 positions. This homology is so striking that it led Frazier (1987) to speculate that this region may be the ligand by which sporozoites mediate their interaction with hepatocytes. This prediction was elegantly confirmed by Cerami et a f . (1992) who demonstrated that recombinant constructs of CSP containing region I1

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Figure 2 Immunogold electron micrographs demonstrating that the CSP binds to micovilli of human and rat hepatocytes in the space of Disse. (A) Human liver section labelled in a triple sandwich with recombinant CSP followed by an antiCSP mAb and visualized with protein A-gold. Aggregates of CSP molecules bind to hepatocyte microvilli within the space of Disse (arrows) and to the lateral membranes of adjacent hepatocytes (arrowheads). The hepatocyte surfaces exposed to the bile canaliculi (UC) and to the sinusoidal membrane of endothelia (EC) are not labelled. H, hepatocyte; N , hepatocyte nucleus; S, sinusoid; E, erthrocyte. Note that the entire surface of the hepatocyte (arrows) except the area exposed to the bile canliculus (BC) is labelled. (B) Higher magnification of a rat liver space of Disse showing CSP aggregates bound to hepatocyte microvilli. H, hepatocyte; EC, endothelial cell. In this section the gold was part of a goat anti-mouse conjugate. (Reproduced from Cerami et al. (1992), with permission of Cell Press.)

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would bind specifically to hepatocytes. This was demonstrated both on paraformaldehyde-fixed, frozen tissue sections of rat liver and on paraformaldehyde-fixed HepG2 (a human hepatoma cell line) cells. Constructs lacking region I1 did not bind and the specific binding was inhibitable with region I1 peptides. The peptides were also shown to be able to inhibit P . berghei sporozoite invasion of HepG2 cells as were antibodies directed against P. berghei region 11. The receptors for this ligand were demonstrated, by electron microscopy using immunogold labelling, to exclusively line the space of Disse in both human and rat liver sections (Figure 2). The space of Disse is the lumen of the liver exposed to the blood and hence the sporozoites. There was no binding to the hepatocyte membranes not in contact with the blood. Therefore, it can be concluded that region I1 of the CSP is a ligand for hepatocytes and this study is perhaps the clearest unequivocal demonstration of adaptive molecular mimicry to date. Robson et al. (1988) have described another malaria protein, which they term thrombospondin related anonymous protein (TRAP), that contains extensive homology with TS and properdin in exactly the same sequence as described for the CSP. TRAP, originally described as a blood-stage molecule, is now also thought to be expressed on the sporozoite and is probably identical to SSP2, another sporozoite antigen (Khusmith et al., 1991; Good et al., 1993). TRAP may play a role in the pathology of falciparum malaria which involves sequestration of the late blood stages in the deep vascular beds. This is a cytoadherence phenomenon and in in v i m models this can be inhibited with both TS and anti-TS antibodies (Roberts et al., 1985). It is highly tempting to speculate that this adherence is mediated at least in part through the TS motif carried by TRAP especially since platelet glycoprotein IV (the TS receptor) is implicated in the process (Barnwell et al., 1985). Whatever the explanation for this degree of homology it is remarkable and is surely more than fortuitous. Also of note is the fact that TRAP possesses an RGD motif which is found as a ligand on many cytoadhesive proteins including TS (Pierschbacher and Ruoslahti, 1984; Ruoslahti and Pierschbacher, 1987). 2.1.2. Elastin Mimicry by Theileria annulata One of the most extensive examples of molecular mimicry so far discovered involves a sporozoite surface antigen (SPAG-1) of the bovine apicomplexan parasite Theileria annulata (Williamson et a f . ,1989; Hall et al., 1992; Boulter et al., 1994). The SPAG-1 molecule is shown diagrammatically in Figure 3A. The molecule has two areas which contain repeats of the motif PGVGV. The most N terminal block contains this motif tandemly repeated 11 times whereas downstream there is a block of six

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I

I [PGVGVI, I

[PGVGVI

I Hydrophoblt dOmalnS, putative signal ana membrane anchor sequences

4

Elastin llke PGVGV repeats contalnlng VGVAPG Units

A

(i)

S E

179 PGVGVPGVGVPGVGVPGVGVPGVGVPGVGV IIIIIIIIIIIIIIIIIIIIIIIIIIIIII 335 PGVGVPGVGVPGVGVPGVGVPGVGVPGVGV PGVGGVPGVGVAPGVGVPGVGVAPGVGV 236 Ill IIIIIII IIIIIIIIII IIIII PGV.GVPGVGV.PGVGVPGVGV.PGVGV 389

(ii)S

588

E 1010

CCCGGTGTAGGTGTTCCAGGAGTTGGTGTTCCAGGA I I I I II II I I II I1 I I II II I I II CCAGGAGTTGGAGTCCCTGGTGTCGGGGTCCCTGGT

GTAGGTGTTCCAGGAGTAGGTGTTCCAGGAGTAGGT I I II IIIIIIII II II II II I I II II GTCGGGGTTCCAGGTGTCGGGGTCCCTGGTGTCGGG GTTCCAGGTGTAGGTGTGCCAGGTGTAGGAGGTGTT IIIIIIIIIII I I II IIIIIIII II II GTTCCAGGTGTCGGAGTCCCAGGTGTT. . .GGAGTC CCCGGAGTTGGCGTTGCACCAGGGGTAGGTGTTCCA I1 I I I I I I I I I I I II I I II I I II CCAGGTGTTGGAGTC . . .CCTGGTGTTGGGGTCCCT GGAGTTGGTGTTGCACCAGGTGTAGGTGTT 761 I I IIIII II I I IIIII II Ill GGTGTTGGCGTC . . .CCTGGTGTTGGAGTT 1183

( iii )

..

424 PGVGVPGVGVPGVGVPGVGVA. , PGVGV . . . . VPGVGGA 456 IIIIIIIIIIIIIIIIIIII: IIIII IIIII.: E 335 PGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVP375

S

B Figure 3 Mimicry of bovine elastin by Theileriu unnulutu surface antigen SPAG-1. (A) Schematic of the structure of the SPAG-1 polypeptide emphasizing the location of the elastin homologous regions. (B) Sequence comparison between SPAG-1 PGVGV repeats and bovine elastin: (i) the most N terminal block of PGVGV repeats; (ii) the DNA sequence comparison over the same region compared in (i); (iii) the more C terminal block of repeats. The VGVAPG motifs are underlined. S denotes the SPAG-1 sequence and E denotes the bovine elastin sequence in all cases. (Reproduced from Hall et al. (1992), with permission from Elsevier Science Publishers.)

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tandem repeats. The motif PGVGV is also repeated in the extracellular matrix protein elastin from a number of species including the host for T . annulata, Bos taurus. The motif occurs 11 times in tandem in bovine elastin and the alignment of the Theileria and bovine sequences is shown in Figure 3B. The homology is striking with only three differences over the N terminal block between the sequences all due to insertions in the Theileria sequence (Figure 3B(i)). Two of the three insertions are due to the presence of an A residue between the basic PGVGV units which allows the generation of a hexapeptide VGVAPG (underlined in Figure 3B). This structure is also present in the downstream repeat block (Figure 3B(iii)) and intriguingly also occurs twice in bovine elastin but downstream of the main repetitive region. Even more interestingly this VGVAPG motif has been demonstrated to be the ligand for an elastin receptor on certain cell types (Blood et al., 1988; Mecham et al., 1989; Robert et al., 1989) including macrophages and monocytes which are favoured targets for T. annuluta infection (Glass et a l . , 1989; Spooner et al., 1989). It is tempting to speculate that this represents a ligand for host cell recognition but unfortunately two lines of evidence argue against this. In the first place inhibition studies with VGVAPG peptides and an anti-VGVAPG monoclonal have proved inconclusive (Brown et al., unpublished observation). Secondly, and more importantly, a second complete SPAG-1 allele has been sequenced which does not contain the VGVAPG motifs, although it does retain the extensive PGVGV mimicry (Hall, unpublished observation). Thus we are left with the extensive elastin mimicry and no good functional explanation other than immune evasion. The evolution of this system is an intriguing question and some clues are perhaps provided by the comparison at the nucleotide level shown in Figure 3B(ii). As can be seen there is a high degree of third position wobble such that the protein sequence is maintained but the nucleotide sequence varies so that it reflects the overall AT richness of the Theileria genome. Consistent with this is the fact that of 43 silent third base changes 18 are A/T for G/C substitutions whereas only three are in the reverse direction. The question remains “Did this parasite acquire this genetic material by convergent evolution or horizontal gene transfer from the host?” However it was acquired, it is perhaps instructive to note that the analogous antigen possessed by the closely related species T. parva does not possess this extensive elastin mimicry but it does have one copy of the motif PGVGV (Nene et al., 1992). Does this represent a contraction of the original repetitive motif acquired by gene transfer from the cow, or is it a reflection of the precursor situation before the original motif was expanded tandemly? I fear we shall never know but the observation is fascinating.

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91

2.1.3. Mimicry of Fibronectin Receptors Several extracellular (e.g. Entamoeba histolytica) and intracellular parasites (e.g. Leishmania spp. and Trypanosoma cruzi) have evolved receptors for fibronectin which they presumably utilize for different purposes. Fibronectin is a high-molecular-weight glycoprotein composed of 220 kDa subunits linked into dimers and polymers by disulphide bonds. It is present in blood, connective tissue and at cell surfaces. It interacts with cells and may mediate cell adhesion by cross bridging. The ligand for cell adhesion has been mapped to a three amino acid motif, RGD, which is also utilized by other integrin receptors (Ruoslahti and Pierschbacher, 1987). (a) Trypanosoma cruzi. Elegant data demonstrating the presence and utilization of fibronectin receptors by a parasite concerns the case of T . cruzi (Ouaissi et al., 1984, 1985, 1986). In this series of studies it was demonstrated that T . cruzi binds to fibronectin and that this interaction mediates host cell interactions. In their rigorous 1986 study Ouaissi and colleagues showed that short peptides containing the fibronectin ligand RGDS bound to the surface of trypmastigotes and, importantly, inhibited invasion of mouse 3T3 fibroblasts. They also demonstrated that monoclonal antibodies to the cell attachment site of fibronectin were inhibitory. Most interestingly these authors demonstrated that BALB/c mice were rendered resistant to T . cruzi infection by immunizing mice with a peptide containing the R G D motif (AVTGRGDSPC) coupled to tetanus toxoid. The suggestion is made that the parasite is able to utilize this as a host recognition system because fibronectin is often in the form of a dimer (or larger polymer) which can form a bridge between the two cells. (b) Leishmania spp. Studies with promastigotes of Leishmania chagasi (Rizvi et a f . , 1988) demonstrated that peptides based on R G D inhibited binding to macrophages, suggesting that this parasite might have evolved a structure similar to this to gain host cell entry. Indeed such a structure appears to reside on the surface protease gp63. This was demonstrated by showing that RGD-containing peptides could inhibit the interaction of macrophages with gp63-coated silica beads (Russell and Wright, 1988). This was originally explained by a predicted R G D motif in gp63. However it was subsequently shown that no RGD was present as sequencing errors had occurred (Button and McMaster, 1988, 1990). The data therefore, although intriguing and suggesting functional mimicry do not as yet have a satisfactory structural basis. (c) Entamoeba histolytica. In an interesting study Talamas-Rohana and Meza (1988) reported a molecule on the surface of Enfamoeba histofyfica that binds to fibronectin. They demonstrated that '251-labelled fibronectin (FN) bound to the surface of live trophozoites in a saturable manner and that the binding could be inhibited competitively by unlabelled fibronectin.

Table 2 Complement binding and regulatory structures mimicked by microorganisms to evade the complement system (andor augment infection).

Complement-bindindregulatory Complement-like functions of microorganism motif of proteins microorganism proteins SCR Block ACP C3 convertase assembly ? Block ACP C3 convertase assembly Block assembly and accelerates ? decay of ACP and CCP C3 convertases

References

Microorganism

Microorganism protein

Trypanosoma cruzi

gP160

Homologous complement protein(s)/ activity CRI, DAF

gp58168

?

87-93 kDa

DAF

Entamoeba histolytica

170 kDa surface galactose binding adhesin

CD59

Blocks MAC

aa 2&55 in CD.59

Candida albicans

p42-CR3

CR3

Binds iC3b but reason unclear

55/60 kDa doublet

CR2

Binds C3d but reason unclear

Shares mAb epitopes Alaei with human CR3 et al. (1993) ? Saxena and Calderone (1990)

CSP, TRAP

Properdin, C6,C7,C8,C9

Binds C3b and then host cells via CRl? Binds host cells directly

Plasmodium fakiparum

Type 1 repeat of thrombospondin also found in properdin, C6,C7,C8 and C9

Cooper (1991) Fischer etal. (1988) Joiner et al. (1988) Braga et al. (1992)

Robson el al. (1988)

Goundis and Reid (1988)

Eimeria tenella

Etp 100

Properdin, C6,C7 ,C8,C9, Factor B

Binds C3b and then host cells via CRl? Binds host cells directly

Type 1 repeat of thrombospondin also found in properdin C6,C7,C8 and C9

Tomley et al. (1991)

Babesia rhodaini

?

CRI?

Binds C3 and acts as bridge to recognize red blood cell?

?

Jack and Ward (1980)

Schistosoma mansoni

?

CRl?

Binds C3b and prevents cascade? ?

Tarleton and Kemp (1981)

Vaccinia

gp35

C4bp

Block CCP C3 convertase assembly

SCR

Kotwall and Moss (1988) Kotwall et al. (1990)

HSV

gc-1 ,gc-2

CRl ,DAF

Disassemble ACP C3 convertase

SCR-like

Seidel-Dugan et al. (1990)

EBV

?

CRl ,DAF,MCP

Factor I cofactor for C3 and C4 cleavage, disassemble ACP C3 convertase

?

Mold et al. (1988)

Abbreviations: CCP, classical pathway; ACP, alternative pathway; TRAP, thrombospondin-related anonymous protein; DAF, decay accelerating factor; SCR, short consensus repeat; MCP, membrane co-factor protein; MAC, membrane attack complex. (Reproduced in modified form from Cooper (1991), with permission of Elsevier Trends Journals.)

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This was confirmed by a triple-layer immunofluorescence assay whereby unlabelled FN was bound to the trophozoites and then incubated first with rabbit anti-human FN serum followed by FITC-labelled sheep anti-rabbit IgG. The molecule responsible for this interaction was identified as a 37 kDa membrane peptide by probing western blots of plasma membrane fractions with 12sI-labelledFN. It was also demonstrated that the amoebae internalized the FN into digestive vacuoles and degraded it. Possibly the most notable observation was that upon interacting with FN the amoebae re-arranged their cytoskeleton resulting in the formation of actin containing “adhesion plaques”, revealed by probing the trophozoites with rhodamine-labelled phalloidin. The detailed molecular basis for this interaction remains to be fully elucidated but it can be speculated that it would provide the amoebae with a useful method of interacting both with cells prior to phagocytosis and also the extracellular matrix through which pathogenic forms must migrate. 2.2. Mimicry of Effectors of the Immune System

2.2.1. Mimicry of Complement Receptors and of Other Modulators of Complement Activation

The functional mimicry of complement receptors and modulating factors is a recurring theme in parasitology (for a review see Cooper, 1991). A few striking examples are outlined below and a more general list is documented in Table 2. Note also that I have not discussed the elegant examples provided by viruses as they are not the main thrust of this article. (a) Plasmodium spp. Region I1 of the CSP as well as being homologous to thrombospondin also possesses homology with the complement-binding region of properdin (Goundis and Reid, 1988) and four of the five terminal complement components (Stanley et al., 1985; Rao et al., 1987; DiScipio et al., 1988; Chakravarti et al., 1989) (Table 2). This has led to the suggestion that perhaps there is another functional role for this mimicry, possibly through binding complement which would then allow interaction with CR1 receptors and thus possibly another route to host cell recognition. A similar suggestion has been made for TRAP in that if it were present on the merozoite surface then it may bind CR1 on red cells via a C3b bridge using the properdin C3b binding domain (Table 2). No data are available to support either of these contentions. (b) Eimeria tenella. Variations of the region I1 motif of the CSP are found repeated five times in a microneme located sporozoite antigen of Eimeria tenella (Clarke et al., 1990; Tomley et al., 1991) (Table 2 ) . Furthermore, this antigen also has another region homologous to factor B

MOLECULAR MIMICRY

95

(amongst other things). The authors of this work speculate that this factor B-homologous region may bind C3b to the surface and that this interaction is then stabilized by the properdin/thrombospondin repeats. The biological reason for this they suggested is to utilize the complement components to facilitate host cell penetration. Direct data to test this are lacking. (c) Babesia rhodaini. A very clear example of the subversion of complement binding was demonstrated in the case of Babesia rhodaini by Jack and Ward (1980) (Table 2) who demonstrated that the ability of this parasite to invade red cells is critically dependent on the presence of complement. They showed that reagents that deplete complement (e.g. cobra venom) delay the onset of parasitaemia in rats whereas the drug suramin (which stabilizes complement levels) increases the parasitaemia. Also, trypan blue (which inhibits C3b receptors) delays parasitaemia. It has also been demonstrated directly that B. rhodaini binds C3 and thus it is believed that this parasite carries surface receptors for complement which facilitate red cell entry by allowing interactions with the red cell via a complement bridge to the C R l receptor. (d) Schistosoma mansoni. Several studies have established that S. mansoni possesses receptors for complement on various life-cycle stages (Santoro et a f . , 1979, 1980; Tarleton and Kemp, 1981) (Table 2). These include receptors for C l q and C3b. Perhaps the clearest demonstration of C3b receptors on adult worms is provided by an indirect assay based on the ability of the adult worms to bind antigen-F(ab’).+omplement complexes specifically (Tarleton and Kemp, 1981). These were then detected by using a second antibody against either the antigen (BSA), the first antibody or complement and the final sandwich was revealed utilizing fluoresceinated Staphylococcus aureus. Specificity controls showing dependence on activated complement (presumably C3b) were included. In addition, it was demonstrated that binding was dependent on the presence of a metabolic inhibitor (2-deoxyglucose) and it was suggested that this was because the complexes are otherwise rapidly cleared. The role and origin (host or parasite) of the receptors remains unclear and these authors suggest that the receptor might bind and inactivate C3b (produced either by the classical or alternative pathway) and prevent the remainder of the complement cascade proceeding to produce C9 and damage the worm. In other words this could be a sophisticated form of evasion of host defence mechanism. (e) Candida albicans. In 1985, Heidenreich and Dierich described the binding of erythrocytes coated with human iC3b and C3d to the germ tubes of Candida albicans. This was considered good evidence for specific receptors on the parasite similar in specificity to CR2 (C3d binding) and CR3 (iC3b binding) (Table 2). These findings have been confirmed (Edwards et a l . , 1986) and extended by observing that certain anti-human

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CR3 monoclonal antibodies (recognizing both the a (OKM-1, Mol and M1/70HL) and possibly the p (7C3) chains of the receptor) bind specifically to the germ tubes (Eigentler et a f . ,1989; Mayer et a f . ,1990). Other specific anti-human CR3 monoclonal antibodies did not bind demonstrating only partial similarity and not identity with the human CR3. Interestingly, the monoclonal 7C3 that recognizes the p chain is directed against a carbohydrate determinant composed of lacto-N-fucopentaose. Eigentler et u f . (1989) used monoclonal OKM-1 to affinity purify presumptive CR3 like receptors and succeeded in isolating three species of molecule ( M , = 50, 100 and 130 kDa) each of which is presumed to have iC3b binding activity. In a more recent study affinity chromatography using C3(H20)-Sepharose was used to extract a 42 kDa molecule (p42-CR3) from lysates of pseudohyphae (Alaei et a f . , 1993). p42-CR3 was shown to cross-react with mAb OKM-1 and rabbit antibodies against it inhibited the binding of iC3b coated sheep erthrocytes to pseudohyphae. When the rabbit antiserum was used in affinity chromatography further components of 66 and 55 kDa as well as p42-CR3 were identified and evidence was presented that these represented glycosylated forms of p42-CR3. The authors do not discuss the relationship (if any) to the OKM-1 binding molecules purified by Eigentler but they do assert that the structure of the C. albicans CR3 receptor is distinct from the human counterpart. The basis for the mAb cross-reactivity and the iC3b binding will hopefully be revealed by cloning the p42-CR3 gene. The C. albicans CR2-like molecules have been studied in depth. Initially, using C3d ligand and monoclonal antibody affinity chromatography to purify proteins from cell lysates, species of 60 and 70 kDa were obtained; ligand activity is believed to reside on the 60 kDa molecule (Calderone et al., 1988; Linehan et a f . , 1988). To obtain large quantities for detailed analysis the same group of workers purified C3d binding proteins from the pseudohyphal culture medium (Saxena and Calderone, 1990). This culture filtrate (simply obtained by filtering the culture through filter paper) was fractionated by preparative isoelectric focusing and assaying fractions for C3d activity. The result was the identification of a pure sample containing a doublet of 60 and 55 kDa. These molecules are thought to be differentially glycosylated forms of the same protein. Analysis of the carbohydrate composition and structure plus the amino acid composition demonstrated that the C. albicans CR2 is distinct from the human receptor. (f) Entamoeba histolytica. Entumoeba histolytica trophozoites that are exposed to human serum do activate complement but are resistant to killing by the C5b-9 membrane attack complex (MAC) (Reed and Gigli, 1990). In a careful study Braga et al. (1992) demonstrated that this inhibition is mediated by interaction of a surface molecule with the termi-

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nal complement components (Table 2). They approached this question by raising a monoclonal antibody (mAb 3D12) which abrogated complement resistance when added either as purified intact IgG or importantly as a Fab fragment. When the analysis was pursued further they discovered that mAb 3D12 recognized a previously characterized surface adhesin which has been shown to be a galactose-specific lectin (Petri et al., 1987, 1989, 1990). They then demonstrated that the purified adhesin inhibited by 90% the lysis of sensitive trophozoites by C5b-9 components. It was shown that the adhesin binds to C8 and C9 and that this binding could be inhibited by mAb 3D12. Sequence analysis and comparison revealed the noteworthy observation of a degree of homology to the host molecule CD59. Furthermore, the non-reduced lectin (composed of a heavy and light subunit) reacted with an antiserum to CD59. CD59 is an inhibitor of the complement MAC found in the membrane of human blood cells (Zhao et al., 1991). Thus it appears that E . histolytica has evolved a complement evasion strategy by mimicking both structurally and functionally the host molecule CD59. (g) Trypanosoma cruzi. The insect stage of T. cruzi, known as epimastigotes are sensitive to the alternative pathway of complement, whereas the trypomastigotes which are infective to mammals are resistant (Kipnis et al., 1981; Schenkman et al., 1986). The mechanisms involved in this evasion are not fully understood but are thought to involve inhibitors of C3 convertase and functional analogues of decay accelerating factor (DAF) (Table 2). One study demonstrated that a purified glycoprotein (gp58/68: non-reduced size M,=58 kDa and, reduced size M,=68 kDa), which is a fibronectidcollagen-binding molecule, was able to inhibit the formation of cell-bound and fluid-phase alternative pathway C3 convertase. However, gp58/68 did not display any other effect such as D A F activity or classical pathway convertase activity. It is suggested that the activity operates by binding to factor B (Fischer et al., 1988). Other workers have demonstrated D A F activities. Joiner et al. (1988) purified a 87-93 kDa fraction from the culture supernatant of trypomastigotes which interferes with the formation and accelerates the decay of the alternative and classical pathway C3 convertases. Another molecule, called gp160, has been purified and shown to bind C3b and block C3 convertase formation. The gene for this is reported to hybridize to the human DAF gene suggesting a structural basis for the observed functional mimicry (Cooper, 1991). Thus T. cruzi trypomastigotes apparently mimic a range of modulators of the complement cascade in order to avoid lysis. 2.2.2. Fc Receptor Mimicry One common strategy that parasites adopt to modulate and possibly avoid the host immune system is to bind immunoglobulin by its Fc portion. Such

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Fc receptors are found on a wide array of parasites including bacteria (see Boyle, 1990a), protozoa (Ferreira de Miranda-Santos and Campus-Neto, 198l), schistosomes (Torpier et a f . , 1979; Tarleton and Kemp, 1981) and viruses (Ogata and Shigeta, 1979; Para et a f . ,1980; Tuckwiller et a f . ,1981). There appears to be a correlation between the presence of Fc receptors on a parasite and pathogenicity. However, the means whereby virulence is promoted as a consequence of the possession of Fc receptors is not fully elucidated but probably involves a range of effects. These include among others complement depletion and inhibition of opsonization (extensively reviewed by Langone, 1982; Widders, 1990). The present discussion will focus on bacterial Fc receptors (principally protein A) and those described on schistosomes. (a) Protein A and protein G . In 1966 Fosgren and Sjoquist described in detail the so called “pseudoimmune” reactivity of Staphylococcus aureus. These authors showed that the ability of most human S. aureus strains to bind non-immune IgG was mediated by a surface protein that had been named protein A by Grov et a f . (1964). In fact the first description of the activity of protein A is now credited to Jensen (1958). Protein A forms the prototype type I bacterial IgG Fc receptor (Boyle, 1990b). This is the first in a series of at least six types numbered I-VI (see Boyle, 1990a). Protein A is present on the surface of all natural human S. aureus isolates and is also secreted into the serum. Variation in the affinity for IgG from different mammalian species is observed (see Table 3 ) and there is also marked variation in affinity for different IgG subclasses (Table 4). The gene for protein A has been cloned and sequenced (Guss et a f . , 1990) and is shown diagrammatically in Figure 4. Peptide mapping and molecular expression studies have revealed that the protein contains 5 IgG binding domains. These occupy more than half the mature molecule and are located in a tandem block in the N-terminal section. They are designated E, D, A, B and C and they are all highly homologous to one another. An “homology gradient” has been noted among the regions such that the neighbouring regions are more homologous than non-neighbours. This has been interpreted to imply evolution by a series of stepwise duplications of the original Fc receptor domain. The other major well-characterized bacterial Fc receptors (types 11-VI) all reside on streptococci (Boyle, 1990a). I will describe briefly a type I11 receptor found in the group G streptococci called protein G and which has been well characterized (see Bjorck and Akerstrom, 1990, for more detail). Protein G binds a wider range of IgG than protein A and in particular has a high affinity for human IgG3. The gene for protein G has been cloned and sequenced and the immunoglobulin binding domains have been mapped. Interestingly there is no homology at all between the IgG binding domains of protein A and protein G. Also of note is the fact that

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Table 3 Reactivity of protein A with different species of IgG. Species

IgG (ng) required to inhibit '"I-PrA binding by 50%

Human Rabbit Pig Goat Sheep cow Dog Rat

13 130 118 13 000 40 000 21 000 100

> 10-5

Reproduced from Boyle (lYYOb), with permission from

Academic Press.

Table 4 Reactivity of protein A with different human IgG subclasses. IgG (ng) required to inhibit '''I-PrA binding by 50%

IgG subclass

~~~

~

Reproduced from Boyle (IYYOb), with permission from Academic Press. STAPHYLOCOCCAL PROTEIN A

S

E

D

A

B

C

Xr

xc

Figure 4 Diagram of the different regions encoded by the gene for staphylococcal protein A. S is the signal sequence; E, D, A , B and C are the homologous, repetitive, immunoglobulin-binding (Fc receptor) regions. Xr is a repctitivc part of the X region involved in binding the cell wall whereas Xc is the region spanning the membrane. (After Guss et al. (1Y90), with permission from Academic Press.)

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protein G binds serum albumin and a2-macroglobulin with very high affinity. Protein A shares the ability to bind a2-macroglobulin but does not bind albumin. The significance of these latter observations is not clear. (b) Schistosoma mansoni. Fc receptors have been demonstrated on larval and adult schistosomula using sheep erythrocyte rosetteing techniques (Torpier et a f . , 1979; Tarleton and Kemp, 1981). The demonstration on adult worms was more difficult than larvae requiring the use of a sensitive sandwich technique utilizing fluoresceinated S. aurew as described above for the complement receptor assays. The Fc receptors found by Torpier on schistosomula (larval forms) are of great interest because they were demonstrated to also bind to P2-microglobulin. The significance of this is that earlier work had shown that host antigens are adsorbed onto schistosomes (Smithers and Terry, 1976) and the identity of some of these components was demonstrated to be major histocompatibility antigens (Sher et a f . , 1978) which are composed of a heavy chain and a light chain identified as P2-microglobulin. Thus a direct receptor for these molecules has been defined. The ability of schistosomes to mimic functional receptors of the host and use them to coat themselves in host components presumably thereby escaping immune detection offers a subtle twist to the molecular mimicry concept. It is also an explanation (albeit incomplete) for the phenomenon of concomitant immunity whereby infected individuals are unable to kill their existing worm burden but are resistant to superinfection. 2.3. Hormones, Hormone Receptors and Serum Protein Receptors

The ability of parasites to exploit the extracellular hormonal signalling mechanisms of the host and exhibit a growth response represents a very subtle and complex form of subversion. A number of examples of possession of receptors by parasites for host hormones exist and may well be regarded as molecular mimicry par excellence. In addition some parasite surface proteins have domains modelled on hormones themselves providing possible modes for cellular recognition and entry. 2.3.1. Steroid Receptors in Candida albicans Candida afbicans possesses cytosolic binding proteins for the mammalian steroid hormones progesterone, corticosterone (Loose et a f . , 1981; Loose and Feldman, 1982; Das and Datta, 1985) and oestrogen (Powell et a f . , 1984; Skowronski and Feldman, 1989). The progesterone and oestrogen receptors are independent protein molecules. In the case of the progesterone receptor it was found that there was also a binding activity in

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another non-pathogenic yeast, Saccharomyces cerevisiae, but that the activity was only 14% of that found in Cundida (Das and Datta, 1985). Pregnant women, women taking contraceptive pills and persons on glucocorticoid therapy have a much enhanced risk of systemic candidiasis. It is tempting to speculate that this pathogenesis is a response to the steroids mediated through the receptor molecules and forms an adaptation of the fungus to the mammalian host. The gene for the high-affinity corticosteroid binding protein has recently been cloned and sequenced (Malloy et a l . , 1993). Comparison of this sequence to the steroid-thyroid-retinoic acid receptor gene superfamily did not reveal any homology and it must be concluded that this represents a strict case of functional mimicry by convergent evolution. 2.3.2. Epidermal Growth Factor (EGF), Low Density Lipoprotein ( L D L ) and Transferrin Receptors in Trypanosoma brucei African trypanosomes grow in the gut of the tsetse fly vector and in the serum of the mammalian host. During these periods of growth the parasite undergoes a series of morphological and metabolic differentiation events. The cycle in the mammal is initiated by the injection of the metacyclic stage into the blood where a transition to rapidly dividing long slender forms occurs. Growth arrest then occurs on transition to short stumpies which are ingested by the fly where they transform in the gut into actively growing procyclics. These migrate to the salivary glands and arrest as the metacyclic stage before transmission and repetition of the cycle. The bloodstream and procyclic forms grown in culture show an absolute requirement for fetal bovine serum. This serum dependence can be explained if it provides a source of essential growth factors and nutrients. Evidence that this is the case is provided below. Strong evidence has been provided by Hide et al. (1989, 1990) that T. brucei possesses a functional homologue of mammalian epidermal growth factor receptor (EGFR) on its surface. These authors demonstrated that polyclonal antibodies both to pure mammalian EFGR and to synthetic peptides modelled on the external domain of EGFR react with a surface molecule of 135 kDa present on procyclics and bloodstream forms. Immunoprecipitates using these antibodies contained a kinase activity consistent with the presence of an EGFR-like molecule. They also demonstrated that a molecule of the same size present in membrane-enriched fractions could be labelled by cross-linking with biotinylated EGF. Furthermore, it was shown that a growth response to E G F was achieved by serum deprived procyclic forms in tissue culture. Taken as a whole, these data suggest that the trypanosomes are functionally mimicking a host cell EGFR as part of their growth regulation strategy. The precise physiological significance however remains unclear.

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There is very good evidence for the uptake of low density lipoprotein (LDL) and transferrin by a process of receptor-mediated endocytosis in bloodstream forms of T. brucei (Coppens et al., 1987). Several lines of evidence support this. 1. Clearance values for these two proteins were two to three orders of magnitude greater than serum albumin. 2. Both proteins bound to the parasites in a saturable manner and this binding was inhibitable only by the homologous ligand. 3. LDL and transferrin colloidal gold conjugates were efficiently taken up and could be visualized by EM in the flagellar pocket and endocytotic vesicles. Presumably these are the routes whereby this parasite obtains important nutrients namely cholesterol and iron. Indeed, these authors stated that trypanosomes cultured in cholesterol-free medium had their growth rate halved and that after 24 hours in delipidized medium LDL uptake was increased by 40%. In a subsequent publication they demonstrated that the use of lipoprotein-depleted serum extended the doubling time in vitro from 14.4 h to 21.1 h and that normal growth was restored by the addition of LDL (Coppens et al., 1988). Using LDL affinity chromatography a 145 kDa glycoprotein has been purified from T. brucei (Coppens et a l . , 1991) and an antiserum raised against this cross reacts with the LDL receptor derived from rat liver (Coppens et al., 1992). Therefore the T. brucei LDL receptor shares some degree of structural homology with its mammalian counterpart in addition to its functional mimicry. It is noteworthy that other examples of receptors for serum proteins have been reported in protozoa. Thus Trichomonas vaginalis has LDL receptors (Peterson and Alderete, 1984), Trypanosoma cruzi bears high density lipoprotein receptors (Prioli et al., 1987) and transferrin receptors are found on Plasmodium fulciparum (Rodriguez and Jungery , 1986). The basis for all these examples of functional mimicry may be reflected in the structure of the molecules when they are cloned and characterized. 2.3.3. Epidermal Growth Factor Motifs in Plasmodium Surface Proteins Two surface proteins expressed on invasive stages of the malaria parasite P. falciparum have been found to contain motifs remarkably similar to domains found in EGF. One of these proteins, Pfs25, resides on the surface of the motile zygote (ookinete) that is formed by fertilization in the mosquito gut. Pfs25 contains four EGF-like domains in tandem and the degree of similarity is striking as shown in Figure 5 (Kaslow et al.,

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CONSENSUS: PFS 2 5 L I N 12 NOTCH EGF LOL RECEPTOR

i n -x-

-

X G x x x, - C x L x L x x D x K x C

-

- -

B

Figure 5 Mimicry of EGF by sexual stage antigen of Plasmodium falciparum Pfs25. (A) Protein structure of Pfs25 arranged to emphasize the relatedness of the four EGF-like domains. Cysteine residues, and other identical or related amino acids are boxed. (B) EGF-like repeat consensus sequence from Pfs25, lin-12 (Greenwald, 1985), notch (Wharton et al., 1985), EGF (Gray et al., 1983; Scott et a l . , 1983) and human LDL-receptor (Sudhof et al., 1985). Cysteine residues are boxed. A double box frames the core of the consensus sequence. x represents any amino acid. -represents an unspecified number of amino acids. (Reproduced from Kaslow et al. (1988), with permission of the publisher.)

1988). The other protein is MSPl which is one of the major surface proteins of the invasive merozoite. This protein is known to be processed during merogony (Holder and Freeman, 1982; Hall et al., 1984; Blackman et af., 1991; Blackman and Holder, 1992). It is synthesized as a 190-210 kDa precursor which is extensively proteolytically processed to produce a 19 kDa C terminal fragment retained on the merozoite surface and which survives into the ring stage following red blood cell invasion. This 19 kDa fragment contains two tandemly repeated EGF domains (Figure 6) and antibodies directed to this region inhibit invasion. It is tempting to speculate that the parasite is mimicking the EGF structure in both proteins to function in binding to and invasion of the host cell. There are many other proteins known to contain these motifs in which it is believed to function in binding and signalling (Apella et af., 1988; Laurence and Gusterson, 1990).

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Pf gp19

putative GPI attachment

Figure 6 Postulated EGF-like domain structure of MSPl gp19. Schematic representation of gp19 based on human epidermal growth factor structure (Cooke et al., 1987) showing conserved residues (within circles, single letter code is used), non-conserved residues (blank circles), inserteddeleted residues (-), Cys residues conserved in P . fakiparum only (*), and semi-conserved Pro residues (+). The P. yoeli protective mAb 302 epitope (Burns et al., 1989) is picked out using brackets. GPI, glycosylphosphatidyl inositol. (Reproduced from Cooper (1993), with permission of Elsevier Trends Journals.)

2.4. Cytoskeletal and Muscle Proteins

2.4.1. Malaria The malaria parasites have an intimate association with their host red cells. The red cell is normally a rigid biconcave disc the integrity of its shape being determined by the complex series of interacting cytoskeletal components. The ability of the parasite to deform the red cell membrane upon invasion and to cause the creation of a parasitophorous vacuole for its residency is indeed remarkable. Furthermore in the case of Plasmodium fakiparum the schizont-infected red cell has protuberences on its surface (the so called “knobs”) that contain parasite proteins. These “knobs” are mediators of the cytoadherence to capillary endothelia causing the sequestration which underlies the cerebral form of the disease. How is this control over the red cell archictecture achieved? Clues to this are provided by the intriguing homologies that exist between certain malaria antigens and cytoskeletal proteins.

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MSPl, the major surface antigen of schizonts and merozoites, has been shown to have homology to the non-helical regions of intermediate filament proteins such as keratin, desmin and vimentin (Cheung et af., 1986). He points out that the common pattern, extending over 50 residues, is a stretch of glycine- and serine-rich repeats with a large hydrophobic residue (such as tyrosine, tryptophan, phenylalanine, valine, or arginine (which has a non-polar “stem”)) every 10-12 residues. It is speculated that this kind of sequence probably characterizes a conserved p-structure. Cheung suggests that this may have evolved in order for the merozoite to interact with the host cell cytoskeleton in order to camouflage itself. Alternatively he suggests that the structure induces some beneficial structural changes to the host membrane. Another antigen PflWRESA (ring-infected erythrocyte surface antigen) has homology to band 3, a red cell anion transporter (Anders et af., 1986, 1988; Holmquist et al., 1988). This antigen is present in merozoites and is deposited in the membrane of the erythrocyte at invasion. It possesses two domains that contain different tandemly repeated structures; one of these is centrally located and the other is at the C terminus. It is the C-terminal structure that possesses homology to a motif at the N terminus of band 3 . The dominant C-terminal repeat unit is EENV which is occasionally interspersed by the related sequence EEYD. Thus the actual sequence (EENVEEYDEENV) provides a tyrosine residue surrounded by several groups of acidic residues. The N-terminal band 3 motif (MEELQDDYEDE) provides a chemically similar structure. Peptides modelled on the RESA sequence block interaction with haemoglobin and also inhibit phosphorylation of band 3. Band 3 interacts, via ankyrin, with the red cell cytoskeleton through structures distinct from the acidic domain mimicked by RESA (Bennett and Stenbuck, 1980). Also the cytoplasmic domain of band 3 can exist in several alternative conformations (Low et al., 1984). These observations led Anders et al. (1988) to suggest the attractive idea that RESA, by disrupting the normal cytoplasmic interactions of band 3 , could induce a conformational change affecting its interaction with ankyrin and thus perturbing the red cell cytoskeleton. This, it is proposed, may increase membrane fluidity and allow the creation of the parasitophorous vacuole during merozoite invasion. Kilejian et af. (1991) performed an elegant study stimulated by an observed homology between one of the knob-associated proteins (KP or KAHRP, knob-associated histidine rich protein) and the spectrin-actin binding domain of the cytoskeletal protein 4.1. The homology extends over 22 residues of a lysine-rich region of protein 4.1 and is shown below with gaps introduced to maximize homology:

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4.1 Lys.Lys.Lys. A r g . Glu.A r g . Leu. Asp. Gly. Glu. Lys. Lys. Lys.His.Lys. A s p . His.-. A s p . Gly. Glu. Lys.

KP

4.1 S e r . Gln. Glu. Glu. Ile. Lys. Lys.His.His.A l a . Ser. Tyr. Gly. A s p . Glu.-. -. Lys.His.His.S e r . Ser.

KP

It is noteworthy that the distribution of charges over this region is also very similar, These authors purified a 30 kDa fragment (fragment 11) of the KP containing this structure and demonstrated that it was able to stably enhance the binding of spectrin to actin. However, it did not have the ability to interfere with the function of protein 4.1 as a mediator of spectrin-actin association. Of considerable interest was their demonstration that when fragment I1 was introduced into normal resealed red cell “ghosts” it was shown to associate with the surface-forming electron-dense knob-like aggregates. It was not conclusively proven that these effects were mediated via the homologous region denoted above and data using synthetic peptides must be obtained to resolve this. The above examples are probably just glimpses at the beauty of the processes whereby through structural mimicry the malaria parasite manipulates the structural integrity of its red cell host to its own advantage. 2.4.2. Schistosoma

Dissous and Capron (1989) have reported a case of shared antigenic determinants between s. mansoni and the snail intermediate host Biomphalaria glabrata. Specifically they demonstrated cross-reactions between snail and schistosome molecules of 39 kDa called Bg39 and Sm39 respectively. Using immunoelectron microscopy they located the epitopes in the schistosome to the muscles and interestingly to vesicles in the epidermal ridges and subtegumental cytons of miracidia. It is suggested that the new sporocyst tegument, created after shedding of the ciliated epidermal plates by the penetrating miracidium, may contain sm39 (Damian, 1991). When the sequence of Bg39 was obtained it was found to be a tropomyosin molecule (Dissous et al., 1990). Homology analysis demonstrated that the Bg39 (tropomyosin) and the schistosome tropomyosin are 65% identical. This is considered by the authors to be higher than expected since they point out that a similar comparison between Trichostrongylus colubriformis and schistosome tropomyosins gives a figure of 60% identity. They argue that the non-muscle tropomyosin presumed to be on the surface of the sporocysts may have evolved as an immune evasion strategy to resist amoebocyte attack. A similar argument has been advanced for the presence of the cytoskeletal protein actin being found in the surface spines of the adult worm tegument (Cohen etal., 1982; Damian, 1991). Obviously in this case it is the mammalian immune response which it is suggested is

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being avoided. The present author feels somewhat sceptical about these arguments and there is a definite onus on the advocates of these suggestions to collect further evidence.

3. CONSEQUENTIAL MIMICRY: AUTOIMMUNITY

Autoimmunity should not occur. Yet the human race is afflicted by a wide range of medical conditions the underlying basis for which appears to be an immune response directed at self. Examples are manifold and include diabetes, myasthenia gravis, rheumatic fever and various other cardiological, neurological and arthritic conditions. Why does this situation exist? What leads to the breakdown of self-tolerance? Indeed why does the body even possess the rather strange capacity to destroy itself immunologically in the first place? A major contribution to answering these questions has been made in recent years by the realization that many microbes mimic their hosts at the molecular level and that this can lead to immunological cross reactions between microbe and host. Indeed many workers take the term “molecular mimicry” to mean the complete hypothesis that explains autoimmunity in terms of shared epitopes. Usually the workers in this field are not interested in the evolutionary reasons (if any) behind this antigen sharing. That aspect of the exercise is usually left undiscussed mainly because there is usually no direct information relating to the issue and secondly because the observations are taken at face value as a starting point for explaining the medical issues. I intend to adopt a similar approach except to say at the outset that I assume this widespread occurrence of mimicry probably reflects a common strategy of immune evasion by these microbes. I will review in depth a selected number of important examples and allude in passing to many more. 3.1. Bacterial Mimicry

3.1.1. Streptococcus pyogenes and Rheumatic Fever Probably the most comprehensive and convincing example of molecular mimicry resulting in a disease is provided by the p-haemolytic group A streptococci and acute rheumatic fever (ARF) (see Froude et al., 1989; Tsuchiya and Williams, 1992 for reviews). The main clinical signs of A R F are chorea, migratory arthritis, acute carditis and valvulitis. All the relevant organs and tissues involved can be demonstrated to share crossreacting determinants with various components of streptococci (Figure 7).

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/

Capsule

Joint

4(

Myocordium

Corbohydrote

Protoplost Membronr

-

I)Volvu'or tirrue

Myocordiol sorcolemmo t 3 Vaaculor lntimo .Skin

Kidney

Figure 7 Schematic representation of mimicry between structures on the group A streptococcus and mammalian tissues. (Reproduced from Froude et af. (1989), with permission of Springer-Verlag.)

The interested reader is referred to the comprehensive survey by Froude et al. (1989) for a more complete picture of the complex and manifold nature of the streptococcal-host cross reactions. Only a few of the more relevant examples will be discussed here. Kaplan (1963) was the first to show that immunization of rabbits with cell walls could elicit antibodies cross reactive with cardiac tissue. These antibodies were subsequently demonstrated to recognize certain regions of the M proteins and the cross reaction was with the sarcolemmal proteins. In terms of the significance to disease Froude et al. report that sera from individuals with ARF recognize a 30 kDa streptococcal membrane protein which can absorb out all the antibodies that react with the cardiac tissue in which they recognize a sarcolemmal protein of 40 kDa (van de Rijn et al., 1977; Froude et al., 1989). Cross reactions to carbohydrates are also thought to be important particularly in patients with rheumatic heart disease (Dudding and Ayoub, 1968). Sera from patients with ARF but with and without rheumatic heart disease were assayed for antibodies to group A carbohydrate. Both groups showed high titres initially but during the subsequent 5 years the patients

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with heart disease maintained these levels whereas in those without disease the antibodies declined dramatically. Also when such patients had cardiac valves surgically removed the anti-group A carbohydrate antibodies declined rapidly (Ayoub et al., 1974). The implication is that the autoimmunity was provoked initially by a streptococcal infection but that this was continually boosted by autologous stimulation from the damaged valve and that this subsided upon removal of the autoantigen. Chorea is another symptom of ARF. This has also been associated with cross reactions between streptococcal antigens and cytoplasmic antigens residing in nerve cells (subthalamic and caudate nucleus neurons) in the brain (Husby et al., 1976). These nerve cells are probably involved in generating choreiform movements. The main point is that cross-reacting antibodies of this type are present in patients exhibiting chorea as a result of A R F and they can be totally absorbed using preparations of group A streptococcal membranes. An important qualification, however, is that 25% of sera from ARF patients not exhibiting chorea also contain these same antibodies suggesting that the causal link is not proven or at least incomplete. Finally molecular mimicry has also been implicated in the migratory arthritis often associated with ARF (Baird et al., 1991). Antibodies that cross react with streptococcal M protein and joint structures were found in patients exhibiting arthritis as a consequence of ARF. These antibodies were capable of complement activation and it is suggested that this is the underlying reason for the acute local inflammation of the joint. Thus molecular mimicry has been implicated in the main pathological lesions of ARF and although a clear causal association is not proven the circumstantial evidence is in my opinion overwhelming. Furthermore, the impressive catalogue of shared determinants between this one organism and its host surely testifies to this as a successful adaptive strategy. 3.1.2. Enterobacteria, H L A B27 and Ankylosing Spondylitis Patients suffering from the non-rheumatoid arthritic condition ankylosing spondylitis (AS) probably had the condition develop as a result of infection with one of a number of gut bacteria. A list of such bacteria includes Shigella, Klebsiella, Salmonella, Yersinia, Campylobacter and E. coli. In addition approximately 95% of Caucasians with AS are HLA B27 positive compared with the normal incidence of this allele of 9%. This led to a hypothesis that these associations were due to molecular mimicry between epitopes on the B27 antigen and parasite molecules. This hypothesis gained some impetus when Schwimmbeck et al. (1987) searched databases and found that the Klebsiella pneumoniae nitrogenase (KPN) shared a hexapeptide QTDRED with the hypervariable region of HLA B27, a

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finding that could occur by chance at a frequency of 1 in 20 million. Synthetic peptides based on these sequences (including flanking amino acids) were tested using sera from 60 patients with AS. It was reported that 29% of these sera reacted with the synthetic peptides spanning this shared sequence and derived from both KPN and B27. The initial and intuitive assumption that these autoantibodies are directed against the mimicked QTDRED motif has proved to be wrong (Ewing et al., 1990; Tsuchiya et al., 1989,1990). The study by Ewing was very comprehensive and clearly demonstrated that octapeptides containing the QTDRED sequence were unreactive with sera from AS patients. It was shown that the main epitopic region in KPN is centred around the sequence 1831CNSRQTDR191 whereas that in B27 is based around &KAKAQTDRT5. The obvious deduction from this is that the common motif QTDR is responsible for the observed cross-reactivity. Although probably true it should be stressed that QTDR alone is very unreactive and upstream sequences from KPN or B27 are essential for antibody recognition. Other attempts to implicate the QTDRED shared motif using sera from patients in Tromso, Norway and Albuquerque, New Mexico failed to find any reactivity with KPN (Tsuchiya et a[., 1989). Indeed autoreactivity to B27 peptide was only observed in the Tromso group. Attempts to find cell-mediated autoreactivity have been uniformly unsuccessful. Because of these uncertainties doubts have been raised about the molecular mimicry hypothesis for AS and it is probably fair to say that the data need to be more carefully considered. Other microbes implicated in the aetiology also share intriguing sequence homologies with B27 hypervariable region, For example, the Yersinia adhesion A protein (YadA, previously called Yopl) contains T D R E and the protein predicted by the Shigella plasmid pHS-2 contains a sequence AQTDRHSL (cf B27 sequence AQTDREDL). It seems most unlikely that these striking homologies between triggering organisms and B27 are meaningless. But what do they mean? The obvious search for meaningful serological cross reactions may be too simplistic and equally the attempts to identify cellmediated autoimmunity may suffer from inadequate assay techniques. Confirmation that B27 itself (as opposed to another gene in linkage disequilibrium with HLA-B27) is intimately involved in the aetiology of the disease was elegantly obtained by creating transgenic rats which developed similar disorders (Hammer et al., 1990). Again evidence of an association with serum autoantibodies and disease was not found but a gene dosage effect was noted. The search goes on and the role of molecular mimicry cannot yet be ruled out. 3.1.3. Mycobacteria, hsp65 and Rheumatoid Arthritis Mycobacteria cause leprosy and tuberculosis, diseases still prevalent in developing countries. Tuberculosis has also seen a resurgence recently in

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the developed world. These organisms too have been shown to share numerous cross-reacting epitopes with their host (reviewed in Thorns and Morris, 1985; Shoenfeld and Isenburg, 1988). The subject of this section will be to focus on the sharing of a T-cell determinant carried on mycobacterial hsp65 and components of the cartilage in joints of Lewis rats. This topic is reviewed comprehensively elsewhere (van Eden et al., 1989) and only the crucial points will be discussed here. When rats are inoculated cutaneuosly with Freund’s complete adjuvant (FCA, a suspension of heat-killed mycobacteria in mineral oil) they develop arthritis 2-3 weeks later and the disease can last for about 2-3 weeks (Pearson, 1956). This experimental disease is called adjuvant arthritis (AA) and is strikingly similar histopathologically to human rheumatoid arthritis. It was long suspected to have an autoimmune basis mainly because it was shown to be transferable to naive recipients by lymphocytes from affected rats (Pearson and Wood, 1964). This initial observation was elegantly confirmed and extended when it was demonstrated that the disease could be transferred to sublethally irradiated animals by a T cell line (called A2) produced from FCA-immunized rats and maintained on whole mycobacteria (Holoshitz et al., 1983). The coup de grace came when it was possible to derive a clone (A2b) from this line which could also transfer the disease (Holoshitz et al., 1984). This clone was typed as of the helper class. This was a remarkable result because it confirmed the autoimmune nature of the disease and significantly pinned down the cause to a single cross-reactive T cell epitope. The epitope that stimulated A2b from the mycobacterium was shown to reside on a heat-shock protein (hsp65) and was finely mapped to a nonapeptide of sequence TFGLQLELT (van Eden et al., 1988; van der Zee et al., 1989). The host epitope resides in the cartilage and is probably associated with the proteoglycan. Unfortunately homology searches were not too fruitful, the most significant being to the link protein with a 4/9 match. Whether this represents the true epitope awaits determination. Evidence that mycobacterial hsp65 has a role in rheumatoid arthritis is circumstantial but corroborative. For example Res et al. (1988) screened T cells from patients’ synovial fluids for stimulation by hsp65 and found marked responses if the joint had been clinically affected for less than three years. Also Bahr et al. (1988) have found raised levels of IgG antibodies against hsp65 in patients with rheumatoid arthritis. It has to be said that the case against mycobacterial hsp65 (and possibly hsp65 from other microbes as it is a ubiquitous phylogenetically conserved molecule) for involvement in rheumatoid arthritis is not made but the clues are indeed tantalizing and I would be surprised if in the final analysis it is not found to play a role. 3.1.4. Hsp60l6.5 and Lyme Disease Lyme disease is a multisystem disorder induced by infection with the spirochaete Borrelia burgdo@ri (Steere et al., 1983). Specific manifestations

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that occur can affect joints, nervous systems, skin, heart and various other organs. Autoimmunity has been implicated in the pathology of this disease since it has been possible to isolate T cell lines from the cerebrospinal fluid of patients that respond to myelin basic protein (Martin et al., 1988a, b). That molecular mimicry may lie behind this disorder was first indicated by the work of Aberer el al. (1989) who screened 11 monoclonal antibodies raised against either B. burgdorferi or B. ermsii. One of these antibodies (H9724) which recognizes the 41 kDa flagellin was shown to cross react with a widely distributed cytoplasmic antigen of Schwann cells, neurons of the CNS, bronchial epithelial cells, smooth and heart muscle, enterochromaffin cells of the gut, adrenal cortex, liver, kidney, pancreas duct epithelium, and joint synovia. Recently this cross-reacting antigen has been identified as hsp60/65 by purifying it using two independent procedures and obtaining the N-terminal amino acid sequence in both cases (Dai et al., 1993). Once again the suggestion of autoimmunity to hsp60/65 as a cause of pathogenesis suggests itself. However, the epitope recognized by both H9724 and sera from five patients has recently been located to a stretch of 12 amino acids (residues 213-224, EGVQQEGAQQPA) within flagellin (Fikrig et al., 1993). There is no significant homology between this sequence and human hsp60/65. Further work is, however, needed in this case to establish that there is a causal relationship between the observed 41 kDa flagellin:Hsp60/65 mimicry and Lyme disease.

3.2. Protozoal Mimicry 3.2.1. Trypanosoma cruzi, Molecular Mimicry and Autoimmune Pathology of Chagas’ Disease Infection with Trypanosoma cruzi causes an autoimmune syndrome known as Chagas’ disease involving pathology of the nervous system particularly the nerves supplying the digestive tract and the heart. The disease exists in three phases. The first stage or acute phase (1-2 months in humans) is usually asymptomatic but is occasionally accompanied by a range of symptoms including, amongst other things, fever and myocarditis. If acute phase pathology is observed then autoantibodies to heart tissue (endocardium, vascular and interstitial tissues (EVI) Cossio et al., 1974a, b) are found suggesting an autoimmune reaction to shared host and parasite antigens (Figure 8). There is then an asymptomatic indeterminate phase which can last many years. This is followed in about 10% cases after 10-30 years by the chronic phase in which severe pathology is found and death by heart failure is common. This is a convenient parasite to study since it infects a wide range of organisms including the laboratory mouse

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Figure 8 EVI (endocardial, vascular and interstitial) immunofluorescence. Serum from chronically infected chagasic patient used to stain cardiac tissue. (Reproduced from Takle and Hudson (1989), with permission of Springer-Verlag.)

in which the pathology is remarkably similar to that found in man (reviewed in Petry and Eisen, 1989). Thus a true in vivo model exists for the human disease something that is a rarity indeed and is to be relished. There is abundant evidence of autoantibodies against the relevant tissues of the host. Thus in addition to EVI antibodies, autoantibodies have been found to Schwann cells (Khoury et af., 1979), laminin (Szarfman et al., 1982), striated muscle (Cossio et af., 1974b) and neurons (Ribiero dos Santos et al., 1979). Autoantibodies have been shown to define shared host-parasite epitopes by cross absorption. More definitively cross-reactive monoclonals have been identified (Wood et af., 1982; Snary et af., 1983). Thus Wood et af. (1982) characterized an IgM monoclonal that recognizes mammalian neurons, cardiac muscle and T. cruzi. Snary et al. (1983) reported two identical IgM anti-T. cruzi monoclonals that stained glia and central and peripheral neurons, the epitope being distinct from that reported by Wood et al. Petry et af. (1987) produced five IgG monoclonals which react with T. cruzi and mouse brain. Two of these (called VESP 6.2 and VESP 8.2) recognize lipid antigens. VESP 6.2 is thought to recognize a sulphated sphingolipid whereas VESP 8.2 recognizes a family of glycolipids reacting specifically with galactosyl ceramides and sulphated galactosyl ceramides containing 2-hydroxy-rich fatty acids. The observation that T. cruzi contains large amounts of 2-hydroxy fatty acids is rather surprising since such glycolipids had been thought to be specific for myelin and

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myelin-producing cells. Galactosylceramide and sulphated galactosylceramide are major components of the myelin sheath, which is a known target in Chagas’ disease. Polyclonal sera from chronically infected mice react with sphingoglycolipids (Petry et al., 1988) and sera from chagasic patients react with sialoglycolipids extracted from T. cruzi (Takaoka et al., 1987). The autoimmune basis for the chronic pathology has been experimentally demonstrated by adoptive transfer experiments in mice. Laguens et al. (1981) demonstrated that transfer of non-adherent splenocytes from chronic mice provoked cardiac symptoms in syngeneic recipients. Other workers have demonstrated that nerve cell pathology can be transferred using parasite free in vitro derived Th cell lines (Hontebeyrie-Joskowicz et al., 1987). Thus quite clearly the host and the parasite share antigens which can form the basis of T cell-mediated autoimmunity.

3.3. Viral Mimicry The aetiology of many diseases considered to be autoimmune is thought to involve a viral component (Barnett and Fujinami, 1992). Notable examples include diabetes and multiple sclerosis and a range of others are shown in Table 5. I will outline a few important experiments and observations. 3.3.1. Multiple Sclerosis, Experimental Allergic Encephalomyelitis ( E AE ) and Hepatitis B DNA Polymerase The pathology of multiple sclerosis is caused by demyelination and this and other central nervous system disorders are thought to be autoimmune in Table 5 Virus disease associations predicted using the molccular mimicry hypothesis. Virus

Host ccll or protein involved Astrocytes (gp41) HLA class I1 antigen U1 snRNP (p68) Macrophages, platelets, PMN Acetylcholine receptor CNS (myelin basic protein) Urea1 tract

Disease

HIV AIDS dementia HIV AIDS Mixed connective disease Influenza B Bovine herpes Bovine shipping pneumonia Herpes simplex Myasthcnia gravis Hepatitis B EAE Experimental allergic uveitis Hepatitis B, baboon virus, AKV murine leukaemia virus Mouse hepatitis Fc receptor MHV persistence Cytomegalovirus HLA-DR Graft rejection Epstein-Barr Collagen Rheumatoid arthritis Reproduced from Barnett and Fujinami (1992), with permission.

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nature. An experimental model for demyelinating CNS syndromes is provided by experimental allergic encephalomyelitis (EAE). This is a disease that can be induced in animals by immunizing them with CNS tissue plus adjuvant. The targets of this T-cell mediated damage are the components of myelin, specifically myelin basic protein (MBP) and proteolipid protein (PLP) (Yasuda et al., 1975; Tuohy et al., 1989; Zanvil and Steinman, 1990). It has been observed that persistent infections with measles virus exacerbated E A E (Massanari et al., 1979). Fujinami and Oldstone (1985) conducted a database search of known viral proteins against the encaphalitogenic sequences of MBP and came up with a hexapeptide identity (Tyr.Gly.Ser.Leu.Pro.Gln) between a known encephalitogenic region of MBP and the DNA polymerase of hepatitis B virus. They synthesized a decapeptide derived from the viral sequence (1le.Gly.Cys.Tyr.Gly.Ser.Leu.Pro.Gln.Glu) and immunized rabbits with this. The result was to elicit antibodies and T cells that recognized native MBP. Most importantly some (4/11) of the rabbits exhibited lesions characteristic of EAE, notably a perivascular infiltration localized to the central nervous system. This supported the hypothesis that molecular mimicry can cause autoimmune disease with experimental evidence. Obviously other factors are involved as indicated by the failure of all the rabbits to show the lesions. 3.3.2. Theiler’s Murine Encephalomyelitis Virus (TMEV) and Antibody-Mediated Demyelination Many examples of autoimmune syndromes exist accompanied by the production of specific autoantibodies. Very rarely, however, have these antibodies been shown to mediate or contribute to the tissue damage in passive transfer or other assays. TMEV causes a demyelinating disease in mice which is thought to be autoimmune in nature (Fujinami and Zurbriggen, 1989). Monoclonal antibodies were raised from TMEV-infected mice and screened for self reactivity and one (H8) was found to react with a major component of myelin, galactocerebroside. Yamada et al. (1990) demonstrated that this H8 monoclonal antibody could augment demyelination in E A E , thus clearly showing that it was possible for autoantibodies to mediate disease. 3.3.3. Myocarditis and Coxsackie B Virus Infection: Another Example of Autoantibody Mediated Pathology In one study (Neu et al., 1987) it was observed that NJ mice infected with coxsackie B3 virus developed myocarditis. This is of medical interest since coxsackie virus infection in humans is believed to lead to myocarditis in

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some individuals. Neu et al. (1987) showed that the affected mice developed autoantibodies to cardiac myosin. Crucially they demonstrated that when this antibody was transferred to uninfected recipient mice they developed myocarditis. Sequence comparison of the coxsackie B3 protein and the cardiac h chain of myosin revealed a very good example of mimicry as shown below with a 7/11 match: Cox B 3

Lys. Ser.Arg. Leu. I l e . Glu. A l a . Ser. S e r . Leu. Asn. CardiachMyosin

Lys. Ser. Arg. Asp. I l e . Gly. A l a . Lys. Gly. Leu. Asn.

When the shared myosin sequence was inoculated into recipient mice, myocarditis was the result accompanied by the production of autoantibodies. One deficiency in this system is that results with the inoculation with the virally derived peptide were not reported and this is much more crucial. Also it was not stated if the autoantibodies found in the infected mice reacted with the region identified by sequence homology. For that matter it was not shown that these pathogenic autoantibodies actually recognize viral components at all. Nonetheless, the observations are intriguing and the passive transfer data are good circumstantial evidence that molecular mimicry can induce pathology in this system. Furthermore, direct evidence of immunological cross reactivty between coxsackie B3 virus and cardiac tissue has been provided using a monoclonal antibody (Srinivasappa et al. , 1986) which strengthens the case considerably.

4. HOW DOES MOLECULAR MIMICRY CAUSE DISEASE?

AUTOIMMUNE CONSIDERATIONS

The observation that in many cases infections with a wide variety of microbes can predispose to a number of autoimmune conditions that are manifest long after the infection has been cleared has given rise to the “hit and run” model (Oldstone, 1987). The central idea of this is that the microbe infects and presents an epitope to the immune system which is similar but not identical to self. This epitope is sufficiently different from self to allow an immune response to develop to it, with presumably severe consequences for the microbe. However, the argument runs, the similarity to self allows the breakdown of self-tolerance and an autoimmune response ensues to the mimicked self-determinant. Once primed by the microbial epitope the autoimmune response is then self-perpetuating and no longer relies on the presence of the original infectious agent. Depending on the nature of the immune response and the location of the mimicked antigen

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characteristic lesions of the particular autoimmune condition will then occur. However, these pathological symptoms may not appear for some considerable time after the initiating event. This model is plausible but leaves a lot of unanswered questions. For example, it has direct implications for the mechanism of self-tolerance. Why does the body have an autoimmune capacity at all? This would seem to be a particularly senseless facility. A remarkable feature of the phenomenon is that the anti-self repertoire seems to be fairly limited and focused, a fact pointed out by Cohen and Young (1991). Also the target antigens are often composed of highly conserved structures such as heatshock proteins, myosin, and a variety of enzymes. One highly imaginative explanation for these observations is the concept of the immunological homunculus expounded by Cohen and Young (1991). This is a somewhat nebulous proposal which has as its centrepiece the notion that the body retains a set of autodominant antigens. The immune system uses this autorecognition to build up an immunological reference map of itself. Each dominant autoantigen is the focus for a balanced network of interacting lymphocytes locked under normal conditions in a dynamic idiotypic equilibrium. The reason for the existence of this system is not abundantly clear but it is proposed that by focusing on a few autoantigens the overwhelming majority of others are relegated to the realms of immunological obscurity and never feature in autoimmunity. The argument is further advanced that under most conditions an immune response to a dominant autoantigen will be effectively buffered by the network and rendered harmless. Only in the case of severe immunological insult will the equilibrium of the network be breached and then serious consequences will ensue. This is an attractive hypothesis and explains the fact that, for example, a wide range of parasites trigger powerful immune responses to heatshock proteins. In the homunculus theory the heatshock proteins would qualify as some of the autodominant antigens of the homunculus. Another very attractive and more explicit hypothesis has recently been advanced (Burroughs et al., 1992; Baum et al., 1993). These ideas were formulated around the details of a rare condition known as primary biliary cirrhosis (PBC). This condition is characterized by autoantibodies against mitochondria. It was noted that there was a high degree of homology between a peptide (EAQGALANIAVDKANLE) from the MHC class I1 invariant a chain of HLA-DR and another peptide (EAEQSLITVEGDKAASME) which forms the lipoyl domain of the E2 subunit of the pyruvate dehydrogenase complex (PDC) of E. coli. The significance of this observation requires some explanation. In the first place, this D R a peptide is believed to be a self-peptide presented by other class I1 molecules. Second, the E2 peptide has high homology with similar subunits in mitochondria and one of the major autoantigens of PBC is mitochondria1 PDC-E2. Third, the

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onset of the disease is frequently associated with recurrent urinary tract infection. Finally, MHC class I1 molecules are inappropriately expressed on the bile duct epithelial cells in PBC. The hypothesis states “that in response to an infection, T cells recognize the lipoyl region of E. coli PDCE2 and other similar enzymes; that these T cells stimulate a conventional antibody response to the infecting immunogens; that molecular mimicry subsequently occurs when these T cells recognize a self-peptide (from HLA-DRa), presented by aberrantly expressed class I1 HLA on biliary epithelial cells; and finally, that this initiates an autoimmune cascade leading to the destruction of the bile ducts in which mitochondria1 autoantibodies and/or T cells may be pathogenetic.” These authors then generalized this concept by suggesting that mimicry of other dominant selfpeptides presented by class I1 MHC inappropriately expressed on particular tissues could underly other autoimmune conditions. To test if their hypothesis has any generality they utilized the DRa motif. They generalized it in the following form [EDKRH]X,[AIVL]X,[GAILV]DKAX,E where [ ] means a set of allowable residues at that position and X means any residue. Note that the DKA motif is invariant. Using a motif search programme PROMOT (Sternberg, 1991) this sequence selected a limited set of proteins from the OWL database of which six are of human origin (Table 6). Four of these six (asialoglycoprotein receptor, insulin receptor, glutamic acid decarboxylase and hsp65) have been implicated as autoantigens in a range of autoimmune conditions. Thus asialoglycoprotein is implicated in chronic active hepatitis; insulin receptor in diabetes; G A D in type I diabetes mellitus; and hsp65 in a range of autoimmune syndromes including PBC. The other identified proteins are all found in pathogens (Table 6), some of which are associated with the onset of autoimmune conditions. This analysis is, to say the least, intriguing and personally I feel that it is extremely significant. A similar analysis with other identified self peptides is eagerly awaited.

5. CONCLUSIONS

The existence of molecular mimicry as an adaptation of parasites to their hosts is an undoubted reality. The examples are manifold and can be conveniently and broadly categorized into four main classes: cytoadhesive proteins and their receptors; effectors of the immune system; hormones, serum proteins and their receptors; and cytoskeletal and muscle proteins. The reasons for such mimicry are often only speculative. The suggestion is made that mimicry exists either to subvert some host function and/or to evade the immune system. Hard evidence for the true functional signifi-

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Table 6 Some proteins bearing the “HLA-DRa” motif, speculated as underlying some autoimmune processes. Sequences were selected using the PROMOT programme from the OWL database by searching for homology to the generalized DRa motif defined in the text. Underlined residues are identical or are conservative substitutions (as defined in the search) to corresponding ones in HLA-DRa. Self T-cell epitope HLA DRa (human) Putative autoantigens Asialogbcoprotein receptor (human) Insulin receptor (human) Glutamic ac’id decarboxylase (human) Hsp65 (human) Vimentin (human) High-mobility group protein HMGl (human)

80

270 526

EAQGALANIAVDKANLE

a p y R w1c ET ELD K A s Q

E R N D IA L K T N GDK A S C E

430 358 155

D K H Y D& S Y D T G D K A L Q C K D D AM&L K G K G D K A Q IE EL RR QVD QL TN GSQE

55

EK GK FE DMAKADKA RYE

Putative environmental antigens 132 EA E Q S 4 I T V E G D K A S M E PDC E2 I 1 ( E . coli) Hsp7O 176 E P T A A A I A Y G L D K A D E G ( Trypanosoma brucei) Fcr V protein 326 2 L Q A K& D E A N A D K A R Y E (Streptococcus) R ~ protein A 57 V I V N U H T A V D K A E S E (Salmonella tvphirnuriurn) _. Periplasmic protein A AADKAAAE 144 P E D K VIJK (Treponernu pallidum) Protein HH LFl DPTLGDKA GHP 1 8 1 E V D P &A(Cytomegalovirus) Pol (HIVI) 709 G I R K V L F L D G I D K A Q D E 158 SN S P T & C Q K F V D K A I & T Pol (MMTV) Reproduced from Baum et ul. (1993), with permission.

cance of particular cases of mimicry is usually lacking but in a few cases compelling explanations are available. Perhaps the best example is that of the region I1 of malaria CSP protein which shows high homology to a cytoadhesive motif found on thrombospondin (and some members of the complement system). This motif has been experimentally demonstrated to mediate binding of the sporozoite to the hepatocyte. This is presumably an example of convergent evolution although other interesting mechanisms such as horizontal gene transfer cannot be ruled out. The existence of molecular mimicry offers a mechanistic explanation for the occurrence of a plethora of autoimmune syndromes associated with microbial infection. The number and range of examples of this phenomenon is now large although the evidence for a direct causal association

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is lacking in most cases. However, in some model syndromes the autoimmune basis has been established and this is also been verified for Chagas’ disease. The data on many more, notably rheumatic fever, are overwhelmingly consistent with this hypothesis. The mechanistic explanation for primary biliary cirrhosis involving mimicry of self-peptides presented on inappropriately expressed class I1 molecules represents a new and important conceptual advance in this field. Much work remains to be done in both the fields of adaptive and consequential mimicry. In the adaptive sphere the emphasis must be to obtain conclusive proof of function for the mimicked structure. The consequentialists are required to establish causation of disease by mimicry. Perhaps a unified concept of molecular mimicry could be evolved?

ACKNOWLEDGEMENTS

I am grateful to Dr Howard Baylis for reading and criticizing the whole of this manuscript. I also further extend my thanks to Howard and the rest of my group for never once complaining while I monopolized the word processor. I am also grateful to the following authors and publishers who allowed me to reproduce material: Kathryn Robson (Figure lB), Carla Cerami (Figure 2), Bengt Guss (Figure 4), David Kaslow (Figure 5 ) , Juan Cooper (Figure 6), John Zabriskie (Figure 7), Leslie Hudson (Figure 8), Michael Oldstone (Table t), Neil Cooper (Table 2), Michael Boyle (Tables 3 and 4), Robert Fujinami (Table 5 ) , Harold Baum (Table 6), SpringerVerlag (Figures 7 and 8, Table l ) , Macmillan Magazines Ltd (Figure l B , Figure 5 ) , Cell Press (Figure 2), Academic Press (Figure 4, Tables 3 and 4), Elsevier (Figure 3B, Figure 6, Table 2 and Table 6) and the FASEB Journal (Table 5). REFERENCES Abercr, E . , Brunncr, C., Suchanek, G . , Kladc, H . , Barbour, A . , Stanek, G. and Lassmann, H . (1989). Molecular mimicry and Lyme borreliosis: a shared antigenic detcrminant between Rorrelia burgdorferi and human tissue. Annals of Neurology 26,152-131. Alaei, S . , Larcher, C . , Ebenbichler, C., Prodinger, Janatova, J . and Dierich, M.P. (1993). Isolation and biochemical characterisation of the iC3b receptor of Cundida albicans. Infection and Immunity 61, 1395-1399. Andcrs, R . F . , Barzaga, N . , Shi, P-T., Scanlon, D . B . , Brown, L.E., Thomas, L.M., Brown, G.V., Stahl, H . D . , Coppcl, R.L. and Kcmp, D.J. (1986). Repetitive sequences in malaria antigens. UCLA Symposium on Molecular and Cellular Biology, New Series 42, 333-342.

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Relationships Between Chemotherapy and Immunity in Schistosomiasis Paul J. Brindley

Molecular Parasitology Unit. and Tropical Health Program. Queensland Institute of Medical Research. The Bancroft Centre. 300 Herston Road. Brisbane. Queensland. 4029. Australia 1. Introduction

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4. Age-related Insusceptibility to Schistosomicides . . . . . . . . . . . . . . . . . . . . . . . . . . . 136 5. Antimonials

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6. Oxamniquine and Hycanthone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . 6.1. Background . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.2. Reduced efficacy in immunosuppressed hosts . . . . . . . . . . . . . . . . . . . . . . . 6.3. Mode of action and drug resistance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.4. Genetics of drug resistance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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................... 7. Praziquantel . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.1. Mode of action . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.2. Reduced efficacy in immunocompromised hosts . . . . . . . . . . . . . . . . . . . . . 7.3. Exposure of surface antigens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.4. Synergy with antibodies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.5. Localization of drug-antibody damage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.6. Molecular characterization of exposed antigens . . . . . . . . . . . . . . . . . . . . . . ............... 7.7. Efficacy in vaccinated animals . . . . . . . . . . . . . . . . . . . ............... 7.8. A complex chain of events . . . . . . . . . . . . . . . . . . . . . . .

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1. INTRODUCTION

Targett (1985) observed that although much contemporary research in tropical medicine and parasitology was concerned with either chemotherapy or immunology of parasitic diseases, little attention had been focused on relationships between chemotherapy and immune responses in parasitic infections. Notwithstanding, a dependence on, or enhancement by, appropriate immune responses is apparently widespread with antimicrobial chemotherapy, as evidenced by the following reports. Pentamidine chemotherapy of visceral leishmaniasis in Brazilian patients is apparently augmented by concurrent administration of interferon (Badaro et al., 1990), antibodies play a role in the elimination of African trypanosomes after chemotherapy with difluoromethylornithine (de Gee et al., 1983; Bitoni et al., 1986), and pyrimethamine, chloroquine, and quinine all show reduced efficacy against malaria in T cell-deficient mice (Lwin et al., 1987). The sites and modes of action of most schistosomicides are not well or not fully understood. However, these anthelmintics often require appropriate immunological responses for full efficacy, at least in experimental infections. A sizeable literature dealing with the relationships between chemotherapeutic efficacy of antischistosomal drugs and host immunological responses to schistosomes is now available, and this literature forms the basis of this review. More general information on the immune dependence of chemotherapy in other parasitic infections is available in Targett (1985) and Doenhoff et al. (1991). 2. SCHISTOSOMIASIS

Schistosomiasis is the most important of the human helminthiases - it is responsible for the death of hundreds of thousands of people each year and for inestimable morbidity (Gibbons, 1992). Various forms of schistosomiasis afflict up to 300 million people in tropical Asia, Africa, South America and the Caribbean, with a further 600 million people at risk of infection in endemic regions (Mahmoud, 1984), and it is spreading. The disease is transmitted by contact with contaminated water and is caused by various species of blood flukes of the genus Schistosoma which live and lay eggs in the veins of the intestines (Schistosoma mansoni, S . japonicum and S. mekongi) or bladder ( S . haematobium and S . intercalatum). Mass and targeted population chemotherapy provide the most effective method for controlling schistosomiasis since no vaccine is available (Butterworth, 1992; Waine et al., in press) and because sewage disposal, snail control

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(molluscicides, drainage), and attempts to change human water contact behaviour have consistently failed, usually through high cost and negligence (Liese, 1986). Ironically, progressive engineering in the form of dammed rivers has favoured the expansion of snail populations and thereby contributed to the spread of schistosomiasis. In the well-characterized exception, a multifaceted approach of all these control strategies successfully eradicated schistosomiasis from the Caribbean island of St Lucia (Jordan, 1985).

3. SCHISTOSOMICIDAL CHEMOTHERAPY

Chemotherapy is employed to treat schistosomiasis and to control its spread (Liese, 1986). Chemotherapy of schistosomiasis began at the time of the First World War when tartar emetic (potassium antimony tartrate) was administered to patients with schistosomiasis haematobia (Christopherson, 1918). The subsequent history of schistosomiasis chemotherapy with successively better drugs including lucanthone, hycanthone, and niridazole has been reviewed over the years in a number of articles (Pellegrino and Katz, 1968; Katz and Pellegrino, 1974; Marshall, 1987; Grove, 1990; Brindley, in press). At present, metrifonate, oxamniquine and praziquantel appear on the World Health Organization’s list of essential drugs: metrifonate is employed for treatment and control of infection with S. haematobium, oxamniquine for S. mansoni, and praziquantel is active against all schistosome species parasitizing humans, including S. japonicum (Goldsmith, 1988). Treatment failures have occurred in schistosomiasis, and in some instances can be ascribed to drug-resistant schistosomes (Katz et al., 1973; Souza el al., 1982). Indeed, resistance to oxamniquine is now present in natural populations of S. mansoni in Brazil. Perhaps more worrying is an ostensibly reliable, recent report of the failure of praziquantel chemotherapy in Africa, which suggests that schistosomes may be developing tolerance to this most important drug (World Health Organization, 1992). Thus a major concern with the widespread use of schistosomicides is the development of drug-resistant parasites. Given the enormous catastrophe that drug resistance to chloroquine and other compounds has become in the treatment and control of malaria, it is also likely that widespread drug resistance to schistosomicides would be disastrous with respect to schistosomiasis. However, chemotherapy may fail in infected individuals for other reasons besides infection with drug-resistant parasites, including noncompliance, drug malabsorption, or inadequate immunological status. Since chemotherapy is required for the foreseeable future for controlling

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schistosomiasis, knowledge of the sites and modes of anthelmintic action including the interrelationship between the immune response and chemotherapy, and of molecular changes underlying the appearance of drug resistance, is critical; not only to ensure the effective use of the limited number of compounds available but ultimately for the rational design of the next generation of schistosomicides.

4. AGE-RELATED INSUSCEPTIBILITY TO SCHISTOSOMICIDES

Schistosomes exhibit inherent insusceptibility to schistosomicidal compounds not only with respect to particular drugs but also with respect to species, strain, sex and age of the parasites (e.g. Hsu et al., 1963; Sabah et al., 1986; Flisser et al., 1989b). Schistosoma japonicum, for instance, is insusceptible to oxamniquine and metrifonate, although these are active against S . mansoni and S . haematobium, respectively (see Brindley, in press). Since many antischistosomal drugs require appropriate immunological responses for development of full anthelmintic efficacy (as discussed below), and because the immunological status of an infected host will obviously change after infection, it is germane to review the age-related insusceptibility of schistosomes to anthelmintics at this point. Sabah et al. (1986) demonstrated that immature S . mansoni were far less susceptible to at least six schistosomicides including potassium antimony tartrate, hycanthone, oxamniquine, niridazole, amoscanate and praziquantel, at doses producing therapeutic cure against adult infections. All six compounds were inactive against infections that had reached the portal blood system, but which were not yet patent. More specifically, hycanthone, oxamniquine, praziquantel and niridazole showed bimodal activity with potent toxicity for schistosomules less than two weeks of age and activity against the adult schistosomes greater than five or six weeks of age, but with little or no effect against worms between these ages. In contrast, amoscanate and potassium antimony tartrate were inactive against schistosomules and immature worms, and full efficacy of these two compounds was seen only against adult S. mansoni more than 5 weeks of age. The study uncovered another interesting grouping of these compounds. Treatment of 3-week-old schistosomes with hycanthone or amoscanate caused a substantial reduction in the fecundity of the adult worms that developed from the drug-exposed worms, although the worms were apparently of the same size as control schistosomes. In contrast, similar exposure of immature parasites to oxamniquine, praziquantel, niridazole, or antimony did not similarly result in reduced fecundity in the resultant patent infections.

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The insensitivity of immature schistosomes to the anthelmintic effect of praziquantel is, as yet, unexplained (Xiao et al., 1985; Sabah et al., 1986). One possible explanation for the resistance of juvenile worms to praziquantel is the lack of a fully developed antibody response, since the chemotherapeutic efficacy against adult schistosomes requires appropriate host immunological responses, as discussed below (Brindley and Sher, 1987). However, transfer of the immune serum from 6- to 7-week-old infections to intact mice carrying 25-day-old schistosome infections did not enhance praziquantel chemotherapy of these infections. Nevertheless, some reaction with the immune serum is evident at the surface of drugtreated 4-week-old worms, as determined by immunofluorescence using immune serum from 7-week-old infections (see Brindley and Sher, 1990). Alternatively, stage-specific developmental differences in the nature of the schistosome tegument could explain the insensitivity of juvenile schistosomes to praziquantel, since electron microscopy reveals only moderate tegumental disruption on 21- to 26-day-old worms perfused out of mice immediately after drug treatment (Shaw, 1990). Thus, although it is possible that the epitopes that are involved in the drug/antibody synergy in praziquantel-sensitive stage schistosomes may not be present or are not exposed in 3- to 4-week-old liver stage worms (Sabah et al., 1985; Flisser et al., 1989b; Shaw, 1990), it is also possible that these developing parasites may be insensitive to praziquantel for other reasons relating to the physiology of the schistosome tegument. As noted, developing schistosomes are less sensitive to praziquantel than sexually mature worms, and moreover females are less sensitive than males. Praziquantel, but not amoscanate, was shown (Cheever and Deb, 1989) to be ovicidal for mature S. japonicurn eggs in mouse tissues and treatment is followed by a marked but transient decrease in eggs passed in the faeces. However, less mature eggs are less affected, and passage of eggs in the faeces of infected mice resumes as they mature. In contrast, other studies indicate that praziquantel does not appear to have an affect on ova in tissues or on the hatching of miracidia but that treatment of infected mice affects the morphology, motility and viability of eggs after they hatch (see Xiao et af., 1991).

5. ANTIMONIALS

As mentioned above, schistosomiasis chemotherapy began with antimony compounds. A number of these have been employed, including tartar emetic (potassium antimony tartrate) (Figure l), stibophen, and antimony sodium gluconate, and they have broad spectrum anthelmintic activity

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I

\

I

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HC-OGb HC-0

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C-OK OH Figure 1 Potassium antimony tartrate. (Reproduced with permission from Mrozik, 1986.)

against the human schistosome species. The principal pharmacological effect of antimony on S . mansoni is at the level of energy metabolism, particularly on the enzyme phosphofructokinase (Mansour and Bueding, 1980). For more than forty years, they were the only effective chemotherapeutic agents for all three important schistosome species, although severe side effects were common (Grove, 1990). Although antimonial compounds are no longer used for treatment of schistosomiasis (but are still employed against other parasitic infections including leishmaniasis (Olliaro and Bryceson, 1993)), it is appropriate to comment on them here because the original results on the immune dependence of schistosomicidal chemotherapy were obtained using antimonials. Doenhoff and Bain (1978) were aware of earlier studies that suggested a link between full efficacy of the immune response and antiparasitic chemotherapy, including observations by Taliaferro on efficacy of quinine against Plasmodium gallinaceum (reviewed in Doenhoff et al., 1991). In their pioneering paper, Doenhoff and Bain (1978) reported that “during screening of schistosomicidal drugs for any immune-dependence of their action in mice it was observed that potassium antimony tartrate was less effective at curing Schistosorna mansoni infections in T cell-deprived animals than in immunologically intact controls”. The CBA strain mice were deprived of T lymphocytes by means of a combination of adult thymectomy (Law et al., 1963) and subsequent administration of rabbit anti-mouse thymocyte antiserum (Levey and Medewar, 1966). Doenhoff et al. (1991) have reported that these combined immunosuppressive

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procedures result in a 90% reduction in the number of circulating T cells in these mice. Four to five weeks later, mice were dosedper 0 s with 100 mg kg-' potassium antimony tartrate (PAT) daily for five consecutive days. When the mice were killed and perfused at days 46 and 67 post-infection, it was found that the efficacy of PAT-chemotherapy was 61% and 87%, respectively, in the intact, control mice but was only 24% and 36% effective in the T cell-deprived mice. Doenhoff and Bain (1978) proceeded to demonstrate the immunedependence of schistosomicidal chemotherapy. They raised heterologous antisera by vaccination of rabbits with a soluble, aqueous extract of adult S . mansoni, and obtained homologous sera from CBA mice six to nine weeks after infection with 200 S. mansoni cercariae. Infected, immunologically intact mice were treated with the immunoglobulin fraction of the rabbit antisera (resulting from precipitation of the serum with 40% ammonium sulphate) (five daily doses of 0.2 ml immunoglobulin solution) at the time of PAT administration. The drug alone reduced the worm loads by 30% to 40%, the immunoglobulin (Ig) alone had no effect, whereas PAT and Ig administered together reduced the worm burden sizes by approximately 60%. When the homologous mouse serum (0.5 ml) was administered intravenously to infected, T cell-deprived mice at the time of PAT treatment, the activity of PAT in the immunologically deprived mice was partially restored. Together, these results demonstrated that the efficacy of PAT against S . mansoni in immunologically deprived mice could be (partially) restored by administration of antisera, and moreover, that PAT and antiparasite immunoglobulin act in synergy in elimination of schistosomes in vivo.

6. OXAMNIQUINE AND HYCANTHONE 6.1. Background

Oxamniquine is considered as a structural analogue of hycanthone and to have a related mode of action because both compounds share common features with the mirasan series of schistosomicides (Richards and Foster, 1969; Archer, 1985; Pica-Mattoccia and Cioli, 1985; Cioli et al., 1993) (Figure 2). Oxamniquine and hycanthone display potent activity against S . mansoni in humans and hycanthone is also active against S . haematobium. S . japonicum is intrinsically insensitive to both drugs (see Brindley, in press). Oxamniquine is still widely used in Brazil, although hycanthone is no longer indicated because of concerns about possible mutagenic effects and because of liver toxicity (Cook, 1990).

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n

'

n

OXAMNIOUINL , R x -cn,oH

-

UK 3883. R = -cn,

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HVCANTHONE, R x LUCANTHONE, RI

- CH,OH -CH,

4-DESMETHVL-LUCANTHONE. R D

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Figure 2 Structural formula of oxamniquine and related drugs. (Reproduced with permission from Pica-Mattoccia and Cioli, 1985.)

6.2. Reduced Efficacy in Immunosuppressed Hosts

Similarly to experimental studies on the immune dependence of schistosomicidal chemotherapy with antimonials, the immune dependence of antischistosomal chemotherapy of hycanthone and oxamniquine has been examined using experimental S. mansoni infections in mice (Sabah et al., 1985; Lambertucci et al., 1989). Because oxamniquine is widely used to control and treat schistosomiasis mansoni and because of the broad interest in defining the mode of action of hycanthone and oxamniquine (above), the results of these reports are reviewed here in detail. The antischistosoma1 effects of the compounds was compared in T cell-deprived and immunologically intact, CBA strain mice (Sabah et al., 1985). Mice were infected with 200 mixed-sex cercariae or 100-150 single-sex (male) cercariae, and treated with 100 mg kg-' hycanthone methane sulphonate by the intramuscular route at 35, 37 and 39 days after infection, or with 80 mg kg-' oxamniquine per os at 42 days post-infection. Schistosomes were perfused from the mice at either 9 or 21 days after the last anthelmintic dose. It was reported that hycanthone reduced the worm load by 93% in immunologically intact mice but was only 77% effective in the T cell-deprived animals, with either mixed sex or single sex infections. Nine days after chemotherapy with oxamniquine, the worm load in intact mice had been reduced by 61% compared to control non-drug treated, immunologically intact mice, whereas oxamniquine treatment of T celldeprived mice had resulted only in a 21% reduction in schistosome numbers. By 21 days after oxamniquine administration, a 92% reduction in intact mice was recorded compared with only 68% reduction in immunosuppressed mice (Sabah et al., 1985).

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As outlined above, an intact immunological response is a necessary cofactor if full efficacy of experimental oxamniquine and hycanthone antischistosomal chemotherapy in mice is to be achieved. Lambertucci et al. (1989) demonstrated that the efficacy of oxamniquine could be enhanced by passive transfer of antischistosome antiserum. These workers raised rabbit a n t i 4 mansoni antisera by immunizing rabbits with antigens from adult schistosomes “agitated in an equal volume of culture medium 199 for 3 h at room temperature” (see also Doenhoff et al., 1987). Immunologically intact CBNLac strain mice were infected with 200 S . mansoni cercariae and treated with 30 mg kg-’ oxamniquine at day 35 after infection. (This dose is subcurative in mice. At day 35 post-infection the schistosomes are not yet fully developed, and interestingly are not fully susceptible even to doses of 80 mg kg-’ or greater, that are curative against mature S . mansoni infections in mice (Jansma et al., 1977; Sabah et al., 1986).) In addition, groups of infected drug-treated and infected, control mice were passively immunized with 0.5 ml rabbit antisera on each of days 35,36,37 and 38 post-infection. Schistosomes were recovered at perfusion of the mice at day 52 post-infection. The results of two experiments showed that the sera from the immunized rabbits reacted synergistically with oxamniquine: reductions in worm load of 58% and 79% were achieved in groups given both oxamniquine and rabbit antisera, in two separate experiments. By contrast, the subcurative dose of oxamniquine reduced the parasite loads by only 6% and 13%. The antiserum alone had a marginal helminthotoxic efficacy (8%) when tested against similar infections. Lambertucci et al. (1989) reported preliminary results of indirect immunofluorescence studies using the rabbit a n t i 4 mansoni antisera: the antibodies reacted to the tubercles on the surface of the worms 9 days after oxamniquine treatment. Unfortunately, no fluorescence micrographs were provided in this publication. It was suggested, however, that the rabbit antisera recognized antigens exposed by oxamniquine on the surface of S . mansoni, a phenomenon that at the time had been observed on the surface of praziquantel-treated schistosomes (Harnett and Kusel, 1986; Brindley and Sher, 1987) (see below). 6.3. Mode of Action and Drug Resistance

This review describes research showing that schistosomicides perform better when accompanied by appropriate host immunological responses. Schistosomicidal chemotherapy and treatment failure in immunocompromised humans has obvious implications for the development of drug resistance, although development of antimicrobicidal resistance may be more rapid in rapidly replicating pathogens such as the malarias (see Lwin

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et al., 1987; Doenhoff et al., 1991). Resistance to drugs in human helminths in general is not common, but has occurred in natural populations of S . mansoni to oxamniquine and hycanthone, and is widespread to veterinary anthelmintics. Notwithstanding that other factors may have been involved, it is conceivable that drug resistance may have been promoted by chemotherapy of immunocompromised patients. Because of the immune dependence of oxamniquine and hycanthone for full efficacy, and because drug resistance to these compounds has appeared, it is timely at this point to summarize what is known about the biochemistry and genetics of oxamniquine and hycanthone resistance. The site and mode of action of oxamniquine and hycanthone are not known unequivocally. To account for its mode of action in schistosomes Cioli et al. (1985, 1993) have proposed that hycanthone is converted enzymically in vivo into a reactive ester, perhaps an acetate, sulphate or phosphate, that alkylates DNA, particularly deoxyguanosine bases (Archer et al., 1990), leading in due course to the death of the drug-sensitive schistosomes through the interruption of nucleic acid synthesis. They argue that the hycanthone-esterifying activity is absent or defective in resistant schistosomes and host tissues. To test the hypothesis a synthetic ester, hycanthone methane carbamate (HCMC), was produced and the action of this against drug-resistant forms of schistosomes was examined. Although more toxic for mice than hycanthone, the workers showed that HCMC killed resistant forms including adult S. japonicum, immature S. mansoni and adult drug resistant S . rnansoni (Cioli et al., 1985). Subsequently, PicaMattoccia et al. (1992) reported that extracts of drug-sensitive S. mansoni and S . rodhani induced binding of tritiated hycanthone to macromolecules, whereas isolates from two independent drug-resistant strains of S . mansoni, and immature S. mansoni, S . japonicum, and control mammalian cells did not (see also Pica-Mattoccia et al., 1988, 1989). They observed that the activity in the extracts was destroyed by boiling and proteinase K, could be blocked by cold hycanthone, its analogue IA-4, and by oxamniquine, whereas the activity was not blocked by other analogues including lucanthone, 4-desmethyl lucanthone, and the oxamniquine analogue UK-3883. A striking correlation was found between the capacity of various drugs to function as substrates for the enzyme activity and their structural and antischistosomal properties. Since the hydroxymethyl group present in hycanthone, IA-4, and oxamniquine is essential for schistosomicidal activity (Archer, 1985), and since analogues with a methyl rather than a hydroxymethyl group (lucanthone, 4-desmethyl lucanthone, UK-3883) are not active against cultured schistosomes (Pica-Mattoccia and Cioli, 1985) and were poor substrates, the enzyme activity ostensibly discriminates between an -H for -OH substitution in an otherwise identical large molecule. All compounds with the hydroxymethyl group (and only those) had both

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schistosomicidal activity and substrate properties. The results provided convincing support for the hypothesis that hycanthone is activated by an enzymatic mechanism present only in drug-sensitive schistosomes.

6.4. Genetics of Drug Resistance

Drug resistance has appeared to both oxamniquine and hycanthone in both natural and laboratory populations of S . rnansoni, and cross-resistance is usually seen with these two compounds and their analogues, but not to unrelated drugs (reviewed by Brindley, in press). Cioli and Pica-Mattoccia (1984) described studies on the genetics of hycanthone resistance in S. rnansoni in mice involving worm transfer techniques to ensure precise matches and cross-fertilization of drug-resistant and drug-sensitive schistosomes. They examined two strains of S. rnansoni: a drug-resistant strain of Puerto Rican origin to which they applied selection pressure for three further generations, and a laboratory-derived, drug-resistant line also of Puerto Rican origin (Jansma et a f . , 1977). Drug-sensitive, parent worms of the latter strain were employed as the drug-sensitive control strain in the analysis. The F, hybrid progeny from crosses between sensitive and resistant schistosomes proved to be sensitive to hycanthone, irrespective of the sex of the resistant parent. The resistance phenotype reappeared in back-crosses and F2progeny. Based on these results, the authors suggested that hycanthone resistance behaved as an autosomal recessive trait. Cioli et af. (1992) have expanded these studies and reported after a thorough in vitro analysis of drug sensitivity of schistosomes that resistance to hycanthone/ oxamniquine is indeed controlled by an autosomal recessive gene. All adult F, progeny from crosses between drug-sensitive and resistant schistomes were killed in vitro by lo4 M hycanthone or 7.2 X lo4 M oxamniquine. Further, numbers of worms of resistant or sensitive phenotypes resulting from backcrosses and in the F2 progeny were close to the Mendelian ratios expected for a trait under the control of a single autosomal recessive gene.

7. PRAZIQUANTEL 7.1. Mode of Action

Praziquantel (PZQ) (Figure 3) exhibits broad spectrum anthelmintic activity against parasitic flatworms, including schistosomes. The exact mode of action of the drug against schistosomes, for which it is indicated

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Figure 3 Praziquantel. (Reproduced with permission from Andrews and Bonse, 1986.)

for all species parasitizing humans (Goldsmith, 1988), is not fully understood (Harnett, 1988; Day et al., 1992). Early effects on adult schistosomes include tetany , hepatic shift, membrane depolarization, and influx of Ca2', followed by damage to the tegument and other organs. In addition, PZQ mediates changes to the surface antigen profile, and appears to inhibit phosphoinositide turnover (Andrews, 1985; Harnett and Kusel, 1986; Weist et al., 1992). However, it is not clear whether these are direct results of exposure to PZQ or the consequence of earlier drug-parasite interactions. Bona fide drug resistance to PZQ has not been documented with natural or laboratory isolates of schistosomes (see Yue et al., 1990), but treatment failures are neither uncommon nor fully explained (e.g. World Health Organization, 1992). 7.2. Reduced Efficacy in lmmunocompromised Hosts

As they had with oxamniquine, hycanthone, and potassium antimony tartrate, Doenhoff and coworkers were the first to report that PZQ was less active against schistosomes in immunocompromised than in normal hosts. In an early study, a suboptimal dose of 40 mg kg-' PZQ three times daily from day 42 post-infection with 100 cercariae was found to have no effect on worm reduction in T cell-derived CBA strain mice (see above) but the worm load in normal mice was reduced by 44% (Doenhoff et al., 1982). In each of three subsequent separate experiments, two involving optimal or higher doses of 3 X or 5 x 250 mg kg-' PZQ, and one involving a suboptimal dose of 40 mg kg-' PZQ, the drug was found to have reduced

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efficacy in T cell-deprived compared with immunologically intact mice (Sabah et al., 1985). In an analysis of the role of intensity of infection on the schistosomicidal action of PZQ, Doenhoff et af. (1987) reported that 250 mg kg-’ PZQ on days 42 and 44 was more effective as infection intensity increased: PZQ killed 90% of the adult S. mansoni in mice infected with 100 cercariae (of mixed sex), 78% of the worms in mice infected with 70 cercariae and only 48% of worms in mice infected with 30 cercariae. Further, they observed effects of age and maturity of S. mansoni infections on PZQ chemotherapy. Six-week-old, single-sex male worms were insusceptible to 3 x 40 mg kg-’ PZQ (4% worm reduction only), whereas this dose reduced the worm load in age-matched, mixedsex infections by 47%. In contrast, 12-week-old single sex worms were as fully susceptible to PZQ (at 3 x 250 mg kg-’) as mixed-sex infections. Similarly, they observed that 6-week-old single-sex female worms were much less susceptible to PZQ than 12-week-old single-sex, female schistosomes. These results, together with earlier studies that showed PZQ to be less effective in T cell-deprived mice, suggested that “elements of the immune response” were involved with praziquantel efficacy. They proceeded to investigate the notion more directly by treating mice with mixed-sex, 5-week-old S. mansoni infections (which are only partially susceptible to killing by PZQ (Sabah et a f . , 1986)) with both PZQ and simultaneous injections of rabbit antisera raised against adult S. mansoni worm antigens. The combined drug plus antiserum treatment reduced the worm load by 68%, whereas the parasite load was reduced by only 38% by PZQ alone. These results strongly suggested that PZQ and humoral antibody acted synergistically in the killing of S. mansoni, although the rabbit antiserum was not tested for its direct helminthotoxic effect. 7.3. Exposure of Surface Antigens

Schistosomes evade host immune responses. The parasite surface acquires erythrocyte and other host antigens which are thought to prevent host antibodies from binding to the tegument of the worms (Smithers and Terry, 1976; Goldring et a f . , 1977). Since PZQ causes vacuolization and disintegration of the schistosome tegument, and given that PZQ chemotherapy was at least partly dependent on the host immune response (Sabah et a f . , 1986), Harnett and Kusel (1986) hypothesized that PZQ may lower the ability of S. mansoni to evade the immune response by increasing the exposure of parasite antigens capable of acting as targets for host antibody, or “antibody-armed cells” at the worm surface, perhaps by disrupting the parasite’s masking coat of host antigens. Granulocytes were known to migrate into schistosomes soon after treatment with PZQ in vivo (Mehlhorn

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ef al., 1981). Preliminary experiments had demonstrated that the integrity of the outer membrane of the schistosome was retained after exposure to PZQ in vitro, since no changes to the rate of loss of '251-labelled wheat germ agglutinin-labelled surface molecules or in the permeability of sodium ["Cr] chromate were detected after incubation of male schistosomes with PZQ. To test the hypothesis, Harnett and Kusel (1986) incubated adult male S. mansoni removed from mice in 10 pg ml-' PZQ for a few minutes at 37" C, then incubated the parasites in rabbit antimouse erythrocyte sera, rabbit anti-S. mansoni schistosomulum serum, or normal mouse serum, and finally in '251-labelled goat anti-rabbit IgG. Some of their results are presented here (Figure 4). PZQ increased the exposure of schistosome parasite antigens, as quantified by radiation levels of several hundred-fold greater than the non PZQ-treated worms. By contrast, the PZQ-mediated exposure of membrane parasite epitopes occurred in the absence of any apparent effect on surface-localized host erythrocyte antigens. In a study undertaken soon after, Brindley and Sher (1987) found similar

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Figure 4 The influence of praziquantel on the parasite and host antigens at the surface of adult male Schistosoma mansoni. Worms from mice were incubated in praziquantel ( a ) or media (b), then probed with rabbit anti-mouse erythrocyte serum (A) or rabbit anti-schistosomulum serum (B). Subsequently, the worms were probed with radio-iodinated anti-rabbit IgG, and the level of adherent radiation determined by scintillation spectroscopy. The results demonstrate that praziquantel causes an increase in cxposure of parasite surface antigens without altering the coat of host erythrocyte antigens adhering to the schistosome surface. (Reproduced with permission from Harnett and Kusel, 1986.)

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results, using a different experimental model involving analysis of the surface of adult S. rnansoni obtained after brief exposure in mice to PZQ. No apparent loss or diminution of the coat of mouse erythrocyte antigen adherent to the surface of both male and female S. mansoni was observed using immunofluorescence, although there was a dramatic increase in the binding of antischistosome antibodies to drug-exposed schistosomes (see below), all of which corroborated the findings of Harnett and Kusel (1986). Since PZQ is lipophilic, and its effect on parasite antigen exposure might result from interaction with hydrophobic regions of the tegumental outer membranes, Harnett and Kusel (1986) incubated male schistosomes with three other lipophilic agents -retinol, Tween 40, and Tween 80. All three of these lipophilic compounds failed to mimic the effect of PZQ on surface antigen exposure, indicating that the mechanism of PZQ-induced exposure of surface antigens was likely to be localized. Reminiscent of the effect of PZQ on the surface antigens of adult schistosomes (Harnett and Kusel, 1986; Brindley and Sher, 1987; Doenhoff et al., 1988), Flisser and McLaren (1989) demonstrated that PZQ treatment in mice induces the exposure of parasite antigens on lung-state (6 days post-infection) S. mansoni schistosomula, yet did not influence the adherent coating of host erythrocyte antigens. In like fashion, Chinese investigators showed, using indirect fluorescent antibody techniques, that PZQ causes the exposure of surface antigens on S. japonicurn, and that the speed, degree and extent of antigen exposure correlates with tegumental damage visualized by scanning electron microscopy (see Xiao et al., 1991). 7.4. Synergy with Antibodies

To assess the role of host humoral responses in the mechanism of action of PZQ against S. rnansoni, Brindley and Sher (1987) compared the efficacy of PZQ in infected B lymphocyte-depleted (p-suppressed) and immunologically intact C3H/HeN mice. (The mice were depleted of B lymphocytes by injecting them from birth with goat anti-mouse IgM antiserum (Lawton et al., 1972).) PZQ was on average only 20% as effective in eliminating adult schistosomes from p-suppressed as from control animals. Specifically, in three of four experiments performed, the drug failed to reduce significantly the worm load in the p-suppressed mice in comparison to non-drug-treated, infected mice. The results were not apparently due to a delay in parasite elimination in the infected B celldepleted mice, because adult worms recovered from these mice as late as 7 weeks after chemotherapy were indistinguishable in number and appearance (light microscopy) from non-drug-treated mice. The efficacy

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Figure 5 Fluorescence micrograph of the surface of adult male Schistosoma mansoni perfused from immunologically intact mouse 1 hour after treatment with praziquantel. The worm was incubated with a fluoresceinated F(ab')* goat anti-mouse IgG reagent and photographed under fluorescent light. Note that most of the immunofluorescence is localized on the spined tubercles. (Reproduced with permission from Brindley and Sher, 1987.)

of PZQ was completely restored by passive transfer of immune serum from donor mice infected for 6 weeks and partially restored with IgG and nonIgG immunoglobulins purified from the same sera. Moreover, IgG and IgM antibodies were detected by immunofluorescence microscopy on the surface of adult schistosomes as early as 1 hour after administration of the drug in vivo. The tubercles of male worms were the major focus of antibody binding in these PZQ-exposed schistosomes (Figure 5 ) . These results formally demonstrated that the mechanism of action of PZQ involves a synergy between PZQ and the humoral immune response of the host, and suggested that the relevant effector antibodies act directly against hidden parasite antigens which become exposed on the surface of the parasites as a consequence of interaction with PZQ. As described above, Brindley and Sher (1987) demonstrated that the defect in PZQ efficacy in B cell-depleted mice could be restored by homologous infection serum, and with immunoglobulin fractions of homologous infection serum. Contemporaneously, Doenhoff et al. (1987) showed that adoptively transferred heterologous antisera, raised in rabbits by vaccination with antigens released by S . mansoni into culture medium, enhanced the cure rate of suboptimal doses of PZQ. Subsequently, Doenhoff et al. (1988) examined a panel of rabbit antisera, raised against different schistosome antigen preparations, for ability to act after passive transfer to infected mice to act in synergy with PZQ chemotherapy. Infected mice were administered with a suboptimal dose of 150 mg kg-' PZQ on days 35 and 37 post-infection, and/or injected intravenously with 0.5 ml rabbit sera

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on each of days 35 to 38. One of the antisera, 1018K, had been raised against an “anodal” antigen. This antigen had been isolated as an immunoelectrophoresis diffusion plate precipitate after polyspecific rabbit antisera had reacted with an S . mansoni vaccine antigen preparation (see Dunne et al., 1986). Whereas PZQ alone reduced the worm load by 33%, the worm load was reduced 98% in the mice given both PZQ and antiserum 1018K. Another serum raised against the same antigen, and sera raised against antigens released by agitating adult worms in culture media, were also found likewise to act synergistically in the model, though not with as much potency as 1018K. These rabbit antisera exhibited no helminthotoxicity when administered to infected mice in the absence of PZQ chemotherapy. By contrast, rabbit antisera raised against deoxycholate detergent extracts and against homogenates of adult worms lacked any synergistic activity with PZQ. When employed to probe immunoblots of detergent-extracts of adult S . mansoni, the monospecific antiserum 1018K recognized an antigen of 27 kDa. Immunoprecipitation of schistosome antigens with antiserum 1018K followed by staining with the non-specific esterase substrate betanaphthyl acetate indicated that the 27 kDa antigen was a non-specific esterolytic enzyme. Other rabbit antisera which also acted in synergy in PZQ-efficacy also recognized the 27 kDa in immunoblots. This antigen has been localized to the dorsal tubercles of male schistosomes (below). 7.5. Localization of Drug-antibody Damage

In a logical extension to experiments of the synergy of antischistosome antibodies and PZQ, Modha et al. (1990) undertook an ultrastructural study using scanning and transmission electron microscopy of S. mansoni adult worms exposed to PZQ and antiserum in vivo. For the study, mice infected 35 days previously with S. mansoni were treated 2 hours before perfusion with 100 mg kg-’ PZQ, a subcurative dose, and/or 1 hour before perfusion with rabbit antischistosome antiserum. The antiserum had been raised against antigens released into culture medium by adult S. mansoni. It was observed that the antiserum alone induced a membrane repair process in both male and female schistosomes, but little other damage. PZQ alone caused the formation of spherical protuberances on the dorsal tubercles of male worms. The damage seen in male worms obtained from mice after combined PZQ/antiserum treatment was much more enhanced than with either treatment alone. In particular, scanning microscopy revealed small blebs distributed over the entire surface of the male worm, and surface protuberances of varying size occurred both over and between the dorsal bosses. Many of the protuberances were not only punctured but

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Figure 6 Scanning electron micrograph (SEM) showing exploded surface protuberances on dorsal tubercles of a male Schistosoma mansoni 2 hours after combined treatment with praziquantel and rabbit a n t i 3 mansoni antiserum of infected mice (top). Higher magnification SEM of an exploded protrusion showing spherical structures within (bottom). (Reproduced with permission from Modha et al., 1990.)

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broken open and spherical structures were evident within them (Figure 6). The enhanced morphological trauma reported for male as compared with female schistosomes is consistent with the observed increase in antibody staining in male worms after PZQ treatment (Figure 5) (Brindley and Sher, 1987; Doenhoff et al., 1988). Modha et al. (1990) concluded that schistosomes exhibit both sexual and regional susceptibility to the immune dependent action of PZQ, a feature noted previously for worms treated with drug alone (Shaw and Erasmus, 1983a,b, 1987). 7.6. Molecular Characterization of Exposed Antigens

Several of the schistosome antigens exposed by PZQ treatment have been characterized (Doenhoff et al., 1988; Brindley et al., 1989). Brindley et al. (1989) examined the surface of PZQ-treated schistosomes by immunofluorescence assay (IF) for reaction with a panel of monoclonal (mAb) and monospecific polyclonal antibodies raised against tegumental or subtegumental antigens from S. mansoni in order to determine which surface antigens were exposed by the drug. Of 21 antibodies tested five were found to react with the surface of male schistosomes obtained by perfusion of infected nude mice 1 hour after PZQ treatment of the mice. Nude mice were used as a source of donor worms for this assay because they were not expected to produce antibodies which would interfere and mask the result of the IF in vitro. Only a minority of the antibodies specific for tegumental components bound to the surface of the parasite which suggested that the damage caused by PZQ to the schistosome tegument was restricted to a limited set of tegumental components. One of the molecules is a 200 kDa glycoprotein abundant in the tubercles and parenchyma of adult S. mansoni (Strand et al., 1982; Aronstein et al., 1986). Only part of the 200 kDa glycoprotein is exposed by PZQ treatment. For instance, the peptide epitope recognized by mAb 305 A411 becomes exposed to the immune response after drug treatment in mice, whereas the peptide epitope recognized by mAb 307 D5/1 on the same glycoprotein does not (Brindley et al., 1989). Exposure of peptide epitopes on this molecule and the subsequent binding of antibodies to the exposed sites appear to be sufficient to effect the synergy between PZQ and the immune response because passive transfer of mAb 305 A4/1, which recognizes one of the exposed epitopes on the glycoprotein, to infected B cell-depleted mice reconstituted the efficacy of PZQ treatment back to normal levels (Brindley et al., 1989). Biochemical characterization of the 200 kDa glycoprotein showed that it is linked to the membrane of the adult schistosome by a glycosylphosphatidylinositol anchor (Sauma et al. , 1991). Conformational changes

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that cause the exposure of the mAb 305 A4/1 epitope may be caused by interactions of PZQ with the lipid bilayer of the schistosome (Harder et al., 1988; Scheppers et al., 1988). Sauma et al. (1991) suggested that the conformational change that exposes only part of this lipid-anchored glycoprotein to the immune response following treatment with PZQ could increase its accessibility to endogenous/exogenous phospholipases, resulting in release of the molecule from the surface of the worm and generation of diacylglycerol or phosphatidic acid. These secondary messengers may then play a role at the worm’s surface, causing the depolarization of the membrane and the influx of extracellular calcium that characteristically follow exposure to PZQ (see Andrews, 1985). Yet, since mAb 305 A4/1 still binds to the tegument at 1 hour after PZQ treatment in vivo (Brindley et al., 1989), it is evident that (at least) some molecules of the 200 kDa glycoprotein remain inaccessible to phospholipases at this time. As noted, a 200 kDa glycoprotein of S. mansoni has been identified as a target of host antibodies in mice which act in synergy with PZQ chemotherapy. Because PZQ is active against all the human schistosomes, Tanaka et al. (1993) examined S . haematobium and S. japonicum for the presence of a cross-reactive homologue of the S . mansoni 200 kDa antigen, by immunoblot analysis using a monospecific polyclonal rabbit antiserum. They detected an antigen of approximately 200 kDa in all three species using the anti-S. mansoni 200 kDa antiserum. However, although epitopes of three mAbs (305, 307,129) specific for the S. mansoni 200 kDa antigen could be detected on S . haematobium, none of the mAbs recognized the S . japonicum homologue. Furthermore, whereas PZQ treatment exposed the 200 kDa antigen to antibody binding at the surface of both S. mansoni and S. haematobium, treatment with PZQ (or with acetone, which renders surface epitopes more accessible) did not expose the 200 kDa antigen on the surface of S . japonicum. If antibodies specific for the 200 kDa surface glycoprotein are involved in the chemotherapeutic efficacy of PZQ against S. mansoni and S . haematobium in humans, the results of Tanaka et al. (1993) would indicate that other antigens besides the 200 kDa glycoprotein are involved in the immune-dependent action of PZQ against S. japonicum. The other well-characterized molecule exposed by PZQ is a 27 kDa surface antigen which exhibits non-specific esterolytic activity (Doenhoff et al., 1988; Brindley et al., 1989). Like the 200 kDa glycoprotein, this esterase is abundant in the tubercles of adult male schistosomes. Transfer of a rabbit antiserum raised against the esterase was found to augment the efficacy of PZQ against 5-week-old S. mansoni infections in mice, against which PZQ is only partially effective (Doenhoff et al., 1987, 1988). In addition, though they are not as well studied, an alkaline phosphatase and a peptide moiety defined by mAb 710 C4/1 on membranous organelles can be exposed on the surface of adult schistosomes by treatment in uiuo with

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PZQ, although adoptive transfer of rabbit antiserum to the alkaline phosphate did not apparently enhance PZQ chemotherapy (Doenhoff et al., 1988; Brindley et af., 1989). Doenhoff et al. (1988) have suggested that PZQ and oxamniquine may expose different antigens on the schistosome surface since a serum raised by “infecting” a rabbit via the ear with S. mansoni cercariae did not act synergistically with oxamniquine, whereas serum from a similarly infected rabbit did act synergistically with PZQ. Finally, as they had observed that loss of spines was a typical feature of schistosomes obtained from PZQ-treated animals, Linder and Thors (1992) have considered the possibility that PZQ-treatment involves exposure of surface spines consisting of “paracrystalline” actin and a subsequent host response against actin through mechanisms established in the host independently from schistosomal infection, including the binding of the actin depolymerizing factor gelsolin (see Chaponnier et al., 1979). Drug-induced exposure of actin was demonstrated using anti-actin antibodies and phallacidin, an actin binding mushroom toxin. Actin spines remained intact at the schistosome surface after in vitro exposure, but spine morphology was lost after in vivo exposure to PZQ. Disintegration of spines in vivo was associated with binding of host antibodies. In vitro spine destruction could be seen in the presence of normal human serum. Moreover, they observed by indirect immunofluorescence that the effect was linked with the calcium-dependent binding of gelsolin.

7.7. Efficacy in Vaccinated Animals

It has been demonstrated that vaccination of mice with radiation-attenuated cercariae enhanced the efficacy of PZQ treatment against challenge infections with S. mansoni. In brief, mice vaccinated with radiation-attenuated cercariae treated with PZQ on day 6 post-challenge exhibited between 79% and 91% PZQ-efficacy whereas naive mice similarly treated with PZQ showed a drug cure rate of between 57 and 66% in three separate experiments. Less marked synergy was detectable when the PZQ was administered on day 1 after challenge infection of vaccinated mice, and this was dependent on the route of PZQ administration. It was seen with intradermal but not intramuscular drug administration (Flisser et al. , 1989a). That the synergy occurred with PZQ-treatment at day 6 rather than at day 1 was an intriguing finding since, in the model, vaccine-induced resistance was known to be a skin phase (i.e. day 1 post-challenge) phenomenon rather than an immune response to the lung-stage schistosomes, i.e. day 6 post-infection-stage parasites. Soon after, Piper et af. (1990) examined the histopathological consequences of the drug-immunological interaction and determined the nature of the damage to the developing

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schistosome. PZQ treatment on day 6 post-challenge facilitated efficient trapping of challenge larvae in the lungs of vaccinated mice, a feature associated with the induction of macrophage-rich inflammatory focal reactions. Whereas parasite trapping in the skin is characteristic of vaccine immunity in the NIMR m o u s e 4 mansoni model, it appears that druginduced exposure of schistosome antigens on lung-stage schistosomules (Hisser and McLaren, 1989) permits the expression of macrophage-mediated killing of the lung stage worms (Piper et al., 1990). The PZQ-host immune response interaction in vaccinated hosts has also been addressed by Chinese researchers with respect to schistosomiasis japonica. A worm reduction rate of 58% was obtained in rabbits immunized with an homogenate of adult S. japonicum and treated with PZQ three weeks after cercarial challenge. In contrast, a reduction of only 12% was obtained in non-vaccinated control animals. In other experiments, rabbits infected twice with 200 cercariae at an interval of 7 weeks were treated with PZQ three weeks after the second infection. A worm reduction rate of 78% was obtained in these rabbits, whereas the drug had no efficacy on rabbits infected only once three weeks previously (see Xiao et al., 1991). Experiments involving PZQ treatment of sexually mature infections in mice vaccinated with radiation attenuated cercariae await to be reported, but it can be expected that they will be instructive. Based on the enhanced performance of PZQ in vaccinated mice against immature schistosomes, vaccination with those antigens that are known to be unmasked by PZQ, including the 200 kDa glycoprotein (Brindley et al., 1989; Tanaka et al., 1993) and the 27 kDa non-specific esterifying enzyme (Doenhoff et al., 1988), prior to PZQ chemotherapy, would probably bolster the performance of PZQ against sexually mature schistosome infections. From the perspective of combined chemo- and immunotherapy, it would be of value to determine whether vaccination and chemotherapy can be combined to reduce morbidity and prevalence of schistosomiasis. In like fashion, it would be informative to assay the effect of vaccination with more well-established candidate antigens (see Richter et al., 1993) some of which including paramyosin (Lanar et al., 1986; Yang et al., 1992) are not exposed by PZQ-treatment (Brindley et al., 1989). 7.8. A Complex Chain of Events

At present, the mode of action of PZQ against schistosomes appears to be a complex chain of events. The immediate effects of PZQ include muscle contraction and metal ion fluxes (Andrews, 1985). Tegumental damage

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follows, exposing antigens to which host antibodies bind (Brindley and Sher, 1987). Subsequently, cellular a n d o r other reactions probably follow which result in the encapsulation and death of the parasites. The nature of the cellular reactions at the end of the above chain of events has not been determined, although granulocytes have been observed migrating into schistosomes in mice within a few hours of PZQ-treatment (Mehlhorn et al., 1981). Brindley and colleagues have indirectly examined several possible mechanisms which may be involved in the killing of PZQ-treated worms. PZQ chemotherapy of experimental infections with S. rnansoni in the B10.D2 osn strain of mice, which bears a defective terminal complement pathway, in the P strain of mice, which has a congenital dysfunction in macrophage activation, and in mice depleted of eosinophils by treatment with anti-interleukin 5 antibody (Sher et al., 1990) was as effective in each case as in control mice (Brindley and Sher, 1990; Brindley, unpublished). Hence, an immunological pathway separate from those requiring activated macrophages, eosinophils, or complement is either involved or at least sufficient to mediate effective chemotherapy. Whatever the mechanism, the same kinds of immunological mediators may be necessary for the anthelmintic efficacy of PZQ against nonschistosome platyhelminths since the drug appears to be less active against infections with the larval cestode Taenia taeniaeforrnis in immunocompromised (B cell-depleted o r athymic) mice than in intact animals (Ballard, Nash and Brindley, unpublished). A synergy between PZQ and the immune response may be required generally for efficacy against other important trematode and cestode parasites that reside in parenteral, though perhaps not in enteral, sites.

8. CONCLUDING REMARKS

Since both praziquantel and oxamniquine apparently require appropriate immunological responses for chemotherapeutic efficacy against S . rnansoni and S. japonicurn in laboratory animals, and since it is not known whether metrifonate is likewise less active against S . haernatobiurn in immunocompromised individuals, full efficacy of each of only three currently available schistosomicides recommended by the World Health Organization may not be achievable in humans in the absence of appropriate immunological responses. Accordingly, schistosomiasis may not be amenable to the currently indicated chemotherapy in immunocompromised patients, including those concurrently infected with HIV. Interestingly, the experimental drug oltipraz is not only effective against S . rnansoni in humans (Bella et al., 1982) but has an antiretroviral activity

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that inhibits replication of HIV in vitro (Prochaska et al., 1993). By contrast, the experimental schistosomicide amoscanate does not apparently exhibit an immune dependence in its antischistosomal action (Sabah et al., 1985). Given that appropriate immune responses including antibodies are necessary for the full efficacy of PZQ in experimental S. rnansoni infections in mice, and given that human IgE responses protect against infection with S. haernatobium (Hagan et al., 1991) (though perhaps not against S . rnansoni or other schistosome species; see Pearce et al., 1991), it may turn out that achieving a semi-immune status either through repeated infection, or perhaps through vaccination, may result in chemotherapeutic cures with dose schedules lower than those necessary to cure non-immune patients. This has certainly been the experience with antimalarial drugs (Targett, 1985). ACKNOWLEDGEMENTS

The financial support of the joint Queensland Institute of Medical ResearcWniversity of Queensland Tropical Health Program is gratefully acknowledged. REFERENCES Andrews, P. (1985). Praziquantel: Mechanisms of anti-schistosomal activity. Pharmacology and Therapeutics 29, 129-156. Andrews, P. and Bonse, G. (1986). Chemistry of anticestodal agents. I n “Chemotherapy of Parasitic Diseases” (W.C. Campbell and R.S. Rew, eds), pp. 447456. Plenum Press, New York. Archer, S. (1985). The chemotherapy of schistosomiasis. Annual Review of Pharmacology and Toxicology 25, 485-508. Archer, S., El-Hamouly, W., Seyed-Mozaffari, A,, Butler, R.H., Pica-Mattoccia, L. and Cioli, D. (1990). Mode of action of the schistosomicide hycanthone: site of DNA alkylation. Molecular and Biochemical Parasitology 43, 89-96. Aronstein, W.S., Lewis, S.A., Norden, A.P., Dalton, J.P. and Strand, M. (1986). Molecular identity of a major antigen of Schistosorna mansoni which cross-reacts with Trichinelia spiralis and Fasciola hepatica. Parasitology 92, 133-151, Badaro, R., Falcoff, E., Badaro, F.S., Carvalho, E.M., Pedral-Sampaio, D., Barral, A., Carvalho, J.S., Barral-Netto, M., Brandely, M., Silva, L., Bina, J.C., Teixeira, R., Falcoff, R., Rocha, H., Ho, J.L. and Johnson, W.D. (1990). Treatment of visceral leishmaniasis with pentavalent antimony and interferon gamma. New England Journal of Medicine 322, 16-21. Bella, H., Rahim, A.G., Mustafa, M.D., Ahmed, M.A.,Wasfi, S. and Bennett, J.L. (1982). Oltipraz - Antischistosomal efficacy in Sudanese infected with

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Schistosoma mansoni. American Journal of Tropical Medicine and Hygiene 31, 775-778. Bitoni, A.J., McCann, P.P. and Sjoerdsma, A. (1986). Necessity of antibody response in the treatment of African trypanosomiasis with alpha-difluoromethyl ornithine. Biochemical Pharmacology 35, 331-337. Brindley, P.J. Drug resistance to schistosomicides and other anthelmintics of medical significance. Acta Tropica (in press). Bnndley, P.J. and Sher, A. (1987). The chemotherapeutic effect of praziquantel against Schistosoma mansoni is dependent on host antibody response. Journal of Immunology 139, 215-220. Brindley, P.J. and Sher, A. (1990). Immunological involvement in the efficacy of praziquantel. Experimental Parasitology 71, 245-248. Brindley, P.J., Strand, M., Norden, A. and Sher, A. (1989). Role of host antibody in the chemotherapeutic action of praziquantel against Schistosoma mansoni. Molecular and Biochemical Parasitology 34, 99-108. Butterworth, A.E. (1992). Vaccines against schistosomiasis: where do we stand? Transactions of the Royal Society of Tropical Medicine and Hygiene 86, 1-2. Chaponnier, C., Borgia, R., Rungger-Brandle, E., Weil, R. and Gabbiani, G. (1979). An actin-destabilizing factor is present in human plasma. Experientia 35, 1039-1040. Cheever, A.W. and Deb, S. (1989). Persistence of hepatic fibrosis and tissue eggs following treatment of Schistosoma japonicum infected mice. American Journal of Tropical Medicine and Hygiene 40,620-628. Christopherson, J.B. (1918). The successful use of antimony in bilharziosis. Administered as intravenous doses of antimonium tartartum (tartar emetic). Lancet ii, 325-327. Cioli, D. and Pica-Mattoccia, L. (1984). Genetic analysis of hycanthone resistance in Schistosoma mansoni. American Journal of Tropical Medicine and Hygiene 33, 80-88. Cioli, D., Pica-Mattoccia, L., Rosenberg, S. and Archer, S. (1985). Evidence for the antischistosomal action of hycanthone. Life Sciences 37, 161-167. Cioli, D., Pica-Mattoccia, L. and Moroni, R. (1992). Schistosoma mansoni: hycanthone/oxamniquine resistance is controlled by a single autosomal recessive gene. Experimental Parasitology 75, 425-432. Cioli, D., Pica-Mattoccia, L. and Archer, S. (1993). Drug resistance in schistosomes. Parasitology Today 9, 162-166. Cook, G.C. (1990). Schistosomiasis: an important cause of colonic, hepatic and urinary-tract disease. In “Parasitic Disease in Clinical Practice”, pp. 121-140. Springer-Verlag, London. Day, T.A., Bennett, J.L. and Pax, R.A. (1992). Praziquantel: the enigmatic antiparasitic. Parasitology Today 8, 342-344. de Gee, A.L.W., McCann, P.P. and Mansfield, J.M. (1983). Role of antibody in the elimination of trypanosomes after DL-a-difluoromethylornithine chemotherapy. Journal of Parasitology 69, 818-822. Doenhoff, M.J. and Bain, J. (1978). The immune-dependence of schistosomicidal chemotherapy: relative lack of efficacy of an antimonial in Schistosoma mansoniinfected mice deprived of their T-cells and the demonstration of drug-antiserum synergy. Clinical Experimental Immunology 33, 232-238. Doenhoff, M., Hamson, R., Sabah, A., Murare, H., Dunne, D. and Hassounah, 0. (1982). Schistosomiasis in the immunosuppressed host: studies on the hostparasite relationship of Schistosoma mansoni and s. bovis in T-cell-deprived and

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hydrocortisone-treated mice. In “Animal Models in Parasitology”, (D.’G. Owen, ed.), pp. 155-169. MacMillan, London. Doenhoff, M.J., Sabah, A.A.A., Fletcher, C., Webbe, G. and Bain, J. (1987). Evidence for an immune-dependent action of praziquantel on Schistosoma mansoni in mice. Transactions of the Royal Society of Tropical Medicine and Hygiene 81, 947-951. Doenhoff, M.J., Modha, J. and Lambertucci, J.R. (1988). Anti-schistosome chemotherapy enhanced by antibodies specific for a parasite esterase. Immunology 65, 507-510. Doenhoff, M.J., Modha, J., Lambertucci, J.R. and McLaren, D.J. (1991). The immune dependence of chemotherapy. Parasitology Today 7 , 16-18. Dunne, D.W., Agnew, A.M., Modha, J. and Doenhoff, M.J. (1986). Schistosoma mansoni egg antigens: preparation of rabbit antisera with monospecific immunoprecipitating activity, and their use in antigen characterization. Parasite Immunology 8, 575-586. Flisser, A., and McLaren, D.J. (1989). Effect of praziquantel treatment on lung-stage larvae of Schistosoma mamoni in vivo. Parasitology 98, 203-21 1. Flisser, A., Delgado, V.S. and McLaren, D.J. (1989a). Schistosoma mansoni: enhanced efficacy of praziquantel treatment in immune mice. Parasite Immunology 11, 319-328. Flisser, A., Elsaghier, A.A.F. and McLaren, D.J. (1989b). Effect of praziquantel on the migration and survival of developmental stages of Schistosoma mansoni in mice. International Journal for Parasitology 19, 665-672. Gibbons, A. (1992). Researchers fret over neglect of 600 million patients. Science 256, 1135. Goldring, O.L., Sher, A., Smithers, S.R. and McLaren, D.J. (1977). Host antigens and parasite antigens of murine Schistosoma mansoni. Transactions of the Royal Society of Tropical Medicine and Hygiene 71, 144-148. Goldsmith, R.S. (1988). Recent advances in the treatment of helminthic infections: ivermectin, albendazole, and praziquantel. In “Parasitic Infections” (J.H. Leech, M.A. Sande and R.K. Root, eds), pp. 327-347, Churchill Livingstone, New York. Grove, D.I. (1990). “A History of Human Helminthology”, pp. 187-295. CAB International, Wallingford, UK. Hagan, P., Blumenthal, U.J., Dunn, D., Simpson, A.J.G. and Wilkins, H.W. (1991). Human IgE, IgG4 and resistance to reinfection with Schhtosoma haematobium. Nature 349, 243-245. Harder, A., Goosens, J. and Andrews, P. (1988). Influence of praziquantel and Ca2+on the bilayer-isotoic-hexagonal transition of model membranes. Molecular and Biochemical Parasitology 29, 55-60. Hamett, W. (1988). The anthelmintic action of praziquantel. Parasitology Today 4, 144-146. Hamett, W. and Kusel, J.R. (1986). Increased exposure of parasite antigens at the surface of adult male Schistosoma mansoni exposed to praziquantel in vitro. Parasitology 93, 401405. Hsu, S.Y.L., Chu, K.Y. and Hsu, H.F. (1963). Drug susceptibility of geographic strains of Schhtosoma japonicum. Zeitschrift fur Tropenmedizin und Parasitologie 14, 37-40. Jansma, W.B., Rogers, S.H., Liu, C.L. and Bueding, E. (1977). Experimentally produced resistance of Schistosoma mansoni to hycanthone. American Journal of Tropical Medicine and Hygiene 26, 926-936. Jordan, P. (1985). “Schistosomiasis: the St. Lucia project”. Cambridge University Press, Cambridge.

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Katz, N. and Pellegrino, J. (1974). Experimental chemotherapy of Schistosomiasis mansoni. Advances in Parasitology 12, 369-390. Katz, N., Dias, E.P., Araujo, N. and Souza, C.P. (1973). Estudo de ume cepa humana de Schistosoma mansoni resistente a agentes esquistossomicidas. Revista da Sociedade Brasileira de Medicines Tropical 7, 381-387. Lambertucci, J.R., Modha, J. and Doenhoff, M. (1989). Schistosoma mansoni: the therapeutic efficacy of oxamniquine is enhanced by immune serum. Transactions of the Royal Society of Tropical Medicine and Hygiene 83, 362-363. Lanar, D.E., Pearce, E.J., James, S.L. and Sher, A. (1986). Identification of paramyosin as schistosome antigen recognized by intradermally vaccinated mice. Science 234, 593-596. Law, L.W., Bradley, T.R. and Rose, S. (1963). Reversal of thymus dependent influence in radiation leukemagenesis of C57BI mice. Journal of the National Cancer Institute 31, 1461-1477. Lawton, A.R., Asofsky, R., Hylton, M.B. and Cooper, M.A. (1972). Suppression of immunoglobulin class synthesis in mice. I, Effects of treatment with antibody to pchain. Journal of Experimental Medicine 135, 277-297. Levey, R.H. and Medewar, P.B. (1966). Some experiments on the action of antilymphoid antisera. Annals of the New York Academy of Sciences 129, 164-177. Liese, B. (1986). The organization of schistosomiasis control programs. Parasitology Today 2, 339-344. Linder, E. and Thors, C. (1992). Schistosoma mansoni: praziquantel-induced tegumental lesion exposes actin of surface spines and allows binding of actin depolymerizing factor, gelsolin. Parasitology 105, 71-79. Lwin, M., Targett, G.A.T. and Doenhoff, M. (1987). Reduced efficacy of chemotherapy of Plasmodium chabaudi in T cell-deprived mice. Transactions of the Royal Society of Tropical Medicine and Hygiene 81, 88-93. Mahmoud, A. A.F. (1984). Schistosomiasis. In “Tropical and Geographical Medicine” (K.S. Warren and A.A.F. Mahmoud, eds), pp. 443-457. McGraw-Hill, New York. Mansour, T.E. and Bueding, E. (1980). The actions of antimonials on glycolytic enzymes of S. mansoni. British Journal of Pharmacology 9, 45W62. Marshall, I. (1987). Experimental Chemotherapy, In “The Biology of Schistosomes from Genes to Latrines” (D. Rollinson and A.J.G. Simpson, eds), pp. 399-430. Academic Press, London. Mehlhorn, H., Becker, B., Andrews, P., Thomas, H. and Frenkel, J.K. (1981). In vivo and in vitro experiments on the effects of praziquantel on Schistosoma mansoni. Arzneimittel Forshung 31, 544-554. Modha, J., Lambertucci, J.R., Doenhoff, M.J. and McLaren, D.J. (1990). Immune dependence of schistosomicidal chemotherapy: an ultrastructural study of Schistosoma mansoni adult worms exposed to praziquantel and immune serum in vivo. Parasite Immunology 12, 321-334. Mrozik, H. (1986). Chemistry of antitrematodal agents. In “Chemotherapy of Parasitic Diseases” (W.C. Campbell and R.S. Rew, eds), pp. 365-399. Plenum Press, New York. Olliaro, P.L. and Bryceson, A.D.M. (1993). Practical progress and new drugs for changing patterns of leishmaniasis. Parasitology Today 9, 32S328. Pearce, E.J., Caspar, P., Grzych, J-M., Lewis, F.A. and Sher, A. (1991). Downregulation of T h l cytokine function accompanies induction of Th2 responses by a helminth parasite, Schistosoma mansoni. Journal of Experimental Medicine 173, 159-166.

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Pellegrino, N. and Katz, N. (1968). Experimental chemotherapy of schistosomiasis mansoni. Advances in Parasitology 6, 233-290. Pica-Mattoccia, L. and Cioli, D. (1985). Studies on the mode of action of oxamniquine and related schistosomicidal drugs. American Journal of Tropical Medicine and Hygiene 34, 112-1 18. Pica-Mattoccia, L., Cioli, D. and Archer, S. (1988). Binding of tritiated hycanthone and hycanthone N-methylcarbamate to molecules of drug sensitive and drug-resistant schistosomes. Molecular and Biochemical Parasitology 31, 87-96. Pica-Mattoccia, L., Cioli, D. and Archer, S. (1989). Binding of oxamniquine to the DNA of schistosomes. Transactionsof the Royal Society of Tropical Medicine and Hygiene 83, 373-376. Pica-Mattoccia, L., Archer, S. and Cioli, D. (1992). Hycanthone resistance in schistosomes correlates with the lack of an enzymatic activity which produces the covalent binding of hycanthone to parasite macromolecules. Molecular and Biochemical Parasitology 55, 167-176. Piper, K.P., Mott, R.F. and McLaren, D.J. (1990). Schistosoma mansoni: histological analysis of the synergistic interaction interaction between vaccine immunity and praziquantel therapy in the lings of mice. Parasite Immunology 12,367-387. Prochaska, H.J., Yeh, Y., Baron, P. and Polsky, B. (1993). Oltipraz, an inhibitor of human immunodeficiency virus type 1 replication. Proceedings of the National Academy of Sciences, USA 90, 3953-3957. Richards, H.C. and Foster, E. (1969). A new series of 2-amino methyltetrahydroquinoline derivatives displaying schistosomicidal activity in rodents and primates. Nature 222, 581. Richter, D., Reynolds, S.R. and Harn, D.A. (1993). Candidate vaccine antigens that stimulate the cellular immune response of mice vaccinated with irradiated cercariae of Schistosoma mansoni. Journal of Immunology 151, 256-265. Sabah, A.A., Fletcher, C., Webbe, G. and Doenhoff, M.J. (1985). Schistosoma mansoni: reduced efficacy of chemotherapy in infected T-cell-deprived mice. Experimental Parasitology 60, 348-354. Sabah, A.A., Fletcher, C., Webbe, G . and Doenhoff, M.J. (1986). Schistosoma mansoni: Chemotherapy of infections of different ages. Experimental Parasitology 61, 294-303. Sauma, S.Y., Tanaka, T.M. and Strand, M. (1991). Selective release of glycosylphosphatidylinositol-anchoredantigen from the surface of Schistosoma martsoni. Molecular and Biochemical Parasitology 46, 73-80. Scheppers, H., Brasseur, R., Goormaghtigh, E., Duquenoy, P. and Ruysschaert, J-M. (1988). Mode of insertion of praziquantel into lipid membranes. Biochemical Pharmacology 37, 1615-1623. Shaw, M.K. (1990). Schistosoma mansoni: stage-dependent damage after in vivo treatment with praziquantel. Parasitology 100, 65-72. Shaw, M.K. and Erasmus, D.A. (1983a). Schistosoma mansoni: The effects of a sub-curative dose of praziquantel on the ultrastructure of worms in vivo. Zeitchshrift fur Parasitenkunde 69, 73-90. Shaw, M.K. and Erasmus, D.A. (1983b). Schistosoma mansoni: dose-related tegumental surface changes after in vivo treatment with praziquantel. Zeitchshrifr fur Parasitenkunde 69, 643-653. Shaw, M.K. and Erasmus, D.A. (1987). Schistosoma mansoni: structural damage and tegumental repair after in vivo treatment with praziquantel. Parasitology 94, 243-254.

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Sher, A., Coffman, R.L., Hieny, S., Scott, P. and Cheever, A.W. (1990). Interleukin 5 is required for the blood and tissue eosinophilia but not granuloma formation induced by infection with Schistosoma mansoni. Proceedings of the National Academy of Sciences, USA 87, 61-65. Smithers, S.R. and Terry, R.J. (1976). The immunology of schistosomiasis. Advances in Parasitology 14, 399-422. Souza, L.C.S., Pedro, R.J. and Deberaldini, E.R. (1982). Use of praziquantel in patients with schistosomiasis mansoni previously treated with oxamniquine and/ or hycanthone: resistance of Schistosoma mansoni to schistosomicidal agents. Transactions of the Royal Society of Tropical Medicine and Hygiene 76,652459. Strand, M., McMillan, A. and Pan, X.Q. (1982). Schistosoma mansoni: reactivity with infected sera and monoclonal characterization of a glycoprotein in different developmental stages. Experimental Parasitology 54, 145-156. Tanaka, T.M., Skubitz, A.P.N. and Strand, M. (1993). Schistosoma: a 200-kDa target antigen is differentially localized in African vs Oriental species. Experimental Parasitology 76, 293-301. Targett, G.A.T. (1985). Chemotherapy and the immune response in parasitic infections. Parasitology 90, 661-673. Waine, G., Becker, B., Kalinna, B., Yang, W., Scott, J., Liu, X., Tiu, W. and McManus, D. Molecular vaccines against schistosomiasis: current status and the challenges ahead. Asia- Pacific Journal of Molecular Biology and Biotechnology (in press). Weist, P.M.,Yining, L., Olds, G.R. and Bowen, W.D. (1992). Inhibition of phosphoinositide turnover by praziquantel in Schistosoma mansoni. Journal of Parasitology 78, 753-755. World Health Organization (1992). Praziquantel shows unexpected failure in recent schistosomiasis outbreak. TDR News 41, 1-2. Xiao, S-H., Catto, B.A. and Webster, L.T. (1985). Effects of praziquantel on different developmental stages of Schistosoma mansoni in vivo and in vitro. Journal of Infectious Diseases 151, 113&1137. Xiao, S-H., Fu, S. and Catto, B.A. (1991). Recent laboratory investigations by Chinese workers on antischistosomal activities of praziquantel. Chinese Medical Journal 104, 599-606. Yang, W., Waine, G.J., Sculley, D.G., Liu, X. and McManus, D.P. (1992). Cloning and partial sequence of Schistosoma japonicum paramyosin: a potential vaccine candidate against schistosomiasis. International Journal for Parasitology 22, 1187-1191. Yue, W-j., Yu, S-h. and Xu, X-j. (1990). Failure to induce resistance of Schistosoma japonicum to praziquantel. South East Asian Journal of Tropical Medicine and Public Health 21, 85-89.

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Regulatory Peptides in Helminth Parasites

’

David W . Halton. Chris Shaw. Aaron G . Maule’ and David Smart’

Comparative Neuroendocrinology Research Group. ‘School of Biology and Biochemistry and 2School of Clinical Medicine. The Queen’s University of Belfast. Belfast BT7 1NN. Northern Ireland. U K ... . . . . 164 1. Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 164 1.1. Historical perspective . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ...................... 166 1.2. Chemistry of regulatory peptides . . .

2. Occurrence and Distribution of Regulatory Peptides in Helminths . . . . . . . . . . . 2.1. General account . . . . . . . . . . . . . . . . . . . . . . . . . . . ................. ................. 2.2. Peptide localization in flatworm parasites . . . . 2.3. Peptide localization in nematodes . . . . . . . . . . . .................

168 168 170 178

3. Quantification and Characterization of Regulatory Peptides in Helminths . . . . . 3.1. General account . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. Flatworm parasites . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ...................... 3.3. Nematodes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

184 184 185 191

4. Isolation and Structure of Helminth Regulatory Peptides . . . . . . . . . . . . . . . . . . . . .................. 4.1. General account . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2. Flatworms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3. Nematodes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

195 195 196 199

5. Evolutionary Aspects of Helminth Regulatory Peptides ..................... 201 5.1. General account . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 201 5.2. Neuropeptide Y (NPY) superfamily peptides . . . . . . . . . . . . . . . . . . . . . 5.3. FMRFamide and related peptides (FaRPs) . . . . . . . . . . . . . . . . . . . . . . . . 6. Functional Aspects of Helminth tory Peptides . . . . . . . . . . . . 6.1. General account . . . . . . . . . ..................................... 207 6.2. Endogenous functions for h regulatory peptides . . . . . . . . . . . . . . . 208 6.3. Exogenous functionsfor helminth regulatory peptides . . . . . . . . . . . . . . . . . 212 7. Future Developments

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Note Added in Proof . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 217 217 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ADVANCES IN PARASITOLOGY VOL34 ISBN & 1 2 4 3 1 7 3 4 4

Copyright0 19‘24 Academic Press Limited A / / rights of reproduction i n any form reserved

DAVID W. HALTON ET AL.

1. OVERVIEW 1.l.Historical Perspective

The discovery by Bayliss and Starling (1902) that injection of intestinal extracts into dogs elicited an increase in the flow of pancreatic juice was the beginning of the science of endocrinology. These crude experiments demonstrated that a substance or substances present in one organ, through the medium of the bloodstream, could elicit profound changes in the activity of another distant organ. Prior to the demonstration of this effect, secretory modulation was believed to be regulated by the nervous system through reflex pathways. Following the discovery of this “secretin”, bioactive substances were sought and detected in many other organs such as the stomach, pancreas, thyroid and pituitary. Indeed, the work of Banting and Best (1922) which led to the discovery of insulin, was a milestone of such research. The finding that the peptide hormones, oxytocin and vasopressin, were actually synthesized in the hypothalamus by neurons and then axonally transported to the neurohypophysis for secretion into the bloodstream to act on distant target tissues, was the breakthrough that led to the concept of neuroendocrine secretion. Early studies on the identification of regulatory peptides used distinct biological activities such as are manifest in bioassays as a means of detection and isolation. Following this “bioassay era”, many novel regulatory peptides were identified and isolated using chemical attributes of the peptides themselves, including the development of a detection system for the presence of a C-terminal amidated amino acid residue (Tatemoto, 1982). The plethora of regulatory peptides which were identified and isolated in the 1970s and 1980s owe their discovery to various combinations of these bio- and chemical assays; an additional factor was the revolution in biochemical analytical techniques and peptide/protein microsequencing technology. Within the space of a decade, regulatory peptide isolation became possible from some tens of grams of tissues rather than the hundreds of kilograms used in pioneer studies. The development of the techniques of radioimmunoassay and immunocytochemistry enabled the evaluation of tissue and circulating concentrations of novel regulatory peptides and the localization of their sources in many tissues. This led to the discovery that many regulatory peptides were produced by central and peripheral neurons as well as by endocrine cells scattered in peripheral organs. The mucosa of the stomach and entire intestine was found to be a particularly rich source of such peripheral endocrine cells. Neurons and endocrine cells have many common cytochemical and microanatomical features. The APUD concept (Amine Precursor Uptake and Decarboxylation) proposed by Pearse (1968), indicated that neurons

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and endocrine cells had common cytochemical features and also, perhaps, a common embryological origin. Although the second feature of the hypothesis has been subject to a considerable amount of debate, the primary assertion appears sound, as additional biochemical features such as enolase enzymes of common subunit structure have been reported. Neurons and endocrine cells thus appear to represent common components representing the two extremes of endogenous signalling cells that use many common messenger molecules. Much recent research points to the origins of peptidergic signalling in invertebrate neurons. Thus, although anatomically quite simple, the nervous systems of many phylogenetically primitive taxa, such as coelenterates, platyhelminths, annelids and nematodes, have also been perceived as somewhat rudimentary in function. However, the single most striking feature to emerge from immunocytochemical and ultrastructural studies of lower invertebrate nervous systems is that their neural components are highly secretory, and generate a wide array of putative messenger molecules for intercellular communication. Indeed, the number of neuroactive substances identified in helminths easily exceeds the described neuronal cell and vesicle types, highlighting a situation that is far more complex than is evident on a structural basis. Primitive metazoan invertebrates have well-developed peptidergic nervous systems, and in the absence of true endocrine or circulatory systems, one might conclude that the peptidergic neuron predates the peptidergic endocrine cell. Indeed, phylogenetic studies would suggest that peptidergic transmission predates the use of the more generally regarded classical neurotransmitters, such as acetylcholine and 5-hydroxytryptamine. For example, regulatory peptides have been identified in bacteria, yeasts, protistans, and even plants (Le Roith et al., 1986). The peptidergic neuron is thus an evolutionarily ancient entity, and one that is abundant in invertebrates, constituting the majority of central and peripheral neurons. It is, therefore, not surprising that in parallel with the discovery of regulatory peptides from vertebrate and, in particular, mammalian sources, numerous peptidic regulatory factors have been isolated from invertebrate sources, most notably from molluscs and insects. Many of these invertebrate regulatory peptides do not appear to have recognizable equivalents in vertebrate neuroendocrine systems and vice versa. However, recent studies with antisera generated to the bioactive, and hence highly conserved amino acid sequences of vertebrate regulatory peptides, have demonstrated the presence of immunoreactive counterparts in invertebrate nervous systems. It is now becoming apparent that whereas both invertebrates and vertebrates may have batteries of specific regulatory peptides, other peptides have evolved and become highly-conserved so early in the process of neurochemical evolution that structurally recognizable

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homologues are present within the nerves of organisms that are as phylogenetically distant as platyhelminths and mammals. 1.2. Chemistry of Regulatory Peptides

Regulatory peptides are a diverse group of biologically active peptides, usually of between 1 and 5 kDa molecular weight and 2-50 amino acid residues in length, that operate as neurohormonal mediators in intercellular communication. In vertebrates, regulatory peptides are synthesized by the endocrine and nervous systems, and, indeed, are also expressed by cells of the immune system; in invertebrates, they are largely elaborated by the nervous system, and most likely function as neurotransmitters, neuromodulators, and as trophic agents. They are derived from larger precursor proteins (preproproteins) whose structures, in common with those of other proteins, are encoded within the genome of the cell and expressed through the sequential processes of transcription, translation and post-translational modification. Of the many posttranslational modifications encountered in regulatory peptides (e.g. cleavage into smaller peptides, glycosylation, sulphation, pyroglutamate formation), one of the commonest is amidation of the carboxy terminus, resulting in a C-terminal amide rather than the usual free acid group. Although this modification occurs commonly in vertebrate peptides, and as mentioned earlier, has been used in the isolation of many of them, it is even more common in peptides of invertebrate origin. The function of this modification is thought to confer resistance to carboxypeptidase attack, although it may have a more fundamental function in charge suppression or receptor interaction. Following removal of the hydrophobic signal sequence, the proprotein products are packaged by the Golgi apparatus into storage vesicles, where most post-translational processing occurs, prior to exocytotic release of contents. In the case of endocrine cells, all of these processes occur in close spatial proximity, whereas in neurons the site of release of the contents of storage vesicles occurs at terminals which may be quite distant from the site of synthesis in the neuronal cell body. The vesicles are transported from the neuronal cell body to the terminals by axonal transport, and their transient accumulation en route leads to the typical “beaded” appearance of peptidergic nerve fibres when subjected to immunocytochemistry,. In contrast, classical transmitters are not specified by the genome, but for each there is a set of proteins involved in transport of metabolic precursors, in enzymatic conversion into active forms, and in re-cycling processes, and these are encoded and synthesized by the normal route of protein synthesis and transported to the terminals by axonal transport. The production of receptors and degradative

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enzymes for both peptidic and non-peptidic transmitters also occurs by this mechanism. There is a great diversity of peptide structure (and presumably also of peptide receptor structure), the basis of which results from regulation of gene expression. For example, through gene duplication, each peptide precursor may encode multiple copies of the same bioactive peptide, as is the case with the precursors of the *FMRFamide-relatedpeptides (FaRPs) (Linacre et al. , 1990; Saunders et al., 1991). In some cases, the duplicated peptides may have subsequently diverged by mutation, giving rise to peptide families of related structure (e.g. neuropeptide Y(NPY) superfamily). The processing pathways may also vary, even between cells expressing the same gene, leading to tissue-specific expression, and an even greater diversity of peptides. Thus, alternate splicing of the primary transcript can result in the production of different species of mRNA from the same gene (e.g. as in the tachykinins, substance P and substance K), and through alternate post-translational modification several different peptides can be produced from the same precursor (e.g. the opioid peptides). All of these features contribute to the multi-messenger potential of the peptidergic cell and are largely responsible for the functional diversity of regulatory peptides in the neuroendocrine system. The actions of regulatory peptides can be slow, both in onset of response and in duration (their effects can last perhaps for minutes or even hours). Following their release, they can diffuse through tissues, as paracrine substances acting on neighbouring cells, or be carried by blood as hormones and so mediate much longer-term effects (e.g. trophic, behavioural) at a distance on target tissues. Most of the specific peptide receptors which have been identified have been shown to belong to the family of G proteincoupled receptors with seven membrane-spanning domains (see Burbach and Meijer, 1992). Thus, it seems that regulatory peptides can mediate changes of not only single function but also of more complex cellular responses that culminate in the functioning of entire systems and associated behaviours. Useful textbook accounts of regulatory peptides can be found in Bradford (1986) and Hall (1992). Interest in the role of regulatory peptides in helminth parasites was sparked some 10 years ago when Gustafsson and her colleagues first identified the peptidergic nature of the nervous system in Diphyllobothrium dendriticum. Since then, a great deal of new knowledge has been gathered *To assist those unfamiliar with abbreviations for amino acids, the three-letter and singleletter equivalent used in this review are as follows: alanine Ala A; arginine Arg R; asparagine Asn N; aspartic acid Asp D; cysteine Cys C; glutamine Gln Q; glutamic acid Glu E; glycine Gly G ; histidine His H; isoleucine Ile I; leucine Leu L; lysine Lys K ; methionine Met M; phenylalanine Phe F; proline Pro P; serine Ser S; threonine Thr T; tryptophan Trp W; tyrosine Tyr Y; valine Val V.

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on helminth neurochemistry, some of which has led to radical departures from conceptions of helminth neurobiology held at that time. It would seem, therefore, an opportune time to review the current state of knowledge on the localization, chemistry, biological actions and phylogenetic relationships of regulatory peptides in helminth parasites.

2. OCCURRENCE AND DISTRIBUTION PEPTIDES IN HELMINTHS

OF REGULATORY

2.1. General Account

Regulatory peptides were first discovered in all of the major groups of helminth parasite in the mid to late 1980s, with findings recorded for the cestode, Diphyllobothrium dendriticum by Gustafsson et al. (1985); the nematode, Ascaris suum by Davenport et al. (1988); the trematode, Fasciola hepatica by Magee et al. (1989); and the monogenean, Diclidophora merlangi by Maule et al. (1989a). Since then, events have evolved in a manner somewhat different from that of other major invertebrate groups, such as the molluscs and insects. In these latter groups, the tendency has been towards measurement of the effects of peptide fractions recovered from a particular organism in bioassay, as the basis for the isolation of novel regulatory peptides. Indeed, the first regulatory peptide to be characterized from a mollusc, the cardioexcitatory tetrapeptide amide, FMRFamide (usually pronounced “fer-merf-amide”), was isolated on the basis of its effects on clam heart (Price and Greenberg, 1977), and the use of various bioassays has led to the characterization of many of the insect regulatory peptides (see Nassel, 1993). Thus, in many cases, the peptides have been functionally characterized to some extent before their tissue localization, or even structure, has been determined. In contrast, the reverse has been generally true for helminths, where localization studies of a particular peptide and its distribution have usually preceded investigations of its structure or function, as has been the case with neuropeptide F (NPF) and FMRFamide-related peptides (FaRPs) (Maule et al., 1991, 1993b). Inevitably, this situation will change as more authentic invertebrate peptide structures are isolated and sequenced from helminths, and their biological actions then determined (see Tables 8 , 9 and Section 6.2.1, p. 208). 2.1.1, Zmmunocytochemistry The essential technique in any study involving localization of regulatory peptides in nerves or endocrine cells is immunocytochemistry (ICC). The

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method, first described by Coons et al. (1941) more than 50 years ago, uses labelled antibodies as probes for localizing tissue constituents (antigens) in situ. An important modification of the original directly labelled antibody procedure was the introduction of the indirect method by Coons et af. (1955). In this method, the primary antibody-binding sites are revealed by application of a secondary antibody, directed against immunoglobulins of the donor species, and which can be labelled with enzyme, metal particle, or fluorescent tags for light microscopy, or with gold spheres to provide immunological probes in ultrastructural studies. While early immunocytochemical procedures were often imprecise and of limited application, recent important improvements in the quality and number of antisera available, and in the specificity of the controls employed, have provided the light and electron microscopist with an extremely useful tool with which to localize biologically important molecules to cellular and subcellular compartments. One further major development in this field has been the application to fluorescence-ICC of confocal scanning laser microscopy (CSLM), with its non-invasive ability to section optically through intact tissues and generate computerized composite images from different levels. In helminths, the technology has been used extensively to map and compare the distribution patterns of neuroactive substances in 3-dimensional reconstructions of nervous systems, and, more recently, to monitor neurochemical changes accompanying parasite ontogeny. For further details on ICC methodologies and CSLM see Johnston etal. (1990), Larsson (1988), Polak and Van Noorden (1986), and Shotton (1989). Much of the ICC of peptides in helminths to date has been performed with heterologous antisera raised against mammalian peptides, and has relied, therefore, on identifying peptides with similar amino acid sequences by virtue of their immunological cross-reactivity. However, although phylogenetic conservation of such sequences allows some extrapolation of data from mammals to helminths, it should be remembered that most of the antibodies used in ICC are directed against a relatively short sequence or epitope of perhaps three to eight amino acid residues. One way of avoiding this problem has been the use of a range of region-specific antisera that have been raised against several highly conserved regions of the peptide in question, thereby adding considerably to the credibility of peptide localization studies. Antibody specificity notwithstanding, the exploration of invertebrate neurobiology by ICC using antisera to vertebrate peptides has been impressive, particularly in identifying homologies between helminth and mammalian peptides; in one instance, this approach led to the discovery of the structure of the first flatworm neuropeptide, neuropeptide F (see Section 4.2.1). Thus, ICC can provide much useful information on helminth neuro-

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biology and, in fact, has established a valuable base for further studies of the chemical nature and function of parasite neuronal substances. One major conclusion to emerge from the ICC work to date is that the nervous system in helminths is chemically far more complex than its relatively simple anatomy might suggest. For example, the total number of helminthpeptide immunoreactivities recorded at the time of writing exceeds 30, and the list continues to grow. The recognized immunoreactive peptides can be arranged into a number of groups, based on homologies in amino acid sequence, and these are listed in Tables 1 and 2. For convenience, the occurrence and distribution of regulatory peptides in the two major groups of helminths, the parasitic flatworms and nematodes, will be considered separately. 2.2. Peptide Localization in Flatworm Parasites 2.2.1. The Flatworm Nervous System

As the major site of regulatory peptide immunoreactivity in flatworm parasites, the nervous system and its organization is of particular significance. In evolutionary terms, the nervous system in these most primitive members of the Bilateria is strategic, since it has developed from a diffuse, radial nerve net to become the first truly bilateral, central nervous system. Although there is a moderate degree of neurocephalization, evident from the aggregation of nerve cells at the anterior end as paired cerebral ganglia, nervous development in flatworms has largely involved the condensation of a superficial nerve plexus into a set of longitudinal medullary nerve cords, interconnected by transverse connectives or commissures. The anterior ganglionic collections of cells perhaps serve as a brain to coordinate peripherally based reflexes, whereas the longitudinal cords and commissures most likely allow for faster and more direct transmission of information. Nerve cells are usually uni- or bi-polar, with some multipolar forms also, and, typically, axons are non-myelinated and distinguished by neurotubules and a wide array of vesicles. These range in appearance and size (approximate mean diameter) from small, clear vesicles (40 nm), through dense-cored vesicles (80 nm) , to large, homogeneously dense vesicles (200 nm). Several types of synapse have been described (single, shared, axo-axonal), and the distribution of synaptic vesicles is usually asymmetric, reflecting a unidirectional mode of conduction. Large accumulations of vesicles are not uncommon in many of the axons and presumably account for the varicosities or beaded appearance shown by many of the nerve fibres in neurochemically stained preparations of the worms. Although there is usually a close spatial relationship between nerve and muscle in

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flatworms, identifiable synaptic connections between the two tissues are very infrequent. It may be that the varicosities described above are synaptoid release sites along the axon, from where myotropic and other factors can diffuse across interstitial material to interact with their target sites. Peripherally, nerve plexuses are usually found innervating the attachment organs (suckers, bothria, bothridia), pharynx (where present), and subtegumental musculature, as well as those muscle fibres that invest the ducts and copulatory structures of the reproductive system. Differentiated nerve endings occur in the tegument, as components of putative sense organs, particularly around the head region, attachment structures and gonopore. 2.2.2. Peptidergic Elements in the Flatworm Nervous System Immunocytochemical methods, using well-characterized antisera directed against a diverse range of vertebrate and invertebrate regulatory peptides, have demonstrated extensive peptide immunoreactivities in flatworm parasites, and have shown that they are restricted largely to cells and fibres of the central and peripheral nervous system (see reviews by Gustafsson, 1990,1992; Halton et al., 1990,1992,1993; Fairweather and Halton, 1991). Immunoreactivities to some 26 mammalian and four invertebrate peptide antisera have been demonstrated thus far, with staining of the nervous system dominated by members of the neuropeptide Y (NPY) superfamily (pancreatic polypeptide (PP), peptide YY (PYY), NPY), including the invertebrate homologue, neuropeptide F (NPF), and by numerous FaRPs; also many of the investigated taxa have been shown to display some tachykinin immunoreactivity (Table 1). The peptidergic portion of the nervous system of adult parasitic flatworms is well-differentiated, and, along with the cholinergic and aminergic components, extends throughout the central (CNS) and peripheral (PNS) nervous systems (Figure la,b). Thus, numerous peptidergic neurons are associated with the paired cerebral ganglia and provide fibre tracts that make up much of the longitudinal cords. The attachment apparatus is invariably well-innervated with peptidergic neurons, often as a plexus of fine fibres distributed throughout the musculature of the bothria, bothridia or suckers; in the ectoparasitic monogeneans, such as Diclidophora merlangi, each clamp of the posterior attachment apparatus is richly innvervated by peptidergic fibres derived from ganglionic masses in the haptor. There is extensive peptidergic innervation to the muscles of the feeding organs (where present) and to the copulatory structures (cirrus/ penidvagina). Peptidergic fibres innervate the walls of the reproductive tract, particularly the ducts of the female system where innervation of the egg-forming apparatus is often extensive (see Section 6.2.3). In Schistosoma

0

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hulin Ahnocorticotropin hormone (ACTH) a-Melanocyte stimulating hormone (a-MSH) Luteinidng hornorereleasing

hormone (LHRH) Growth hormone-releasing factor (GRF) Human chorionic gonadompin (hCG) Prolactin Oxymcin Vasatocin Urntensin I MVERTEBRATE Newpeptide F (NPR FMRFamide RFamide GNFFRFamide small cardiac peptide B (SCPd Eledoisin AUatostatin

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* +. present: 0.absent. References I. M a h (personal communication) 2. Reuter (1987) 3. Reuter (1988) 4. Maule cr 01. (1989a) 5. Maule er 01. (1989b) 6. Mauk craL (1%) 7. Mauk er a/.(1996%) 8. Maule er a/. (19921) 9. Smart et a/. (1993a) 10. ~rownlee( p e ~ n a communication) l 11. McKay cral. (1991~) 12. Pan er a/. (1994) 13. Magee cr aL (1993) 14. Banon el aL (1993)

15. B s h and Gupta (1988) 16. Gupta and Basch (1989) 17. Thorndyke and Whitfield (1987) 18. Richard CI aL (1989) 19. Magee PI a/. (1989) 20. Magee er aL (1991a) 21. Made (personal wmmunicalion) 22. McKay et el. (1991b) 23. Halton er aL (1987) 24. McKay el al. (199Oa) 25. McKay el aL (1990b) 26. McMichael-Phillips cr aL (1991)

27. Gustafsson (1987) 28. Duvaux-Mire1 er aL (1990)

29. Skuce cr aL (199Oa) 30. Skwe er aL (199Ob) 31. Bmwnlee cr a1 (1993a) 32. Fainveather ct a/. (1990) 33. Hallon (personal communication) 34. GustPfsson era/. (1985) 35. Gustafwn cr aL (1986) 36. W i k p (1986) 37. WiligRn ct aL (1986) 38. Gustafsson and Wikgren (1989) 39. Wkm and T h o d y k e (1990) 40. Guslafsson (1991) 41. Gustafsson el aL (1993) 42. El0 ef aL (1993)

43. McKay PI a/. (1992) 44. Marks CI aL (1992) 45. M m b n al. (1993a) 46. M m b cr al. (1993b) 47. Brownlee cr OL (1992) 48. Kumnzawa and Moriki (1986) 49. Failweather cr a/. (1988) 50. McKay cr al. (1991a) 51. HrOrova cr aL (1993) 52. Maulc er aL (1991) 53. Maulc er af.(1992h) 54. Made ef aL (1993a) 55. Made PI aL (1993b) 56. Failweather ct a/. (1991)

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Er AL.

Figure 1 Confocal scanning laser microscope (CSLM) fluorescence images of neuropeptide F (NPF) immunoreactivity (IR) in the central (CNS) and peripheral (PNS) nervous systems of whole-mount preparations of certain helminth parasites. (a) Moniezia expunsa scoledneck region, showing IR in longitudinal nerve cords (arrows) and associated connectives. A, acetabulum. Scale bar = 100 pm. (b) Diclidophora merlangi, showing IR in the PNS of the haptor region. C, clamp. Scale bar = 200 pm. (c) Himusthlu leptosoma redia, showing IR in the anterior ganglia (AG) and longitudinal cords of the CNS, as well as in the network of varicose fibres of the PNS. Note the immunoreactive cercaria (unlabelled arrow) emerging from the birth pore. Scale bar = 200 pm. (d) Ascaris suum anterior end, showing IR in CNS, including the anterior nerve ring (ANR), ventral nerve cord and ganglion (VNC), lateral nerve cord (LNC) and ganglion (LG). Note IR in the sensory cephalic papillary neurons (CPN). Scale bar = 100 pm.

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mansoni, an extensive plexus of peptidergic fibres and associated cell bodies has been observed in the lining of the gynaecophoric canal, indicative, perhaps, of a sensory function in the orientation of male and female pairing (Skuce et al., 1990a). A neuropeptide involvement in contact communications is also suggested by the peptide-rich innervation of the dorsal tubercles in the male S . mnnsoni, and by peptideimmunoreactivities (notably substance P) in the ciliated, putative sensereceptors that are scattered throughout the general body tegument of both sexes (Gustafsson, 1987; Basch and Gupta, 1988; Skuce et a f . , 1990a). Similar findings of substance P-immunoreactive nerve endings have been described from the tegument of the D. dendriticum plerocercoid by Gustafsson et af. (1993). Mapping the distribution pattern of peptidergic neurons in some flatworm parasites has shown that it more closely resembles that of the cholinergic system than the aminergic (5-HT) system, e.g. in D.merlangi (Maule et af., 1990a); similarly, in Moniezia expansa, peptide immunostaining has revealed a neuronal subtype, distinct from serotoninergic neurons, with a disposition comparable to those in the cholinergic system (Maule et a f . , 1993a). From work on nervous systems in higher organisms, it appears that individual neurons typically contain both classical, small molecule, transmitters and one or more peptides, and that this combination of co-transmitters, with their different release requirements and timecourses of action, very probably contributes to the complexity of signalling in the brain (for further information, see Hokfelt, 1991). Co-localization studies, such as those described above, indicate that there is every likelihood that neuroactive peptides co-exist with classical transmitters in helminths, although this necessitates confirmation by the application of double antibody studies and stringent controls at the ultrastructural level. Using electron immunogold-labelling procedures, the subcellular distribution of peptide immunoreactivity in flatworms has been shown to be localized exclusively to dense-cored vesicles, occurring both randomly in axons as well as concentrated in axonal swellings, or varicosities, throughout the central and peripheral nervous systems (Halton et af., 1991; Maule et al., 1992a,b; Brennan et al., 1993b; Brownlee et af., 1994b). Immunogold staining of neuronal cell bodies in the CNS of M. expansa, using the neuropeptide F (M. expansa) antiserum, NPF 792(1), has revealed regional differences in the peptide immunoreactivity of dense-cored vesicles (Brennan et a f . , 1993a). Thus, counts of the gold-probes showed that the heaviest labelling for NPF was in vesicles in the neck region of the cell, with fewer of the gold spheres over those vesicles occupying the perikaryon region, and none over the contents of the granular endoplasmic reticulum or Golgi apparatus, Since the antiserum requires an amidated C-terminus, these regional differences in the immunostaining in the cell body indicate that

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the NPF peptide undergoes post-translational amidation only after it has been packaged through the Golgi and prior to it entering the axon. In D. merlangi and M . expansa, the ultrastructural demonstration of multiple peptide antigens, using immunogold procedures, has revealed an overlap of PP, NPF and FMRFamide immunoreactivities in the same population of dense-cored vesicles, suggesting an apparent homogeneity of antigenic sites for these three peptides throughout the contents of the vesicles (Brennan et al., 1993a,b). However, the results from preabsorption experiments in which NPF ( M . expansa) prevented vesicle labelling with either PP or FMRFamide antisera, and the failure of PP and FMRFamide to block the NPF immunostaining, indicate that any apparent co-localization of these three peptides is due largely to cross-reactivity,and that most, if not all, of the immunostaining is due to NPF or an NPF-like peptide. In addition to the adult parasites, peptidergic elements have been demonstrated in the nervous systems of all of the life-cycle stages. These include the miracidia and cercariae of Schistosoma mansoni (Skuce et al., 1990b); the rediae and cercariae of Cryptocotyle lingua and Himasthla leptosoma (Pan et al., 1993) (Figure lc); the cercariae of Cercaria emasculans and Sanguinicola inermis (Pan et al., 1994; McMichael-Phillips et al., 1991); the metacercariae of Diplostomum sp. and Cotylurus erraticus (Barton et al., 1993); and, where examined, the metacestode stages of tapeworms, e.g. the procercoid and plerocercoid of D . dendriticum (Gustafsson et al., 1985, 1986; Wikgren, 1986; Wikgren et al., 1986); the cysticercoid of H. diminuta (Fairweather et al., 1988); and the tetrathyridium of Mesocestoides corti (Hrtkova et al., 1993). In all of the developmental stages (e.g. from miracidium to metacercaria), the entire larval central nervous system has been found to be immunoreactive for peptides, mirroring the situation found in the adult worm. However, in some instances (e.g. Diphyllobothrium), ontogeny is accompanied by marked changes in the levels of certain peptides, reflecting perhaps a stage-specific expression of peptide and an involvement in morphogenesis (see section 6.2.2). A number of non-neuronal sites of peptide immunoreactivity have been recorded from ICC studies of parasitic flatworms, including cells in the prostate glands of Echinostoma caproni and F. hepatica (Richard et al., 1989; Magee et al., 1989); tegumental cells in E. liei, Hymenolepis diminuta and Triloculariaacanthiaevulgaris (Thorndyke and Whitfield, 1987; Kumazawa and Moriki, 1986; Fairweather et al., 1990); and the mature interproglottid glands of M . expansa (Maule et al., 1990c; Shaw and Johnston, 1991). The functional implications of some of these findings are discussed in Section 6.3. For further details of the distribution pattern of peptidergic staining in

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a particular flatworm species, see the appropriate references cited in Table 1. 2.2.3. Peptides and the Reproductive System in Flatworm Parasites Many flatworm parasites are impressively fecund, displaying a prodigious egg output and a reproductive biology of marked complexity. They are the first group of metazoans to have developed complex reproductive structures comprising, basically, gonads and an intricate series of muscularized reproductive ducts and associated glands. As already stated, most of the regulatory peptides that have been demonstrated in flatworms have been found in the central and peripheral nervous systems, and so not surprisingly peptidergic elements occur also in those components of the PNS that innervate the reproductive system, most notably the musculature of the gonoducts and accessory structures (see Fairweather and Halton, 1991, 1992; Gustafsson, 1990, 1992; Halton et al., 1990, 1993). Thus, in the male system, immunoreactivities for PP-like peptides and FMRFamide have been found in the nerve plexuses innervating the musculature of the seminal vesicle, cirrus (or penis), cirrus sac and genital atrium. In the cestodes, Proteocephalus pollanicolu and M . expansa, strong immunostaining for NPYPP-like peptides was found in the neuromusculature of the vaginal sphincter and cirrus, reflecting perhaps a role in insemination, insofar as the sphincter probably serves to hold the cirrus or prevent escape of spermatozoa (Marks et al., 1993a; Maule et al., 1993a). Peptide innervation of the gonads has also been reported, with interesting species-differences in the nature of the peptide demonstrated. For example, in the pseudophyllidean, D. dendriticum the testicular follicles have been found to be lined by nerves immunoreactive for peptide histidine isoleucine (PHI), whereas innervation of the testes in the tetraphyllidean, T. acanthiaevulgark was immunoreactive for vasoactive intestinal peptide (VIP) (Gustafsson et al., 1986; Fairweather et al., 1990). PHI and VIP in mammals are encoded by the same gene and perform essentially similar physiological functions, so in platyhelminths there may be species-specific or, indeed, stage-specific expression of the two peptides. There are numerous reports of immunoreactivites for several peptides in the female reproductive apparatus of flatworms, with members of the NPY peptide superfamily (NPY, PP, PYY, NPF), tachykinins (substance P, neurokinin A, eledoisin), FMRFamide, and gastridcholecystokinin having been recorded in fibres and associated nerve cell bodies (somata) in the walls of the oviduct, vitelline duct and reservoir, ovovitelline duct, and uterus (Magee et al., 1989; Skuce et al., 1990a; McKay et al., 1990a, 1991a,b; Maule et al., 1990b). However, in all flatworm parasites examined the most intense peptide immunoreactivity has been found in cells and fibres associated with the egg chamber or

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.uAL.

ootype, a finding which points to an involvement of peptides in the egg-forming sequence (see Section 6.2.3). 2.3. Peptide Localization in Nematodes

2.3.1. The Nematode Nervous System The use of periodic acid-fuchsin (PAF) as a neurosecretory stain in the 1960s and early 1970s demonstrated the presence of presumptive peptidergic neurons in several nematode species, including Phocanema decipiens, Ascaris suum, Haemonchus contortus, Brugia pahangi and Dipetalonema (now Acanthocheilonema) viteae (Davey, 1966; Rogers, 1968; Delves et al., 1989). However, the use of ICC for the localization of specific peptide immunoreactivities, the complete reconstruction of the nervous system of Caenorhabditis elegans (White et al., 1986), and the demonstration that the nervous systems of other nematodes are comparable with that of C. elegans, both in terms of the number of neurons and their morphology (Stretton et al., 1991), revolutionized peptide-localization studies in nematodes, enabling the immunostaining of individual neurons in different nematodes to be compared directly. For this reason, this section will include results derived from free-living nematodes (notably C. elegans) along with data from parasitic forms. The nematode nervous system of central, peripheral and enteric components is composed of relatively few (approximately 300) neurons. These are arranged as anterior (circumpharyngeal) and posterior (circumanal) nerve rings with associated ganglia, connected by ventral, dorsal, lateral and sublateral nerve cords which proceed the length of the animal. The nerve cords are connected by a number of commissures, the pattern of which is repeated along the length of the worm, effectively dividing the worm into “segments” (Stretton et al., 1992). In addition, there are neurons which innervate various sense organs (e.g. the cephalic papillae, the amphids and phasmids), the pharynx, and certain reproductive structures (e.g. the vulva and spicules). A diffuse peripheral nerve net, such as is present in flatworms, has not been found in any of the nematodes examined.

2.3.2. Peptidergic Elements in the Nematode Nervous System A number of reports appeared in the mid to late 1980s describing the presence of several peptide immunoreactivities, notably FMRFamide, in various species of nematode, including Goodeyus ulmi, A . suum, C . elegans, Panagrellus redivivus and Heterodera glycines (Leach et al., 1987;

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Davenport et al., 1988; Atkinson et al., 1988; see Table 2). These studies were confined mainly to the demonstration of a single peptide immunoreactivity in a few species, or a few peptide immunoreactivities in a single species, and it was not until Sithigorngul et al. (1990) published the results of the first major immunocytochemical “screen”, using a large number of antipeptide antisera in a nematode, that the extent and diversity of peptide innervation in nematodes became apparent. They tested antisera to 42 known vertebrate and invertebrate regulatory peptides on A. mum and demonstrated the presence of 12 peptide immunoreactivities, eight of which were specific in that immunostaining could be abolished by preabsorption of the antiserum with the evoking antigen. Studies by other workers have since confirmed and extended these results in A. mum (Brownlee et al., 1993b,c, 1994a; Smart et al., 1992a, 1993a,b; see Table 2). Table 2 shows that there are inconsistencies in reports of the presence of some peptide immunoreactivities, either in the same species (e.g. NPY or substance P in A. mum) or between different species (e.g. adipokinetic hormone (AKH) in P. redivivus or adrenocorticotropic hormone (ACTH) in G. ulmi, neither of which was found in A. suum). Species differences in immunoreactivity may reflect differences in the neurochemistry of different nematodes, despite the fact that the basic anatomical form of the nematode nervous system is similar in many species. Conflicting reports of the presence of a particular peptide immunoreactivity in a single species may be related to differences in the antisera used, or in the methods used to prepare the tissue for immunostaining or to visualize bound antiserum. Even when the presence of a particular peptide is confirmed in separate studies, use of antisera with different specificities may lead to inconsistencies in the localization of that peptide. Some 26 peptide immunoreactivities have been demonstrated in nematodes, the majority in A. mum. However, this may not be a true reflection of the number of different peptides present. For example, it is possible that a single antiserum may cross-react with several related peptides, as appears to be the case with the FaRPs and leucokinin-like immunoreactivities in A. suum (Cowden et al., 1993; Smart ef al., 1993b), or several antisera may cross-react with different epitopes on the same peptide. FaRPs are the most common peptide-immunoreactivities demonstrated in nematodes, having been shown to be present in eight different species. FaRP-immunoreactivity was demonstrated in the nerve rings and nerve cords of the free-living nematode, C. ulmi (Leach et al., 1987), and has also been shown to be present in the nerve rings, nerve cords, ventral and lateral ganglia, and in neurons around the vulva, in P. redivivus, C. elegans and H . glycines (Atkinson et al., 1988). Similar results have been obtained for FaRP-immunostaining in adults and developmental stages of B. pahangi and Dirofilaria immitis (Warbrick et al., 1992).

+ +++++++

a-Melanocyte stimulating hormone (a-MSH) Luteinizing hormone releasing hormone (LHRH) Corticotropin-releasingfactor (CRF) Calcitonin gene-releasing peptide (CGRP) Atrial natiuretic peptide (ANP) Chromogranin A (CGA) KGQELE KELTAE Mammalian gonadotropin-releasing hormone (MGnRH) Salmon gonadotropin-releasing hormone (SGnRH) INVERTEBRATE FMRFamide RFamide KNEFIRFamide (AFl) KHEYLRFamide (Am) SDPNFLRFamide (PF1) SADPNFLRFamide (PF2) L-11 L-128 Small cardiac peptide B (SCPB) Adipokinetic hormone (AKH) Leucokinin (LK) Allatostatin SALMFamide * present; 0, absent.

+,

+ + + + + + + + + + + + + + + + + + + + +

+ + + + + +

o o

10. 11. 12. 13. 14.

+ + +

+ +

Schinkmann and Li (1992) McIntyre and Horvitz (1985) Leach et al. (1987) Kerboeuf and Dubois (1981) Davenport et al. (1991)

182

DAVID W. HALTON H A L .

Initial studies on A . suum demonstrated FaRP-immunoreactivity in the anterior nerve ring, cephalic papillary, lateral and rectal ganglia, in the nerve cords and pharyngeal neurons (Davenport et al., 1988), and these results have been greatly extended by Stretton and co-workers (Sithigorngul et al., 1990; Cowden et al., 1993). This group demonstrated that at least 60% of the neurons in A . suum could be immunostained using antisera specific for RFamide, although only 10% of the neurons in C. elegans were immunostained with the same antisera. In A. suum, FaRP-immunoreactivity was demonstrated in both left and right lateral ganglia (20-28 pairs of cells), both cells in the dorsal, and four out of five cells in the subdorsal ganglia; seven pairs of cells, and three asymmetric neurons in the ventral ganglion were immunostained, as were six of the seven pairs of cells in the subventral ganglion, and three out of 13 cells in the retrovesicular ganglion. In addition, FaRP-immunostaining was localized in cells of the tail ganglia, in the nerve cords, and in some 18 pharyngeal neurons which were observed to elaborate a network of fine, immunoreactive processes over the surface of the pharynx. The results of FaRP-immunostaining in A . mum have been confirmed by Brownlee et al. (1993b,c, 1994a), who, in addition, demonstrated FaRP-immunoreactivity in a plexus of nerves on the outer surface of the female gonoduct. The results obtained with antisera specific for RFamide must, however, be interpreted with some caution, since, for example, immunostaining in A . mum with RFamide antisera is similar to that produced with certain antisera directed against members of the NPY superfamily of peptides (Sithigorngul et al., 1990; Brownlee et al., 1993b,c, 1994a; and see Figure Id). Thus, antisera raised against PYY and the C-terminal hexapeptide amide of PP produced intense immunostaining throughout the nervous system of A . suum (Brownlee et a l . , 1993b,c, 1994a). The four invertebrate members of the NPY superfamily which have been characterized to date (neuropeptide F (NPF) from M . expansa, Artioposthia triangulata, Helix aspersa and Aplysia californica), and the related peptide YF (PYF) from Loligo vulgaris, all have the sequence RXRFamide (where X is P (proline) or T (threonine)) at their C-terminus (Maule et al., 1991; Curry et al., 1992; Leung et al., 1992; Rajpara et al., 1992; Smart et al., 1992d), and peptides related to this family have been partially characterized in A. suum (Smart et af., 1992c, see below). It is possible, therefore, that RFamidespecific antisera visualize both the NPY superfamily-related peptides and FaRPs in this nematode. Further evidence for this interpretation is the fact that immunostaining with antisera raised against various members of the NPY superfamily, and which do not cross-react with FMRFamide, is slightly less extensive than that obtained with RFamide antisera; also, chromatographic studies (see Sections 3.3.1 and 3.3.3) have demonstrated the presence of members of both peptide families in A. mum. Finally,

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double-immunogold staining of axons in the anterior nerve ring of the worm has revealed an apparent co-localization of both PP and FMRFamide antigenic sites in dense-cored vesicles, and antigen preabsorption studies showed that there was little or no cross-reactivity between the PP and FMRFamide antisera (Brownlee et al., 1994~). Recently, immunoreactivity to a further two peptides, unrelated to FaRP or NPY superfamilies, but showing similar patterns of immunostaining, has been demonstrated in A. s u m . The first of these was immunoreactivity towards the rat chromogranin A-derived hexapeptide KGQELE, which has been found throughout the nervous system of A. suum, including the anterior and posterior nerve rings and associated ganglia, nerve cords, amphidial and papillary neurons, and nerves in the pharynx and gonoduct (Smart et al., 1992a). The KGQELE-immunoreactive peptide has subsequently been isolated and sequenced (see Section 4.3.2). The other peptide family for which extensive immunoreactivity has been found in A. mum is the allatostatins (Smart et al., 1993a), a group of peptides first isolated from insects (see Nassel, 1993). In A. suum, allatostatin immunoreactivity was found in the nerve rings and asociated ganglia, in the nerve cords, papillary, amphidial and pharyngeal neurons. In contrast to the immunostaining for FaRPs, NPY-related, or KGQELElike peptides, allatostatin-immunoreactivity was not apparent in the nerves of the gonoduct. Allatostatin-immunostaining in A. suum was shown to be abolished by preincubation of the antiserum with either allatostatin I, 11, 111, or IV, indicating that the antiserum was cross-reacting with an A. suum peptide that has similar epitopes to the conserved mid-to-C-terminal region of these four allatostatins (LYXFGLamide, where X is a small aliphatic residue). Other peptide immunoreactivities in A. suum and other nematode species have a more restricted distribution than those described above, being confined mainly to certain nerves in the nerve rings and nerve cords. For further information on these peptides and the parasites examined see Table 2. Surprisingly, there is only one report of a peptide immunoreactivity being demonstrated by ICC in non-neuronal tissue, that of a-endorphin in the gonads of Heligmosomoides polygyrus (Kerboeuf and Dubois, 1981). The demonstration of AKH-immunoreactivity in P. redivivus is of some significance, since the nematode contains a peptide that exhibits AKH-like bioactivity (Davenport et al., 1991). As in flatworm parasites, a number of nerves in nematodes appear to contain several peptide immunoreactivities (notwithstanding the cautionary note above) and, in some cases, stain for small-molecule transmitters as well, raising the likelihood of their co-localization and possible interaction. Such apparent biochemical heterogeneity of the helminth nervous system suggests there already exists a fair degree of sophistication in neurotransmission at this level of organization.

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DAVID W. HALTON € T A L .

3. QUANTIFICATION AND CHARACTERIZATION OF REGULATORY PEPTIDES IN HELMINTHS

3.1. General Account

Once a regulatory peptide has been identified within a helminth parasite, either through its activity in a specific bioassay system, or as a result of its cross-reactivity with antisera under immunocytochemical or immunoassay conditions, the next logical step is its quantification, isolation and structural characterization. Regulatory peptides have been quantified most commonly using immunofluorescence detection systems, radioimmunoassay (RIA) or enzymelinked immunosorbent assay (ELISA) techniques. These systems generally work well when peptides of known structure are being quantified. However, since the degree of cross-reactivity of an unidentified peptide with any particular antiserum is unknown, only the relative amount of the peptide-immunoreactivity can be assessed. Another variable is the particular extraction method employed. It is, therefore, necessary to carry out a range of extraction procedures so as to identify the method which generates the highest yield of peptide. However, the optimal method identified is unlikely to extract 100°/~of the peptide from the tissue and will, assuming full cross-reactivity in the assay system, underestimate the quantity of peptide in the tissue. Although such quantification methods must be viewed with caution, they can provide useful information on the relative abundance of regulatory peptides in different tissues and in different species. During the process of purification, information is gained on the characteristics of the peptide being isolated. This information can be in a number of forms, including its activity in a bioassay system, e.g. its activity in an isolated receptor-membrane assay which has known ligand structureactivity requirements; its cross-reactivity with specific peptide antisera with known binding requirements; and, its structural characteristics. The latter are generated during the purification procedure which most commonly uses a range of chromatographic techniques. Chromatographic analyses can provide information on the size of the particular molecule, as well as details of its chemical nature, such as its charge, hydrophobicity and aromaticity. In this way, characterization data can be accumulated on helminth peptides for which detection systems are available, and it often provides useful information not only on the more abundant peptides prior to sequence analysis, but also on peptides which, for whatever reason, cannot be isolated in sufficient quantities to allow primary structural analyses.

REGULATORY PEPTIDES IN HELMINTH PARASITES

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3.2. Flatworm Parasites

Peptide quantification in the platyhelminths has been carried out using a range of peptide antisera, most commonly raised against vertebrate regulatory peptides in radioimmunoassay methodologies. The quantities measured can not be taken as absolute, but they provide valuable information on the relative abundance of a particular peptide epitope within worm extracts. Since most of the regulatory peptides identified in animal tissues to date have been basic in nature, acidic extraction media have generally provided the highest yields of peptide. In this respect, acidified ethanol has consistently proved to be the optimal extraction medium. The purification of neuropeptides from flatworm parasites has not been easy. The worms are generally small and rarely available in the quantities required to isolate sufficient peptide for sequencing. Moreover, their nervous tissue is almost impossible to dissect, so that extractions are most often carried out on whole worms. This necessitates a number of purification steps which, in turn, adversely affects the final yield of pure peptide. This limiting factor has largely accounted for the fact that few flatworm peptide sequences have been deduced to date. Nonetheless, improvements in technology, notably high performance liquid chromatography (HPLC) and sensitive automated microsequencers, have radically improved yields of peptides during isolations, and reduced the quantities required for sequencing. Quantification and characterization studies have been carried out on a number of different peptide families, and these will be discussed below. 3.2.1. Neuropeptide Y ( N P Y ) Superfamily In mammals, the NPY superfamily, also known as the pancreatic polypeptide (PP)-fold superfamily of peptides, comprises three members: PP, localized to pancreatic endocrine cells; peptide YY (PYY) localized primarily to mucosal endocrine-like L cells of the lower intestine; and NPY which occurs mainly in the central and peripheral nervous systems. All of the vertebrate family members consist of 36 amino acid residues (except chicken PYY which has 37 residues) and are C-terminally amidated. They appear to possess an intramolecularly helical structure, referred to as the PP-fold, and they share certain key amino acid residues that are probably essential for receptor interaction or biological activity. The immunocytochemical results presented in Section 2.2.2 show clearly that Cterminally-directed PP antisera have identified widespread immunoreactivity throughout the central and peripheral nervous systems of all of the parasites examined to date (Table 1). However, results are much less

186

DAVID W. HALTON ET AL.

conclusive when the evidence for the presence of NPY-immunostaining in platyhelminths is examined. Under the competitive conditions of RIA, non-specific cross-reactivities are less frequently encountered. In this respect, the radioimmunoassay of parasitic flatworm extracts has indicated consistently that they possess a native neuropeptide that cross-reacts, under competitive radioimmunometric conditions, with C-terminally directed PP antisera, but not with C-terminally directed NPY antisera (see below). The only monogenean parasite for which neuropeptide characterization data are available is Diclidophora merlangi (Maule et al., 1989c, 1992~). Using a C-terminally directed PP antiserum, extracts of whole specimens of D . merfangi were found to contain 44.9 ng g-' equivalents of PPimmunoreactivity. Following chromatographic analyses, a single native PP-immunoreactive peptide was identified, with a molecular weight similar to that of NPY superfamily peptides. The native parasite peptide did not cross-react with either an N-terminally directed PP antiserum, or with a range of NPY antisera, indicating that this monogenean neuropeptide was PP-like only in its C-terminal region and not NPY-like. PP-immunoreactivity has also been detected radioimmunometrically in digenean trematodes, including Schistosoma mansoni (Shaw and Johnston, 1991), Fasciola hepatica (Magee et al., 1991a), and Haplometra cylindracea (McKay et al., 1990b). In all cases, the most abundant peptideimmunoreactivity was recorded using PP antisera (see Table 3). Chromatographic analyses detected a single molecular form of PP-immunoreactivity Table 3 Quantification of NPY superfamily peptides in parasitic platyhelminths by radioimmunoassay. ~~

~

~~

Peptide immunoreactivity (ng equivalentdg wet weight of tissue) Species PP NPF Reference 52.1 Maule et al. Diclidophora merlangi 44.9 (1989c, 1992) nt Fasciola hepatica (host) (Rat) 41.1 Magee et al. (1991a) 41.8 (Cattle) nt nt 38.4 (Sheep) Schistosoma mansoni nt 22.9 Shaw and Johnston (1991) Haplometra cylindracea 220.0 McKay ef al. (1990b) nt Moniezia expansa 192.8 Maule et al. (1991) 114.0 Hymenolepis diminuta 79.8 McKay et al. (1991a) nt Proteocephalus p ollanicola nt 58.7 Marks et al. (1993b) Note: It should be noted that all of the PP-immunoreactivities were obtained using antiserum PP221 which was raised to the C-terminal hexapeptide amide of bovine PP. The NPF-immunoreactivities were obtained using antiserum NPF792 which was raised to the C-terminal decapeptide amide of NPF ( M . expama). nt, not tested.

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187

in extracts of all three species. Following gel-permeation chromatography, the PP-immunoreactive peptides from S. mansoni and F. hepatica were found to have comparable elution positions to those of bovine PP and mouse PP, respectively, suggesting similar molecular sizes (Magee et al., 1991b). Although the size of the PP-immunoreactive peptide in H . cylindracea was not determined, data from analytical HPLC analyses showed that its hydrophobic properties resembled those of the PP-like peptide in D.merlangi (McKay et al., 1990b). In cestodes, large amounts of PP-related peptide-immunoreactivities have been measured in extracts of Hymenolepis diminuta (McKay et al., 1991a) and Moniezia expansa (Maule et al., 1991) (see Table 3). The immunoreactivity in M . expansa was found to comprise a single molecular species in sufficient quantities to enable purification and sequencing (Maule et al., 1991). The native parasite peptide, unlike NPY which terminates C-terminally in a tyrosinamide residue, possesses a C-terminal phenylalaninamide, and is designated neuropeptide F (NPF) (see Section 4.2.1 for details). With knowledge of the primary sequence of NPF ( M . expansa), it was then possible to generate specific NPF antisera which did not cross-react with known mammalian NPY superfamily peptides (Maule et al., 1992b). These antisera have been used to quantify and characterize NPF-immunoreactivity in other tapeworm genera, e.g. the proteocephalidean, Proteocephalus pollanicola (Marks et al. , 1993b). Thus, following gel-permeation chromatography, P . pollanicola was found to contain a single molecular form of NPF-immunoreactivity with an apparent molecular weight of 44004700 daltons. Under RIA conditions, only the Cterminally directed NPF antiserum cross-reacted with the P . pollanicola peptide, showing that, although it was similar in its C-terminal region to NPF ( M . expansa), N-terminal differences were present. However, analytical HPLC analyses resolved two peaks of NPF-immunoreactivity , both of which were more hydrophilic than NPF ( M . expansa). The resolution of two immunoreactive peptides may not, of course, be indicative of more than one NPF-like peptide in the nervous system of P. pollanicola. It is possible, for example, that the proteocephalidean NPF-like peptide has an oxidizable methionine residue, such that the oxidized and reduced forms would be chromatographically resolved; alternatively, one of the peaks may be a degradation product of the parent molecule.

3.2.2. Tachykinins The tachykinins constitute a large family of structurally related neuropeptides that share a common C-terminal pentapeptide amide (Phe-X-GlyLeu-Met NH2) (where X is a variable residue) and similar biological activities. The first and best-known member of this family to be isolated

188

DAVID W. HALTON ETAL.

and sequenced was the undecapeptide, substance P (SP), followed by neurokinin A (NKA) and neurokinin B (NKB). There is immunocytochemical evidence for the widespread occurrence of tachykinin-immunoreactivities in the nervous systems of parasitic platyhelminths (see Table l ) , including SP (-Phe-Phe-Gly-Leu-Met NH2), NKA (-Phe-Val-Gly-Leu-Met NH2), and eledoisin (-Phe-Ile-Gly-Leu-Met NH2) which was isolated originally from the Mediterranean octopus, Eledone moschaca. To date, only SPand NKA-immunoreactivities have been detected radioimmunometrically in extracts of parasitic platyhelminths. Tachykinin immunoreactivity which was cross-reactive with both SP and NKA antisera has been identified in extracts of D . merlangi, although nearly twice as much NKA-immunoreactivity was recorded (Maule et al., 1989b). Although the SP-immunoreactivity had an apparent molecular size similar to mammalian SP, that of the NKA-like peptide was of the order of 1000 daltons and appeared to be smaller than bovine NKA (1297 daltons) (Maule et al., 1989b). Analytical HPLC analyses resolved two native parasite tachykinins, one of which cross-reacted with a far-Cterminally directed SP antiserum, whereas the other cross-reacted with an NKA antiserum. Radioimmunoassays which employed other SP and NKA antisera established that the native parasite tachykinins differed from their mammalian analogues. Both SP and NKA-irnmunoreactivities have been detected in extracts of F. hepatica (Magee et al., 1991a) and H . cylindracea (McKay et al., 1990b) (Table 4). In the liver fluke, both the SP and the NKA-immunoreactivities co-eluted, following gel-permeation chromatography, although the SP was present in higher concentrations. Following semipreparative HPLC, the tachykinins from F. hepatica were resolved into three peptides, all of which cross-reacted with a SP antiserum. and one of which also cross-reacted Table 4 Quantification of tachykinin-immunoreactivities in parasitic platyhelminths by radioimmunoassay. Peptide immunoreactivity (ng equivalent@ wet weight of tissue) Species Substance P NKA Reference Diclidophora merlangi 0.94 2.1 Made et al. (1989b) Fasciola hepatica (host) (Rat) 0.68 nt Magee et al. (1991a) 0.32 nt (Cattle) (Sheep) 0.36 nt Haplometra cylindracea 1.08 0.15 McKay et al. (1990b) Hymenolepis diminuta 0.14 nt McKay et al. (1991a) Note: It should be noted that all of the substance P and NKA-immunoreactivities were recorded using antisera GSPlO and NKA570, respectively (Maule et al., 1989b). nt, not tested.

REGULATORY PEPTIDES IN HELMINTH PARASITES

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with a NKA antiserum. The possibility that one of these peptides was an oxidation product of the major immunoreactive peptide in the extract cannot be discounted, such that two tachykinin-like molecules appeared to be present in F. hepatica. Extracts of H . cylindracea were found to contain a single peptide which was cross-reactive with a C-terminally directed SP antiserum, together with two peptides that cross-reacted with an NKA antiserum. Analytical HPLC analyses indicated that the SPimmunoreactive peptide in H . cylindracea differed from mammalian and amphibian SP-like peptides, and that one of the NKA-like peptides had similar elution profiles to those of the frog tachykinin, kassinin. Although both SP- and NKA-immunoreactivities have been detected immunocytochemically in cestodes (Table l), no cestode tachykininimmunoreactivity has been characterized as yet. However, the SPimmunoreactive peptide in H. diminuta was quantified by RIA (McKay et al., 1991a) (Table 4).

3.2.3. Vasoactive Intestinal Polypeptide and Peptide Histidine Isoleucine Vasoactive intestinal polypeptide (VIP) and peptide histidine isoleucine (PHI) are 28- and 27-amino acid residue peptides, respectively. In mammals, the two peptides occur on the same prohormone, such that proteolytic processing generates both peptides. VIP and PHI immunoreactivities have been identified immunocytochemically in parasitic platyhelminths (see Section 2.2.3 and Table 1). PHI-immunoreactivity has been quantified radioimmunometrically in F. hepatica (Magee et al., 1991a), but no PHI was detected in extracts of H . cylindracea (McKay et al., 1990b) (Table 5 ) ; VIP-immunoreactivity could not be detected in acid-ethanol extracts of either species. Limited PHI- and no VIP-immunoreactivities were recorded in extracts of H . dirninuta (McKay et al., 1991a) (Table 5 ) . There is only one report Table 5 Quantification of VIP- and PHI-immunoreactivities in parasitic platyhelminths by radioimmunoassay.

SDecies Fasciofa hepatica (host) (Rat) (Cattle) (Sheep) Haplometra cylindracea Moniezia expansa Hymenolepis diminuta

Peptide immunoreactivity (ng equivalentdg wet weight of tissue) VIP PHI Reference 0 1.59 Magee et al. (1991a) 0 1.17 0 0.69 McKay et al. (199Ob) 0 0 0.52 0.80 Shaw and Johnston (1991) 0 0.09 McKay et af. (1991a)

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DAVID W. HALTON ET AL.

of the chemical characterization of these peptides from a cestode, namely M. expansa (Shaw and Johnston, 1991). Acid ethanol extracts of the worm were subjected to N- and C-terminal VIP and C-terminal PHI RIAs and found to contain immunoreactivities in all three assay systems (see Table 5). A single immunoreactive peak, which co-eluted with both synthetic porcine VIP and PHI, was recorded with each of the antisera, following gel permeation chromatography. Analytical HPLC analyses of these immunoreactivities resolved two peptides, one of which was cross-reactive with both VIP antisera, the other cross-reacting with the PHI antiserum. Further HPLC analyses could not distinguish the VIP- and PHIimmunoreactivities present in M . expansa from those of the host (sheep) intestine. The apparent similarities in both chemical and immunochemical properties of these peptides led Shaw and Johnston (1991) to speculate that parasite and host possessed identical peptides (see Section 6.3). 3.2.4. Gastrin-releasing Peptide

Gastrin-releasing peptide (GRP)-immunoreactivities have been quantified in extracts of F. hepatica recovered from three different hosts (rat, 0.12; cattle, 0.28; sheep, 0.11 ng equivalentdg wet weight of tissue) (Magee et al., 1991a), H . cylindracea (1.5 ng equivalentdg wet weight of tissue) (McKay el al., 1990b), and in H . diminuta (0.22 ng equivalentdg wet weight of tissue) (McKay et al., 1991a). Chromatographic analyses of the GRP-immunoreactivity from H . cylindracea identified two separate molecular forms (McKay et al., 1990b), but their molecular size was not determined. Since GRP occurs in two distinct forms in mammals, one consisting of 27 amino acid residues the other of 10, the finding of two GRP-immunoreactive peptides in H . cylindracea was taken as evidence for the proteolytic processing of a larger biologically active peptide to a smaller active C-terminal fragment by the parasite (McKay et al., 1990b). Although this remains a possibility, it is possible that the smaller fragment may have been generated during the extraction process. Alternatively, the two parasite GRP-like peptides may be distinct peptides. Clearly, sequence information may reveal which of these possibilities is likely to be correct. 3.2.5. Glucagon

Glucagon is a 29-amino acid residue peptide produced by the pancreatic A cells in mammals, and is involved in the mobilization of hepatic glycogen stores. Limited glucagon-immunoreactivity has been detected immunocytochemically in two trematode species (see Table 1). Using a C-terminally directed glucagon-antiserum, 0.45 ng equivalents of glucagon-

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191

immunoreactivity/g wet weight of tissue were measured in an extract of H . cylindracea, and chromatographic analyses identified a single molecular form which was more hydrophilic than the glucagon-immunoreactivity present in lung tissue of its frog host (McKay et a l . , 1990b). 3.2.6. Opioid Peptides Opioids in vertebrates are derived from three types of precursor molecule, including pro-opiomelanocorticotropin (POMC), proenkephalin and prodynorphin. Related peptides have been demonstrated immunocytochemically in platyhelminths (Venturini et al., 1983; Wikgren and Reuter, 1985). Using RIA, three POMC-derived peptides have been detected in extracts of S . mansoni (Duvaux-Miret et al., 1990). Peptides cross-reactive with antisera to adrenocorticotropic hormone (ACTH), amelanocyte-stimulating hormone (a-melanotropin) and P-endorphin were detected in four stages (cercariae, schistosomula, adults and miracidia) of the schistosome life cycle (Duvaux-Miret et al., 1992b). HPLC characterization of the parasite P-endorphin-immunoreactive peptide demonstrated that this peptide was highly homologous to human P-endorphin (DuvauxMiret et al., 1990). The occurrence of immunoreactivities to the three main POMC-derived peptides in S. mansoni led the authors to hypothesize that the parasite possesses a POMC-gene. In this respect, oligonucleotide probes specific for conserved regions of the genes encoding POMC identified related sequences in the parasite genome (Duvaux-Miret et al., 1992b).

3.3. Nematodes The quantification and characterization of regulatory peptides in nematodes presents a situation somewhat different from that in the platyhelminths. For example, the nervous system of nematodes is generally far less extensive than in trematodes and cestodes, and it is usually without the diffuse peripheral elements that characterize that of the flatworms. In addition, the larger nematodes allow dissection of specific organ-systems, such as pharynx, gonad, intestine and body wall, although the procedure can be a singularly tedious one. The anterior and posterior portions of nematodes, e.g. those of the first and last one-centimetre length of mature Ascaris s u m , invariably yield a preparation that is rich in nervous material, relative to whole worms (Sithigorngul et al., 1990). To date, quantification and partial characterization studies have been performed for three peptide families in nematodes.

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DAVID W. HALTON E T A L .

3.3.1. Neuropeptide Y ( N P Y ) Superfamily Immunocytochemical studies have demonstrated abundant immunoreactivity directed against mammalian PP in A. suum (see Section 2.3.2, Brownlee et a f . , 1993b,c, 1994a; Sithigorngul et al., 1990). Immunoreactivity towards the related peptide, NPY in this parasite was less abundant than that for PP (Brownlee et af., 1993b,c, 1994a), or was absent (Sithigorngul et a f . , 1990). The use of RIAs specific for either PP or NPY have shown significant amounts of both peptide immunoreactivities in various tissues of A . mum, including the anterior and posterior portions of the worm, the intestine, body wall and pseudocoelomic fluid (PCF; Smart et af., 1992~). Interestingly, the gonoduct was found to contain large amounts (c. 200 ng g-' tissue) of PP-immunoreactivity, but no NPY-immunoreactivity (Table 6). The results have also revealed some disparity with those obtained by immunocytochemistry (ICC). Thus, RIA showed more NPYimmunoreactivity than PP-immunoreactivity in the heads and tails, the reverse being true with ICC. Moreover, the use of RIA demonstrated both PP and NPY-immunoreactivities in the intestine and ovary whereas none had been detected in these tissues with ICC (Smart et al., 1992c; Brownlee et ul.). These results indicate that some of the peptide immunoreactivity in this parasite may be non-neuronal, as neither the intestine nor the ovary is innervated. In this respect, the use of RIA has detected significant (26-35 ng ml-') quantities of both PP and NPY-immunoreactivities in the PCF. It was thought that these amounts of peptide immunoreactivity could not be derived from leakage of the peptide from cut tissues into the PCF when it was collected. It has been speculated that the PCF of pseudocoelomates may serve a circulatory function (Barnes et af., 1993). In this respect, the demonstration of peptide-immunoreactivity in the PCF may indicate that this tissue functions as a rudimentary endocrine system (Smart et al., 1992~). Table 6 Quantification of PP- and NPY-immunoreactivities (IR) in tissues of

A . suum.

Tissue PP-IRa NPY-IRa Heads and tails 16.9 k 3.6 43.6 f 9.9 ( n = 4) Body wall 3.1 f 0.3 17.4 +_ 1 . 1 ( n = 5) Intestine 47.3 6.1 5.4 +_ 4.6 (n = 4) Testes nd nd ( n = 4) Ovaries 2.2 k 0.5 9.5 f 1.3 ( n = 5) Gonoduct 201.2 f 115 nd ( n = 2) Pseudocoelomic fluid 25.8 & 5.8 34.8 f 2.4 ( n = 4) From Smart et al. (1992~). a Values given in ng g-' tissue (ng ml-' for pseudocoelomic fluid), mean f SE nd, not detected (< 1 ng g-'); n = number of samples.

*

REGULATORY PEPTIDES

IN HELMfNTH PARASITES

193

Analysis of the PP and NPY-immunoreactivities from A . suum, using reverse-phase HPLC, has revealed a considerable degree of molecular heterogeneity in these peptides, with resolution of at least five different PP-immunoreactivities and at least three different NPY-immunoreactivities (Smart et a f . , 1992~).These peptides all eluted at different positions from the same HPLC column, indicating that there were at least eight NPY superfamily-related peptides in A. suum. The peptides differed from those of the host, insofar as they did not co-elute with porcine NPY or PP. All invertebrate members of the NPY superfamily sequenced to date possess a C-terminal RFamide (see Maule et al., 1991; Curry et al., 1992; Leung et a f . , 1992; Rajpara et al., 1992). It is possible, therefore, that some of the peptides that have been detected are members of the Ascaris AFfamily of peptides (Stretton et al., 1991; see below). However, the peptides detected by Smart et al. (1992~)appeared to be more hydrophobic than those of the AF-family peptides; also, the antisera used to detect both NPY and PP-immunoreactivities do not cross-react with FMRFamide. These authors concluded that the PP and NPY-immunoreactivities in A . mum belong to a family of peptides different from the AF-family. 3.3.2. Leucokinins The leucokinins (LKs) are a family of eight related peptides, isolated originally from extracts of the heads of the cockroach, Leucophaea maderae, and have myotropic action on visceral muscle (Cook et al., 1989, 1990). LKs are all octapeptides, with bioactivity residing in a common core of residues at the mid- to C-terminus, sequence FXSWGamide, where X is a variable residue (Nachman et al., 1990). Several nerves in A . suum have been shown to be immunostained with an antiserum raised against LK-V (Smart et a f . , 1993b). This antiserum cross-reacted in RIA with all the Cterminal variants of the LK family that have been characterized to date. In addition, the LK immunostaining in A . suum could be abolished by preabsorbtion of the antiserum with different LKs, suggesting that the antiserum was directed towards common (presumably C-terminal) epitopes on the LKs, and that components in the nervous system of the worm possessed similar epitopes. The amounts of LK-immunoreactivity detected in the tissues of A . suum are somewhat lower than those of NPY superfamily peptides (Tables 6 and 7), and this is consistent with the differences in the levels of immunostaining observed with antisera towards PP and LK. As with the NPY superfamily peptides, small but significant amounts of LK-immunoreactivity were detected in the PCF and in noninnervated tissues, such as gut and gonad. Although the LKs were not analysed by HPLC, comparison of specific binding versus dilution curves for different tissues in A. suum with those of LK standards demonstrated

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DAVID W. HALTON E T A L .

Table 7 Quantification of leucokinin (LK) immunoreactivity in tissue extracts from Ascaris mum. Tissiie

Heads and Body wall

Guts

Testes

tails

Ovaries Pseudocoelomic fluid"

LK-IR (ng g-'

5.2 f 0.57 5.9 f 0.56 14.6 k 2.62 4.8 f 1.06 2.6 f 0.3 1.6 k 0.16 tissue) (17) (15) (15) (16) (22) (24) From Smart et al. (1993b). Values are mean k SEM ( n ) normalized for sample dilution; a Value in ng ml-'.

that the LK-immunoreactivityin heads and tails, ovaries, PCF and body wall diluted in parallel with standard; on the other hand, the LK-immunoreactivity in the gut and testes did not dilute in parallel with the standards or with each other. This suggests that A. mum possesses a number of LK-immunoreactive components, some of which may not be homologous to the insect LKs. The multiplicity of LK-immunoreactive components in A. mum extracts has been confirmed by HPLC analysis (Smart et al., unpublished observation).

3.3.3. FMRFamide-related Peptides (FaRPs) Peptides with the sequence FXRFamide (where X is a small, aliphatic residue) are one of the most abundant regulatory peptide families in the animal kingdom, with representatives in virtually all groups of Metazoa (Price and Greenberg, 1989; Walker, 1992). Not surprisingly, the FaRPs are by far the best studied of the invertebrate neuropeptides and, in this respect, helminths are no exception. Where examined, antisera directed towards FMRFamide or RFamide have produced abundant immunostaining in all nematodes (see Table 1; Section 2). More than 60% of the neurons in A. suum were recorded as stained with antisera against RFamide by Cowden ei al. (1993), although the figure was substantially less (10%) for C . elegans (Schinkmann and Li, 1992). However, these results must be interpreted with some caution in view of the possible crossreactivity of RFamide-specific antisera with peptides related to the NPY superfamily, which are also present in nematodes (see above). Some 13 FaRPs have been identified in A suum (Stretton et al., 1991; Cowden et al., 1993). These include the peptides, AFl and AF2, both of which have been sequenced (see Section 4.3.1 and Table 9). Differences in the sequences of AF1 and AF2 indicate that one peptide is not derived by proteolytic processing of the other, and this may be the case for other FaRPs in A. mum (Cowden et al., 1993). Chromatographic analysis of FaRPs in A. mum indicated that different subsets of peptides were located in different tissues and different sexes. For example, the pharynx con-

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195

tained seven FaRPs, including AF1 and very small amounts of AF2, whereas the head minus the pharynx contained all members of the group (Cowden et al., 1993). In males, greater amounts of A F l , and at least two other peptides, were found in the heads than in the tails, and at least three other peptides were found in greater amounts in the tails than in the heads, despite the fact that both heads and tails contained similar numbers of neurons. Removal of the vas deferens from the male tail prior to extraction and HPLC analysis resulted in the loss of one FaRP peak, suggesting a non-neuronal source of some of the FaRP-immunoreactivity . Differences in the amounts of FaRPs extracted from female heads and tails were harder to interpret, as the heads contained far more neurons than the tails. The study by Cowden et al. (1993) also demonstrated the qualitative and quantitative effects of different extraction techniques on the recovery of FaRP-immunoreactivity from both A . suum and C. elegans. Thus, the use of acidified methanol generally led to a greater yield of FaRP-immunoreactivity from these nematodes than did the use of acetone, both in terms of the number of different peaks of immunoreactivity, and the amount of peptide assayed. However, use of acetone led to the extraction of some FaRPimmunoreactive peaks from C. elegans which were not extracted with acidified methanol. Although six FaRPs have been identified in C. elegans by gene sequence analysis (Rosoff et al., 1992), chromatographic analysis of FaRPs from this nematode suggests that at least eight peptides are present. At least three FaRPs have been identified in P. redivivus, two of which have been sequenced (Geary et al., 1992b; see Section 4.3.1, and Table 9). Sequence data are available for only two families of nematode peptides, namely the FaRPs (Stretton et al., 1991; Geary et al., 1992b; Rosoff et al., 1992) and TE-6 (Smart et al., 1992b; see Table 9). The FaRPs of A . suum and C. elegans, and other peptide families (leucokinins and NPY superfamily) in A. s u m , have been analysed by HPLC and/or quantification studies. The results have shown marked differences in the amounts of various peptides in different tissues and/or sexes, and have demonstrated possible non-neuronal sources of peptide immunoreactivity. Since the FaRPs, NPY superfamily peptides, and leucokinins each comprise several members, their relative quantification will require the use of monospecific antibodies, or HPLC and RIA with antibodies of known cross-reactivity. 4. ISOLATION AND STRUCTURE OF HELMINTH REGULATORY PEPTIDES 4.1. General Account

The structural characterization of parasitic helminth regulatory peptides has progressed only slowly, following their discovery in the nervous system

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of D . dendriticum by Gustafsson et al. (1985). Many attempts were made in the late-1980s to purify to homogeneity sufficient amounts of peptide from helminth extracts to enable primary structural determinations to be made. However, most of these attempts resulted only in the generation of data on the immunochemical and structural characteristics of these messenger molecules (see Section 3). As stated in Section 3.2, the purification of platyhelminth regulatory peptides has been limited by a number of factors, not least the large amounts of tissue required, and by the fact that, in general, whole-worm extracts have to be used and, as such, include much extraneous tissue. Nevertheless, although rather small in comparison with the number of known peptide sequences from other invertebrate groups, the list of fully characterized regulatory peptides from helminths is beginning to grow. To date, the partial primary structure of one, and the full primary structures of four platyhelminth regulatory peptides have been elucidated. In all cases, the parasites were extracted initially in acidified ethanol and the peptides were purified by sequential gel permeation and reverse-phase HPLC fractionation, with subsequent peptide identification by radioimmunoassay (RIA). Nematode regulatory peptides have been isolated and sequenced from A. mum and from the closely related freeliving nematodes, P. redivivus and C. elegans, and comprise seven different FaRPs and a novel regulatory peptide, designated TE-6. The structures of two other nematode FaRPs have been deduced from the nucleotide sequences of a cloned nematode gene. 4.2. Flatworms

4.2.1. Neuropeptide Y (NPY) Superfamily Peptides Consistently, the most abundant regulatory peptide-immunoreactivity in parasitic platyhelminths has been recorded using C-terminally directed mammalian PP antisera in RIA (see Section 3.2.1). Immunocytochemical methodologies localized this immunoreactivity to the central and peripheral nervous systems of all of the flatworms examined (see Section 2.2.2 and Table 1). It was the abundance and apparent ubiquity of this immunoreactivity which led to a number of attempts to purify and sequence the “native” platyhelminth neuropeptide that was responsible for this cross-reactivity with PP antisera. Following acid ethanol extraction of 432 g of F. hepatica, and a number of chromatographic steps, the partial sequence of a PP-immunoreactive peptide was obtained by Magee et al. (1991b). The PP-antiserum employed in the purification procedure was C-terminally directed, and required a C-terminally amidated aromatic residue for binding. Unfortunately, only

Table 8 The primary structuresa of platyhelminth regulatory peptides. NPY superfamily peptides (NPF-like) Fasciola hepatica Moniezia expansa Artioposthia triangulata

PSVQEVEKLLHVLDRNG-KV-AE-

Reference - - - - - - - -b

NH2

PDKDFIVNPSDLVLDNKAALRDYLRQINEYFAIIGRPRF

NH2

KWHLRPRSSFSSEDEYQIYLRNVSKYIQLYGRPRF

NH2

----

Magee et al. (1991b) Maule et al. (1991) CUT’ et al. (1992)

FMRFamide-Related Peptides (FaRPs) Moniezia expansa Artioposthia triangulata

GNFFRF NH2 RYIRF NH2

Maule et al. (1993b) Maule et al. (19941

aAmino acid sequence shown using single letter notation (see p. 167). bWhile the number of amino acid residues which constitute the F. hepatica peptide is unknown, the partial structure is aligned so as to display the maximum homology with NPF ( M . expansa).

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DAVID W. HALTON E T A L .

sufficient peptide was purified to enable sequencing of the amino-terminal 24 amino acid residues (Table S), rather than the C-terminal region of the molecule where most of the homology in NPY superfamily peptides resides. Comparison of the N-terminal 24 amino acid residues of the F. hepatica PP-immunoreactive peptide with mammalian PPs revealed very limited homology, and confirmed that any homology that existed between this flatworm peptide and mammalian PPs must reside in the C-terminal region. Indeed, there was no conclusive evidence that this F. hepatica peptide was an invertebrate NPY superfamily peptide. At the time of writing, there is no sequence information available on any other trematode regulatory peptide. The first complete sequence of a parasitic platyhelminth neuropeptide was obtained from an acid-alcohol extract of 1 kg of the large ruminant tapeworm, M. expansa (Maule et al., 1991). The peptide comprises 39 amino acid residues and has a molecular mass of 4594 daltons, terminating C-terminally in a phenylalaninamide residue. It was fully cross-reactive with C-terminally directed PP antisera and displayed C-terminal structural homology with vertebrate PPs (Table 8). Since this tapeworm peptide displayed significant sequence homology with the vertebrate NPY superfamily of peptides, but terminated in a phenylalaninamide (where phenylalanine = F, using the single letter notation), this tapeworm peptide was designated neuropeptide F (NPF). Thus, NPF (M. expansa) was the first invertebrate member of the NPY superfamily to be sequenced. Subsequently, an NPF peptide was isolated and sequenced from the turbellarian, Artioposthia triangulata (Curry et al., 1992; see Table 8). NPF ( A . triangulutu) is a 36-amino acid residue peptide with a molecular weight of 4433 daltons and has a C-terminal pentapeptide amide identical to that of NPF (M. expansa). Using specific NPF antisera, positive immunostaining has been demonstrated in the nervous systems of all flatworm species examined to date, including over 20 different parasite species (Maule et al., in press a). Indeed, NPFs have been identified in most of the major invertebrate phyla, such that they are now believed to constitute a ubiquitous invertebrate neuropeptide family (see Section 5.2). 4.2.2. FMRFamide- Related Peptides (FaRPs) Initial immunocytochemical evidence for the presence of PP-related peptides in flatworms was thought to be due to the non-specific crossreactivity of some PP antisera with FaRPs. This view was not surprising since FaRPs had been identified in many invertebrate groups, including flatworms (see Table 1). While the sequencing of NPF from two flatworm species confirmed the presence of NPY superfamily peptides in their nervous systems, the only evidence for the occurrence of FaRPs in

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platyhelminths was immunocytochemical. As NPF-related peptides and FaRPs both terminate C-terminally in RFamide, the non-specific crossreactivity of C-terminally directed FaRP antisera with NPF-related peptides appeared a distinct possibility. The sequencing of NPF, therefore, called into question the occurrence of FaRPs in platyhelminths. Recently, two novel FaRPs have been isolated and sequenced from platyhelminth species, one from M . expansa and the other from A. triangufata (Table 8; Maule et a f . , 1993b, 1994). The FaRP from M. expansa has a molecular weight of 785 daltons and is a hexapeptide amide, with the amino acid sequence GNFFRFamide. This peptide was deemed to be C-terminally amidated by the high degree of cross-reactivity (>80%) it showed with the amide-requiring FMRFamide antiserum that was employed in its purification. The FMRFamide antibody was also used in the purification of a novel pentapeptide amide, RYIRFamide from A . triangufata. These data have established unequivocally that FaRPs occur in the nervous systems of the platyhelminths. Interestingly, only a single FaRP species was identified in acid-alcohol extracts of each of these flatworm species, whereas taxa from other invertebrate groups have been found to possess families of FaRPs (Walker, 1992). Of course, other FaRPs, that were not recovered following the acidified-ethanol extraction methods employed in these studies, may be present in the nervous systems of these flatworms. 4.3. Nematodes

4.3.1. FaRPs The majority of investigations into the occurrence of regulatory peptides in nematodes have concentrated on FaRPs. Indeed, chemical characterizations of nematode FaRP-immunoreactivities have identified families of these peptides in tissue extracts of the worms. To date, seven different FaRPs have been purified and sequenced directly from nematode extracts (Table 9), two of which have been isolated and sequenced from acidmethanol extracts of freeze-powdered heads of A . suum (Cowden et a/., 1989; Cowden and Stretton, 1993). These peptides, designated AFl and AF2, were found to be the most abundant FaRP-immunoreactivities in acid-methanol extracts of A. suum. However, up to 11 other FaRPs have been identified as occurring in this nematode species, although, some of these may be the proteolytic by products of larger fragments (see Section 3; Stretton et al., 1991; Cowden et a f . , 1993). Two FaRPs have been isolated and sequenced from P . redivivus (Table 9; Geary et a f . , 1992b). Both of these peptides were sequenced from the

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DAVID W. HALTON E T A L .

Table 9 The primary structuresa of nematode regulatory peptides. Ascaris suum

FMRFamide-related peptides KNEFIRF NH2 (AFI) KHEYLRF NH2 (AM)

Panagrellus redivivus

SDPNFLRF SADPNFLRF Caenorhabditis elegans SDPNFLRF SADPNFLRF SQPNFLRF ASGDPNFLRF AAADPNFLRF PNFLRF AGSDPNFLRF PNFMRY

NHz (PFl) NH2 (PF2) NH2 NHZ NH2 NH2 NHZ N H ~ ~ NH2' NH2'

Reference Cowden et al. (1989); Cowden and Stretton (1993) Geary et al. (1992b) Rosoff et al. (1992, 1993)

Ascaris suum TKOELE Smart et al. (1992b3 aAmino acid sequences shown using single letter notation (see p. 167). bSequence deduced from gene and identified by mass determinations of an isolated peptide. 'Sequences deduced from gene only. ~

~~

major FMRFamide-immunoreactive peak that was identified following HPLC/RIA analyses of 300 g of acetone-extracted whole worms. It would appear, therefore, that the most abundant FaRPs present in A. suum and P. redivivus are structurally quite different. However, different extraction procedures were employed for the two species, such that it is impossible to conclude if the most abundant FaRPs present have been sequenced. Another approach employed in the identification of nematode regulatory peptides has been the analysis of the nucleotide sequences derived from a cloned precursor gene from C. elegans (Rosoff et al., 1992). In all, the sequence of seven FaRPs from two alternatively spliced transcripts of a C. eleguns gene (flp-1)were deduced. Subsequently, six of these peptides were isolated, five of which have been sequenced and one other identified following mass determinations, indicating that these FaRPs are expressed in vivo by C. eleguns (Table 9; Rosoff et al., 1993). Interestingly, C. eleguns peptides, SDPNFLRFamide and SADPNFLRFamide are identical to those sequenced from P. redivivus by Geary and colleagues, and were the most abundant FaRPs detected following acetone extractions of the two species. Although the presence of both of these peptides in extracts of P. redivivus and C . elegans suggests that they may occur throughout freeliving nematode species, their presence in parasitic nematodes remains to be established. It should be noted that there is no RIA or sequence evidence to indicate the presence of FMRFamide per se in either

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nematodes or platyhelminths. It would appear that immunocytochemical demonstrations of FMRFamide-immunoreactivities in helminths (see Section 2.3.2 and Table 2) are due to the cross-reactivities of FMRFamide-antisera with either other FaRPs or NPF-related peptides. 4.3.2. TE-6 Using acid-ethanol extracts of 680 mg of the gonoduct of A . mum, Smart et al. (1992b) isolated and sequenced a novel peptide with the amino acid sequence, TKQELE, designating it TE-6. This peptide was initially identified immunocytochemically , using an antiserum directed towards KGQELE, a sequence which flanks the C-terminus of pancreastatin in rat chromogranin A. The failure to detect other chromogranin A fragments, a number of which are known to possess sequence or chemical homologies with TE-6, in the A. mum extract intimated that TE-6 was completely unrelated to chromogranin A-like peptides and was not, therefore, derived by proteolytic processing of a nematode chromogranin A-related precursor (Smart et al., 1992b). It was also found that TE-6 had no structural homology with any other known class of vertebrate or invertebrate peptides, indicating that it may represent a novel class of regulatory peptide.

5. EVOLUTIONARY ASPECTS OF HELMINTH REGULATORY PEPTIDES 5.1. General Account

Phylogeny may be defined as the determination of the degree of relatedness between organisms and their divergence times from common ancestral forms. As regulatory peptides are ubiquitous within the nervous systems of all metazoans so far examined, and are obviously of early evolutionary origin, they represent ideal models in the study of phylogeny. Since the primary structures of regulatory peptides are readily obtained, either by molecular biological or protein analytical techniques, comparisons of amino acid sequences and peptide precursor and gene structures are possible between species. By employing antisera to highly conserved and often bioactive regions of vertebrate regulatory peptides in immunocytochemical studies on invertebrate nervous systems, many have been found to demonstrate immunoreactive counterparts in respresentatives of even the most primitive phyla known to possess neurons. Several such immunoreactive counterparts have been isolated and sequenced from invertebrates

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and protochordates, using biochemical analytical techniques. For example, a gastrin/cholecystokinin immunoreactive peptide, named cionin, has been isolated from the ganglia of the sea-squirt, Ciona intestinalis. The discovery of this peptide suggested that cholecystokinins, rather than gastrins, were the prototypes of this peptide family (Johnsen and Rehfeld, 1990). Several insect neuropeptides, e.g. the sulfakinins, which exhibit C-terminal homology with vertebrate gastrins/cholecystokinins, are probably more distant relatives of these vertebrate peptides. Whereas some invertebrate neuropeptides possess primary structural features which readily identify them as invertebrate analogues, and perhaps ancestral forms of vertebrate equivalents, others appear to have no recognizable equivalents in vertebrates. It may be that, in some cases, the millions of years of molecular evolution separating these groups have resulted in drastic changes in primary structure so that no readily identifiable homologies remain; on the other hand, the process of molecular evolution may have produced novel vertebrate neuropeptides with no equivalents in invertebrates, and vice versa. In any case, it is currently apparent that several families of vertebrate regulatory peptides have exceptionally long evolutionary histories. One such family is the NPYPP superfamily of regulatory peptides.

5.2. Neuropeptide Y (NPY) Superfamily Peptides

During studies of the presence and distribution of peptides in the nervous systems of flatworms using antisera to conserved regions of vertebrate regulatory peptides, it was discovered that in all of the species investigated, the predominant immunoreactivities were obtained with antisera to vertebrate NPY superfamily peptides (comprising NPY, pancreatic polypeptide (PP), and peptide YY (PYY)) (Tables 1 and 2). Since vertebrate regulatory peptides are believed to have arisen as neuropeptides in lower invertebrates, such as coelenterates and platyhelminths, and since at this level of animal organization there is no true circulatory system and therefore no true endocrine glands, the nervous system necessarily performs all somatic integrative functions. Interestingly, insulin-like peptides are present in neurosecretory regions of the insect brain, whereas in mammals, and in most other groups of vertebrates, the peptide is localized exclusively in true endocrine cells of the pancreatic islets. It is not unreasonable to expect, therefore, the invertebrate analogue of the NPY superfamily to resemble most closely, in primary structural terms, vertebrate NPY (brain neuropeptide) rather than vertebrate PP (pancreas endocrine peptide). However, the isolation and structural characterization of the invertebrate analogue, neuropeptide F (NPF) has shown that this is not the case.

Table I0 A comparison of the full primary structuresa of neuropeptide F (NPF) (Moniezia expansa) with those of: A. bovine pancreatic polypeptide (PP); B. NPF (Artioposthia triangulata); and, C . NPF (Helix aspersa) and NPF (Aplysia californica).

N-terminus A.

M . expansa Bovine PP

PDKDFIVNF'SDLVLDNKAALRDYLRQINEYFAIIGRPRF APLEPEYPGDNATPEQMAQ-AAELRR-INMLT---Y

B. M. expansa A . triangulata

PDKDFIVNPSDLVLDNKAALRDYLRQINEYFAIIGRPRF KWKLRPRSSFSSEDEYQI---NVSK-IQLY-----

C. M. expansa H . aspersa A . californica

PDKDFIVNPSDLVLDNKAALRDYLRQINEYFAIIGRPRF STQMLSPPERPREFRHPNE--Q--KEL---Y--M--T-DNSEMLAPP-RPEEFTSAQQ--Q--AAL---YS-Y-----

aAmino acid sequences shown using single letter notation (see p. 167). -,Denotes amino acid residues homologous with those of NPF ( M . expansa).

C-terminus NH2 NH2

Reference Made et al. (1991) Chance et al. (1979)

Curry et al. (1 992) NH2 NH2 NH2

Leung et al. (1992) Rajpara et al. (1992)

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NPF was isolated originally from the cestode, Moniezza expansa by Maule et al. (1991), using an antibody directed to the C-terminal hexapeptide amide of mammalian PP (i.e. LTRPRY NH2), and was found to comprise 39 amino acid residues, with homology to mammalian PP concentrated in the C-terminal domain of the molecule (Table 10). Thus, in both cases, residues 17 and 10 from the C-terminus are tyrosine (Y) and residues 4 and 2 from the C-terminus are arginine (R); the C-terminal residue is an aromatic tyrosine (in PP) or phenylalanine (in NPF), both of which are amidated. Subsequently, NPFs were isolated from the land planarian, Artioposthia triangulata (Curry et al., 1992), from the gastropod mollusc, Helix aspersa (Leung et al., 1992), and from the sea hare, Apfysia californica (Rajpara et al., 1992) comprising 36, 39 and 40 residues, respectively. While, as expected, the cestode NPF (M. expansa) shows significant homology with the turbellarian NPF (A. triangulata), with nine of the 20 C-terminal residues in identical positions and the C-terminal pentapeptide amide fully conserved, the degree of homology between NPF ( M . expansa) and the molluscan NPFs (H. aspersa and A . californica) is quite remarkable. Of the 20 residues in the C-terminal domain of the three NPFs, 13 are identical, and of the remaining seven residues, six are conservative substitutions due to single base-pair mutations (Table 10). The possibility of this high degree of homology occurring by chance, through convergent evolution, is infinitesimally small. Rather, it is more likely that the cestode and molluscan NPFs have evolved from a common ancestral precursor molecule that was encoded by the same ancestral gene some 1400 million years ago. This interpretation squares well with current evolutionary thinking which places flatworm ancestors, rather than coelenterates, as the central root stock in the evolution of the Bilateria, and from which are derived modern-day flatworms and molluscs (Barnes et af., 1993). The homology of invertebrate NPF with vertebrate family members resides in the C-terminal region, and in particular the C-terminal tetrapeptide amide, RPRFamide, which is identical to the homologous region of most known amphibian PPs (McKay et al., 1990~).On the other hand, the analogous site of all vertebrate NPYs, from fish to mammals, is RQRYamide. This finding is somewhat puzzling in view of the perception that PP is the most recently evolved member of this peptide family. It is possible, however, that PP in higher vertebrates has not arisen from duplication and mutation of the NPY gene, as is proposed in many schemes (Larhammer et al., 1993), but rather the family members in vertebrates have a common ancestor in NPF. Support for the theory that NPY and PP genes are derived from one another is evident in the common structural organization in their precursors, a feature shared with the third vertebrate member of the family, PYY. Recent cloning of NPF cDNA from A.

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REGULATORY PEPTIDES IN HELMINTH PARASITES

HUMAN NEUROPEPTIDE Y PRECURSOR SIGNAL PEPTIDE 1281

NEUROPEPTIDE Y 1361

C-EXTENSION [CPON] 130)

97 Ala

Tvr

Gly

Lys

Arg

Total number of residues

92 SIGNAL PEPTIDE (21)

NEUROPEPTIDE F (401

A

C-EXTENSION (28)

APLYSIA NEUROPEPTIDE F PRECURSOR Figure 2 A schematic drawing comparing the structures of the precursor peptides encoded by the human NPY gene and the Aplysia (molluscan) NPF gene. The

number of amino acid residues that comprise the signal peptides, NPY/NPF peptides, and the C-terminal extensions are shown inside brackets. The Nterminus of each peptide is generated by signal-peptide cleavage (at AlaTyr for NPY; Ala-Asp for NPF), and the Gly and Lys-Arg residues mark the sites for amidation (NH2) and cleavage, respectively, at the C-terminus of NPY and NPF. Note that an additional potential tribasic cleavage site is present within the predicted C-terminal extension peptide of Aplysia NPF (arrow head). CPON, Carboxy-terminal peptide of NPY. (After Maule et al., in press.)

californica by Rajpara et al. (1992) has revealed a precursor organization which is similar to that of vertebrate NPY family members (Figure 2), confirming the presence of a highly conserved genetic structure, and also adding weight to the assertion that NPF is the authentic invertebrate phylogenetic analogue of the NPY superfamily. As already mentioned, the primary structural homologies between invertebrate NPFs and vertebrate family members reside in the C-terminal region. NPY and PYY interact with two common receptor subtypes, designated Y1 and Y2, although a specific NPY receptor subtype, Y3 has been demonstrated recently (Grundemar et al., 1993). The structureactivity requirements of Y1 and Y2 differ, in that the Y2 receptor recognizes C-terminal fragments of both peptides, whereas the Y1 subtype requires both ends of each molecule for recognition. On the basis of this evidence, one might speculate that the Y2 subtype is similar in specificity to the invertebrate NPF receptor, and that in vertebrates this subtype

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HALTON E ~ A L .

represents the most primitive type. Indeed, Y2 receptors occur in the most primitive areas of mammalian brain, whereas the Y l receptors predominate in the cortical regions. Clearly, phylogenetic studies on regulatory peptides provide interesting insights into the evolution of all components of this type of signalling system, from mRNA and precursor organization and processing to receptor subtype evolution. Thus, the NPY peptide superfamily provides a useful model to illustrate the evolution of a regulatory peptide family from primitive invertebrates to higher vertebrates, and summation of current data substantiates the hypothesis that vertebrate regulatory peptides of the NPY superfamily arose as invertebrate neuropeptides. Moreover, the early origin, continued abundance and apparent persistence of members of this peptide family throughout the neuroendocrine tissues of motile metazoan life forms indicate that these peptides serve in fundamental functions, and that deletion or mutation of genes specifying their production have been lethal. 5.3. FMRFamide and Related Peptides (FaRPs)

Although proctolin and adipokinetic hormone (both from insects) were the first invertebrate regulatory peptides to be discovered (Nassel, 1993), FMRFamide isolated originally from the mollusc, Macrocallista nirnbosa by Price and Greenberg (1977) has attracted a much greater research effort, and is generally regarded as the archetypal invertebrate neuropeptide. The primary structures of FMRFamide-related peptides (FaRPs) have been determined from a variety of molluscs, arthropods, annelids, and helminths, including nematodes, cestodes and turbellarians. In vertebrates, the presence and status of FaRPs remain unclear, with only a single putative relative having been isolated from chicken brain (Dockray el al. , 1983). However, regions of the vertebrate pro-opiomelanocorticotropin (POMC) precursor display remarkable primary structural homology to FMRFamide, particularly the region encoding methionine-enkephalin. Thus, in contrast to NPF, there does not appear to be an abundant and obviously related analogue to this invertebrate peptide family in vertebrate neuroendocrine tissues. The apparent abundance and widespread distribution of FMRFamide immunoreactivity in many invertebrates would seem to be due to such antisera cross-reacting with NPF. Interestingly, in M . expansa, where both NPF and a FaRP (GNFFRFamide) have been isolated from the same extract, the NPF was found to be present in an approximately 40-fold greater amount than the FaRP (Maule et al., 1993b). From all current data, FaRPs appear to have reached their zenith of abundance in molluscan nervous systems, in contrast to NPF/NPY superfamily peptides, which appear to represent the most abundant

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neuropeptides in the nervous systems of organisms from platyhelminths to mammals. The isolation of authentic FMRFamide from the polychaete annelid, Nereis virens, a representative of a group that is considered, phylogenetically, to predate the molluscs, led to the speculation that this FMRFamide represents the ancestral peptide from which the diverse molluscan and arthropod forms arose. However, FMRFamide itself was not found in extracts of M . expansa, nor, and perhaps more importantly, was it found in extracts of the free-living turbellarian, Artioposthia triangulata. Thus far, in both of these species, FaRPs are represented by single peptides: the hexapeptide, GNFFRFamide ( M . expansa) and the pentapeptide, RYIRFamide ( A . triangulata) (Maule et al., 1993b, 1994). Although much further isolation work is required, particularly on additional species of turbellarian, in order to ascertain the structure of the ancestral peptide, it is possible to conclude from the current helminth primary structural data that this will not prove to be authentic FMRFamide. In the nematodes, Ascaris suum, Panagrellus redivivus and Caenorhabditis elegans, FaRPs have been isolated and appear to be present as a number of different molecular variants in each species; molecular biology in C . elegans has located these FaRPs on a single common gene, fEp-l (Rosoff et al., 1992). Although isolation of the total FaRP complement of each species, with the possible exception of C. elegans, is incomplete, there does appear to be a difference in molecular structure between FaRPs in parasitic and free-living forms, a finding which may be related to differences between the parasitic and free-living life-styles. The quest for helminth neuropeptides is still in its infancy, but the limited information available at present has produced some valuable insights into the origins and primary structural evolution of these ubiquitous and essential messenger molecules. Since regulatory peptides are produced from discrete genes, whose mutation rates and similarities can, in part, be determined from the primary structures of the final products of their expression, they present the evolutionary biologist with useful model systems to study the phylogenesis of metazoan life forms; with the advent of more primary structural information, they will likely provide useful clues in systematics. 6. FUNCTIONAL ASPECTS OF HELMINTH REGULATORY PEPTIDES 6.1. General Account

From the literature, it is all too evident that very few regulatory peptides have been tested in helminths, so that demonstrable evidence of their

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bioactivities is largely lacking. Thus, in general, the functions of many of the peptides demonstrated in helminths, either by immunocytochemistry (Tables 1 and 2) or by isolation and sequencing (Section 4) have been inferred from their tissue localization. For example, their abundance in the central and peripheral nervous systems suggests important roles in the motor, sensory and behavioural activities of helminths, whereas peptide immunoreactivities associated with the gonads and egg-forming apparatus are perhaps indicative of a variety of hormonal-like actions in reproduction. In some cases, monitoring the neuroanatomical and neurochemical changes during parasite ontogeny has revealed that certain neuropeptides are expressed by groups of neurons at particular times in development, suggesting an involvement in morphogenesis. However, as yet, there are no confirmed data on the precise endogenous roles of regulatory peptides in helminths, or of their exogenous actions in the host-parasite relationship. The only experimental evidence of their functional involvement in helminth biology is in the few documented accounts of nerve-muscle physiology in nematodes, described below. 6.2. Endogenous Functions for Helminth Regulatory Peptides

6.2.1. Motility The only helminth neuropeptides to be investigated in any detail, using assay systems in which their functions could be isolated from possible interactions with other modulatory substances, are the two FMRFamiderelated peptides (FaRPs), AF1 and AF2, isolated from Ascaris mum. Stretton et al. (1992) have shown that synthetic copies of both of these peptides have potent biological activity on the neuromuscular system of A. suum.The first of these peptides to be isolated and sequenced, AF1, has been shown to cause local paralysis of locomotory waveforms when injected into A. suum,suggesting a role in muscle control (Stretton et a l . , 1991). Using intracellular recording techniques, the application of low concentrations of the peptide (1-100 nmol) were found to be excitatory, insofar as they abolished slow membrane potential oscillations in certain dorsal and ventral inhibitory neurons in the parasite. Stretton et al. (1992) believe the effect is due largely to the presence of AF1 receptors on the inhibitory motor neurons. Although synaptic transmission was not blocked, the input resistances of the inhibitory motor neurons were markedly reduced by AFl, to the extent that they were presumed to no longer function in locomotory control (Cowden et al., 1989). Excitatory motor neurons in the worm were not apparently affected directly by AF1. All of these data indicate a possible neuromodulatory role for AF1 in the

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control of locomotion. The injection of AF2 into A. suum also blocked locomotory movements in the region of the injection site (Cowden and Stretton, 1993). These workers have also shown AF2 to have multiple, concentration-dependent effects on dorsal-muscle strip tension in A. m u m , with concentrations as low as 10 PM producing relaxation, followed by contraction and rhythmic activity. These changes were accompanied by hyperpolarization and hypopolarization, respectively (Cowden and Stretton, 1993). Other workers have also demonstrated an excitatory action for AF2, in that it elicits marked increases in the spontaneous rhythmicity of muscle cells adjacent to the nerve ring in A. suum (HoldenDye et al., 1993). Although, nothing has yet been documented on the nature of the receptors involved in the actions of the two FaRPs in A. suum, it would seem that they reside on nerve rather than muscle (Stretton et al., 1992); whether there are separate AF1 and AF2 receptor types is not clear. The finding of peptide immunoreactivities, such as for FMRFamide, PP, TE-6, VIP, and allatostatin, in known motor neurons in nematodes raises the possibility that some of them may have a role in the control of worm motility (Sithigorngul et al., 1990; Smart et al., 1992a, 1993a,b; Warbrick et al., 1992). Thus, FMRFamide was shown to produce an apparent inhibition of motility in Panagrellus redivivus, using a sedimentation bioassay, suggesting an effect on muscle activity, although other effects on sensory perception and the integration of stimulus and response could not be ruled out (Smart et al., 1990). Interestingly, the effects of FMRFamide in this bioassay were observed only at relatively high concentrations of peptide (335 VM), reflecting perhaps difficulties associated with the exogenously applied peptide crossing the nematode cuticle to access its sites of action (Geary et al., 1992a). Smart et al. (1990) also demonstrated an apparent stimulation of motility in P. redivivus by pentagastrin and angiotensin I, using the same bioassay. 6.2.2, Development Evidence of regulatory peptide involvement in cestode morphogenesis comes from work exploring neurosecretory (peptidergic) elements as inductive factors in cell proliferation and differentiation - an area of research deserving further attention, in view of the remarkable regenerative capacity of the flatworm nervous system (see Bagunh et al., 1989). Thus, Gustafsson and co-workers found that when the inactive, encysted plerocercoid of D. dendriticum (from fish) was cultured in vitro at 38°C (i.e. approximating that of the natural gull host or of an experimentally infected hamster), there was, accompanying considerable motility in the worm, a pronounced elevation in the secretory activity of the nervous

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system, with at least a 10-fold increase in the number of demonstrable peptidergic neurons, compared with untreated plerocercoids (Gustafsson and Wikgren, 1981; Gustafsson et af., 1983). This sudden increase in peptide secretion is compatible with a switch in the cestode’s physiology from plerocercoid to adult, triggered by the rise in temperature, and is consistent with the need for strong muscular activity in migration and attachment in the host gut. Of special interest during this plerocercoid/ adult transformation process are the small cardiac peptide B (SCPB)immunoreactive neurons that can be found around the bothridial musculature in the actively working scolex of the adult worm, but which are absent (or perhaps inactive) from the passive scolex of the plerocercoid stage (Gustafsson and Wikgren, 1989). In nematodes, a number of presumptive peptidergic neurons have been identified in several species, using periodic-acid fuchsin (PAF) staining (see Section 2.3.1). In Dirofifariaimmitis, such changes have been correlated with the moulting cycle (Delves et af., 1989), suggesting a peptide involvement in ecdysis. However, a number of other, non-peptide factors have also been implicated in the control of moulting in nematodes, including noradrenaline (Goh and Davey, 1984), ecdysteroids (Barker and Rees, 1990), and juvenile hormone (Davey, 1988). It would seem, therefore, that the moulting process may involve a number of different neurotransmitters and/or neuromodulators; it also illustrates graphically how the effects of a single transmitter substance at the physiological or behavioural level cannot be considered in isolation of those of other transmitters. 6.2.3. Reproduction One area of helminth activity in which peptide immunoreactivities have been implicated is reproductive function, and in particular the mechanisms that regulate the assembly of the large numbers of eggs produced by these parasites. The most detailed studies have been made on the flatworms, F. hepatica, S. mansoni and D. merlangi. How events in egg formation in flatworm parasites are controlled have yet to be determined, but the mechanisms involved undoubtedly reside in the nerve and muscle elements that invest the ootype wall and associated ducts. More precisely, it is the innervation of these structures that likely initiates and coordinates the successive events of egg assembly. In this respect, a consistent finding from immunocytochemical mapping of peptide immunoreactivities in flatworm parasites is that the innervation of their reproductive systems is predominantly peptidergic. Moreover, the peptidergic cells corresponding to these nerve plexuses are invariably associated with the ootype/Mehlis’ gland complex.

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In F. hepatica, for example, three peptidergic cells (nerve plexus 1, NP1) occur at the entrance to the egg chamber and at the confluence of the oviduct and vitelline duct, with another group of two cells (nerve plexus 2, NP2) positioned at the exit of the chamber and in association with the uterine valve (Magee ef al., 1989); a similar situation exists in S. mansoni (Skuce et al., 1990a). These groups of peptide-immunoreactive cells correspond to nerve plexuses described originally in these two worms by Gonnert (1955, 1962), and their strategic position may reflect a functional involvement in regulating the movement of egg material into and out of the egg chamber or ootype. No immunostaining for regulatory peptides has been recorded in the Mehlis’ glands per se, but immunoreactivities for PP and FMRFamide have been found in what are presumed to be nerve cells scattered among the Mehlis’ cells. These peptidergic cells, numbering around 50, are unipolar in shape, with axons extending into the muscle layers of the ootype wall (Magee et al., 1989). In the monogenean D . merlungi a heterogeneous population of some 100 uni-, bi-, and multipolar neurons, interconnected by axonal extensions, converge on the ootype wall and associated ducts (Figure 3) (Maule et al., 1990b). Again, these peptidergic cells are distinct from the Mehlis’ gland cells, and are immunoreactive for members of the neuropeptide Y (NPY) superfamily and for FMRFamide, as well as for allatostatin (Maule et al., 1990b; Smart et al., 1993a). Their positioning at the exit of the vitelline reservoir and around the ootype strongly suggests, as in F. hepatia and S. mansoni, that they have a peptidergic involvement in egg assembly and reproductive function. The ultrastructure of these cells is consistent with that of a secretory cell engaged in the synthesis and export of protein via dense-cored vesicles (Halton et al., 1991). The vesicles fill the axonal extensions of the cell and come to accumulate in the swollen cell terminals that form synaptic appositions with the smooth muscle fibres of the ootype wall. Electron microscopic double-immunogold labelling has revealed an identical distribution of pancreatic polypeptide (PP) and FMRFamide immunoreactivities in the dense-cored inclusions, which, from preabsorption experiments using neuropeptide F (NPF) ( M . expansa), can be explained by cross-reactivity of these C-terminally directed antisera with an NPFrelated peptide (Brennan ef al., 1993a,b) (see also Section 2.2.2). Moreover, using antisera directed to the intact synthetic NPF ( M . expansa) (residues 1-39) or to the C-terminal decapeptide (residues 30-39) showed that the distribution pattern of immunostaining for NPF in D. merlangi, both at light- and electron-microscopic levels, mirrored exactly the staining patterns for PP and FMRFamide (Maule et al., 1992a). As mentioned in Section 5.2, NPF is an evolutionarily ancient molecule and it likely subserves important neuronal functions in helminth parasites. Such functions have yet to be investigated experimentally but may, in

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PEPTIDE, CELLS

Figure 3 Schematic diagram showing the close spatial relationship of peptideimmunoreactive cells with the muscle of the proximal ducts of the female reproductive apparatus of D. merlungi. Note that the S1 and S2 cells comprise Mehlis’ gland, and that, once formed, the egg passes from the ootype into the uterus (arrow). (After Made et ul., 1990b.)

concert with other mediators, e.g. the ubiquitous FaRPs, include regulation of the duct muscles and sphincters involved in bringing to the ootype the components of each egg, its assembly and shaping, and its release, when formed, to the uterus; additionally, these peptides may exert a paracrine-like influence on the secretory activity of the Mehlis’ gland (Halton et al., 1991, 1993). 6.3. Exogenous Functions for Helminth Regulatory Peptides

In addition to any endogenous role, peptides resulting from de novo synthesis in endoparasitic helminths may be released and interact with the physiological systems of the host. For example, Duvaux-Miret et al. (1992b) have shown that, under physiological conditions, both adult and intramolluscan stages of Schistosoma mansoni secrete P-endorphin and

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adrenocorticotropic hormone (ACTH) and that, in effect, these two proopiomelanocorticotropin (P0MC)-derived peptides mask the worm from host immunosurveillance, and so enhance parasite survival. Thus, there is experimental evidence to support the view that the released peptides operate either by direct action on host immune cells or through their conversion into known immunosuppressive substances, such as amelanocyte-stimulating hormone (a-MSH). In this way, it would seem that the parasite escapes host-immune reactions through molecular mimicry, by using highly conserved peptide signal molecules that it has in common with both vertebrate and invertebrate hosts. The possibility that helminths can synthesize and release host-like molecules is perhaps best exemplified by the plerocercoid stage of Spirometra mansonoides, which releases a substance that accelerates growth of the host rodent (Mueller, 1966). The substance, known as the plerocercoid growth factor (PGF), displays many of the biological, immunological and molecular properties of mammalian growth hormone (GH), with the important exception that it does not possess the antiinsulin/diabetogenic activities of G H (Phares, 1987, 1992). This finding could be of immense clinical significance, but as yet the structure of PGH is unknown. Since most known endoparasitic helminths inhabit the vertebrate gastrointestinal tract, whose physiological function is controlled by a battery of neurohormonal peptides, it is likely that their peptide secretions induce significant pathophysiological conditions in the host. For example, it has been speculated that the vasoactive intestinal polypeptide/peptide histidine isoleucine (VIP/PHI)-immunoreactive peptides present in the mature interproglottid glands in Moniezia expansa (see Section 2.2.2) may be released into the sheep-host’s intestine to interact with mucosal-cell receptors, thereby inducing fluid and electrolyte secretion such as is normally mediated by the endogenous mammalian VIP peptide (Shaw and Johnston, 1991). If the parasite peptides contribute to the production of the watery diarrhoea associated with M. expansa infections, their release into the host intestine would have survival value for the parasite in promoting expulsion of ripe proglottids in host-derived secretions. Similar implications for host pathophysiology are evident in the finding of a VIPlike molecule in the tegument of the intestinal trematode, Echinostoma liei (Thorndyke and Whitfield, 1987). Other examples which implicate helminth-derived peptides in parasite survival strategies are those in which helminth infections modify host behaviour to the benefit of the parasite. Although relatively little information is available on the neuromodulatory mechansims that mediate parasite-induced behavioural changes in the host, there is evidence that in some instances they involve increased production of immunosuppressive

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opioid and related neuropeptides, both by the parasite and also as a result of parasite-induced changes in endogenous opioid activity of the host (Kavaliers and Colwell, 1992). Nematodes, such as Ostertagia ostertagi and Haemonchus contortus cause significant alterations in the plasma levels of host hormones, particularly gastrin and cholecystokinin (Nicholls et al., 1988; Fox et al., 1989); adult Dirofilaria immitis have been shown to alter vascular reactivity in infected dogs through an action on edothelial cell function (Kaiser et al., 1989). Although the mechanisms underlying these changes have yet to be elucidated, and may be related to parasite pathogenicity, it is possible that they may involve the release of peptides from the parasites to modulate the behaviour of the host to the advantage of the parasite. Clearly, both host and parasite-derived peptides are an integral component of an intimate physicochemical relationship between host and parasite. This is especially true for endoparasites, where parasite-host interactions involve a multiplicity of signal molecules, including regulatory peptides, that naturally mediate in the host immune and neuroendocrine systems (Blalock, 1989). The impact of helminth infections on this complex communication network is only beginning to be addressed, and is beyond the scope of this review, but as expected, often involves profound biochemical, physiological, immunological, pathological and behavioral changes in the host. For reviews on this neuroimmunomodulatory aspect of host-parasite interactions, reference should be made to Duvaux-Miret et al. (1992a); Fairweather et al. (1992) and de Jong-Brink (1992).

7. FUTURE DEVELOPMENTS

It is highly likely that future developments in regulatory peptide research in helminths will centre around exploiting the potential usefulness of the helminth nervous system as a target for chemotherapy (see review by Geary et al., 1992a). After all, many of the currently successful anthelmintics, e.g. ivermectin, levamisole and praziquantel, operate apparently by disrupting parasite neuromusculature, and thereby locomotory behaviour. However, until helminth regulatory peptides and their receptors have been fully characterized and a physiological function assigned to them, their value as potential drug targets remains uncertain. To this end, a structured programme of research into several areas of helminth neurobiology needs to be pursued. It is essential that work on the structural characterization and localization of authentic helminth regulatory peptides remains an important focus. Once the primary structure of a helminth peptide has been deduced,

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then research towards exploring its physiology, mode of action, and pharmacology, using synthetic copies of the peptide and its analogues becomes feasible. Knowledge of the peptide structure also means that region-specific antisera can be generated, and these in turn enable the exact distribution of the particular peptide messenger to be mapped immunocytochemically and its abundance determined by specific immunoassay. So far, only a handful of helminth peptides have been isolated and sequenced (see Tables 8 and 9), and their cellular distribution determined immunocytochemically with specific antisera. In all cases, their identities were determined on the basis of their cross-reactivities with antisera to known mammalian (e.g. neuropeptide F (NPF) with pancreatic polypeptide (PP) antisera; TE-6 with chromagranin A antisera) or invertebrate (FaRPs with FMRFamide antisera) peptides. It is possible that important bioactive peptides exist which are unique to helminths, but which exhibit little or no cross-reactivity with peptides from other taxa; they would have real potential for therapeutic exploitation. Finally, the use of peptide sequence information in conjunction with polymerase chain reaction (PCR) technology would enable the design and synthesis of oligonucleotide probes to screen cDNA libraries, thereby gaining insight into the structural organization of peptide genes and the processing of their encoded precursor prepropeptides. With structural information available on helminth peptides, it is possible to synthesize these native peptides and their analogues and to explore some of their functional characteristics. From their distribution patterns, the most obvious site of peptide action is the neuromuscular system in helminths. To date, however, only the two Ascaris FaRPs (AFI, AF2) have been investigated experimentally, although useful helminth models are now being used for examining the way in which the numerous “chemical addressing” messenger molecules work in an anatomically simple neural system. Direct information about ion-channel function in helminth muscle, using voltage-clamp and patch-clamp techniques, is now possible in S. rnansoni by a method that isolates individual muscle fibres in dispersions of cells from the worm (Blair et al., 1991). The mode of action of regulatory peptides is via specific receptors linked to ion channels, o r through G-protein associated second messenger generation whereby they are able to trigger a cascade of cellular reactions. Since it is the properties and the mode of action of the peptide receptors which will be instrumental in determining the tissue response, peptide receptors in helminths provide an attractive target in anthelmintic discovery. However, research into peptide receptors in invertebrates in general is in its infancy, and none has been documented so far for helminths. The use, for example, of radiolabelled peptide ligands to localize binding sites at both light and electron microscopic levels would help identify the sites of

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action, and may contribute information on peptide function. Receptor binding assays will allow important key sequences within the helminth peptides to be identified, and these could then be modified and screened for antagonistic properties. Synthetic peptide derivatives could also be used to isolate and purify the peptide receptors themselves, using affinity chromatography, electrophoresis or functional receptor expression techniques. Whereas most of the information on the nerve-muscle functioning of classical transmitters has been derived from pharmacological experiments using high-affinity receptor antagonists, such drugs, with few exceptions (e.g. cholecystokinin (CCK), opiate, tachykinin antagonists), are not available for peptides. This is one reason why so little is known about the physiological actions of peptides. Moreover, peptide antagonists present problems, not least their susceptibility to biodegradation. In this respect, the synthesis of novel non-peptide ligands as receptor antagonists, such as has been achieved for mammalian CCK receptors (see Hokfelt, 1991), would be an essential step if peptide receptors in helminths are to be exploited therapeutically in anthelmintic discovery. As mentioned earlier (Section 3.2) the purification of peptides from tissue extracts relies heavily on the amount of both the particular peptide in question and the tissue-type and, in the case of helminth parasites, the availability and size of the particular species in which it occurs. Unfortunately, these quantitative constraints mean that current biochemical methods allow only the more-abundant peptide messengers to be isolated and sequenced. The likely development of more sensitive assay techniques and the improvement of purification procedures will enable less abundant peptide species to be structurally characterized. However, the small size of most helminths, and the general inaccessibility of their nervous systems, will always present technical limitations on the use of biochemical extraction and isolation procedures for helminth neuropeptides. For this reason, the use of recombinant DNA techniques to search for genes encoding peptide precursors will likely increase, enabling workers to deduce indirectly amino acid sequences of peptides that prove impossible to isolate biochemically. Moreover, since peptide genes may encode more than one peptide, the information gained on nucleotide sequences could be used to predict the existence of novel peptide species in the parasites. The cloning and sequencing of peptide precursor genes from cDNA expression libraries has already proved to be successful in a number of lower invertebrate groups, including coelenterates (Darmer et al., 1991), and has been used recently to reveal peptide sequences in C . eleguns (Rosoff et al., 1992). Finally, the extension of this technology to helminthpeptide receptor cloning and expression would expose any structural and pharmacological features that are distinct from those of host receptor homologues, and so provide a basis for developing selective anthelmintics.

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In conclusion, it is likely that the next few years will witness major advances in regulatory peptide research in helminths, through the interaction of a multidisciplinary range of newly developing techniques in biochemistry, cytochemistry, electrophysiology, molecular biology, peptide chemistry and pharmacology. It is anticipated that these advances will have important implications in anthelmintic discovery, hopefully leading to the development of selectively acting agents for controlling helminth parasites.

NOTE ADDED IN PROOFS In Section 5.3 (FMRFamide and Related Peptides (FaRPs)) on page 207, a statement is made which refers to the differences in the primary structures of FaRPs from parasitic and free-living nematodes, which may be related to their different life-styles. Recently, the authors have established that the Ascaris suum FaRP, AF2, is present in the free-living nematode, Panagrellus redivivus (Maule et al., 1994, Parasitology, in press). In addition, a novel FaRP, KSAYMRFamide, has been isolated from this species and has been found to be myoactive in A . suum somatic muscle (Maule et al., 1994, Biochemical and Biophysical Research Communications, in press). These data suggest that the complement of FaRPs in parasitic and free-living nematode species is not as radically different as initial isolation studies suggested. This is in all probability due to the different extraction techniques employed in these studies on parasitic ( A . suum) and free-living ( P . redivivus and Caenorhabditis elegans) nematode species. The full complement of FaRPs in a single species remains to be elucidated.

REFERENCES Atkinson, H.J., Isaac, R.E., Harris, P.D. and Sharpe, C.M. (1988). FMRFamidc immunoreactivity within the nervous system of the nematodes Panugrellus redivivus. Caenorhabditis elegans and Heterodera glycines. Journal o f Zoology London 216, 663471. Baguna, J., Salo, E. and Romero, R. (1989). Effects of activators and antagonists of the neuropeptides substance P and substancc K on cell proliferation in planarians. International Journal of Developmental Biology 33, 261-264. Banting, F.G. and Best, C.H. (1922). Journal of Laboratory and Clinical Medicine 5 , 256-271. Barker, G.C. and Rees, H. (1990). Ecdysteroids in nematodes. Parasitology Toduy 6,384-387.

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Barnes, R.S.K., Calow, P. and Olive, P.J.W. (1993). “The Invertebrates: a New Synthesis”. 2nd edn. Blackwell Scientific Publications, Oxford. Barton, C.L., Halton, D.W., Shaw, C., Maule, A.G. and Johnston, C.F. (1993). An immunocytochemical study of putative neurotransmitters in the metacercariae of two strigeoid trematodes from rainbow trout (Oncorhynchus mykiss). Parasitology Research 79, 389-396. Basch, P.F. and Gupta, B.C. (1988). Immunocytochemical localization of regulatory peptides in six species of trematode parasites. Comparative Biochemistry and Physiology 91C, 565-570. Bayliss, W.M. and Starling, E.H. (1902). The mechanism of pancreatic secretion. Journal of Physiology 28, 325-353. Blair, K.L., Day, T.A., Lewis, M.C., Bennett, J.L. and Pax, R.A. (1991). Studies on muscle cells isolated from Schistosoma mansoni: a Ca2+-dependent K+ channel. Parasitology 102, 251-258. Blalock, J.E. (1989). A molecular basis for bidirectional communication between the immune and neuroendocrine systems. Physiological Reviews 69, 1-32. Bradford, H.F. (1986). “Chemical Neurobiology”. W.H. Freeman, New York. Brennan, G.P., Halton, D.W., Maule, A.G. and Shaw, C. (1993a). Electron immunogold-labelling of regulatory peptide immunoreactivity in the nervous system of Monieria expansa (Cestoda: Cyclophyllidea). Parasitology Research 79, 409-415. Brennan, G.P., Halton, D.W., Maule, A.G., Shaw, C., Johnston, C.F., Moore, S. and Fairweather, I. (1993b). lmmunoelectron microscopical studies of regulatory peptides in the nervous system of the monogenean parasite, Diclidophora merlangi. Parasitology 106, 171-176. Brownlee, D.J.A., Fairweather, I., Rogan, M.T., Johnston, C.F., Shaw, C. and Halton, D.W. (1992). The localisation of neuropeptide immunoreactivities in the hydatid organism, Echinococcus granulosus. Regulatory Peptides 39, 269. Brownlee, D.J.A., Thorndyke, M.C. and Johnston, C.F. (1993a). Immunocytochemistry and immunogold labelling of SALMFamide immunoreactivity in the nervous system of the trematode, Schistosoma mansoni. Regulatory Peptides 47,97. Brownlee, D.J.A., Fairweather, I . , Johnston, C.F., Smart, D., Shaw, C. and Halton, D. W. (1993b). Immunocytochemical demonstration of neuropeptides in the central nervous system of the roundworm, Ascaris suum (Nematoda, Ascaroidea). Parasitology 106, 305-316. Brownlee, D.J.A., Fairweather, I. and Johnston, C.F. (1993~).Immunocytochemical demonstration of neuropeptides in the peripheral nervous system of the roundworm Ascaris suurn (Nematoda, Ascaroidea). Parasitology Research 79, 302-308. Brownlee, D.J.A., Fairweather, I . , Johnston, C.F. and Shaw, C. (1994a). Immunocytochemical demonstration of peptidergic and serotoninergic components in the enteric nervous system of the roundworm, Ascaris m u m (Nematoda, Ascaroidea). Parasitology 108, 89-1 03. Brownlee, D.J.A., Brennan, G.P., Halton, D.W., Fairweather, I . and Shaw, C. (1994b). Ultrastructural localisation of FMRFamide- and pancreatic polypeptide-immunoreactivities within the central nervous system of the liver fluke, Fasciola hepatica (Trematoda: Digenea). Parasitology Research 80, 117-124. Brownlee, D.J.A., Brennan, G.P., Halton, D.W., Fairweather, I. and Shaw, C. (1994~).Ultrastructural localisation of pancreatic polypeptide- and FMRFamideimmunoreactivities within the central nervous system of the nematode, Ascaris suum (Nematoda, Ascaroidea). Parasitology (in press).

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