Molecular Biology of Spirochetes

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MOLECULAR BIOLOGY OF SPIROCHETES

NATO Science Series

A series presenting the results of scientific meetings supported under the NATO Science Programme.

The series is published by IOS Press and Springer Science and Business Media in conjunction with the NATO Public Diplomacy Division. Sub-Series I. II. III. IV. V.

Life and Behavioural Sciences Mathematics, Physics and Chemistry Computer and Systems Sciences Earth and Environmental Sciences Science and Technology Policy

IOS Press Springer Science and Business Media IOS Press Springer Science and Business Media IOS Press

The NATO Science Series continues the series of books published formerly as the NATO ASI Series.

The NATO Science Programme offers support for collaboration in civil science between scientists of countries of the Euro-Atlantic Partnership Council. The types of scientific meeting generally supported are “Advanced Study Institutes” and “Advanced Research Workshops”, although other types of meeting are supported from time to time. The NATO Science Series collects together the results of these meetings. The meetings are co-organized by scientists from NATO countries and scientists from NATO’s Partner countries – countries of the CIS and Central and Eastern Europe. Advanced Study Institutes are high-level tutorial courses offering in-depth study of the latest advances in a field. Advanced Research Workshops are expert meetings aimed at critical assessment of a field, and identification of directions for future action. As a consequence of the restructuring of the NATO Science Programme in 1999, the NATO Science Series has been re-organized and there are currently five sub-series as noted above. Please consult the following web sites for information on previous volumes published in the series, as well as details of earlier sub-series: http://www.nato.int/science http://www.springeronline.nl http://www.iospress.nl http://www.wtv-books.de/nato_pco.htm

Series I. Life and Behavioural Sciences – Vol. 373

ISSN: 1566-7693

Molecular Biology of Spirochetes

Edited by

Felipe C. Cabello

New York Medical College, Valhalla, New York, USA

Dagmar Hulinska

Czech Republic National Institute of Public Health, Prague, Czech Republic

and

Henry P. Godfrey

New York Medical College, Valhalla, New York, USA

Amsterdam • Berlin • Oxford • Tokyo • Washington, DC Published in cooperation with NATO Public Diplomacy Division

Proceedings of the NATO Advanced Research Workshop on Molecular Biology of Spirochetes Prague, Czech Republic 5–8 December 2005 © 2006 IOS Press. All rights reserved. No part of this book may be reproduced, stored in a retrieval system, or transmitted, in any form or by any means, without prior written permission from the publisher. ISBN 1-58603-665-3 Library of Congress Control Number: 2006933007 Publisher IOS Press Nieuwe Hemweg 6B 1013 BG Amsterdam Netherlands fax: +31 20 687 0019 e-mail: [email protected] Distributor in the UK and Ireland Gazelle Books Services Ltd. White Cross Mills Hightown Lancaster LA1 4XS United Kingdom fax: +44 1524 63232 e-mail: [email protected]

Distributor in the USA and Canada IOS Press, Inc. 4502 Rachael Manor Drive Fairfax, VA 22032 USA fax: +1 703 323 3668 e-mail: [email protected]

LEGAL NOTICE The publisher is not responsible for the use which might be made of the following information. PRINTED IN THE NETHERLANDS

Molecular Biology of Spirochetes F.C. Cabello et al. (Eds.) IOS Press, 2006 © 2006 IOS Press. All rights reserved.

v

Preface This book is a compilation of presentations at a NATO Advanced Research Workshop on the “Molecular Biology of Spirochetes” held at the Czech Republic National Institute of Public Health, Prague, Czech Republic, December 5–8, 2005. This meeting was supported by the NATO Programme for Security Through Science, the United States National Institutes of Health, Office of Rare Diseases, the United States Institute of Allergy and Infectious Diseases, the Czech Republic National Institute of Public Health, Czech Republic and New York Medical College, Valhalla, New York, U.S.A. It was organized to foster the exchange of experience among scientists from NATO countries in North America, Western and Eastern Europe. This type of encounter is valuable because diseases produced by spirochetes, including Lyme borreliosis, syphilis and leptospirosis, are on the rise worldwide, and because the biology of their causative organisms, their epidemiology, and clinical presentation display important variations in different geographical areas. For example B. burgdorferi sensu lato produces approximately 20,000 cases of Lyme borreliosis a year in the United States and 60,000 cases in Europe, but B. burgdorferi sensu stricto, B. afzelii and B. garinii are transmitted by different vectors and have different reservoirs and clinical presentations in these different geographic areas. Awareness and better understanding of these variations by researchers in the field is thus highly relevant to improvements in their prevention and treatment, and critical for improvement of human health. The meeting was organized with oral presentations by major speakers and poster sessions by students and postdoctoral fellows from Eastern Europe. This volume includes not only the presentations of the major speakers but also several additional presentations by investigators who were invited but were unable to attend. For many reasons (including meeting organization and funding limitations), this volume does not intend to represent a comprehensive coverage of all aspects of spirochete biology. It rather focuses on a series of state of the art presentations of the research taking place in the laboratories of the contributors. As such, we hope that it may be useful as an introduction to those individuals entering in the burgeoning field of spirochete research. We would also like to believe that the meeting and this book will serve as a stimulus for researchers in the field to widen collaborations and exchanges between investigators in the different geographical areas where spirochetal diseases are common since these interactions can only be of benefit to the field. Finally, we would like to thank the participants who risked the cold weather to attend the meeting, the authors who despite their inability to attend were willing to submit chapters to this book, the funding institutions mentioned above, and in particular, Drs. Phil Baker, Patti Rosa, Tom Schwan, Frank Gherardini, Ms. Marylin E. Kunzweiler (United States National Institute of Allergy and Infectious Diseases), Dr. Jorge Benach (Stony Brook University), Ms. Mary C. Demory (United States National Institutes of Health), Dr. Marina Cinco (University of Trieste), Dr. Michael Norgard (University of Texas Southwestern Medical Center) and Dr. Ira Schwartz (New York Medical College), whose efforts were critical to our securing some of the

vi

funds that supported the meeting, and TestLine, Qiagen, BioConsult, Roche, Biowestern, Generi BioTech and BagMed who provided additional support for this meeting. We would also like to thank Ms. Leonor Delgado for editorial assistance and Ms. Harriett V. Harrison for her outstanding and continuing assistance in organizing the meeting and in preparing the manuscripts that compose this book. Felipe C. Cabello New York Medical College Valhalla, New York, USA Dagmar Hulinska Czech Republic National Institute of Public Health Prague, Czech Republic Henry P. Godfrey New York Medical College Valhalla, New York, USA July, 2006

vii

Contents Preface Felipe C. Cabello, Dagmar Hulinska and Henry P. Godfrey

v

Introductory Overview Dissemination and Persistence are Pathogenic Events Common to All of the Major Human Spirochetal Infections Gary P. Wormser

3

Part 1. Molecular Genetics of Spirochetes Transposon Mutagenesis of Infectious Borrelia burgdorferi B31: A Pilot Study Douglas J. Botkin, April Abbott, Jerrilyn K. Howell, Mary Mosher, Philip E. Stewart, Patricia A. Rosa, Hiroki Kawabata, Haruo Watanabe and Steven J. Norris

The Isolation and Characterization of Isogenic Mutants in Infectious Borrelia burgdorferi J. Seshu, Maria Labandeira-Rey, M. Dolores Esteve-Gassent, Magnus Höök and Jonathan T. Skare Motility Gene Regulation and Chemotaxis in Borrelia burgdorferi Nyles W. Charon, Melanie Sal, Michael R. Miller, Richard G. Bakker, Chunhao Li and Md. Abdul Motaleb

Targeted and Random Mutagenesis in Leptospira biflexa: Application for the Functional Analysis of Iron Transporters Hélène Louvel, Simona Bommezzadri, Nora Zidane, Paula Ristow, Zoé Rouy, Claudine Medigue, Caroline Boursaux-Eude, Isabelle Saint Girons, Christiane Bouchier and Mathieu Picardeau

Antibiotic Resistance in Borrelia burgdorferi: Applications for Genetic Manipulation and Implications for Evolution D. Scott Samuels Development of Treponeme Genetic Systems Howard K. Kuramitsu and Caroline E. Cameron

13

25 42

50

56 71

Part 2. Genomics and Diversity Comparative Genomics of Borrelia burgdorferi Sherwood R. Casjens, Wai Mun Huang, Eddie B. Gilcrease, Weigang Qiu, William D. McCaig, Benjamin J. Luft, Steven E. Schutzer and Claire M. Fraser

79

viii

Treponema Genomics George M. Weinstock, David Šmajs, Petra Matĕjková, Michal Strouhal, Thomas J. Albert, Steven J. Norris, Timothy Palzkill and Erica J. Sodergren Comparative Analysis of Pathogenic Leptospira Genomes Richard L. Zuerner, Dieter M. Bulach, Torsten Seemann, Ross L. Coppel and Ben Adler Leptospira interrogans: Genomics and “Immunomics” Ana L.T.O. Nascimento

Genotypic Variation and Borrelia burgdorferi Pathogenesis Ira Schwartz, Guiqing Wang, Radha Iyer, Caroline Ojaimi, Darya Terekhova, Sabina Sandigursky, Gary P. Wormser and Dionysios Liveris

Multilocus Sequence Analysis (MLSA) as an Alternative to Whole DNA/DNA Hybridization (WDDH) in Borrelia burgdorferi sensu lato Taxonomy Guy Baranton and Danièle Postic

Diversity and Variability of Protein-Encoding Genes of Borrelia burgdorferi sensu lato and Implications for Pathogenesis and Diagnosis of Lyme Borreliosis in Europe Bettina Wilske, Volker Fingerle and Ulrike Schulte-Spechtel Are Borrelia recurrentis and Borrelia duttonii the Same Spirochaete? Sally J. Cutler, Julie C. Scott and David J.M. Wright Genotyping of Borrelia burgdorferi sensu lato in Russia Edward I. Korenberg, Valentina V. Nefedova, Irina A. Fadeeva and Nataliya B. Gorelova

Ecological and Genetic Diversity Within the Leptospiraceae Family: Implications for Epidemiology Yulia V. Ananyina, Anna P. Samsonova, Evgeny M. Petrov, Igor A. Shaginyan, Marina Yu. Chernukha, Marina S. Zemskaya and Yulia S. Alyapkina

Characterization of Borrelia burgdorferi sensu lato from Czech Patients and Ticks by Culture and PCR-Sequence Analysis Dagmar Hulinska, Martin Bojar and Václav Hulinsky

Infection of Ixodid Ticks, Mosquitoes and Patients with Borrelia, Bartonella, Rickettsia, Anaplasma, Ehrlichia and Babesia in Western Siberia, Russia Olga Morozova, Vera Rar, Yana Igolkina, Andrey Dobrotvorsky, Igor Morozov and Felipe C. Cabello

96 101 115 124

135

146 159 174

200

208 221

Part 3. Gene Expression Genetic Studies of the Borrelia burgdorferi bmp Gene Family Felipe C. Cabello, Lidiya Dubytska, Anton V. Bryksin, Julia V. Bugrysheva and Henry P. Godfrey

235

ix

Porins of Borrelia Marija Pinne, Yngve Östberg, Roland Benz and Sven Bergström

250

Use of Green Fluorescent Protein Transcriptional Reporters to Study Differential Gene Expression by Borrelia burgdorferi Christian H. Eggers, Melissa J. Caimano and Justin D. Radolf

264

The Telomere Resolvase ResT and Evolution of the Borrelia Genomes George Chaconas

292

Regulation of Expression of the Integrin Ligand P66 in Borrelia burgdorferi Melisa S. Medrano, Paul Policastro, Tom G. Schwan and Jenifer Coburn

Hairpin Telomeres of Linear Bacterial Chromosomes and Plasmids: How to Make Them Wai Mun Huang, Qiurong Ruan and Sherwood R. Casjens

281

299

Part 4. Interactions of Spirochetes and Hosts Blood-Induced Transcriptional Changes in Borrelia burgdorferi Rafal Tokarz and Jorge L. Benach

Roles of Leptospiral Outer Membrane Proteins in Pathogenesis and Immunity David A. Haake

Genetic Analysis of Attachment of Borrelia burgdorferi to Host Cells and Extracellular Matrix Nikhat Parveen and John M. Leong

Borrelia burgdorferi and Ixodes scapularis: Exploring the Pathogen-Vector Interface Utpal Pal, John F. Anderson and Erol Fikrig The Lyme Disease Spirochete Erp Protein Family: Structure, Function and Regulation of Expression Brian Stevenson, Tomasz Bykowski, Anne E. Cooley, Kelly Babb, Jennifer C. Miller, Michael E. Woodman, Kate von Lackum and Sean P. Riley

Lyme Disease Spirochetes Evade Innate Immunity by Acquisition of Complement Regulators, Factor H, and FHL-1 Reinhard Wallich, Peter F. Zipfel, Christine Skerka, Michael Kirschfink, Markus M. Simon, Brian Stevenson, Susan M. Lea and Peter Kraiczy

Outer Surface Lipoproteins of Borrelia burgdorferi: Role in Virulence, Persistence of the Pathogen, and in Protection Against Lyme Disease Markus M. Simon, Nico Birkner, Rinus Lamers and Reinhard Wallich

311 323 333 345 354

373

383

Localization of Lyme Disease and Relapsing Fever Spirochetes in Mammalian Hosts Infected with Different Borrelia Species, Strains, and Serotypes Diego Cadavid

393

Author Index

399

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Introductory Overview

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Dissemination and Persistence are Pathogenic Events Common to All of the Major Human Spirochetal Infections Gary P. WORMSER 1 Division of Infectious Diseases, Department of Medicine of New York Medical College, Valhalla, NY 10595 Abstract. In all of the major human spirochetal infections, the fundamental pathogenetic event underlying the most serious complications of these diseases is documented or presumed hematogenous dissemination of the spirochete from the site of inoculation to distant sites. Lyme borreliosis is attractive for study of spirochetal dissemination for a variety of reasons including: the availability of a large patient base, the ability to identify patients early during the course of infection, and the ability to culture Lyme borrelia readily in vitro. Tick feeding per se does not directly lead to blood stream invasion by Lyme borrelia. The genotype of the strain of Borrelia burgdorferi introduced by the tick, however, does appear to be an important determinant of dissemination in humans and other mammals. Recent evidence suggests that host factors affect both the development of infection and subsequent dissemination of this spirochete as well. In one United States study, independent risk factors for hematogenous dissemination of B. burgdorferi included having a first episode of Lyme borreliosis and being more than 55 years of age. In another United States study, infection due to the least invasive genotype of B. burgdorferi was associated with carriage of the HLA class II allele DRB1*0101. All of the major human spirochetal infections are also characterized by persistence of the spirochete in mammalian hosts, and depending on the specific spirochete and host involved, this phenomenon may be closely linked to the pathogenesis of important clinical manifestations and to communicability to uninfected hosts. In conclusion, spirochetemia and persistence are common and important pathogenetic features of the major human spirochetal infections. Keywords. Borrelia burgdorferi, Lyme disease, spirochetes, dissemination, persistence

Spirochetes are important causes of infection throughout the world. What might be regarded as the major spirochetal infections of humans are listed in Table 1 [1]. An estimated 12 million cases of syphilis occur annually worldwide with the majority of cases occurring in developing countries [2]. In industrialized countries, Lyme borreliosis is also a common spirochetal infection, at least in parts of North America and Europe [3, 4].

1 Corresponding Author: New York Medical College, Division of Infectious Diseases, Munger Pavilion, Room 245, Valhalla, NY 10595, USA; E-mail: [email protected].

G.P. Wormser / Dissemination and Persistence are Pathogenic Events

4

Spirochetal infections have quite diverse modes of transmission. For example, syphilis is spread predominantly through intimate physical contact between humans, whereas Lyme borreliosis can only be acquired through the bite of an infected tick. Clinical manifestations are also diverse with prominent cutaneous abnormalities in syphilis [5], the endemic treponematoses [6], Lyme disease [7] and rat bite fever due to Spirillum minus [8]. In contrast, cutaneous lesions are usually absent in leptospirosis [9] and relapsing fever [10]. All of these infections, however, share certain features central to the pathogenesis of their most serious complications or that play a pivotal role in communicability and spread of infection. Documented or presumed spirochetemia is a fundamental aspect of the pathobiology of these infections (Table 1). In syphilis, for example, if Treponema pallidum were confined to the site of entry into the skin, then even in untreated patients, this infection would be a short-lived, self-limited, almost trivial skin condition rather than a multisystem disease associated with substantial morbidity and even mortality [11, 12].

Table 1. Bacteremia and persistence in the major human spirochetal infections. Infection

Bacteremia

Persistence

Syphilis

Yes

Yes

Yaws

Yes

Yes

Endemic syphilis

Yes

Yes

Pinta

Yes

Yes

Leptospirosis

Yes

Yes (especially kidney)

Relapsing fever

Yes

Yes

Lyme disease

Yes

Yes

Rat bite fever

Yes

Yes

Another important feature that the major spirochetal infections share is the ability to persist in an untreated host (Table 1). Differences do exist among the listed spirochetal infections as to duration and sites of persistence; in some cases, such as leptospirosis, persistence occurs principally in non-human rather than human hosts [9]. One of the great enigmas concerning the persistence of these predominantly extracellular bacteria is that it may occur despite an intense humoral and cellular immune response to the microorganism. Among the major human spirochetal infections, syphilis has been the best documented and may be the only one capable of persisting for as long as decades [5]. Obviously the impact of persistence on the natural history of syphilis and the other major spirochetal infections has been dramatically altered by the timely use of antibiotic therapy to treat these infections. No convincing evidence exists for persistence of any of these infections after appropriate antibiotic therapy [13]. Potential mechanisms for persistence include location in an anatomic sanctuary protected from the sterilizing effects of the immune response, antigenic variation or modulation by the spirochete to avoid the immune response, antigenic concealment

G.P. Wormser / Dissemination and Persistence are Pathogenic Events

5

through lack of expression of surface antigens or acquisition of an outer coating of host-derived materials such that key surface antigens are prevented from being recognized by the immune system, or immunomodulation of the host by the spirochete such that an effective immune response is abrogated [12, 14-18]. Antigenic variation seems to be the mechanism for persistence adopted by the relapsing fever spirochetes [19]. Leptospira have found a sanctuary in the urinary tract [9, 20]. The mechanism(s) for persistence in the other major spirochetal infections is less well understood and conceivably could be multifactorial with the spirochetes adopting more than one of the strategies discussed above. The reader is referred elsewhere for reviews of much of the available information on how T. pallidum [12] or Lyme borrelia [14] might persist. Most of the serious consequences of human spirochetal infections arise from spread from the site of entry in the skin or mucous membranes to visceral locations. Despite the obvious importance of clarifying the natural history and mechanisms for hematogenous dissemination, this issue has been difficult to study directly due to a number of logistical issues, such as the inability to cultivate many of the spirochetal agents in vitro. Lyme borreliosis is attractive for study of hematogenous dissemination because the infection can be identified clinically in its early stages based on the characteristic appearance of the cutaneous lesion (called erythema migrans) that occurs at the site of deposition of the spirochete into the skin, or even earlier if the patient were to recognize and remove an attached infected tick. Lyme borrelia can be grown successfully in vitro, and a sufficiently large number of patients with this infection exist to conduct meaningful clinical research studies.

Table 2. Yield of blood cultures in representative studies in which a low volume of blood was cultured. Author

Year

Material

Volume

Patients

Yield

USA Benach [21]

1983

Citrated blood

4 mL

36

5.6%

Steere [22]

1984

Citrated plasma

8x coverage) of the saprophyte L. biflexa serovar Patoc strain Patoc1 consists of a 3.6megabase large chromosome and a 278-kilobase small chromosome with a total of 3801 predicted coding genes, 60% of which are conserved with those of L. interrogans.

52

H. Louvel et al. / Targeted and Random Mutagenesis in Leptospira biflexa

2. Targeted Mutagenesis in L. biflexa In this work, the ability of Leptospira to use various iron sources for its physiological role requirement was studied. We show that Leptospira spp. can acquire iron from different sources, including the siderophores aerobactin and ferrichrome. However, the uptake systems of these molecules remain to be determined. The outer membrane of Leptospira is relatively impermeable [5], and the acquisition of molecules may mainly rely on the active transport through dedicated outer membrane receptors. In gramnegative bacteria, the transport of iron sources into the periplasm against their concentration gradients is mediated by the inner membrane complex TonB, ExbB, and ExbD. The energy is transduced to the outer membrane receptor, also called TonBdependent receptor. Analysis of the genomes indicated that L. biflexa and L. interrogans contain eight and 12 genes, respectively, whose products share homology with TonB-dependent receptors. However, substrate specificity is difficult to characterize based solely on sequence analysis of the receptors. By random transposon mutagenesis in L. biflexa, we recently identified mutants (see below) with insertions in a gene (LEBIa1620) encoding a protein that shares homology with FecA [7]. Besides FecA, we have attempted to disrupt the other putative genes encoding TonB-dependent receptors by allelic exchange in L. biflexa. Gene inactivation of LEBIa2634, LEBIa2704, and LEBIa2712 resulted in a wild-type phenotype in iron-depleted medium supplemented with different iron sources. The lack of a phenotype in these mutants could be due to functional redundancy with another iron uptake system. Disruption of LEBIa3308 resulted in a mutant that was impaired in its ability to use desferrioxamine as an iron source. Interestingly, the mutant was also impaired in its ability to utilize ferrichrome, while the transport of aerobactin, another hydroxamate siderophore, was unaffected. These results are evidence that LEBIa3308 encodes the receptor protein for ferrioxamines and ferrichrome in L. biflexa. Finally, we failed to obtain double crossover events in LEBIa0145, LEBIa2087, and LEBIa3054. This may indicate that these genes are essential for the survival of L. biflexa. Leptospira have an absolute requirement for vitamin B12, which is usually transported via TonB-like systems in other gram-negative bacteria. Since vitamin B12 is a co-factor for enzymes of major biological processes, inactivation of its receptor should result in non viable mutants. Amino acid comparisons of TonB-dependent receptors of heme, hemoglobin, siderophores, and vitamin B12 revealed a highly conserved domain containing the FRAP and NPNL amino acid box [2, 11]. This conserved domain was found in some TonB-dependent receptors from Leptospira, including LEBIa2087 and LEBIa0145.

3. L. biflexa Mutants Isolated by Random Insertional Mutagenesis An important approach for investigating metabolism processes is the generation of large numbers of mutant bacteria. Electroporation of L. biflexa with plasmid vector pSC189 [3] containing both the hyperactive transposase C9 [6] and transposon terminal inverted repeats flanking a kanamycin resistance gene resulted to approximately 5000 transformants per Pg of DNA. To improve expression of the Himar1 transposase, the hyperactive transposase C9 was fused to spirochetal promoters. L. biflexa was electroporated with plasmids pSHT and pSFLT expressing the mariner transposase from the L. interrogans hsp10 and flgB promoters, respectively. We found that the transposition of Himar1 from donor plasmids pSHT and pSFLT was about 5-fold more


53

efficient than that of Himar1 from pSC189, i.e., 2 to 3.5 104 transformants per Pg of DNA were obtained with plasmids pSHT and pSFLT. Southern blot hybridization and sequencing of the Himar1 insertion sites of 50 randomly chosen kanamycin-resistant colonies showed the transposition to be random and stable [7]. Similar to other hosts, the mariner transposon targets the dinucleotide sequence TA, which is duplicated upon cut-and-paste insertion. This target site is abundant in the L. biflexa genome with an average G+C content of approximately 39%. The availability of the whole genome sequence of L. biflexa should greatly facilitate genetic analyses in Leptospira. For example, genes disrupted by transposon insertion mutagenesis can be rapidly identified in mutants with interesting phenotypes through sequence analysis of the flanking regions and comparison with genome sequence. To investigate iron transporters, 6000 L. biflexa transposon mutants were screened onto medium with and without hemin, thus allowing the identification of 15 hemin-requiring mutants, and the putative genes responsible for this phenotype were identified. Twelve mutants had distinct insertions in a gene encoding a protein that shares homology with TonB-dependent receptor FecA, involved in ferric citrate transport. The L. biflexa fecA mutants were impaired in their ability to use iron citrate [7], and also iron sulfate and aerobactin as an iron source. Interestingly, among hydroxamate siderophores, aerobactin forms a distinct subfamily of siderophores that is derived from citrate, and therefore aerobactin and iron citrate share a similar structure that could be recognized by the same receptor. We also identified two mutants with a Himar1 insertion into feoAB-like genes, the product of which is required for ferrous iron uptake in many bacterial organisms [7]. Interestingly, another mutant exhibited a Himar1 insertion into a two-component system that could be involved in the regulation of iron uptake. Finally, by screening for manganese-requiring mutants, two new genes of unknow function, including a putative manganese transporter, were also identified. Phenotypic characterization of these mutants extends our understanding of the biology of Leptospira spp., which remains largely unknown.

4. Conclusions The availability of the whole-genome sequence of L. biflexa should shed light on the evolution of genomes in Leptospira and reveal ways in which virulent pathogens can evolve. The genome content reflects the bacterial lifestyles, and results from the distinct ongoing process of genome optimization in saprophytes and pathogens. The possession of specialized iron transport systems for the saprophyte L. biflexa and the pathogen L. interrogans may thus reflect the various iron sources they may encounter in their diverse habitats. Based on our findings, a model for iron uptake in Leptospira can reasonably be proposed (Figure 1). The analysis of the genome of the pathogen L. interrogans has allowed the identification of 12 putative TonB-dependent receptors, while L. biflexa possesses eight putative TonB-dependent receptors. This difference suggests that pathogenic species are able to use a wider panel of iron sources, in comparison to L. biflexa. The pathogens may also present redundancy in their genome content. Like in gram-negative bacteria, periplasmic binding proteins may shuttle ironcontaining complexes from TonB-dependent receptor to cytoplasmic membrane ATPbinding cassette (ABC) transporters, that in turn deliver them in the cytoplasm. The pathogen L. interrogans may obtain iron from its association with heme and thus


54

Red blood cells

3308

?

2634

?

2712

?

3054

2087

0145

Outer mb

?

2704

?

Heme proteins

Desferrioxamine Ferrichrome

Iron sources

Iron citrate Aerobactin Iron salts

1620

Vitamin B12

Hemolysins

TonB-dependent recept

Siderophores RTX toxin transporter

Inner mb ExbB ExbD TonB

Hemin uptake system (HmuSTUV)

Siderophore typeABC transporter (?)

Metal type- ABC transporter

FeoAB

Figure 1. Schematic representation of iron acquisition systems in L. biflexa. Analysis of the draft genome sequence of the saprophyte L. biflexa suggests the presence of eight putative TonB-dependent receptors. The L. biflexa genome contains five TonB loci that could be involved in the formation of the ExbB-ExbD-TonB complex (only one TonB system is represented in this figure). By mutagenesis in L. biflexa, we isolated and characterized the function of two TonB-dependent receptors (LEBIa3308 and LEBIa1620) and the FeoAB system. Failure to obtain knock-out mutants and genetic organization suggest that LEBIa0145 is the TonBdependent receptor of either vitaminB12 or hemin. A metal type-ABC transporter was found in the L. biflexa genome and the HmuSTUV transport proteins may be involved in the periplasmic transport of hemin. An undescribed system may also exist for the periplasmic transport of siderophores (indicated in italic text). Leptospira could also release heme and hemoglobin from host red blood cells by the secretion of hemolysins. There is no evidence of siderophore synthesis by Leptospira spp. The molecules participating in each step of the transport process have not been identified and may involved reductases, periplasmic proteins, and permeases. In the cytoplasm, iron can be stored in bacterioferritin and Dps proteins.

secretes hemolysins to lyse red blood cells and liberates this metal. Surprisingly, L biflexa, a non pathogenic species, has putative hemin uptake and hemolysin secretion systems and may use a similar mechanism, a property hitherto described exclusively for pathogenic bacteria. However, the L. biflexa genome does not contain orthologs to the L. interrogans sphingomyelinase hemolysins, which may be involved in the typical vascular damage seen in the acute disease. It is rather questionable whether hemin is available to saprophytes during their living in the environment. The role of the genes of L. biflexa that encoded hemolysins and a putative hemolysin secretion system in iron acquisition remains to be elucidated. Leptospira also possess uptake systems that use siderophores produced by other bacteria or fungi. Bacterial iron homeostasis is best understood in E. coli, a bacterium phylogenetically distant from Leptospira. This study will serve as a basis for further study on iron acquisition systems in Leptospira. The application of our random transposon mutagenesis system to saprophytic and pathogenic strains will be particularly useful for discovering new genes involved in iron uptake and regulation.


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References [1]

Bourhy, P., H. Louvel, I. Saint Girons, and M. Picardeau. 2005. Random insertional mutagenesis of Leptospira interrogans, the agent of leptospirosis, using a mariner transposon. J. Bacteriol. 187: 3255í3258. [2] Bracken, C. S., M. T. Baer, A. Abdur-Rashid, W. Helms, and I. Stojiljkovic. 1999. Use of heme-protein complexes by the Yersinia enterocolitica HemR receptor: histidine residues are essential for receptor function. J Bacteriol 181: 6063í6072. [3] Chiang, S. L., and E. J. Rubin. 2002. Construction of a mariner-based transposon for epitope-tagging and genomic targeting. Gene 296: 179í185. [4] Cruveiller, S., J. Le Saux, D. Vallenet, A. Lajus, S. Bocs, and C. Medigue. 2005. MICheck: a web tool for fast checking of syntactic annotations of bacterial genomes. Nucleic Acids Res. 33: W471í479. [5] Haake, D. A. 2000. Spirochaetal lipoproteins and pathogenesis. Microbiology 146:1491-1504. [6] Lampe, D. J., M. E. Churchill, and H. M. Robertson. 1996. A purified mariner transposase is sufficient to mediate transposition in vitro. EMBO J 15: 5470í5479. [7] Louvel, H., I. Saint Girons, and M. Picardeau. 2005. Isolation and characterization of FecA- and FeoBmediated iron acquisition systems of the spirochete Leptospira biflexa by random insertional mutagenesis. J. Bacteriol. 187: 3249í3254. [8] Nascimento, A. L., A. I. Ko, E. A. Martins, C. B. Monteiro-Vitorello, P. L. Ho, D. A. Haake, S. Verjovski-Almeida, R. A. Hartskeerl, M. V. Marques, M. C. Oliveira, C. F. Menck, L. C. Leite, H. Carrer, L. L. Coutinho, W. M. Degrave, O. A. Dellagostin, H. El-Dorry, E. S. Ferro, M. I. Ferro, L. R. Furlan, M. Gamberini, E. A. Giglioti, A. Goes-Neto, G. H. Goldman, M. H. Goldman, R. Harakava, S. M. Jeronimo, I. L. Junqueira-de-Azevedo, E. T. Kimura, E. E. Kuramae, E. G. Lemos, M. V. Lemos, C. L. Marino, L. R. Nunes, R. C. de Oliveira, G. G. Pereira, M. S. Reis, A. Schriefer, W. J. Siqueira, P. Sommer, S. M. Tsai, A. J. Simpson, J. A. Ferro, L. E. Camargo, J. P. Kitajima, J. C. Setubal, and M. A. Van Sluys. 2004. Comparative genomics of two Leptospira interrogans serovars reveals novel insights into physiology and pathogenesis. J. Bacteriol. 186: 2164í2172. [9] Ren, S., G. Fu, X. Jiang, R. Zeng, H. Xiong, G. Lu, H. Q. Jiang, Y. Miao, H. Xu, Y. Zhang, X. Guo, Y. Shen, B. Q. Qiang, X. Q., A. Danchin, I. Saint Girons, R. L. Somerville, Y. M. Weng, M. Shi, Z. Chen, J. G. Xu, and G. P. Zhao. 2003. Unique and physiological and pathogenic features of Leptospira interrogans revealed by whole genome sequencing. Nature 422: 888í893. [10] Rubin, E. J., B. J. Akerley, V. N. Novik, D. J. Lampe, R. N. Husson, and J. J. Mekalanos. 1999. In vivo transposition of mariner-based elements in enteric bacteria and mycobacteria. Proc Natl Acad Sci U S A 96: 1645í1650. [11] Simpson, W., T. Olczak, and C. A. Genco. 2000. Characterization and expression of HmuR, a TonBdependent hemoglobin receptor of Porphyromonas gingivalis. 182: 5737í5748. [12] Ussery, D. W., and P. F. Hallin. 2004. Genome update: annotation quality in sequenced microbial genomes. Microbiology 150: 2015í2017.


56

Antibiotic Resistance in Borrelia burgdorferi: Applications for Genetic Manipulation and Implications for Evolution D. Scott SAMUELS 1 Division of Biological Sciences and Biomolecular Structure and Dynamics Program, The University of Montana, Missoula, Montana, USA Abstract. Antibiotic resistance is not a clinical problem with Lyme disease, but it has been extensively employed to genetically dissect the causative agent Borrelia burgdorferi. The first selectable marker was a coumermycin A1-resistant gyrB allele, which encodes a subunit of DNA gyrase, a target of several antibiotics. The utility of coumermycin A1 resistance has been compromised by technical and genetic barriers; resistance to other antibiotics has replaced the gyrB marker. Fluoroquinolones are another class of antibiotics that target DNA gyrase, as well as its homolog topoisomerase IV. Fluoroquinolone resistance in B. burgdorferi maps to parC, which encodes a subunit of topoisomerase IV, suggesting that this enzyme is the primary target of fluoroquinolones in Borrelia. A fluoroquinoloneresistant parC allele has been fashioned into a counter-selectable marker, a genetic tool used to select for the loss of DNA. One of the second-generation selectable markers is a heterologous aadA gene that confers resistance to spectinomycin and streptomycin, which target the small subunit of the ribosome. Selection with spectinomycin failed due to a high frequency of mutants in the population. These had mutations in 16S rRNA and were able to compete with wild type in vitro. This lack of a significant fitness cost for the mutant may contribute to the spread of antibiotic resistance. Keywords. Antibiotic resistance, Borrelia burgdorferi, Lyme disease, genetics, mutants

Introduction Antibiotic resistance in clinical isolates is a colossal public health concern [1í3]. Fortunately, there have been no reported cases of antibiotic resistance in Lyme disease patients. However, antibiotic resistance has been used as a selectable marker for molecular genetic studies of Borrelia burgdorferi and, because of technical limitations, is likely the only viable genetic marker that can be used for selection. Rosa, Tilly, and

1 Corresponding author: Division of Biological Sciences, The University of Montana, 32 Campus Dr., Missoula, MT 59812-4824, USA: E-mail: [email protected].

D.S. Samuels / Antibiotic Resistance in Borrelia burgdorferi

57

Stewart [4] is recommended as a recent and fairly comprehensive review of molecular genetics in B. burgdorferi. Antibiotics are broadly defined as organic substances that are either toxic or growth-inhibitory for organisms. Infectious disease therapy depends on the selective toxicity of antibiotics for the pathogen, so antimicrobial agents target a structure or function only found in a specific organism or group of organisms. For antibiotics that are specific for bacteria, these targets include cell wall synthesis (beta-lactams, glycopeptides, and cyclic lipopeptides), the cell membrane (polymyxins), protein synthesis (aminoglycosides, and the closely related spectinomycin, tetracyclines, macrolides, streptogramins, ketolides, and lincosamides), folate synthesis (sulfonamides and trimethoprim), RNA synthesis (rifamycins), DNA synthesis (fluoroquinolones and coumarins), and others [3, 5]. Many infectious diseases have been vanquished by these and other antibiotics, which have significantly extended and improved the lives of an enormous number of people. Unfortunately, resistance to these antibiotics is proving to be a “new apocalypse” [1]: methicillin-resistant Staphylococcus aureus, vancomycin-resistant S. aureus, vancomycin-resistant enterococci, macrolide-resistant Streptococcus, penicillin-resistant pneumococci, and multidrug-resistant tuberculosis [1í3, 6]. There are several causes for the escalation in antibiotic resistance, but the overuse of antibiotics, especially in agriculture, which accounts for well over half of the antibiotic use in the United States, is likely the primary factor [7]. A recent study found that the majority of soil bacteria sampled are resistant to many types of antibiotics: most bacterial strains tested were resistant to about seven different antibiotics, and not a single antibiotic had efficacy against all the bacterial strains [8]. Hence, antibiotic resistance is pervasive in the environment and will likely spread because of the promiscuity of bacteria sharing their genes [6, 9í12]. There are several mechanisms of antibiotic resistance: the antibiotic can be either kept out or pumped out of the cell, the antibiotic can be either destroyed or modified, or the target of the antibiotic can be changed. The mechanisms discussed in this chapter will focus on target mutation [5]. The mutated targets in antibiotic-resistant B. burgdorferi include DNA gyrase [13], its cousin topoisomerase IV [14], and the ribosome [15]. Two of these mutants have been developed into molecular tools for genetic experiments in the laboratory [16, 17], and the third has proved to be a hindrance [18].

1. History of Coumermycin A1 Resistance and its Application as the First Selectable Marker We isolated the first genetically defined antibiotic-resistant mutants of B. burgdorferi in 1993 [13]. Background (or “spontaneous”) mutants (as opposed to those induced with a mutagen) were selected with the coumarin antibiotic coumermycin A1, which targets the B subunit of DNA gyrase, a type II topoisomerase in prokaryotes [19, 20]. Type II topoisomerases are enzymes that alter DNA topology by breaking and resealing both strands of the double helix. DNA gyrase is a tetramer comprising two A subunits (GyrA) and two B subunits (GyrB). The A subunit is involved in the double-stranded nicking and resealing reactions, while the B subunit is responsible for providing energy through ATP hydrolysis. The gyrB and gyrA genes are in an operon proximal to the replication origin [21] near the center of the linear chromosome in B. burgdorferi [16, 22í24].

58

D.S. Samuels / Antibiotic Resistance in Borrelia burgdorferi Thr-165 Arg-136 Gly-77

ATP

Asn-107 Asp-105

Figure 1. A region of the N-terminal domain of GyrB from Escherichia coli with a non-hydrolyzable ATP analog and residues homologous to those altered in coumarin-resistant mutants highlighted in black: Gly-77, Asp-105, Asn-107, Arg-136, and Thr-165 are homologous to Gly-74, Asn-102, Gly-104, Arg-133, and Thr162 in B. burgdorferi. Coordinates are courtesy of Dale Wigley [26].

DNA gyrase has proved to be a popular drug target because of its essential nature as well as structural and functional differences with the eukaryotic type II topoisomerases that were exploited. The clinically effective fluoroquinolone antibiotics target the A subunits of DNA gyrase and its homolog topoisomerase IV (see section 2 below). On the other hand, coumermycin A1 and the other coumarin antibiotics (such as novobiocin) never achieved widespread clinical application because of several pharmacological limitations, such as solubility and toxicity [19, 20]. We mapped the coumermycin A1-resistant mutations to a conserved arginine residue in GyrB [13]. This residue (Arg-133 in B. burgdorferi is homologous to Arg-136 in Escherichia coli) makes contact with coumarin antibiotics [25], which interact with GyrB at the ATPbinding site (Figure 1), and is mutated in several other coumarin-resistant prokaryotes (Table 1) [27í34]. Note that this arginine is numbered 133 in our publications (GenBank accession AF017075) [13, 16], but corresponds to Arg-138 in the B. burgdorferi genome sequence (BB0436) [22]. This discrepancy is because our collaborator Wai Mun Huang observed that there is no reasonable Shine-Dalgarno sequence for a ribosome binding site except downstream of the start codon assigned during genome annotation. 1.1. gyrB Mutants Resistant to Coumermycin A1 Arg-133 was mutated to either isoleucine or glycine in coumermycin A1-resistant B. burgdorferi; the mutants had a mild growth phenotype [13]. Curiously, the homologous arginine (or lysine in some bacterial species) is mutated to serine, cysteine, histidine, leucine, isoleucine, glutamine, glutamic acid, and threonine in other coumarin-resistant microorganisms (Table 1) [27í34]. (Three of the nine species in which coumarinresistant mutations have been isolated are spirochetes!) The particular mutations depended on which of the six arginine codons was present in a particular species: the AGA of B. burgdorferi gyrB is mutated to ATA or GGA in background mutants [13], although mutations to AGU or AGC (serine), AAA (lysine), and ACA (threonine) would only require a single base change (and, as we discovered, do confer resistance to coumermycin A1 [35]). These observations provoked the question of which amino acid


59

substitutions would confer resistance. We used site-directed mutagenesis of this codon to demonstrate that any substitution conferred resistance: the 19 single Arg-133 mutants conferred between sixfold and 560-fold resistance to coumermycin A1 [35].

Table 1. GyrB mutations conferring coumarin resistance. Organism

Mutation (conserved Arg or Lys in bold)

Reference

Escherichia coli

Arg-136 to Ser, Cys, His, Leu, or Ala; Asp-73 to Glu; Gly-77 to Ala or Ser; Ile-78 to Ala or Leu; Gly-164 to Val; or Thr-165 to Ala or Val

[27, 28, 36]

Haloferax alicantei

Arg-137 to His, Asp-82 to Gly, Ser-122 to Thr (triple mutant)

[29]

Streptococcus pneumoniae

Ser-127 to Leu

[37]

Staphylococcus aureus

Arg-144 to Ser or Ile; Gly-85 to Ser; Ile-102 to Ser; Ser-128 to Leu; Thr-173 to Asn or Ala; or Ile-175 to Thr; or double mutants, including with Ile-56 to Ser

Bartonella bacilliformis

Arg-184 to Gln; Gly-124 to Ser; or Thr-214 to Ala or Ile

[30]

Treponema denticola

Lys-136 to Glu or Thr

[31]

Brachyspira hyodysenteriae

Gly-78 to Ser or Cys; or Thr-166 to Ala

[38]

Thermoplasma acidophilum

Arg-136 to His

[33]

Borrelia burgdorferi

Arg-133 to Gly or Ile, or any other substitution; or double and triple mutants, including with Asn-102 to Asp and/or Gly-104 to Asp; or other mutations at Gly-74 or Thr-162

[32, 34]

[13, 35]

We next hypothesized that a second-site mutation would increase resistance. To test this hypothesis, we plated Arg-133 to Ile mutants, which were marked with a second silent mutation in the Arg-133 to Ile codon (ATT instead of the background single ATA mutation), in higher levels of coumermycin A1. Two types of second-site mutations were isolated: either Asn-102 to Asp or Gly-104 to Asp. These residues are opposite Arg-133 on a lid covering the ATP-binding site (Figure 1) [26], but move distal to the protein when a coumarin binds [25]. Both mutants yielded higher levels of resistance, and the experiment yielded another question: would all three mutations confer even greater resistance? The advantage of a “super-resistant” allele for developing coumermycin A1-resistant gyrB into a selectable marker would be that one could use very high levels of coumermycin A1 to minimize the number of background mutants in genetic experiments. The additional motivation was that more mutations might decrease homologous recombination of the marker into the chromosomal gyrB locus. Therefore, one of the second-site mutations was transformed into the other double mutant. The resulting triple mutant did in fact have even higher levels of resistance. Note that the colloquial description of this triple mutant allele, “NGR,” is derived from the one letter codes for the three amino acids that were mutated

60


(N102D/G104D/R133I) and became a shortcut when writing the mutations on a multitude of culture tubes every week. 1.2. Development and Downfall of gyrB as a Selectable Marker The final step in fashioning the triple mutant gyrB allele into a selectable marker for genetic studies was to map the transcriptional start site for gyrB (and presumably the gyrBA operon [24]) in collaboration with Rich Marconi [39]. The gyrB cassette was amplified as the upstream regulatory region, and the entire open reading frame with the appropriate restriction enzymes incorporated into the primers. These coumermycin A1resistant gyrB cassettes were used for many years as the only selectable marker available. Despite problems and inconveniences, the coumermycin A1-resistant gyrB was seminally employed to demonstrate genetic transformation [16] and recombination of DNA into a heterologous site [40]. In addition, coumermycin A1 resistance was used to show that short oligonucleotides could serve as transformation substrates for bacteria [41]. Besides for illustrating the feasibility of reverse genetic experiments, several genes were disrupted with gyrB to probe their in vitro function, including ospC [42], guaB [43], oppAIV [44], gac [45], rpoS [46], and chbC [47]. The problems of the gyrB marker include extensive screening of transformants, which is due to homologous recombination into the chromosomal gyrB locus [40, 45], the large size, which we propose prevented its use in demonstrating transduction despite exhaustive efforts by former graduate student Christian Eggers [48], and pleiotropic effects, which are due to perturbed levels of DNA supercoiling. Regarding the latter issue, gyrB mutants, which have relaxed DNA supercoiling [27], have increased expression of groEL [49] and ospC [50]. Furthermore, our preliminary data suggest that the coumermycin A1-resistant gyrB allele suppressed the phenotype of at least one mutant in our laboratory. We had discovered Gac, an architectural DNA-binding protein in B. burgdorferi [24] and were curious about its function. The gac gene is uniquely embedded in the gyrA gene, which encodes the A subunit of DNA gyrase, so generating a null gac mutant without disrupting the essential gyrA gene was a challenge. The gyrB marker was used to mutate the first two methionine codons of gac, resulting in abolition of Gac synthesis without dramatically affecting GyrA [45]. Unfortunately, the gac mutants had no recognizable phenotype. However, more recent efforts using a kanamycin resistance cassette (see below) resulted in gac mutants with a growth phenotype that was readily suppressed during outgrowth of the cultures. We are currently introducing the gac mutations linked to kanamycin resistance into coumermycin A1-resistant cells to test our hypothesis that the gyrB mutation suppresses the gac mutant phenotype (and we are creating a conditional gac mutant using an inducible promoter). Gac has HU-like activity [24] and there is precedence for suppressing an HU mutant defect with a coumarin-resistant gyrB allele [51, 52]. Other experiments have revealed genetic suppression of cell division gene defects by gyrB mutations [53, 54]. 1.3. Further Developments in Genetic Manipulation A major breakthrough was when Jim Bono, a postdoctoral fellow in Patti Rosa’s laboratory, fused a strong constitutive B. burgdorferi promoter to an exogenous gene, aphI, which confers resistance to the aminoglycoside kanamycin [55]. Concurrently, Felipe Cabello’s laboratory discovered that the ermC gene conferred erythromycin


61

resistance to B. burgdorferi, and they fused a native promoter to a gfp allele [56] (although that allele does not function well in B. burgdorferi [57]). The new antibiotic resistance markers shattered the barriers to many genetic experiments (see [4] for review) and the promoter fusion method, first introduced to B. burgdorferi by Chuck Sohaskey when he was in Alan Barbour’s laboratory [58], has been wielded several times since [17, 18, 57, 59í64]. A caveat regarding the ermC marker is that some Institutional Biosafety Committees have balked at its use because erythromycin can be prescribed to treat Lyme disease in certain situations, although the discovery of natural erythromycin-resistant mutants should allay these concerns [65]. However, two other hybrid selectable markers, conferring resistance to streptomycin [18] and gentamicin [59], are now popular with molecular borreliologists. The other vital advance was the construction [57, 66], or identification [56], of shuttle vectors that replicate in B. burgdorferi. Currently, there are a suite of plasmids available for genetic studies: these are based on either pGK12, which is derived from the Lactococcus lactis plasmid pWV01 [56], pBSV2, which is derived from cp9 [66], or pCE320, which is derived from a cp32 [57]. Some of these plasmids are compatible during replication in B. burgdorferi [18]. More recently, several next-generation genetic tools have been developed: transposon mutagenesis [62, 63], inducible promoters [64, 67, 68], and a counter-selectable marker [17].

2. Fluoroquinolone Resistance and its Application as a Counter-selectable Marker Many, but not all, bacteria have two type II topoisomerases: DNA gyrase and topoisomerase IV. DNA gyrase introduces negative supercoiling into DNA, and topoisomerase IV decatenates replicated DNA as well as relaxes supercoiled DNA [69]. Topoisomerase IV is also a tetramer comprised of two A subunits, called ParC, and two B subunits, called ParE (for their role in partitioning of DNA to daughter cells). Fluoroquinolones, potent antibiotics that are widely used, target type II topoisomerases [20, 70]. Resistance to fluoroquinolones usually maps to a small region in the A subunits of DNA gyrase (GyrA) and topoisomerase IV (ParC). The primary target of fluoroquinolones, defined as the type II topoisomerase that is mutated in firststep fluoroquinolone-resistant strains, is usually DNA gyrase in gram-negative bacteria and topoisomerase IV in gram-positive bacteria, although this also depends on the particular fluoroquinolone [20, 70]. To our knowledge, no data had ever been reported on the primary target in spirochetes. Therefore, we used a genetic approach to identify the antibiotic target by selecting for mutants of B. burgdorferi that are resistant to various fluoroquinolone antibiotics [14]. 2.1. Fluoroquinolone Mechanism of Action How fluoroquinolones work is not completely understood [71, 72]. DNA topoisomerases catalyze changes in DNA topology by a nucleophilic attack with their active site tyrosine on the phosphodiester bond between nucleotides. A transesterification results in one or two covalent bonds between one or both strands and the topoisomerase. Type II topoisomerases, DNA gyrase, and topoisomerase IV attack both strands of the DNA substrate. These enzyme-linked DNA strands serve as a gate to pass other DNA strands resulting in the eponymous changes in DNA topology. The topoisomerase mechanism is completed by a second transesterification that restores the

62


integrity of the DNA. The fluoroquinolone antibiotics are thought to stabilize the covalent complex between the type II topoisomerase and DNA. These result in blockage of transcribing RNA polymerases and replicating DNA polymerases as well as in double-stranded breaks. Therefore, these antibiotics are thought to turn topoisomerases into poisons. An antibiotic-sensitive enzyme in the cell is postulated to be sufficient to cause DNA damage and cell death [71, 72]. Consequently, resistant mutations in gyrA are recessive [73]. The story with topoisomerase IV and parC mutations is not as straightforward. Results in E. coli have demonstrated codominance and gene dosage effects [74, 75]. However, parC is not the primary target of the fluoroquinolones in E. coli, although experiments in Staphylococcus aureus, where topoisomerase IV is the primary target, demonstrated fluoroquinolone-resistant parC was dominant [76]. A caveat to this latter report is that parC is the second gene in an operon with parE, and fluoroquinolone-resistant parEC was recessive, suggesting that expression of parC alone may be attenuated and result in lack of genetic interaction. 2.2. parC Mutants Resistant to Fluoroquinolones Mutants are difficult to obtain because fluoroquinolone resistance is recessive (which is one reason that fluoroquinolones are clinically successful chemotherapeutic agents). We isolated several fluoroquinolone-resistant mutants of B. burgdorferi: all of them had one of five different mutations (at three codons) in parC, the gene encoding the A subunit of topoisomerase IV, and none had mutations in gyrA [14]. This provided strong support that topoisomerase IV is the primary target of fluoroquinolone antibiotics in B. burgdorferi. The mutations were Thr-69 to Lys or Arg, Ser-70 to Pro, and Glu-73 to Gly or Lys. Thr-69 and Glu-73 are highly conserved residues that are hot spots for fluoroquinolone-resistant mutations in other bacteria (although the homolog of Thr-69 is a Ser in almost all other bacteria). The parC mutants, like the gyrB mutants, had a mild growth phenotype; they were between fourfold and 75-fold more resistant than wild-type, depending on the particular mutation and fluoroquinolone tested, with the exception that none of the mutants were resistant to the fluoroquinolone ciprofloxacin [14]. In addition, we were not able to isolate ciprofloxacin-resistant mutants. We do not know why the mutants are susceptible to ciprofloxacin (class II), but resistant to the more the potent fluoroquinolones sparfloxacin (class III), moxifloxicin (class IV), and Bay-Y3118 (experimental) [77].

Figure 2. Alignment of a portion of the ParC and GyrA proteins from B. burgdorferi (Bb), Streptococcus pneumoniae (Sp), and E. coli (Ec). The numbers at the top refer to the amino acid residues of the ParC protein of B. burgdorferi and indicate residues that are mutated in the fluoroquinolone-resistant strains (Thr69, Ser-70, and Glu-73). The asterisk (*) indicates the highly conserved serine that confers fluoroquinolone resistance when mutated; the plus (+) indicates the conserved acidic residue that is also often mutated. This figure is reprinted from Galbraith et al., Antimicrob. Agents Chemother. (2005) [14].


63

Threonine, at position 69 in ParC from B. burgdorferi, is a conservative substitution for serine, but the homologous residue in GyrA is Gln-86 (Figure 2). Wildtype B. burgdorferi are less susceptible to fluoroquinolones than most other bacteria [77, 78]. We hypothesize that the presence of a glutamine, instead of the conserved serine that is often mutated in fluoroquinolone-resistant strains, is responsible for both the natural lack of fluoroquinolone susceptibility and the primary target preference for topoisomerase IV [14]. 2.3. Development of parC as a Counter-Selectable Marker Recent advances (described above), including new selectable markers for introducing recombinant DNA, have allowed researchers to exploit the awesome power of genetics to study B. burgdorferi. However, another essential component of a genetic system, besides useful selectable markers, is a counter-selectable marker. This allows us to select for the loss of genetic material from the experimental organism. A counterselectable marker has been developed for Leptospira [79]. One strategy for counterselection involves using a merodiploid with a recessive antibiotic-resistance allele on the chromosome and a dominant antibiotic-sensitive allele on a plasmid. The merodiploid is susceptible to the antibiotic, unless the plasmid is lost. Therefore, plasmid loss is selected with the antibiotic. Although the story is complicated, we hypothesized that fluoroquinolone resistance in B. burgdorferi is recessive, because mutants were not readily isolated compared to other antibiotics such as coumermycin A1, spectinomycin, and the aminoglycosides [13, 15]. Therefore, we constructed a counter-selectable marker for B. burgdorferi using parC alleles. In Borrelia, parC appears to be in an operon with parE and lack its own promoter [22]. We have previously used the gyrBA promoter to drive transcription of a selectable marker (see above). Therefore, we fused the gyrBA promoter to the parC open reading frame. This hybrid fluoroquinolone-sensitive cassette was cloned into the shuttle vectors pBSV2 [66] and pKFSS1 [18], generating the plasmids pBSCSM and pKFCSM that confer resistance to kanamycin and streptomycin, respectively, and susceptibility to fluoroquinolones [17]. As a proof of principle, pBSCSM was transformed into the Glu-73 to Lys parC mutant that is resistant to several fluoroquinolone antibiotics [14] and the presence of the plasmid was selected for with kanamycin. The transformants were then plated with or without a fluoroquinolone antibiotic (Bay-Y3118) and with or without kanamycin. Kanamycin selects for the presence of the plasmid, and Bay-Y3118 selects against the presence of the plasmid. Ten colonies were picked from the kanamycin plate and ten from the Bay-Y3118 plate; no colonies were obtained in the presence of both kanamycin and Bay-Y3118. Plasmid maintenance was assayed by xenodiagnosis in E. coli and by PCR by using plasmid-specific primers. All ten colonies from the kanamycin plate possessed the plasmid and none of the ten colonies assayed from the Bay-Y3118 plate demonstrated evidence of the plasmid [17]. As controls, the parental plasmid pBSV2, which lacks gyrBAp::parC, does not confer fluoroquinolone susceptibility to the parC mutant, and pBSCSM cannot be selected against in the parental wild-type strain. pKFCSM has not yet been assayed, but its parental plasmid pKFSS1, which confers resistance to spectinomycin and streptomycin, is less stable than pBSV2 [18], leading us to hypothesize that it may be more readily cured upon counter-selection with fluoroquinolone. These preliminary data suggest that the hybrid counter-selectable marker functions in the cell and support our hypothesis that fluoroquinolone-resistant parC is recessive.

64


3. Spectinomycin Resistance and its Implications for the Evolution of Antibiotic Resistance We continued to develop and refine genetic tools to study B. burgdorferi on a molecular level. In the 1990s, coumermycin A1 resistance was the only available selectable marker, and shuttle vectors had neither been identified nor constructed. Our experimental excursions eventually led us to assemble a hybrid spectinomycin- and streptomycin-resistant cassette [18], now one of several selectable markers in the molecular toolbox for borreliologists [16, 55, 56, 59, 60]. This antibiotic resistance marker has been used to disrupt genes [80-83] and to complement mutants [67, 84í88]. 3.1. Development of aadA as a Selectable Marker We originally attempted to construct a shuttle vector by cloning the coumermycin A1resistant selectable marker into a variety of broad-host-range plasmids: pRKY55 (a derivative of pTJS133) [89], pUFR047 (a derivative of pSa747) [90], pBBR1MCS [91], and pDSK600 and pVLT35 (derivatives of pRSF1010) [92, 93]. None of these recombinant plasmids replicated independently in B. burgdorferi for us. However, when we transformed with pVLT35 carrying a coumermycin A1-resistant gyrB, a single crossover recombination event resulted in the insertion of the intact recombinant plasmid in the B. burgdorferi chromosome and the consequent duplication of the gyrB gene [94], which has been observed by others [95]. The recombined plasmid also carried the aadA gene [96], which confers resistance to spectinomycin and streptomycin. Preliminary data suggested that the transformant had low-level resistance to these antibiotics, which motivated us to fuse a B. burgdorferi promoter to the aadA gene, as previously described [55], and clone it onto the extant shuttle vector pBSV2 [66]. The new plasmid, pBV102, conferred a high level of resistance to both spectinomycin and streptomycin to B. burgdorferi. Therefore we replaced the aphI open reading frame, which encodes kanamycin resistance, on pBSV2 with the aadA open reading frame, thus creating pKFSS1 [18]. pKFSS1 conferred resistance to spectinomycin and streptomycin and could be comaintained in transformants with its parental plasmid pBSV2 or with pCE320; selection of B. burgdorferi transformants with spectinomycin failed, resulting in many colonies that did not contain the plasmid [18]. Note that the hybrid cassette also confers resistance to spectinomycin and streptomycin in E. coli, but many common laboratory strains are resistant to streptomycin. Because of these limitations, spectinomycin is used to select in E. coli, and streptomycin is used to select in B. burgdorferi. 3.2. Spectinomycin-Resistant Mutants Have a Low Fitness Cost We were curious why spectinomycin selection failed, yielding large numbers of spectinomycin-resistant B. burgdorferi colonies [18]. We first hypothesized that these clones had a mutation in the small subunit of their ribosomes, a well-established target for spectinomycin. Spectinomycin-resistant mutations have been described in the S5 protein or the 16S rRNA of several organisms. All of the B. burgdorferi spectinomycinresistant clones had mutations in the 16S rRNA gene, encoding either A1185 to G or C1186 to U [15]. These mutants were over a 1000-fold more resistant to spectinomycin than wild type. We also isolated mutants resistant to the closely related aminoglycoside


8 6 4 2 0 WT

16S 16S 16S C1186U A1185G A1402G

S12 K88E

Antibiotic-resistant colonies (%)

B.

10

Doubling time (h)

A.

50

65

100

generations

50 40 30 20 10 0 WT + WT + C1186U A1185G

WT + K88E

WT + A1402G

Figure 3. Growth rate and competition of antibiotic resistant mutants of B. burgdorferi compared to wildtype (WT). A. The doubling time of the wild-type strain is not significantly different from the doubling times of the antibiotic-resistant mutants, although the kanamycin- and gentamicin-resistant mutant (16S A1402G) grows slightly slower; values are means plus the standard error of the means (SEM) for a total of five replicates in three independent experiments. B. The two spectinomycin-resistant mutants (C1186U and A1185G), but neither the streptomycin-resistant mutant (K88E) nor the kanamycin- and gentamicin-resistant mutant (A1402G) successfully compete with the wild-type strain during 50 to 100 generations of coculture; values are means ± SEM for three to five independent experiments.

antibiotics kanamycin, gentamicin, and streptomycin, which also target the small subunit of the ribosome. The kanamycin-resistant and gentamicin-resistant clones both had an A1402 to G mutation in their 16S rRNA and were about 100-fold resistant to either antibiotic [15]. The clones selected in streptomycin were about tenfold resistant and had mutations in rpsL, encoding Lys-88 to either Arg or Glu in the S12 protein [15]. All of these mutations in B. burgdorferi had precedence in other organisms. The frequency of the spectinomycin-resistant mutants was 6 × 10–6, which was about 100fold higher than the frequency of the aminoglycoside-resistant mutants. We next hypothesized that there was a large subpopulation of spectinomycinresistant cells with a 16S rRNA mutation in the parental strain, a highly passaged B31, and that the high frequency was a result of a lower fitness cost for the 16S rRNA C1186 to U and A1185 to G mutations conferring spectinomycin resistance. Again, there was precedence in other organisms, historically in E. coli and more recently in Chlamydia psittaci [97]. Growth assays (Figure 3A) demonstrated that the spectinomycin-resistant mutants grew at the same rate as wild-type in vitro without selection; however, a streptomycin-resistant strain with a Lys-88 to Glu mutation in the S12 protein also grew at the same rate [15]. To assess fitness, competition assays followed mixed cultures of wild type and antibiotic-resistant mutants over 50 or 100 generations [15]. The competition assays (Figure 3B) showed that the spectinomycinresistant mutants were maintained in the co-culture at close to the starting frequency for 50 generations and were still a significant presence in the culture after 100 generations, while the aminoglycoside-resistant mutants were greatly diminished at 50 generations and absent from the culture at 100 generations [15]. These results suggested that there was little fitness cost associated with spectinomycin resistance, especially with the C1186 to U mutation.


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3.3. Antibiotic Resistance in the Clinic and in the Laboratory Antibiotic resistance has not yet become a recognized problem in treating Lyme disease. In addition, spectinomycin and the other antibiotics discussed in this chapter are not used to treat B. burgdorferi infections. However, our results indicate that B. burgdorferi can become resistant by mutation of antibiotic targets. Other resistance mechanisms, such as preventing an antibiotic from entering a cell, pumping an antibiotic out of a cell, or inactivating an antibiotic, have also not been identified in clinical isolates; however, there is no a priori reason that antibiotic-resistant mutants cannot emerge in Lyme disease patients. Moreover, our data on spectinomycin resistance suggest that certain antibiotic-resistant mutants have minimal fitness cost and thus may be able to survive even in the absence of the antibiotic, which may have a profound effect on the evolution and dissemination of resistance [98, 99]. Although discussion of antibiotic resistance in Lyme disease patients is purely conjectural, there are profound implications for developing genetic markers used for experimentally manipulating B. burgdorferi. Resistance to aminoglycosides [18, 55, 59, 60] has been extensively exploited in these endeavors [4]. The high frequency of spectinomycin-resistant mutants in a population of B. burgdorferi cells constrains selection with the aadA cassette to E. coli [18] and serves as a caveat in augmenting the genetic tools for progressively elaborate molecular experiments.

Acknowledgments I thank Christian Eggers and Amanda Ng for their thoughtful and critical reading of this manuscript; the past and current members of my laboratory, especially Janet Alverson, Sharyl Bundle, Dan Criswell, Betsy Eggers, Christian Eggers, Kristi Frank, Kendal Galbraith, Mike Gilbert, Scott Knight, Craig Kuchel, Meghan Lybecker, Kathy Mach, Mike Mazzotta, Elizabeth Morton, Amanda Ng, Virginia Tobiason, and Beth Todd; and my colleagues, especially Steve Lodmell, Mike Minnick, Nyles Charon, Mike Norgard, Justin Radolf, Kit Tilly, Rich Marconi, Xiaofeng Yang, George Chaconas, Phil Stewart, Chuck Sohaskey, Wai Mun Huang, Claude Garon, Cathy Lawson, Thad Stanton, Melissa Caimano, Bob Gilmore, Darrin Akins, Jon Skare, Aravinda de Silva, John Leong, Ira Schwartz, Mathieu Picardeau, Jim Miller, Steve Norris, Alan Barbour, David Haake, Erol Fikrig, Tom Schwan, Patti Rosa, Joe Hinnebusch, Karl Drlica, Tony Maxwell, Peter Heisig, and Christian Eggers. Our research is supported by a grant from the National Institutes of Health (AI051486) and was supported by a grant from the National Science Foundation (MCB-9722408).

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Development of Treponeme Genetic Systems a

Howard K. KURAMITSU a,1 and Caroline E. CAMERON b Department of Oral Biology, State University of New York at Buffalo, Buffalo, NY, USA b University of Washington, Seattle, WA 98195, USA Abstract. Electroporation of Treponema denticola allowed for construction of monospecific mutants in this oral spirochete. The subsequent construction of shuttle plasmids enabled the expression of heterologous genes in these organisms. More recently, a comparable expression system has been developed in T. phagedenis. Keywords. Treponema denticola, shuttle plasmids, electroporation, mutants, Treponema phagedenis

Introduction Treponemes have been recognized as being responsible for a number of human diseases including syphilis [1]. In addition, a variety of these organisms are associated with the common human malady periodontitis [2]. The relatively fastidious nature of these organisms has made it difficult to cultivate and examine them in the laboratory. This has been especially true in attempting to apply molecular genetic approaches in defining the virulence properties of these organisms. This review will describe the development of genetic systems in the past decade for examining the physiological properties of selected treponemes. One of the spirochetes associated with periodontitis, Treponema denticola, can be cultivated in the laboratory and has served as a model organism for noncultivable members of this genus [2]. Biochemical approaches identified several potential virulence factors of these organisms including a major outer surface protein, Msp [3], as well as a serine protease, PrtP [4]. However, the absence of mutants defective in these proteins made it difficult to verify the role of each of these factors in pathogenicity. For this reason, initial attempts were carried out to develop a gene transfer system in these organisms.

1 Corresponding Author: Howard K. Kuramitsu, Department of Oral Biology, SUNY at Buffalo, Buffalo, NY 14214; E-mail: [email protected].

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H.K. Kuramitsu and C.E. Cameron / Development of Treponeme Genetic Systems

1. Gene Transfer in T. denticola Initially, markers for detecting transfer of heterologous plasmids into T. denticola were sought by examining the antibiotic sensitivity of two strains of the organism, ATCC 33520 and 35405 [5]. This analysis suggested that either erythromycin or chloramphenicol might allow for the detection of gene transfer into these organisms. Therefore, the broad-host range plasmid encoding chloramphenicol resistance, pKT210, was electroporated into strain 33520 and transformants were identified [5]. Although some modification of the plasmid was detected, this demonstration indicated that it was possible to introduce genetic material into T. denticola following electroporation.

2. Mutant Construction in T. denticola The demonstration of gene transfer into T. denticola 33520 suggested that electroporation might be useful to construct monospecific mutants in these organisms. Therefore, since the background of spontaneous chloramphenicol resistant mutants in T. denticola appeared to be higher than erythromycin resistant mutants, the ermFermAM cassette [6] was utilized to interrupt T. denticola genes. Initially, mutagenesis of the flgE gene of strain 35405 was attempted since inactivation of this gene coding for the flagellar hook protein would lead to an easily recognizable nonmotile phenotype. Following electroporation of an flgE gene fragment interrupted with the erm cassette into strain 35405, Ermr colonies were identified that appeared to be nonmotile on agarose plates [7]. Confirmation of the correct double cross-over integration event indicated that this procedure could be used to construct monospecific mutants in T. denticola. Subsequently, a number of different laboratories have used this procedure to construct defined mutants of the organism [8]. Most recently, this approach has been utilized to identify T. denticola genes that are involved in synergistic biofilm formation with Porphyromonas gingivalis [9]. This was also one of the first reports examining biofilm formation by a spirochete. The availability of a system to inactive genes in T. denticola also allows for the identification of factors that are involved in the potential virulence of these organisms. For example, inactivation of the prtP gene coding for the serine protease dentilisin of strain 35405 followed by inoculation into mice has demonstrated that the protease is a likely virulence factor in these organisms [10].

3. Heterologous Gene Expression in T. denticola Since a number of pathogenic treponemes cannot be readily cultivated in the laboratory, the ability to express genes from these organisms in a genetically tractable treponeme could provide a means to investigate genes from the former organisms. Therefore, the utilization of T. denticola for such purposes was investigated. In order to construct a shuttle plasmid for use in T. denticola, a replication region from a naturally occurring plasmid was sought. In this regard, two plasmids were identified, pTD1 and pTS1, and characterized in two different strains of T. denticola [11, 12]. Therefore, based upon the nucleotide sequence of plasmid pTS1 [13], a putative replication region was identified in this plasmid and was used to construct a shuttle plasmid that could be utilized in both T. denticola and Escherichia coli [14]. The plasmid also contained the ermFermAB


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cassette immediately downstream from the promoter region of the T. denticola prtB gene. Using this shuttle plasmid, pKMR4PE, it was possible to express the flaA gene from T. palllidum in T. denticola 33520 [14]. More recently, Limberger and coworkers [15] have constructed a similar plasmid expressing chloramphenicol resistance and have expressed the fliG gene from T. pallidum in strain 33520. Interestingly, shuttle plasmids could be successfully introduced into strain 33520, which harbors the naturally occurring plasmid pTD1, but not in strain 35405, which does not contain a plasmid. However, the molecular basis for this difference has not yet been elucidated. It was also of interest to determine if nontreponemal genes could be expressed in T. denticola as reporter genes. Therefore, the lacZ gene from E. coli coding for ȕgalactosidase activity was engineered downstream of the erm cassette in plasmid pKMR4PE and introduced into strain 33520. On X-gal agarose plates, the transformants appeared dark blue while the control strain was colorless (Figure 1). Direct assays for enzyme activity also showed a significant increase in the strain harboring the lacZ-containing plasmid relative to 33520 with the shuttle plasmid alone. These results indicated that some nontreponemal genes could also be expressed in T. denticola and that the lacZ gene could be used as a reporter gene in these organisms. More recently, the same shuttle plasmid was utilized to express the green-fluorescence protein gene in T. denticola 33520 although the resulting fluorescence was not as stable nor as strong as the protein expressed in E. coli [16]. However, it may be possible to express stronger fluorescence in these organisms using more recently constructed gfp genes.

4. Complementation Analysis in T. denticola One of the advantageous of developing a shuttle plasmid system is the ability to express genes in the organism for complementation of either mutations or naturally occurring defects. For complementation of Ermr mutants in strain 33520, a shuttle plasmid based upon the pKMR4PE plasmid but expressing coumerymycin resistance was developed [17]. This plasmid was utilized to express the T. denticola flgE gene in the strain 33520 flgE mutant and was demonstrated to restore the motility of the mutant. More recently, a similar shuttle plasmid expressing a T. pallidum fliG gene was utilized to complement a fliG mutation in strain 33520 [15]. Therefore, these systems can be valuable in characterizing genes isolated from noncultivable treponemes such as T. pallidum. Because of the inability to establish the pTS1-based shuttle plasmids in strain 35405, it was not possible to complement mutants constructed in this strain. A comparable interference strategy was also used to confirm a role for the leucine-rich repeat protein, LrrA, in the motility of T. denticola [18]. When the lrrA gene from strain 35405 was introduced into strain 33520, which is motile and does not contain a homolog of the lrrA gene, the resultant transformant was attenuated in motility. This confirmed a role for the LrrA protein in the motility of T. denticola as suggested from the properties of an lrrA mutant in strain 35405.

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A

B

Figure 1. lacZ expression from T. denticola 33520. A, colonies on X-gal agarose plates containing pKMR4PE-lacZ; B, shuttle plasmid alone. Arrows indicate location of colonies.

5. Expression of Heterologous Treponeme Genes in T. phagedenis Since T. pallidum and T. denticola have been isolated in humans, it was of interest to develop a gene transfer system in a treponeme which is not a normal human colonizer since such an organism would be expected to be physiologically distinct from those inhabiting the human host. T. phagedenis is one such organism and also is very similar to T. denticola in its G+C ratio [19]. Therefore, the shuttle plasmids developed for use in T. denticola might also function in T. phagedenis as well. Utilizing T. phagedenis Kazan, it was indeed possible to demonstrate the transformation of shuttle plasmid pKMR4PE into this organism (Yamada and Kuramitsu, unpublished results). Therefore, the same shuttle plasmid system used in T. denticola was evaluated in T. phagedenis for expression of heterologous treponeme genes. One of the potential virulence properties of T. pallidum is its ability to attach to the extracellular matrix, ECM, including the glycoprotein laminin [20]. Recently, one of our laboratories has isolated a gene from T. pallidum coding for the laminin-binding adhesin Tp0751 [20]. Confirmation of the role of this adhesin in such binding was sought by expression of a laminin-binding domain of Tp0751 in the non-lamininbinding T. phagedenis. Using a derivative of the T. denticola-E. coli shuttle plasmid pKMR4PE, a 159 bp DNA fragment from the tp0751 gene containing its ribosome binding site, putative signal sequence, and amino acids involved in laminin binding [21] was introduced into the shuttle plasmid. Transformation of T. phagedenis following the electroporation protocol developed for T. denticola [14] allowed for the identification of transformants harboring the correct chimeric plasmid. Northern blot analysis allowed for the identification of the mRNA corresponding to the T. pallidum insert (data not shown). To determine if T. phagedenis expressing the laminin-binding domain of Tp0751 could mediate binding to laminin, laminin-coated glass slides were incubated with the transformant and binding compared with the organism containing only the shuttle plasmid under dark field microscopy. These results indicated that the construct expressing the laminin-binding domain, but not that containing only the shuttle plasmid, bound to the laminin-coated slides. Quantitation of these results by


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counting the number of organisms in six separate fields revealed that there was a significant increase in attachment when the Tp0751 laminin-binding domain was expressed in T. phagedenis (Figure 2). These results confirmed the laminin-binding activity of the Tp0751 adhesin and also indicated that T. phagedenis can serve as a convenient treponeme host for the expression of genes from some noncultivable treponemes.

Number of Treponemes/Field

100

WT pKMR 751/pKMR

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p95% identical based on hybridization. One hopes that in the future, comparison of these sequences will shed light on which virulence factors are responsible for the unique host ranges and infection phenotypes. In an initial study, using restriction enzyme digestion of segments of the genome produced by long-range PCR as the basis for comparison, only four differences were found between the syphilis and yaws bacteria, and each of these were only single genes (E. Sodergren et al., unpublished). This result is consistent with the high degree of conservation seen by hybridization and suggests that the phenotypic differences may reside at the single nucleotide level. Moreover, all four differences were in or adjacent to tpr genes, fortifying the notion that these are changes affecting virulence genes that give the distinctive treponemal infections. Similar results were found in studies of other treponemes in the table (M. Strouhal et al., unpublished). To probe the differences more deeply, hybridization to oligonucleotide arrays was performed. In this case the oligonucleotide array was constructed at NimbleGen and comprised 29-mers spaced seven nucleotides apart along the genome. Comparison of two syphilis strains showed slightly more than 300 single nucleotide polymorphisms (SNPs), while comparison of syphilis to yaws bacteria showed of the order of 1000 SNPs and at least several times this number were found in comparing the human and rabbit syphilis strains (P. Matejkova et al., unpublished). For a sense of scale, the syphilis-yaws comparison indicates about 1 SNP per gene, yet only a subset of these are expected to cause nonconservative changes in protein structure, so only a fraction of the genes are affected.

3. Conclusions In the century since T. pallidum was identified there have been notable successes in the struggle to control syphilis and understand this bacterium. Notable among these are the invention of the first antimicrobial treatments, such as Salvarsan, by Paul Ehrlich, the discovery of the exquisite sensitivity of T. pallidum to penicillin, and even a Nobel Prize in 1927 for treatment of advanced syphilis by infection with Plasmodium. However, the genome sequence published in 1998 ushered in a whole new era, the early stages of which are described here. We can certainly expect the future to hold further discoveries about not only syphilis and T. pallidum, but also the other fascinating close relatives.

References [1] [2] [3] [4]

Cox, D.L. Culture of Treponema pallidum. Methods Enzymol, 1994. 236: 390–405. Norris, S.J., et al. Treponema and other human host-associated spirochetes, in Manual of Clinical Microbiology, P.R. Murray, Editor. 2003, ASM Press: Washington, D.C., Chapter 61. Waugh, M. The centenary of Treponema pallidum: on the discovery of Spirochaeta pallida. Skinmed, 2005. 4(5): 313–315. Fraser, C.M., et al. Complete genome sequence of Treponema pallidum, the syphilis spirochete. Science, 1998. 281(5375): 375–388.

100 [5] [6] [7] [8] [9] [10] [11] [12] [13] [14]

G.M. Weinstock et al. / Treponema Genomics Weinstock, G.M., et al. The genome of Treponema pallidum: new light on the agent of syphilis. FEMS Microbiol Rev, 1998. 22(4): 323–332. Weinstock, G.M., et al. Identification of virulence genes in silico: infectious disease genomics, in Virulence Mechanisms of Bacterial Pathogens, K.A. Brogden, et al., Editor. 2000, ASM Press: Washington, D.C., 251–-261. Mathers, D.A., et al. The major surface protein complex of Treponema denticola depolarizes and induces ion channels in HeLa cell membranes. Infect Immun, 1996. 64(8): 2904–2910. Fenno, J.C., K.H. Muller, and B.C. McBride Sequence analysis, expression, and binding activity of recombinant major outer sheath protein (Msp) of Treponema denticola. J Bacteriol, 1996. 178(9): 2489–2497. Norris, S.J., D.L. Cox, and G.M. Weinstock. Biology of Treponema pallidum: correlation of functional activities with genome sequence data. J Mol Microbiol Biotechnol, 2001. 3(1): 37–62. McKevitt, M., et al. Systematic cloning of Treponema pallidum open reading frames for protein expression and antigen discovery. Genome Res, 2003. 13(7): 1665–1674. McKevitt, M., et al. Genome scale identification of Treponema pallidum antigens. Infect Immun, 2005. 73(7): 4445–4450. Brinkman, M.B., et al. Reactivity of antibodies from syphilis patients to a protein array representing the Treponema pallidum proteome. J Clin Microbiol, 2006. 44(3): 888–891. Smajs, D., et al. Transcriptome of Treponema pallidum: gene expression profile during experimental rabbit infection. J Bacteriol, 2005. 187(5): 1866–1874. Seshadri, R., et al. Comparison of the genome of the oral pathogen Treponema denticola with other spirochete genomes. Proc Natl Acad Sci U S A, 2004. 101(15): 5646–5651.


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Comparative Analysis of Pathogenic Leptospira Genomes Richard L. ZUERNER a,*,1 , Dieter M. BULACH b,c,*, Torsten SEEMANN c, Ross L. COPPELb,c,d and Ben ADLER b,c,d a Bacterial Diseases of Livestock Research Unit, National Animal Disease Center, USDA Agricultural Research Service, Ames, IA 50010, USA b Australian Bacterial Pathogenesis Program, Department of Microbiology, Monash University, VIC 3800, Australia c Victorian Bioinformatics Consortium, Monash University, VIC 3800, Australia d Australian Research Council Centre of Excellence in Structural and Functional Microbial Genomics, Department of Microbiology, Monash University, VIC 3800, Australia Abstract. The presence of two chromosomes makes Leptospira unusual amongst its closest relatives in the bacterial world. The Leptospira genome is in a state of flux, as indicated by the presence of many chromosomal rearrangements that alter genetic organization between individual serovars. It is therefore somewhat remarkable that at least two Leptospira loci (LPS biosynthetic genes and the S10spc-alpha ribosomal protein operon) form large, extended operons that are among the longest bacterial operons reported to date. Insertion sequences (IS) that are distributed throughout Leptospira genomes contribute to the formation of rearrangements. These elements can transpose and disrupt the integrity of genes, or alternatively, can activate cryptic genes by providing promoter activity to genomic sequences downstream of the insertion site. Bioinformatics and experimental functional analyses were used to characterize the L. interrogans genomes and thus gain insight into this organism’s biology. Quantitative analysis of the L. interrogans serovars Lai and Copenhageni genomes showed that these bacteria are proficient in environmental sensing and response, and in nutrient transport. These data support epidemiological evidence that L. interrogans is transmitted primarily by passage through environmental sources. Few pseudogenes were detected in either strain, suggesting that there is sufficient selective pressure to maintain a highly functional genome. However, several genes were identified that are complete in one strain but have frameshifts in the other that may affect phenotype. Further differences in phenotype may also result from gene acquisition, and we found several large, serovar-specific gene clusters. Analysis of an ECF locus from L. interrogans serovar Pomona is used to show how RT-PCR and expression vectors can be used to localize promoters in L. interrogans. Antisera produced against recombinant fusion proteins were used to detect invasion of lung, liver, and kidney during experimental infection of hamsters with serovar Pomona. These data are consistent with some of the clinical manifestations of severe leptospirosis and

1 Corresponding Author: Richard L. Zuerner, Bacterial Diseases of Livestock Research Unit, National Animal Disease Center, USDA Agricultural Research Service, Ames, IA 50010, USA. * Denotes first co-authors.

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R.L. Zuerner et al. / Comparative Analysis of Pathogenic Leptospira Genomes help to illustrate how genomic analysis can aid in the understanding of these pathogenic bacteria. Keywords. Leptospira, genomics, insertion sequences, recombinant proteins

Introduction Leptospirosis is one of the most common and widespread zoonotic diseases known [1]. This disease is caused by pathogenic species of Leptospira; most mammalian species are susceptible to infection [2]. Infection occurs through exposure of contaminated body fluids, principally urine from infected animals, enabling the bacteria to gain access through mucous membranes or abrasions. Following dissemination through the blood, Leptospira concentrates in liver and kidney. Release of viable bacteria through the urine is an important part of the transmission process, leading to infection of other animals by either direct exposure, or by contamination of water sources that are then a broader source of infection [2]. Periodic epidemics of human leptospirosis coincide with seasonal flooding in urban areas and are principally associated with environmental contamination from urine originating from chronically infected maintenance hosts [3]. Leptospirosis is manifested in one of two forms, as either a chronic infection with low mortality or an acute infection with high mortality [2]. Chronic Leptospira infections in maintenance host species result in little apparent disease in adults but can induce reproductive failure (abortion, stillbirth, and weak offspring) during gestation. Normal maintenance hosts are a persistent source of infection for wildlife, domesticated species, and humans. For example, rats chronically infected with L. interrogans are one of the most common sources of environmental contamination leading to human leptospirosis outbreaks during urban flooding [3]. Chronic Leptospira infections in livestock, especially cattle, can result in economic losses due to reduced herd vitality and lost milk production, and may be a source of health risk for individuals in the livestock industry. Accidental exposure of non-maintenance hosts to contaminated fluids, either directly or from environmental sources, can result in acute infection. The clinical symptoms resulting from accidental exposure to pathogenic Leptospira can range from a mild, influenza-like disease to an acute, severe infection resulting in death from multiple organ failure. The same strains that cause chronic infections in their normal maintenance hosts can cause severe infections in nonmaintenance hosts [2]. Progress in analyzing pathogenic Leptospira has been slow for several reasons. Leptospira is fastidious, and these bacteria utilize fatty acids and fatty alcohols as carbon and energy sources. Although some strains can grow in a defined, protein-free medium, protein, in the form of bovine serum albumin, is often needed to bind free fatty acids to overcome toxicity [4]. Leptospira grows slowly, and primary isolation from clinical samples is often difficult. Few tools for genetic manipulation of pathogenic strains are available and are limited to transposon mutagenesis [5, 6]. Methodology enabling the generation of gene-specific mutations is still lacking, and there are no methods for genetic complementation. Thus, genetic analysis of pathogenic Leptospira has relied on gene isolation and sequencing, and more recently, genomic analysis. In this paper, we discuss developments in the genomic analysis of pathogenic Leptospira leading to new information on the biology of these bacteria.

R.L. Zuerner et al. / Comparative Analysis of Pathogenic Leptospira Genomes

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1. Genetic Organization Although Leptospira shares a common ancestry with Brachyspira, Borrelia, and Treponema, genetic organization among these bacteria is quite different. The L. interrogans genome is nearly 5 Mbp in length and is composed of two circular chromosomal replicons present at nearly equal molar ratios [7]. Early studies suggested that the larger chromosomal replicon (CI) resembled a typical bacterial chromosome, containing all copies of ribosomal rRNA genes and a typical replication origin [8, 9]. The small chromosomal replicon (CII) is a constant feature among Leptospira, and these early studies showed the presence of a unique copy of asd, a gene essential for cell wall biosynthesis [8]. Further characterization of both replicons lagged until genomic sequencing data for two L. interrogans serovars, Lai and Copenhageni, were reported [10, 11]. Through genomic sequence analysis, we now have a much better and detailed understanding of the genetic content of L. interrogans; these data provide a foundation for identifying virulence traits, antigenic proteins, and metabolic potential that previous mapping studies could not generate. Genomic sequence analysis revealed that all tRNA and most housekeeping functions are encoded on the CI replicon [10, 11]. As with many other bacteria, the Leptospira replication origin coincides with a sharp deflection in the GC content of the leading strand, also known as GC skew. Although there is a sharp deflection in the GC skew in the CII replicon, the genetic content surrounding this region is different from that of CI. Instead of genes typical of chromosomal replication origins (for example, dnaA and gidA) on CII, the GC skew is located adjacent to parA and parB, two genes associated with plasmid and chromosomal partitioning. This finding suggests that L. interrogans utilizes the parAB gene system for segregation of the small chromosome.

2. Genomic Analysis To provide an accurate and detailed comparison of the L. interrogans serovar Lai and Copenhageni genome sequences, we modified the annotation by applying consistent criteria for all encoded proteins, including determining the consensus amino terminus through BLASTP analysis, and removed several small putative coding sequences (CDS) that lacked credible upstream translation initiation sequences. By revising the annotation of these two genomes, we established a format useful for comparison of additional Leptospira genome sequences as they become available. Furthermore, this process created a platform for consistent annotation of Leptospira genome sequences, making comparative analysis easier and more accurate. The revised annotation of both strains is summarized in Table 1. The CDS line in this table shows what we define as the “functional genome” in comparison to the total coding potential of these bacteria (that is, no pseudogenes, gene fragments, or transposable elements). The L. interrogans genome contains a small proportion (2–3%) of pseudogenes. By comparison, many pathogenic bacteria have a significantly higher proportion of pseudogenes. This finding suggests that there is strong selective pressure on L. interrogans to maintain gene integrity. Both strains have IS elements distributed around the CI replicon; serovar Lai also has IS elements on CII [10, 11]. Based on hybridization analysis, the L. interrogans serovar Pomona genome has more copies of


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Table 1. Essential features of the L. interrogans genome. Serovar Copenhageni

Serovar Lai

Feature Size (Kb)

CI

CII

CI

CII

4,280

350

4,330

359

Protein coding sequences CDS 1

3,110

274

3,114

272

With assigned function

1,813

159

1,799

157

Conserved hypothetical

496

35

497

34

Unique hypothetical

801

80

818

81

Pseudogenes

65

5

109

17

Transposases

76

0

90

11

3,251

279

3,313

300

37

0

37

0

2

0

1

0

Total Transfer RNA genes Ribosomal RNA genes 23S

1

16S

2

0

2

0

5S

1

0

1

0

not including transposases or pseudogenes

the elements IS1500 [12], IS1501 (data not shown), and IS1502 [9] than either serovar Lai or Copenhageni. This may contribute to the large number of rearrangements that differentiate these strains (see below). Quantitative analysis of L. interrogans CDS was done using the clusters of orthologous genes (COGs) approach [13, 14]. The L. interrogans genome encodes a large number of proteins associated with environmental sensing and response, and diverse transport functions (Figure 1). These findings are consistent with the survival requirements of bacteria that routinely pass through water or other environmental niches between mammalian hosts. Two of the largest groups in L. interrogans are the poorly characterized conserved hypothetical proteins (R and S). Few genes in either serovar are unique. In serovar Lai, there are 54 serovar-specific genes not present in serovar Copenhageni. Conversely, there are 31 serovar Copenhageni-specific genes not present in serovar Lai. In both strains, several of the serovar-specific genes are found as clusters, with some extending approximately 40 kb in length. The presence of these clustered, serovar-specific genes suggests they may have been acquired through horizontal gene transfer. In both serovars, most of the unique CDS encode hypothetical proteins, so it is unclear how they may


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Figure 1. COG analysis of the L. interrogans genome. Each segment represents the proportion of the genome encoding proteins in each COG category. General COG categories (starting at the top of the chart and going clockwise) include: information storage and processing (COG J, A, K, L, and B), cellular processes and signaling (COG D, Y, V, T, M, N, U, and O), metabolism and transport (COG C, G, E, F, H, I, P, and Q), and poorly characterized groups R and S.

affect phenotype. Copenhageni-specific genes that have putative functions include a ferrodoxin related protein, tautomerase, and an AcrR family transcription factor, while serovar-specific genes in Lai include a nucleotidyltransferase, proteic killer gene system, and a gene encoding a stability toxin. Further diversification between serovars Lai and Copenhageni is achieved through the generation of pseudogenes. There are only six pseudogenes common to both serovars, with all other CDS being intact in the other serovar. Many of the serovarspecific pseudogenes occur in CDS encoding hypothetical proteins, making it difficult to assess their potential role in altering phenotype. However, several serovar-specific pseudogenes are in CDS encoding proteins having presumed functions based on their homology with better-characterized proteins. For example, serovar Copenhageni has frameshift mutations (pseudogenes) in genes encoding glutathione transferase, LigC, a TonB dependent receptor, and a flavoprotein. Pseudogenes in serovar Lai are found in CDS encoding a TPR-repeat protein, a Na+/H+ antiporter, a phospholipase D like lipoprotein, an adenylate-guanylate cyclase, and a phospholipid synthase. Mutations in some of these genes, for example, mutation of the adenylate-guanylate cyclase may have broad impact on gene expression, as this group of proteins participates in global gene regulation.

3. Genetic Rearrangements Several studies have shown that genetic organization in L. interrogans is quite variable. The larger (CI) replicon undergoes extensive rearrangements (inversions, deletions, and

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Figure 2. The CI replicons from L. interrogans serovars Pomona and Icterohaemorrhagiae are represented as horizontal lines. The lines drawn between the two maps denote the relative position of identical genes. Lines that intersect or diverge indicate rearrangements between the two chromosomal replicons. The sigE locus is localized in serovar Pomona near rrlB.

insertions), which dramatically alter global genetic organization of closely related strains [8]. Figure 2 is a comparison of the genetic maps of two serovars of L. interrogans showing the presence of extensive rearrangements throughout the CI replicon. This propensity for rearrangement is likely to be limited by viability, given the absence of rearrangements in two key loci. Specifically, there is no evidence of rearrangement in the LPS [15] and S10-spc-alpha [16] loci, each forming large operons that generate some of the longest bacterial transcripts known. Studies comparing the restriction endonuclease digestion patterns of L. interrogans serovar Pomona isolates showed the presence of several polymorphisms that may affect important phenotypic characteristics including antigenicity, or adaptation to a particular host species [17]. DNA hybridization studies indicate that serovars Icterohaemorrhagiae and Pomona share about 84% similarity in genetic composition [18] and have therefore undergone considerable divergence. In contrast, the two L. interrogans strains for which genomic sequencing has been completed, serovars Lai and Copenhageni, share about 98% of their genetic composition [10, 11, 18]. Comparison of the CI replicons from L. interrogans serovars Lai and Copenhageni reveal the presence of a large inversion (Figure 3A) that coincides with a rearrangement between two copies of an IS element [10]. The role of IS elements in generating chromosomal rearrangements in L. interrogans was first suggested in a study describing IS1500 [12]. Localization of some IS1500 insertions showed these often coincide with regions of the genome that have undergone large rearrangements [12]. These data are also consistent with studies that


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Figure 3. Linear representation of the genomic sequences, both CI (A) and CII (B), of L. interrogans serovars Lai (top) and Copenhageni (bottom). Regions of sequence similarity were identified by using Megablast and visualized using ACT. Black lines denote regions of highly similar sequence between the map lines drawn in grey.

have used IS elements as epidemiological tools to identify serovars [17]. In contrast to the variability seen in the CI replicon, genetic organization in CII appears to be more stable (Figure 3B), and recombination between the CI and CII replicons is not apparent.

4. Insertion Sequences The role of IS elements in altering chromosome organization in Leptospira spp. has been suggested by several studies. IS elements are a group of elements that can transpose to new sites in the genome [19]. Many IS elements follow a classic structural organization, consisting of a central “unique” region that is often flanked by terminal inverted repeats. This central region often contains one or more genes that encode proteins by catalyzing the transposition of the element [19]. Most IS elements generate a small duplication at the integration site. This sequence duplication results from enzymatic repair to staggered ends formed at the insertion site during transposition. Insertion of an IS element into a gene usually inactivates that gene and can often cause polar mutations within operons by disrupting transcription of genes located downstream of the insertion site [19]. Some IS elements can promote transcription of sequences adjacent to the insertion site, thereby activating cryptic genes [20]. Insertion elements may also influence genetic organization by providing targets for homologous recombination. Several IS elements from Leptospira spp. have been described, including IS1500 [12], IS1501 [21], IS1502 [9], IS1533 [22], and an IS5-like element [23]. Identification of the IS3-like element, IS1500, resulted in the development of a useful tool for epidemiological studies [17]. Variation in the hybridization patterns or PCR products using IS1500-based probes suggests that these elements are active and can transpose in L. interrogans. We recently demonstrated the spontaneous transposition of IS1501 during analysis of in vitro selected antigenic variants of L. interrogans serovar Pomona [21]. In that study, novel transpositions were discovered in an LPS biosynthetic gene,

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Figure 4. Transposition of IS1501 into the LPS biosythesis locus of L. interrogans serovar Pomona. Antigenic variants of L. interrogans serovar Pomona were selected in vitro and novel copies of IS1501 were identified. A common insertion in OrfP35 (black arrow), a gene near the 5' end of the LPS locus was identified in both variants. The insertion site is shown as an open box in the parent locus, and the novel insertion of IS1501 is shown as a grey arrow in the mutant locus. Sequences at the parent and mutant loci are shown with the 3-bp duplication highlighted in uppercase letters.

and alterations in the LPS were detected. IS1501 generates a 3-bp duplication of the target site during transposition (Figure 4). Transcriptional analysis at two of the insertion sites showed that IS1501 directs transcription into adjacent downstream sequences. Therefore, this element is capable of acting as a mobile promoter. Because of this promoter activity, IS1501 can disrupt the coding region of a gene without inducing a downstream polar mutation.

5. Functional Genomics To illustrate the application of genomic data, we describe two studies whereby genes identified through genome sequencing formed the basis for further analyses leading to a better understanding of Leptospira biology. First, we describe a locus cloned from L. interrogans serovar Pomona that has homology to the ECF (extra cytoplasmic factor) family of proteins [24, 25] and may play a role in regulating cellular responses to the extracellular environment. Second, we describe studies leading to identification and characterization of the outer membrane protein LipL21 and its expression during experimental infection. These studies applied sequence data to develop strategies that would be untenable in a pre-genomics era. Many bacterial species regulate genes associated with the extracellular environment by coordinating the process of transcription initiation. Often, the promoters of these genes are recognized by alternative ı factors belonging to the ECF family [24, 25]. ECF-regulated genes can affect host-parasite interactions, and there is a growing body of literature showing that inactivation of ECF encoding genes can reduce bacterial virulence. For example, inactivation of either of two ECF-like genes of Salmonella typhimurium results in significantly reduced virulence [26, 27]. During an ongoing project in use to identify genes in L. interrogans through sequencing random clones, we identified a genetic locus that spans genes encoding an ECF-like protein and two potential transmembrane proteins. Analysis of this locus may help us analyze how L. interrogans responds to its extracellular environment to regulate gene expression.


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Figure 5. A map of the 3,659 bp HaeIII fragment from L. interrogans serovar Pomona. Key restriction sites are noted above the horizontal line representing the fragment. Closed arrows indicate genes encoded on the fragment and open boxes indicate the various plasmid inserts mentioned in the text. Note that fragment 669595 contains the promoters for both hsa and sigE.

BLAST analysis of sequences obtained from randomly picked cloned fragments of the L. interrogans serovar Pomona genome led to identification of an ECF encoding gene on plasmid pKB25. Analysis of this sequence revealed an open reading frame that encoded a novel ECF (extra cytoplasmic factor) related protein and was designated sigE. This gene corresponds to loci LA0876 and LIC12757 from L. interrogans serovars Lai and Copenhageni, respectively. Sequences flanking sigE were isolated using a PCR-based genome walking method [16], resulting in the isolation of clones 671RH and 673RH. In total, these three cloned fragments spanned a 3,659 bp HaeIII fragment containing four genes (Figure 5). Two genes occur downstream of sigE, both lacking homologs in GenBank, and were designated dshA and dshB (for downstream of igE hypothetical gene). In L. interrogans serovar Pomona, these genes may represent pseudogenes due to the presence of an inframe stop codon that is not present in orthologous genes from serovars Lai (LA0878) and Copenhageni (LIC12756). Upstream of sigE is a gene with a region of limited similarity (E value = 0.083) to the Leptospira borgpetersenii ysp1 gene, a gene adjacent to a sphingomyelinase encoding gene, sphA [28]. The L. interrogans gene is designated hsa (for homolog to sphA adjacent gene) and corresponds to LA0875 (serovar Lai) and LIC12758 (serovar Copenhageni). Analysis of L. interrogans RNA by RT-PCR showed that sigE, dshA, and dshB are co-transcribed (data not shown). This transcript may end at a potential transcription termination stem-loop structure is predicted to occur downstream of dshB. The sigE and hsa genes are separated by 62 bp and are divergently transcribed. Promoter activity for both sigE and hsa was tested in Escherichia coli by inserting a 457 bp amplicon with the sigE-hsa intergenic region and part of both coding regions upstream of the promoterless chloramphenicol acetyltransferase gene of pKK232-8 [29]. This insert, placed in both orientations in pKK232-8, conferred chloramphenicol resistance while the intact vector did not, showing that promoter activity for both genes functioned in E. coli.

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Figure 6. Comparison of the SigE protein to ECF-ı factors. Proteins were aligned with SigE and shaded either black (identical residue in all sequences), dark grey (some sequences share an identical residue with SigE), or light grey (some sequences share a similar residue with SigE). Sequences shown with accession numbers are: Lin_sigE (this work, AF143504.); Bac_sigV (Bacillus subtilis SigV, O05404); Bac_sigX (B. subtilis SigX, P35165); Bac_yhdM (B. subtilis YhdM, CAA74497); Cac_sigX (Clostridium acetobutylicum SigX, AAC12856); Pae_sigX (Pseudomonas aeruginosa SigX, AAD11567); and Rho_rpoE (Rhodobacter spharoides RpoE, AAB17906). A predicted helix-turn-helix structure is found in segment 4.2.

The SigE protein shares several motifs with ECF-ı factors (Figure 5). Like other ECF-ı factors, SigE lacks region 1, a region thought to inhibit DNA binding in the absence of core RNA polymerase [25]. The most conserved portion of ı factors, including SigE, is region 2, which is subdivided into four regions: region 2.1 may bind core RNA polymerase; region 2.2 is a conserved domain of unknown function; region 2.3 may affect DNA melting; and region 2.4 is thought to bind to the -10 sequence of ECF-regulated promoters (Figure 5) [25]. Like other ECF-ı factors, region 3 of SigE is much smaller than primary ı-factors. In primary ı-factors, region 3 residues may bind DNA and interact with the core RNA polymerase [15]. Region 4 is thought to bind –35 sequences and often has a DNA binding helix-turn-helix motif [25, 30]. SigE also has a predicted helix-turn-helix motif within region 4 (Figure 6). The Hsa and DshA proteins each have regions with predicted transmembrane domains and these proteins may be exposed to the extracytoplasmic environment. There are three potential transmembrane domains in Hsa and one in DshA. The DshB protein had no recognizable motifs that might suggest function. Alignment with homologs from L. interrogans serovars Lai and Copenhageni suggest this gene in serovar Pomona may be a pseudogene created by introduction of an in-frame stop codon. Further analysis is required to determine the cellular locations and possible functions of these proteins. The identification of an ECF-ı factor-encoding gene in L. interrogans suggested that this bacterium uses alternative sigma factors to control transcription of selected genes. This is a key finding in understanding how Leptospira control gene expression, especially in response to alterations in the extracellular environment. Moreover, considering the current understanding of fluid nature of genome layout in Leptospira, this strategy may be an effective strategy to maintain control of genes as they are moved to different locations. In the context of bacterial pathogenesis, the identification and characterization of genes controlled by ECF-ı factors in Leptospira may lead to the


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identification of novel virulence factors, given the importance of this class of genes in other pathogenic bacteria. In a second example using genomics to characterize pathogenic Leptospira, a draft sequence of L. borgpetersenii was used as a database to search for matches to peptide sequences obtained from the L. interrogans serovar Lai outer membrane [31], resulting in the identification of the LipL21 CDS [32]. PCR primers were designed that were used to amplify a portion of the LipL21 CDS leading to the construction of a Histagged fusion protein that was produced in E. coli. His-tagged LipL21 fusion protein was gel purified and used to immunize rabbits and the production of homologous

A

C

B

D

Figure 7. Detection of L. interrogans serovar Pomona in hamster tissue. Paraffin-embedded tissue from hamster lung (A), kidney (C), and liver (D) after infection with L. interrogans serovar Pomona were processed for antigen detection. Sections were incubated with anti-LipL21, followed by a secondary antibody tagged with a fluorescent dye, then illuminated with ultraviolet light. Tissue nuclei were counterstained with DAPI. Arrows point to fluorescence due to the presence of LipL21. For comparison, a section of lung stained with silver and examined by light microscopy (B) reveals the presence of L. interrogans.

antisera. Subsequent immunoblot analysis of Leptospira lysates showed that LipL21 is a major outer membrane protein that is well conserved across pathogenic Leptospira species, but not produced by saprophytic species [32]. Immunoblot analysis using patient sera and sera from experimentally infected hamsters revealed the presence of anti-LipL21 antibodies. These data suggested that LipL21 is expressed during infection. To directly test for LipL21 expression during mammalian infection, we used anti-LipL21 antisera in indirect immunofluorescence studies to analyze tissue sections from hamsters experimentally infected with L. interrogans serovar Pomona. Analysis of lung, kidney, and liver tissue showed the presence of Leptospira in all tissues by


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silver stain (Figure 6 and data not shown), and the LipL21 antisera bound to bacteria in each of these tissues, showing that this protein is expressed during infection in several different organs. The bacteria distribute differently in these tissues, being localized in tight junctions between hepatocytes, having a diffuse distribution in the kidney, or present in the alveolar space of the lung. Detection of Leptospira in the lung, in particular, is consistent with pulmonary hemorrhage, a clinical manifestation severe leptospirosis [33].

6. Conclusions Genome sequence analysis of pathogenic Leptospira is providing valuable data that can be rapidly applied to understand more about the biology of these bacteria. Accurate comparison of genomic sequence data relies on establishing common criteria for gene identification and the realization that genome annotations are dynamic data sources that need periodic revisions as new information becomes available. By revising genome annotations of L. interrogans serovars Copenhageni and Lai, we created databases by using common criteria useful for comparative analysis with existing genome sequences and established a foundation for future studies as more bacterial genomes become available. Gene discovery combined with functional analysis is making it possible to explore the biology of pathogenic Leptospira and helping to identify potential vaccine candidates and potential virulence factors. Combined, these studies will facilitate rational, technology-based development of new methods for the control and prevention of leptospirosis.

Acknowledgements We thank Paul Cullen and David Haake for the generous gift of anti-LipL21 sera, Paul Hauer for providing hamster tissues, and David Alt, Ami Frank, Richard Hornsby, and Amanda Toot for their excellent technical support throughout this project.

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Leptospira interrogans: Genomics and “Immunomics” Ana L. T. O. NASCIMENTO 1 Centro de Biotecnologia, Instituto Butantan, São Paulo, SP, Brazil Abstract. Leptospirosis, an emerging infectious disease, is a worldwide zoonosis of human and veterinary concern. Caused by pathogenic spirochaetes of the genus Leptospira, the disease presents greater incidence in tropical and subtropical regions. Humans can be infected by exposure to chronically infected animals and their environment. The genome sequence of Leptospira interrogans serovar Copenhageni was recently reported. It contains a broad array of genes encoding for regulatory system, signal transduction, and methyl-accepting chemotaxis proteins, conforming to the organism’s ability to respond to diverse environmental stimuli. A large number of exported lipoproteins and transmembrane outer membrane proteins were identified that may be involved in leptospiral pathogenesis and protective immunity. Comparative analysis with the Leptospira interrogans serovar Lai genome revealed that, despite genetic similarity, there are structural differences, including a large chromosomal inversion. The leptospiral genome sequence, combined with bioinformatics tools, offered a unique opportunity to search for immune targets to be used for vaccine or diagnostic kit development. Out of a hundred recombinant proteins tested, sixteen were recognized by antibodies present in sera from patients diagnosed with leptospirosis and might be useful for these purposes. The most important results obtained within genome sequences, comparative genomics, and outer membrane genome-derived protein expressed in E. coli are reviewed here. Keywords. Genome, Leptospira, leptospirosis, vaccine, diagnosis, recombinant proteins

Introduction Spirochetes are motile, helically shaped bacteria, which include the genera Leptospira, Leptonema, Borrelia and Treponema. Borrelia and Treponema are the causative agents of Lyme disease, relapsing fever, and syphilis. Leptospira consists of a genetically diverse group of pathogenic and non-pathogenic or saprophytic species [1]. Leptospirosis is an emerging infectious disease of human and veterinary concern. The disease presents greater incidence in tropical and subtropical regions [1, 2]. The transmission of leptospirosis has been associated with exposure of individuals in close proximity to wild or farm animals [3]. Recently the disease became prevalent in cities 1 Corresponding author: Ana L.T.O. Nascimento, Centro de Biotecnologia, Instituto Butantan, Avenida Vital Brazil, 1500, CEP 05503-900, São Paulo, SP, Brazil. E-mail: [email protected]

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with sanitation problems and large populations of urban rodent reservoirs that contaminate the environment through their urine [4]. The incidence of leptospirosis remains underestimated in part due to the broad spectrum of signs and symptoms that patients may present. Children primarily show fever, vomiting, headache, diarrhea, and abdominal and generalized muscle pain, whereas adults have fever, headache, anorexia, muscle pain, and constipation [4, 5]; 5 to 15% of the cases evolve more severely, presenting hemorrhages with renal and hepatic failure, a condition known as Weil's syndrome [4], with a mortality rate of 5 to 40%. Leptospirosis also has a great economic impact in the agricultural industry since the disease affects livestock, inducing abortions, stillbirths, infertility, reduced milk production, and death [3, 4]. Environmental control measures are difficult to implement because of the long-term survival of pathogenic leptospires in soil and water and the abundance of wild and domestic animal reservoirs [1]. Currently available veterinary vaccines are based on inactivated whole cell or membrane preparations of pathogenic leptospires. These types of vaccine confer protective responses through induction of antibodies against leptospiral lipopolysaccharide [6]. However, these vaccines fail to induce long-term protection against infection and do not provide cross-protective immunity against leptospiral serovars not included in the vaccine preparation. The large number of pathogenic serovars (>230) imposes a major limitation on the production of a multiserovar component vaccine and the development of immunization protocols based on whole cell or membrane preparations. Protein antigens conserved among pathogenic serovars may contribute to overcome these limitations. The genome sequences of L. interrogans serovars Copenhageni and Lai have been published [7, 8], and comparative genome analysis between the two serovars was performed [9]. The main features found through genome analysis of the L. interrogans serovar Copenhageni that should contribute to the understanding of leptospiral physiology and pathogenesis are reviewed here. The results obtained through “genome data mining”, gene cloning, and protein expression are discussed.

1. Genome Features The L. interrogans serovar Copenhageni genome consists of two circular chromosomes with a total of 4,625,429 base pairs: chromosome I (CI) with 4,277,169 bp and chromosome II (CII) with 350,181 bp [7, 9]. The L. interrogans genome has one rrf gene, two rrl genes and two rrs genes coding for 5S, 23S, and 16S rRNA, respectively. As in other parasitic strains, L. interrogans serovar Copenhageni has only one rrf (5S) gene, which is located close to the origin replication region as described before for other strains of L. interrogans [7, 10]. Phylogenetic studies based on 16S rDNA sequences, using Leptonema as an outgroup, were performed, and the resultant analysis showed that Leptospira are split into two well-supported monophyletic groups, one of them formed by the pathogenic strains (e.g., L. interrogans) and the other formed by the non-pathogenic strains (e.g., L. biflexa). At the base of the clade of the pathogenic strains, L. inadai and L. fainei form a well-supported assemblage. Considering a constant divergence rate of 1 to 2% per 50 million years for the 16s rDNA [11], separation time between the two main assemblages (L. interrogans versus L. biflexa) was estimated to be approximately 590 to 295 million years ago [7].


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Based upon the localization of the gene metF, which encodes for the essential enzyme methylene tetrahydrofolate reductase [12], the small replicon of L. interrogans was previously suggested to be a second chromosome. A thorough genome sequence annotation revealed that genes encoding enzymes for metabolic pathways, such as glycolysis and the tricarboxylic acid cycle, as well as the enzymes for biosynthesis of amino acids and cofactors, are also spread between the two chromosomes. Sequence analysis of CII shows that an almost complete operon of genes coding for protoheme biosynthesis pathway is present (hemAIBCENYH). Although no homolog of the gene encoding for uroporphyrinogen III synthetase (hemD) was found, experimental evidence has shown that the hemC gene is able to cope with hemD activity [13]. L. interrogans therefore has the capability to synthesize protoheme de novo. In addition, thirteen genes clustered in CII coding for the cobalamin biosynthesis pathway were identified (cobC, cobD, cbiP, cobP, cobB, cobO, cobM, cobJ, cbiG, cobI, cobL, cobH, cobF) [7]. Orthologs of cobGKN genes, known to be involved in the cobalamin pathway [14], were not found. However, predicted coding sequences inside this operon in CII (LIC20133 and LIC20135) could perform these steps. Other genes present in the genome, such as LIC11145, LIC13354, LIC12391, and LIC10522, could also cope with these activities. The presence of cysG, in CI, may also be a cobalt-inserting enzyme in the B12 pathway [7, 15]. The remaining genes involved in this biosynthesis were found in CI (cysG/hemX/cobA, cobT/cobU, cobS). Indeed, comparable growth curves for L. interrogans serovar Lai, L. interrogans serovar Copenhageni, and L. biflexa serovar Patoc were obtained in either the presence or absence of vitamin B12 on the EMJH culture medium [7]. Thus L. interrogans, unlike the spirochetes Borrelia burgdoferi and Treponema pallidum, have the complete repertoire of genes for de novo synthesis of protoheme and cobalamin. The functional link between the two replicons strengthens the concept that the small replicon is indeed a second chromosome.

2. Comparative Genomics 2.1. L interrogans serovars Copenhageni and Lai Comparative genome analysis of L interrogans serovars Copenhageni and Lai showed that they are highly conserved and similar in size (~ 4.3 Mb and ~ 350 kb for CI and CII, respectively). The average nucleotide identity between the two serovars is 95%; the average nucleotide identity between pairs of predicted protein coding genes that are orthologs is 99%; and the numbers of ortholog pairs are 3079 and 261 for CI and CII, respectively [9]. Chromosomes CII in both serovars are collinear, but for the CI chromosome, a large inversion was detected. Flanking the inversion breakpoints, two identical copies of an IS element, in opposite orientation, were identified in serovar Lai. In serovar Copenhageni, 56 shotgun clones that span the inversion breakpoints and are anchored in non-repetitive portions of the sequence unequivocally confirmed the assembly. Taken together, the data suggested that the rearrangement took place in the Lai genome [9]. In addition, a 54 kb insertion was found in the Lai genome, accounting for the differences in genome organization. The Copenhageni and the Lai genomes contain 3728 and 4768 predicted open reading frames, respectively. The difference in the number of structural genes is mainly because the Brazilian Sequencing Group did not consider predicted coding sequences less than or equal to 150 bp in length that lacked significant homologs. The Copenhageni and the Lai genomes contain 64 and

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118 unique predicted coding genes, respectively. Eighty-one out of a hundred-eighteen structural genes unique to serovar Lai are located in the 54 kb insertion mentioned above [9]. 2.2. L. interrogans and Spirochetes A comparison between the L. interrogans genome and the genomes of the spirochetes B. burgdorferi and T. pallidum yielded the following results: 1167 (31%) of the genes in L. interrogans Copenhageni are found in B. burgdorferi and/or in T. pallidum; 666 (41%) of the genes in B. burgdorferi are found in the Copenhageni genome; 589 (57%) of the genes in T. pallidum are found in the L. interrogans genome. Three hundred sixty-two predicted genes were found to be shared among all three spirochaetes, of which 45 are hypothetical [7] (detailed list available at http://aeg.lbi.ic.unicamp.br/world/lic/). A thoroughly analysis of these common genes should contribute to the understanding of spirochete biology. 2.3. Energy Metabolism L. interrogans does not utilize glucose as a carbon and energy source under normal laboratory conditions [4], and it was thought that the glycolysis pathway was absent or incomplete. Surprisingly, the genome sequence revealed a complete glucose utilization route. Thus, the question arises as to why leptospires do not metabolize glucose as their energy source. In an attempt to answer this question, the genome was thoroughly searched, and only one glucose uptake system, a glucose/sodium symporter that is dependent on a sodium gradient across the bacterial membrane, was found within the leptospiral genome. In addition, no sugar ABC-transporter was identified, and an incomplete phosphoenolpyruvate-protein phosphotransferase system (PTS) was present with no B or C sugar permease components [9]. Taken together, these observations could explain the difficulties in utilization of glucose as a source of energy under certain growth conditions. Leptospira utilizes beta-oxidation of long-chain fatty acids as the major energy and carbon source [16], and as expected, a complete route was found. Glycerol metabolism genes are also present, including those encoding a glycerol-3-phosphate transporter, a glycerol uptake facilitator protein, glycerokinases, and a glycerol-3-phosphate dehydrogenase, which suggested that glycerol and fatty acids are obtained through phospholipid degradation. Two key enzymes of the pentose-phosphate pathway are missing, glucose-6-phosphate 1-dehydrogenase and 6-phosphogluconate dehydrogenase. L. interrogans probably obtains most of its reducing power for macromolecular biosynthesis via a membrane nicotinamide nucleotide transhydrogenase driven by a proton motive force [9]. L. interrogans generates ATP using an F0F1 type ATPase that is encoded by several genes organized in a single operon, atpBEFHAGDC. This operon has the same genetic organization as most of the eubacteria, in contrast to the ATP synthases found in B. burgdorferi and T. pallidum, which are of the V1V0-type [17, 18].


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3. Regulatory Systems and Signal Transduction A vast array of genes encoding for regulatory systems that enables Leptospira to respond to environmental signals was identified in the genome. There are 80 genes encoding components of the phosphorylation-mediated signal transduction pathway: 29 histidine kinases (HK), 30 response regulators (RR), and 18 hybrid kinase/regulators (HK/RR). Nineteen of the histidine kinases are located in the inner membrane, nine are cytoplasmic, and one is probably found in the periplasm, as predicted by the PSORT program [7, 19]. The response regulators are the cytoplasmic effectors of the message, which become functional after being phosphorylated by the cognate histidine kinase. The RRs may possess a second effector domain, which will perform its ultimate function, such as the DNA-binding helix-turn-helix domain (HTH). Other domains found in L. interrogans RRs are the GGDEF and EAL motifs, which correspond to putative diguanylate cyclase and phosphodiesterase domains, respectively [7].

4. Motility and Chemotaxis The motility and chemotaxis apparatus of L. interrogans is complex as its genome contains at least 79 putative motility-associated genes. All genes are well conserved among L. interrogans, T. pallidum, and B. burgdorferi, and 42 genes were found to be common to all three genera. However, the leptospiral genome contains multiple copies of a number of motility-associated genes, accounting in part for the higher number. In addition, the L. interrogans genome contains 11 putative genes encoding methylaccepting chemotaxis proteins (MCPs), which is roughly twice as many as in T. pallidum and B. burgdorferi. Forty-eight of the 79 motility-associated genes are located in 14 gene clusters varying in size from two to eight genes. Thus, as in T. pallidum and B. burgdorferi, the majority of the structural and functional motility genes are organized in operons. However, it seems that the operons suffered extensive rearrangements because they are usually smaller, corresponding to portions of the major Treponema and Borrelia operons [7, 9]. These differences might be associated with the high capacity of pathogenic leptospires to survive and adapt to a variety of environments and hosts.

5. Outer Membrane Proteins An important focus of current leptospiral research is the identification of outer membrane proteins (OMPs). Due to their location, leptospiral OMPs are likely to be relevant in host-pathogen interactions and consequently have the potential ability to stimulate heterologous immunity. The L. interrogans genome contains at least 263 predicted genes encoding for potentially surface-exposed integral membrane proteins, 250 of which were previously unknown. The leptospiral genome contains homologues of SecY and other secretory proteins involved in exporting proteins with signal peptides across the cytoplasmic membrane. Genes encoding for signal peptidases I (LIC11233, LIC10478), the lipoprotein biosynthesis pathway (LIC11063, LIC12389, and LIC12556, LIC13250), and the proteins involved in transport and incorporation of

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lipoproteins into the outer membrane (LIC12545, LIC10232, LIC12429, and LIC10233) were identified [7, 9]. One hundred eighty-four predicted coding sequences in the L. interrogans genome were found to have a lipoprotein signal peptidase cleavage site [9]. As shown by their lipobox motifs, all the predicted lipoprotein-coding sequences conform to the rule previously described [9, 20, 21]. Eighty-four outer membrane proteins with transmembrane domains were identified, including TolC orthologs, factor CzcC, TonB-dependent outer membrane proteins, and porin [9, 22]. Outer membrane proteins that are conserved among pathogenic serovars may serve as vaccine that avoids the limitations of currently available whole-cell preparations.

6. Searching for Immune Targets: “Immunomics” The advent of whole-genome sequencing has made an impressive impact on the microbial field landscape. The complete genomic sequence of Neisseria meningitidis serogroup B offered a new strategy for the identification of vaccine candidates [23]. This landmark approach, called reverse vaccinology, has been applied in the last few years, revolutionizing the vaccine research area [24, 25]. The design of vaccines is based on bioinformatic tools for the prediction of potential antigens in silico, hence narrowing down the universe to be tested. In addition, this approach has the advantage of revealing proteins independent of their abundance and without the need of growing the microorganism in vitro [26]. A first high-throughput screening aimed at identifying candidates for vaccine or diagnostic test evaluation was recently published [27]. These studies described 16 new leptospiral membrane-associated proteins selected from the genome of L. interrogans serovar Copenhageni [7, 9]. The rationale for the choice of the predicted coding sequences, described by Gamberini and colleagues [27], is that surface-associated molecules are potential targets for inducing immune responses. The PSORT program (http://psort.nibb.ac.jp/) [19] was used to predict the localization of the coded proteins within the bacterium. Public and custom sequence-specific search algorithms were used for identification of sequence motifs including lipoprotein cleavages sites, transmembrane domains and signal peptides (http://www.cbs.dtu.dk/services/TMHMM) (http://www.cbs.dtu.dk/services/SignalP) [28, 29]. Putative proteins, homologous to surface proteins previously characterized as virulence factors in other organisms, were searched for by blast analysis (http://www.ncbi.nlm.nih.gov/BLAST/) [30]. The in silico approach resulted in a large number of genes covering ~20 % of the total number of predicted proteins in the genome. From these sequences, the selection was focused mainly on hypothetical, unknown proteins, having either signal peptide sequences or lipobox motifs [7, 9]. Genes encoding proteins with known cytoplasmic functions were excluded. Of the 206 selected coding sequences, more than 97% were amplified. The correct sequences were confirmed by DNA sequencing and 175 genes (84%) were effectively cloned into pENTR. The DNA inserts were transferred by recombination from pENTR to pDEST17 expression vector. This E. coli vector expresses the recombinant proteins with six histidine residues at the N-terminus, which allows a rapid purification of the protein by metal chelation chromatography. By using this approach, the authors have successfully expressed and purified 150 recombinant proteins. Purified proteins were screened for reactivity by immunoblotting with serum from patients diagnosed with leptospirosis. The recombinant lipoprotein LipL32 was employed as a positive control


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since it was shown to be immunogenic and highly conserved among Leptospira pathogenic serovars [31, 32]. Antibodies present in serum from convalescent leptospirosis patients recognized 16 proteins of 100 tested. These comprise: four probable new lipoproteins, six leptospiral conserved hypothetical proteins, four conserved hypothetical proteins, one hypothetical protein, and one peptidoglycanassociated membrane protein [27].

Whole genomic sequence

Computer prediction of surface-exposed proteins (2000ORFs)

In silico selected protein candidates (206 ORFs)

Cloning and express recombinant proteins: 175 ORFs cloned; 150 recombinant proteins expressed

Immunoblotting: 16 proteins

Figure 1. Strategy used for identification of vaccine candidates from the whole genome sequence.

Protein expression in the most prevalent pathogenic serovars of L. interrogans is an important requirement for leptospiral vaccine candidates. The conservation of the selected proteins was evaluated against protein extracts from several L. interrogans serovars: Canicola, Icterohaemorrhagiae, Copenhageni, Bratislava, Hardjo, Autumnalis, Pomona, Pyrogenes, Grippotyphosa, and the nonpathogenic strain L. biflexa serovar Patoc. Four of 10 proteins tested proved to be highly conserved among the pathogenic leptospires. Most interesting, none of these four proteins were present in the nonpathogenic L. biflexa strain, suggesting that they may be relevant for pathogenesis [27]. The protective immune activity of these proteins is currently under investigation in our


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laboratories. Figure 1 summarizes the experimental approach used in the work performed by Gamberini and colleagues [27].

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Levett, P. N. (2001) Leptospirosis. Clin. Microbiol. Rev. 14: 296–326. Levett, P. N. (2003) Usefulness of serologic analysis as a predictor of the infecting serovar in patients with severe leptospirosis. Clin. Infect. Dis. 36: 447–452. Bharti AR, Nally JE, Ricaldi JN, Matthias MA, Diaz MM, Lovett MA, Levett PN, Gilman RH, Willig MR, Gotuzzo E, Vinetz JM; Peru-United States Leptospirosis Consortium. (2003) Leptospirosis: a zoonotic disease of global importance. Lancet Infect. Dis. 3: 757–771. Faine, S., Adler, B., Bolin, C., and Perolat, P. (1999) Leptospira and Leptospirosis, 2nd Edition. Melbourne, Australia: MediSci. Plank, R., and Dean, D. (2000) Overview of the epidemiology, microbiology, and pathogenesis of Leptospira spp. in humans. Microbes Infect. 2: 1265–1276. de la Pena-Moctezuma, A., Bulach, D.M., Kalambaheti, T., and Adler, B. (1999) Comparative analysis of the LPS biosynthetic loci of the genetic subtypes of serovar Hardjo: Leptospira interrogans subtype Hardjoprajitno and Leptospira borgpetersenii subtype Hardjobovis. FEMS Microbiol. Lett. 177: 319– 326. Nascimento ALTO, Verjovski-Almeida S, Van Sluys MA, et al. (2004) Genome features of Leptospira interrogans serovar Copenhageni. Braz. J. Med. Biol. Res 37: 459–477. Ren SX, Fu G, Jiang XG et al. (2003). Unique physiological and pathogenic features of Leptospira interrogans revealed by whole-genome sequencing. Nature, 422: 888–893. Nascimento ALTO, Ko AI, Martins EAL et al. (2004). Comparative genomics of two Leptospira interrogans serovars reveals novel insights into physiology and pathogenesis. J. Bacteriol. 186:2164– 2172. Fukunaga M & Mifuchi I (1989). Unique organization of Leptospira interrogans rRNA genes. J. Bacteriol. 171: 5763–5767. Ochman H, Elwyn S & Moran NA (1999). Calibrating bacterial evolution. Proceedings of the National Academy of Sciences of the USA, 96: 12638–12643. Bourhy P & Saint Girons I (2000). Localization of the Leptospira interrogans metF gene on the CII secondary chromosome. FEMS Microbiology Letters, 191: 259–263. Guegan R, Camadro JM, Saint Girons I & Picardeau M (2003). Leptospira spp. possess a complete haem biosynthetic pathway and are able to use exogenous haem sources. Molecular Microbiology, 49: 745–754. Rodionov DA, Vitreschak AG, Mironov AA & Gelfand MS (2003). Comparative genomics of the vitamin B12 metabolism and regulation in prokaryotes. Journal of Biological Chemistry, 278: 41148– 41159. Spencer JB, Stolowich NJ, Roessner CA & Scott AI (1993). The Escherichia coli cysG gene encodes the multifunctional protein, siroheme synthase. FEBS Letters, 335: 57–60. Henneberry, R. C., and C. D. Cox. (1970). Beta-oxidation of fatty acids by Leptospira. Can J Microbiol 16: 41–5. Fraser, C. M., Casjens S., Huang W. M., et al. (1997). Genomic sequence of a Lyme disease spirochaete, Borrelia burgdorferi. Nature 390:580–6. Fraser, C. M., Norris S. J., Weinstock G. M., et al. (1998). Complete genome sequence of Treponema pallidum, the syphilis spirochete. Science 281:375–88. Nakai, K., and Kanehisa, M. (1991) Expert system for predicting protein localization sites in gramnegative bacteria. Proteins 11: 95–110. Haake, D.A. (2000). Spirochaetal lipoproteins and pathogenesis. Microbiology 146 :1491–504. Setubal JC, Reis M, Matsunaga J, Haake DA (2006) Lipoprotein computational prediction in spirochaetal genomes. Microbiology 152: 113–21. Haake, D A., Champion C.I, Martinich C., et al. (1993). Molecular cloning and sequence analysis of the gene encoding OmpL1, a transmembrane outer membrane protein of pathogenic Leptospira spp. J Bacteriol 175:4225–34. Pizza M, Scarlato V, Masignani V, et al. (2000) Identification of vaccine candidates against serogroup B meningococcus by whole-genome sequencing. Science 287: 1816–1820.


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[24] Wizemann TM, Heinrichs JH, Adamou JE, et al. (2001) Use of a whole genome approach to identify vaccine molecules affording protection against Streptococcus pneumoniae infection. Infect. Immun. 69:1593–8. [25] Rappuoli, R. (2001) Reverse vaccinology, a genome-based approach to vaccine development. Vaccine 19: 2688–2691. [26] Adu-Bobie, J., Capecchi, B., Serruto, D., Rappuoli, R., and Pizza, M. (2003) Two years into reverse vaccinology. Vaccine 21: 605-610. [27] Gamberini M, Gomez RM, Atzingen MV, Martins EA, Vasconcellos SA, Romero EC, Leite LC, Ho PL, Nascimento AL. (2005) Whole-genome analysis of Leptospira interrogans to identify potential vaccine candidates against leptospirosis. FEMS Microbiol Lett. 244:305-13. [28] Krogh A., Larsson B., von Heijne G.and Sonnhammer EL.(2001) Predicting transmembrane protein topology with a Hidden Markov Model: Application to complete genomes. J. Mol. Biol. 305: 567-580. [29] Bendtsen JD., Nielsen H., von Heijne G. and Brunak S. (2004) Improved Prediction of Signal Peptides: SignalP 3.0. J. Mol. Biol. 340: 783–795. [30] Altschul FF., Madden TL., Schäffer AA., Zhang J., Zhang Z., Miller W. and Lipman DJ. (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 25 (17): 3389–3402. [31] Dey, S., Mohan, C.M., Kumar, T.M., Ramadass, P., Nainar, A.M., Nachimuthu, K. (2004) Recombinant LipL32 antigen-based single serum dilution ELISA for detection of canine leptospirosis. Vet. Microbiol. 103: 99-106. [32] Haake, D.A., Suchard, M.A., Kelley, M.M., Dundoo, M., Alt, D.P., Zuerner, R.L. (2004) Molecular evolution and mosaicism of leptospiral outer membrane proteins involves horizontal DNA transfer. J. Bacteriol. 186: 2818-28.


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Genotypic Variation and Borrelia burgdorferi Pathogenesis Ira SCHWARTZ a,b,1 , Guiqing WANG a, Radha IYER a, Caroline OJAIMI a, Darya TEREKHOVA a, Sabina SANDIGURSKY a, Gary P. WORMSER b and Dionysios LIVERIS a a Department of Microbiology & Immunology, New York Medical College, Valhalla, NY 10595, USA b Division of Infectious Diseases, Department of Medicine, New York Medical College, Valhalla, NY 10595, USA Abstract. Lyme disease, the most commonly reported arthropod-borne disease in the United States, is caused by infection with the spirochete, Borrelia burgdorferi. The acute stage of the infection has a varied presentation, ranging from mild, localized disease (characterized by a skin lesion) to highly symptomatic, disseminated disease. We hypothesize that this is due, in part, to B. burgdorferi genotypic variation. By combining PCR amplification of the 16S-23S rDNA spacer with restriction fragment length polymorphism (RFLP) analysis, several genotypes were identified among B. burgdorferi clinical isolates obtained from either skin or blood of early Lyme disease patients. Hematogenous dissemination in humans is associated with a distinct genotype and disease severity and spirochete burden was also associated with this same genotype in a murine model of Lyme disease. A genomic approach was undertaken to elucidate the differences in genome content and/or gene expression that may result in disease variability. Comparative transcriptional profiling of two clinical isolates with distinct genotypes (invasive and attenuated) was performed using whole genome arrays. A total of 78 ORFs had significantly different expression levels in the two isolates. Nearly 25% of the differentially expressed genes are predicted to be localized on the cell surface, implying that these two isolates have considerably different cell surface properties. Comparative genome hybridization demonstrated that genotypic variation largely results from differences in plasmid content and/or sequence and revealed several plasmid-encoded candidate genes that are uniquely absent in attenuated strains. A number of genes identified in these investigations are currently under further study by genetic analysis to substantiate a possible role in virulence. Keywords. Borrelia burgdorferi, pathogenesis, comparative genomics

1

Lyme

disease,

genotypic

variation,

Corresponding Author: Department of Microbiology & Immunology, New York Medical College, Valhalla, NY 10595 USA; E-mail: [email protected], Phone: 914-594-4658.

I. Schwartz et al. / Genotypic Variation and Borrelia burgdorferi Pathogenesis

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Introduction Lyme borreliosis is a global arthropod-borne zoonosis, occurring throughout the Northern hemisphere [1, 2]. It is caused by infection with spirochete Borrelia burgdorferi sensu lato [2, 3]. This group of organisms is comprised of at least 12 genomic species [3í5]. Three of these are responsible for the majority of human disease worldwide; B. burgdorferi sensu stricto in the US and Europe and Borrelia garinii and Borrelia afzelii in Eurasia [3, 6, 7]. Globally, it has been noted that symptoms of Lyme borreliosis vary with geographic location; neurologic and chronic skin manifestations predominating in Europe and arthritic symptoms being more common in the US [1, 2]. It has been proposed that these differences may result from differences in pathogenesis between the infecting spirochetal species [3, 8, 9]. Lyme disease is the most frequently reported arthropod-borne disease in the US and all Lyme disease is caused by B. burgdorferi sensu stricto (referred to simply as B. burgdorferi throughout this chapter) [3, 10]. A characteristic skin rash, erythema migrans (EM) occurs in >75% of patients [1]. Furthermore, 70-80% of patients with EM are symptomatic at presentation (i.e., report at least one symptom such as fever, headache, neck or joint pain) and over 50% can be shown to have disseminated infection based on having a positive blood culture for B. burgdorferi or the presence of multiple EM skin lesions [11]. Thus, early Lyme disease in the US has a varied presentation, ranging from no symptoms to disseminated infection with multiple symptoms. Variation in symptom profile or in spirochete dissemination could be related to differences in the host and/or in the infecting strain of B. burgdorferi. One hypothesis is that the distinctiveness in disease manifestations may be the result of genotypic variation of the infecting strain of B. burgdorferi. A corollary of this hypothesis is that not all B. burgdorferi genotypes circulating in nature are capable of causing human disease.

1. Molecular Typing of B. burgdorferi Many methods have been employed for the molecular typing of B. burgdorferi. These include serotyping, multi-locus enzyme electrophoresis, ribotyping, pulsed-field gel electrophoresis (PFGE), randomly amplified polymorphic DNA and sequencing of various target genes (reviewed in [3]). We combined PCR amplification of the proximal portion of the 16S-23S rDNA spacer with restriction digestion of the

Table 1. Genotype distribution of B. burgdorferi isolates cultivated from skin or blood of early Lyme disease patients.1 RST1

RST2

RST3

Total

Skin

81 (28%)

129 (44%)

83 (28%)

293

Blood

53 (42%)

57 (45%)

17 (13%)

127

1

Cultivated from patients presenting with EM at the Lyme Disease Diagnostic Center, New York Medical College, 1991-2002.

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I. Schwartz et al. / Genotypic Variation and Borrelia burgdorferi Pathogenesis

amplified product to produce a rapid assay that could be employed with infected wildlife, tick and human specimens and did not require prior cultivation of B. burgdorferi or genomic DNA isolation [12, 13]. Initially, PCR-RFLP typing of 217 B. burgdorferi isolates originally cultivated for skin biopsies or blood of early Lyme disease patients showed three major RFLP types which are designated RST1, RST2and RST3 [14]. Currently, 420 B. burgdorferi isolates cultivated from early Lyme disease patients with EM between 1991-2002 have been typed by this method and the results are presented in Table 1. The distribution of genotypes was significantly different in skin and blood (P

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