Nature Reviews Microbiology (June 2006 Vol 4 No 6)

EDITORIAL Cause and effect Identifying the causative agent of an infectious disease is a notoriously tricky, but vitall...

Author: David O’Connell

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EDITORIAL Cause and effect Identifying the causative agent of an infectious disease is a notoriously tricky, but vitally important, business. When faced with new potential pathogens, the principles fostered by Koch’s postulates are as relevant today as they were over a century ago.

…Koch’s postulates, and the scientific rigour they foster, still provide an invaluable service in efforts to prove that a microorganism is the cause of a disease.

All microbiologists interested in infectious diseases are aware of the various statistics used to emphasize the threat microbial infection poses to human health, statistics that are regurgitated daily for conference audiences, funding bodies and journalists. One particularly interesting statistic, however, states that new infectious diseases are estimated to be emerging at the rate of one per year. In light of the potential ramifications of this statistic for global public health, a relevant question that requires consideration is: what constitutes a new infectious disease? In the early days of microbiology, the attention of scientists and clinicians was primarily focused on diseases that presented with ‘textbook’ clinical symptoms and signs, that is, easily recognizable and distinguishable conditions. Many of the infectious diseases that preoccupy today’s practitioners do not possess such obvious hallmark symptoms or signs. Patients can remain asymptomatic for decades, but these individuals act as reservoirs for some of our most deadly infections. Modern clinical advances, including transplantationrelated immunosuppression, have blurred the distinction between commensals and pathogens, and this fuzziness prevents a consensus being reached about what actually constitutes a pathogen1. These issues aside, more than 120 years after they were first proposed, Koch’s postulates still remain the gold standard for any investigation that sets out to prove the aetiology of an infectious disease. Although advances in microbiology since the nineteenth century have led to modern reintepretations2,3, Koch’s postulates, and the scientific rigour they foster, still provide an invaluable service in efforts to prove that a microorganism is the cause of a disease. Take a recent case in point. Patients with chronic granulomatous disease (CGD) are subject to recurrent infections with a range of bacterial and fungal pathogens. In many cases, however, patients present with clinical symptoms that are indicative of an infection for which no obvious pathogen can be identified. In these cases, is disease caused by a known microorganism that eludes detection, or is a novel pathogen to blame? A recent study published in PLoS Pathogens4 describes the story of one CGD patient suffering from recurring lymphadenitis of unknown aetiology. The paper reports on the isolation and characterization of a new bacterium,

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Granulobacter bethesdensis, from the lymph nodes of this patient. More importantly, by applying Koch’s postulates with commendable rigour, the authors were able to convincingly establish a causal link between this novel organism and the infection in the patient — the first reported case of an invasive human disease caused by the Acetobacteraceae. Another recent example provides more food for thought. Infection of the root canals of human teeth (commonly polymicrobial in nature), can lead to inflammation and the destruction of the periradicular tissues. A paper recently published in the Journal of Clinical Microbiology reports, for the first time, the detection and identification of an archaeal phylotype in a proportion of infected root canals5. This is an intriguing finding because although Archaea are recognized as a component of the human microbiota, it is generally assumed that this domain does not cause human disease. In this example, however, does an association of Archaea with an infected site prove that these organisms are actively contributing to disease? Are any of Koch’s postulates fulfilled? By demonstrating an association between Archaea and infection, these findings certainly support a hypothesis that members of this domain can be human pathogens. An association, however, represents only one step on the journey to proof, and more research will be required before causality can be conclusively established in this case. In the fight against newly emerging infectious diseases, the accurate identification of the offending aetiological agent will be essential if preventative and therapeutic measures are to be implemented effectively. It will serve the microbiological community well if Koch’s postulates remain the gold standard to aim for when determining microbial cause and effect. 1. Falkow, S. What is a pathogen? ASM News 63, 359–365 (1997). 2. Falkow, S. Molecular Koch’s postulates applied to bacterial pathogenicty — a personal recollection 15 years later. Nature Rev. Microbiol. 2, 67–72 (2004). 3. Fredericks, D. N. & Relman, D. A. Sequence-based identification of microbial pathogens: a reconsideration of Koch’s postulates. Clin. Microbiol. Rev. 9, 18–33 (1996). 4. Greenberg, D. E. et al. A novel bacterium associated with lymphadenitis in a patient with chronic granulomatous disease. PLoS Pathogens 2, e28 (2006). 5. Vianna, M. E. et al. Identification and quantification of archaea involved in primary endodontic infections. J. Clin. Microbiol. 44, 1274–1282 (2006).

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RESEARCH

HIGHLIGHTS I N N AT E I M M U N I T Y

Finding flagellin Bacterial flagellin is a known ligand for the Toll-like receptor TLR5. However, several recent papers have now revealed that in addition to the TLR5 pathway, which responds to extracellular flagellin, host macrophages can respond to cytosolic flagellin through members of the NOD-like receptor (NLR) family. The recognition of pathogenassociated molecular patterns by host TLRs is a key component of innate immunity and much has been learned about TLRs and their signalling pathways over the past decade.

More recently, attention has turned to the role of non-TLRs in innate immunity, including the cytoplasmic NLR family. Details of the NLR signalling pathways are beginning to emerge and NLRs are known to be involved in secretion of the proinflammatory cytokine interleukin1β (IL-1β) by macrophages. IL-1β is produced initially as a zymogen that is activated for secretion by caspase 1. In Salmonella typhimurium infection, the NLR protein Ipaf was known to be involved in caspase 1 activation and IL-1β secretion but, until now, the S. typhimurium ligand for Ipaf was unknown. Two independent groups led by Gabriel Núñez and Alan Aderem investigated the nature of the innate immune response to S. typhimurium infection. Both groups confirmed that Ipaf was required for IL-1β production and caspase 1 activation in macrophages. Additionally, they both found that S. typhimurium mutants that either lack or have mutated flagella did not stimulate caspase 1 activation or IL-1β secretion, suggesting that flagellin is the S. typhimurium ligand for Ipaf. As flagellin is also a known ligand for TLR5, the involvement of TLRs was examined — both groups found that S. typhimurium could stimulate caspase 1 activation and IL-1β secretion in TLR5-deficient macrophages, and in wild-type macrophages, and in addition Franchi et al. found normal levels of caspase 1 activation

and IL-1β secretion in tolerant macrophages that are refractory to TLR stimulation. Taken together, these results suggest that macrophages sense flagellin through a TLR5independent pathway that relies on the cytoplasmic sensor Ipaf. Further confirmation that Ipaf senses flagellin in the cytosol independently of TLR5 comes from the fact that both groups also demonstrated that purified flagellin delivered to the cytosol triggered caspase 1 activation in wild-type but not Ipaf-deficient macrophages. The mechanism by which flagellin accesses the cytosol during infection remains to be completely elucidated, however genetic evidence presented by Miao et al. suggests that it is transferred directly into the host cell cytoplasm by the virulence-associated type III secretion system. These results are echoed by results published recently in two independent papers, one in PLoS Pathogens and one in Journal of Experimental Medicine, which indicate that an NLR is also involved in cytosolic sensing of Legionella pneumophila flagellin through a TLR5-independent, caspase-1-dependent pathway. Sheilagh Molloy ORIGINAL RESEARCH PAPERS Franchi, L. et al. Cytosolic flagellin requires Ipaf for activation of caspase-1 and interleukin 1β in Salmonellainfected macrophages. Nature Immunol. 30 April 2006 (doi:10.1038/ni1346) | Miao, E. A. et al. Cytoplasmic flagellin activates caspase-1 and secretion of interleukin 1β via Ipaf. Nature Immunol. 30 April 2006 (doi:10.1038/ni1344) | Ren, T. et al. Flagellin-deficient Legionella mutants evade caspase 1 and Naip5-mediated macrophage immunity. PLoS Pathogens 3 e18 (2006) | Molofsky, A. B. et al. Cytosolic recognition of flagellin by mouse macrophages restricts Legionella pneumophila infection. J. Exp. Med. 203, 1093–1104 (2006)

RESEARCH HIGHLIGHTS ADVISORS ADRIANO AGUZZI University Hospital of Zürich, Zürich, Switzerland NORMA ANDREWS Yale University School of Medicine, New Haven, CT, USA ARTURO CASADEVALL The Albert Einstein College of Medicine, Bronx, NY, USA

RITA COLWELL University of Maryland Biotechnology Institute, Baltimore, MD, USA STANLEY FALKOW Stanford University School of Medicine, Stanford, CA, USA TIMOTHY FOSTER Trinity College, Dublin, Ireland

NATURE REVIEWS | MICROBIOLOGY

KEITH GULL University of Oxford, Oxford, UK NEIL GOW University of Aberdeen, Aberdeen, UK HANS-DIETER KLENK Philipps University, Marburg, Germany

BERNARD MOSS NIAID, National Institutes of Health, Bethesda, MD, USA JOHN REX AstraZeneca, Cheshire, UK DAVID ROOS University of Pennsylvania, Philadelphia, PA, USA

PHILIPPE SANSONETTI Institut Pasteur, Paris, France CHIHIRO SASAKAWA University of Tokyo, Tokyo, Japan ROBIN WEISS University College London, London, UK

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RESEARCH HIGHLIGHTS A DA P T I V E I M M U N I T Y

Private repertoire blues

…it is the unique make-up … of the cross-reactive memory CD8+ T cells … that determines the response to heterologous infection…

The diversity of the mammalian immune system is one of its most powerful attributes. Activation of cell-mediated immunity by foreign antigens induces a diverse range of T cells, each clone bearing a T-cell receptor (TCR) with unique specificity. But sometimes, for reasons that have so far been unclear, the T-cell response to viral infection is restricted, consisting of only a limited range of T-cell clones. New work by Liisa Selin, Raymond Welsh, Markus Cornberg and colleagues reveals that the unique composition of an individual’s memory T-cell pool may lie at the heart of this dysfunctional immune response. The authors suspected that activation of cross-reactive memory T cells might be implicated in generating a restricted T-cell response and they used a mouse model of virus

infection to investigate this further. They first showed that epitopes from lymphocytic choriomeningitis virus (LCMV NP205–212) and Pichinde virus (PV NP205–212) are crossreactive: NP205-specific CD8+ T cells that are activated by primary infection with LCMV or PV produced IFN-γ in response to the heterologous NP205 peptide. Sequence analysis of the Vβ portion of the TCR (a region of the TCR that varies among T cells) showed that NP205-specific CD8+ T cells activated during acute LCMV or PV infection have a broad and diverse TCR repertoire. TCRs from NP205-specific CD8+ T cells obtained from LCMV-infected mice revealed hundreds of different TCR Vβ sequences or clonotypes. Once acute infection with LCMV or PV resolves, a pool of memory CD8+

B AC T E R I A L P H Y S I O LO GY

Precious metal Riboswitches are conformational switches in complex folded RNA domains that are induced by binding to specific small molecules, and which lead to a switch in gene-regulatory function. In a recent issue of Cell, Eduardo Groisman and his team at the Howard Hughes Medical Institute in St Louis, USA, report on the first

example of a riboswitch that senses, and responds to, a metal ion. Magnesium (Mg2+) ions are abundant in biological systems and are required for a wide variety of cellular processes and structures. Cells maintain cytoplasmic Mg2+ levels within a narrow range, but little is known about the mechanisms through which

…the first example of a riboswitch that senses, and responds to, a metal ion.

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T cells is established, including the cross-reactive NP205-specific CD8+ T cells. The authors went on to show that these cross-reactive T cells can have a profound influence on the memory T-cell repertoire. In PV-immune mice that are infected with LCMV, proliferation of cross-reactive PV NP205-specific memory CD8+ T cells results in a dominant NP205 response. But in contrast to the diverse CD8+ T-cell population induced by NP205 in acute infection, the TCR repertoire of NP205-specific CD8+ T cells after heterologous LCMV challenge is limited and dominated by specific Vβ clonotypes. These results indicate that only a small subset of the cross-reactive NP205-specific CD8+ T cells proliferate after heterologous infection. This oligoclonal TCR repertoire can favour the generation of viral escape mutants as shown by the isolation of an LCMV NP205-variant virus from PV-immune mice 8 months after LCMV challenge. Using adoptive transfer experiments, Cornberg et al. revealed

cells sense and maintain these Mg2+ levels. Salmonella enterica serovar Typhimurium (S. typhimurium) contains the Mg2+-responding PhoP–PhoQ twocomponent regulatory system and several Mg2+ transporters. One of these transporters is MgtA, which mediates Mg2+ influx. The mgtA gene is regulated by the transcriptional activator PhoP in response to extracytoplasmic Mg2+ sensed by the sensor kinase PhoQ. In addition to the PhoP–PhoQ system, it was suspected that mgtA is regulated by another, Mg2+-sensing, system, as mgtA transcription still responded to Mg2+ in an S. typhimurium strain lacking PhoQ and harbouring a Mg2+-independent PhoP mutant. This Mg2+-sensing mechanism is what Cromie et al. set out to investigate. First, they used RT-PCR to examine the levels of mgtA mRNA in wild-type S. typhimurium strains that had been grown in the presence of different Mg2+ concentrations. They found that the mgtA transcript reaches the coding region only when S. typhimurium experiences very low Mg2+ concentrations, despite mgtA transcription being initiated at Mg2+

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RESEARCH HIGHLIGHTS that it is the unique make-up (or the private specificity) of the crossreactive memory CD8+ T cells of individual mice that determines the response to heterologous infection, although so far the mechanisms that favour the growth of some of these cells over others remain unknown. The cross-reactive profiles of private T-cell repertoires might explain why some patients mount oligoclonal T-cell responses to HIV or hepatitis C virus infection and seem to be prone to the development of viral escape mutants. Finally, these results have important implications for vaccine design, as, in certain individuals, immunizing peptides could generate a pool of cross-reactive memory CD8+ T cells that respond to subsequent heterologous virus challenge with a restricted T-cell repertoire.

IN BRIEF B AC T E R I A L P H Y S I O LO GY

Definition of the bacterial N-glycosylation site consensus sequence Kowarik, M. et al. EMBO J. 13 April 2006 (doi:10.1038/ sj.emboj.7601087)

In eukaryotes, the consensus sequence for N-linked glycosylation is N–X–S/T (where X represents any amino acid except proline). In this work, Kowarik et al. investigated the substrate requirements for bacterial N-glycosylation in vivo and found the Campylobacter jejuni protein glycosylation machinery recognizes the consensus sequence D/E–Y–N–X–S/T (where X and Y can be any amino acid except proline). In addition to having an extended consensus sequence, Kowarik et al. demonstrated that the bacterial glycosylation system also shows increased stringency and specificity compared with eukaryotes. These results have implications for the biotechnological production of glycoproteins in bacteria. VIROLOGY

Role of the α/β interferon response in the acquisition of susceptibility to poliovirus by kidney cells in culture

Shannon Amoils

Yoshikawa, T. et al. J. Virol. 80, 4313–4325 (2006)

ORIGINAL RESEARCH PAPER Cornberg, M. et al . Narrowed TCR repertoire and viral escape as a consequence of heterologous immunity. J. Clin. Invest. 13 April 2006 (doi:10.1172/JCI27804)

concentrations 100-fold higher. Further experiments showed that the 5′-UTR region of mgtA is necessary, and sufficient, to respond to Mg2+. In a next step, Groisman and colleagues used the Mfold program to predict the secondary structure that might be adopted by the mgtA 5′-UTR. They identified two potential mutually exclusive structures: one including two stem-loop structures, A plus B, and an alternative stem-loop structure, C. Using S. typhimurium mutant strains where mgtA transcription initiated at different positions, it was found that stem loop B might be the structure formed when Mg2+ levels are high, and might be responsible for transcription stopping before the mgtA coding region. On the other hand, formation of stem loop C, the alternative to stem loops A plus B, might be favoured when Mg2+ levels are low. Synthesizing the full-length mgtA 5′UTR, and treating the RNA with RNases and chemicals to probe its structure after incubation in the presence of different Mg2+ concentrations, Cromie et al. showed that Mg2+ can modify the structure of the mgtA 5′-UTR.


Stem loop A was found to be crucial for Mg2+ sensing, and for the Mg2+promoted changes taking place in stem loops B and C. Finally, using an in vitro transcription system, they demonstrated that the mgtA 5′-UTR responds to Mg2+ by affecting the ability of RNA polymerase to stop transcription. So, the expression of MgtA is controlled by Mg2+ in two steps — the initiation of mgtA transcription by PhoP, which is controlled by PhoQ in response to extracytoplasmic Mg2+, and the early stopping of mgtA transcription by its 5′-UTR riboswitch, which responds to cytoplasmic Mg2+ — in contrast to most genes that are controlled by riboswitches, which are typically only regulated by their respective riboswitches. Although this is the first evidence of a cation-responsive riboswitch, it is likely that additional ion-regulated riboswitches exist. Annie Tremp ORIGINAL RESEARCH PAPER Cromie, M. J., Shi, Y., Latifi, T. & Groisman, E. A. An RNA sensor for intracellular Mg2+. Cell 125, 71–84 (2006)

In vivo, poliovirus, the causative agent of poliomyelitis, has a strict neurotropism, replicating at only a few sites including the brain and spinal cord. Fortunately for researchers working on poliovirus, the pioneers of early laboratory techniques for poliovirus cultivation, including John Enders and Renato Dulbecco, discovered that this neurotropism was not strictly observed in vitro and that cell lines of monolayer cultures derived from almost any primate tissue are susceptible to poliovirus infection. However, the molecular basis for the acquisition of poliovirus susceptibility by cultured cells has always been unknown. Now, reporting in the Journal of Virology, Yoshikawa et al. have shown that changes in the antiviral activity of the interferon response are likely to be the most important factors determining the acquisition of poliovirus susceptibility. ANTI-INFECTIVES

Co-expression of virulence and fosfomycin susceptibility in Listeria: molecular basis of an antimicrobial in vitro–in vivo paradox Scortti, M. et al. Nature Med. 23 April 2006 (doi:10.1038/nm1396)

The so-called ‘in vitro–in vivo’ paradox for antibiotics refers to discrepancies between the resistance to a particular compound that is observed using in vitro susceptibility tests and the treatment outcomes that are observed for this compound in the clinic; that is, antibiotics that have poor efficacy in vitro can be successfully used to treat infections in vivo. In this Brief Communication in Nature Medicine, the authors present a molecular mechanism that could explain the paradox in fosfomycin treatment of Listeria monocytogenes infection. In in vitro susceptibility tests L. monocytogenes is resistant to fosfomycin, yet Scortti et al. demonstrate that in mice fosfomycin does have anti-listerial activity. They found that L. monocytogenes uptake of fosfomycin is dependent on the Hpt transporter and its regulator PrfA. PrfA is only weakly expressed under in vitro conditions but is highly induced in vivo, therefore explaining why L. monocytogenes is resistant to fosfomycin in vitro, but not in vivo.

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RESEARCH HIGHLIGHTS PA R A S I T O L O G Y

E N V I R O N M E N TA L M I C R O B I O L O G Y

Who’s HOSTing who? How the obligate intracellular pathogen Toxoplasma gondii acquires the nutrients that are necessary for its survival and growth has long been an open question. Reporting in Cell, Isabelle Coppens and colleagues now describe a novel mechanism by which T. gondii gains access to key molecules: sequestration of organelles of the host-cell endocytic pathway. Infection of host cells with T. gondii involves internalization of the parasite into a membrane-bound compartment known as the parasitophorous vacuole, which — in contrast to most other pathogen-containing compartments — does not fuse with host-cell endocytic organelles. T. gondii therefore avoids the degradation that would result from sequential fusion with compartments of the endocytic pathway; however, in this way, it is not exposed to the proteins and lipids, such as cholesterol (for which T. gondii is auxotrophic), that enter the host cell through this pathway. Coppens and colleagues, however, had previously observed that cholesterol is somehow transferred from host endocytic organelles to the parasitophorous vacuole, so they set out to examine the localization of host endocytic organelles in T. gondiiinfected cells, using fluorescence microscopy and transmission electron microscopy. After infection with T. gondii, host endolysosomes were found to accumulate around the parasitophorous vacuole, which was also shown to be surrounded by networks of host microtubules. This cytoskeletal reorganization was found to result in the formation of invaginations — referred to as host organelle-sequestering tubulo-structures (HOST) — of the membrane of the parasitophorous vacuole. The authors propose that the tubular structure of HOST allows them to function as conduits for the delivery of endolysosomes to the parasitophorous vacuole. Furthermore, HOST were found to be coated with parasite proteins, including the granule protein GRA7, which can constrict the HOST conduits, thereby preventing exit of the host organelles and sequestering them within the parasitophorous vacuole. The authors postulate that the contents of these organelles would then undergo hydrolysis, and cholesterol and other important lowmolecular-weight molecules would be released across the membrane of the intact organelles and absorbed by the parasite. Not only is this an unexpected finding of a novel process of nutrient acquisition by a parasite, but it is also a unique mechanism for the unidirectional transport and sequestration of host-cell organelles. Davina Dadley-Moore ORIGINAL RESEARCH PAPER Coppens, I. et al. Toxoplasma gondii sequesters lysosomes from mammalian hosts in the vacuolar space. Cell 125, 261–274 (2006) FURTHER READING Gruenberg, J. & and Gisou van der Goot, F. Toxoplasma: Guess who’s coming to dinner. Cell 125, 226–228 (2006) WEB SITE Isabelle Coppens’s homepage: http://faculty.jhsph.edu/?F=Isabelle&L=Coppens

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A global unculture… Two reports recently published in Nature shed new light on two processes only recently identified as being major contributors to global carbon and nitrogen cycling. Both processes — the anaerobic oxidation of ammonium and methane — were thought to be non-existent in nature but are, in fact, catalysed by unrelated microorganisms that have yet to be grown in pure culture. The obligate anaerobic ammonium oxidation (anammox) reaction, which uses nitrite as the primary electron acceptor, is

accountable for up to 50% of oceanic nitrogen loss. Although identified in 1999, the planctomycete-like bacteria that catalyse this process have proven difficult to study owing to their extremely slow growth. Now, Strous et al. have applied the advantages of environmental genomics to gain insight into the unusual biology of these important microorganisms. Anammox bacteria have a unique prokaryotic organelle (the ‘anammoxosome’) surrounded by ladderane lipids that contain hydrazine oxidoreductase,

The photograph shows Marc Strous extracting a mud sample from the Twentekanaal, a canal in the Netherlands. These anoxic sediments were used as the inoculum for the enrichment of an anaerobic methane-oxidizing microbial consortium. Photograph courtesy of Radboud University Nijmegen.

F U N G A L PAT H O G E N E S I S

Global control Researchers have identified the regulator of the phase transition from the non-pathogenic hyphal form to the pathogenic yeast form in dimorphic fungal pathogens, according to a paper published recently in Science. Six dimorphic ascomycetes including Blastomyces dermatitidis and Histoplasma capsulatum are typically found in the environment but can cause disease in humans if spores are inhaled. Temperature is the key environmental cue for fungal dimorphic switching, with the transition occurring with a shift from 25°C to 37°C, but the key outstanding question in the field has been what regulates this phase transition. In this work, Julie Nemecek, Marcel Wüthrich

and Bruce Klein set out to identify the master regulator that controls the switch from the hyphal to the yeast form. Previous work in the Klein laboratory had shown that Agrobacterium tumefaciens DNA could be transformed randomly into the B. dermatitidis genome and so Nemecek et al. devised an insertional mutagenesis assay in which A. tumefaciens DNA was transformed into a B. dermatitidis reporter strain. Blue/white selection identified seven transformants and the transformant chosen for further analysis had an 86% reduction in transcription of the yeast-phasespecific reporter gene (BAD1) compared with the parental strain. Further analysis of the mutant

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RESEARCH HIGHLIGHTS an enzyme that combines nitrite and ammonia in a one-to-one mechanism. Using an anoxic laboratory bioreactor that contained a complex microbial community dominated by the anammox bacterium Kuenenia stuttgartiensis, the authors sequenced over 1 gigabase of extracted DNA. These sequences data allowed the researchers to assemble the genome of K. stuttgartiensis, one of the first complete genome sequences of an organism not available in pure culture. Analysis of the genome revealed the presence of over 200 genes directly involved in anammox catabolism and respiration, as well as candidate genes responsible for ladderane biosynthesis and biological hydrazine metabolism. This large number of genes is also indicative of an unexpected degree of metabolic versatility in the microorganism. Environmental sequence analysis was also used to analyse the genomes of a microbial consortium that coupled the direct oxidation of methane to denitrification of nitrate in the absence of oxygen. In this separate study, Marc Strous and colleagues enriched for microbial life derived from the anoxic sediments of a freshwater canal over a

16-month period. Analysis of the culture revealed the presence of two different types of microorganism, a bacterium belonging to a novel phylum without any documented cultured species, and an archaeon distantly related to marine methanotrophic archaea. Interestingly, similar sequences were also identified in freshwater ecosystems from different global locations, strongly suggesting that the process of microbial anaerobic methane oxidation coupled to denitrification has worldwide ecological significance. The contribution of microorganisms to global biogeochemical cycles is well established; however, important aspects pertaining to their biology have remained ill-defined or overlooked. These two studies represent significant advances in addressing these gaps and clearly show the power of genomics in elucidating the biology of environmentally important, yet ‘unculturable’, microorganisms.

phenotype revealed a pleiotropic set of defects, including the inability to undergo the switch from the hyphal to the yeast form. The authors went on to map the site of insertion in the B. dermatitidis genome and identified the disrupted gene as an open-reading frame (ORF) encoding a 1274-residue protein. Sequence analysis revealed that this ORF encodes a hybrid histidine kinase with homology to the Saccharomyces cerevisiae histidine kinase SLNI. Knocking out the ORF by allelic replacement induced the same pleiomorphic defects as observed in the original transformant and so the authors named the gene DRK1, for dimorphism-regulating histidine kinase. In addition to having a homologue in the S. cerevisiae genome, DRK1 is also conserved in the other dimorphic fungal pathogens for which extensive sequence information is available, including H. capsulatum and Coccidioides posadasii. RNA interference was used to silence DRK1

expression in B. dermatitidis and H. capsulatum and it was found that in H. capsulatum, as in B. dermatitidis, DRK1 is involved in dimorphic switching and virulence-gene expression. Finally, in vivo analysis showed that the pathogenicity of DRK1-silenced strains of both B. dermatitidis and H. capsulatum was reduced in murine models of infection. This study represents a milestone in research for those interested in dimorphic fungal pathogens as it not only identifies DRK1 as a key global regulator that controls the hyphalto-yeast transition, virulence-gene expression and pathogenicity but also finally confirms that the transition to the yeast form is a requirement for pathogenicity. Sheilagh Molloy


David O’Connell ORIGINAL RESEARCH PAPERS Strous, M. et al. Deciphering the evolution and metabolism of an anammox bacteria from a community genome. Nature 440, 790–794 (2006) | Raghoebarsing, A. A. et al. A microbial consortium couples anaerobic methane oxidation to denitrification. Nature 440, 918–921 (2006)

ORIGINAL RESEARCH PAPER Nemecek, J. C., Wüthrich, M. & Klein, B. S. Global control of dimorphism and virulence in fungi. Science 312, 583–588 (2006)

Immuno-electron microscopy showing non-fusogenic disruption of the EEV outer membrane, which exposes the IMV particle to the cell surface. Image reproduced with permission from Law et al. © (2006) National Academy of Sciences, USA.

VIROLOGY

A novel state of undress Enveloped viruses penetrate target cells by fusing their single lipid membrane with the target cell membrane and releasing the infective naked viral core. But some virus forms such as the extracellular enveloped virus (EEV) of Vaccinia virus have two lipid membranes, and therefore cell entry requires the removal of both lipid barriers. New work from Geoffrey Smith’s laboratory at Imperial College London has uncovered a unique way in which EEV sheds its outer lipid membrane, providing novel insights into virus entry and also a new target for antiviral therapy. The authors studied the binding and entry of EEV to cells by immuno-electron microscopy and observed that the outer EEV membrane ruptures at the site of cell contact. This disruption, however, takes place in the absence of membrane fusion and the shed EEV outer membrane remains draped over the underlying single-enveloped virion, which is called an intracellular mature virus (IMV). The single membrane of IMV subsequently fuses with the target cell membrane and the virus core enters the cell. Because disruption of the EEV outer membrane takes place only on contact with a cell, and not a synthetic substrate, the authors proposed that outer membrane dissolution required the interaction between ligands on the virus and the target cell surface. A series of experiments identified the virus glycoproteins and, and cellular surface polyanions, known as glycosaminoglycans, as the required ligands. This is the first example of the removal of a virus membrane without fusion, which Smith and colleagues term ligand-dependent nonfusogenic viral membrane dissolution. The authors went on to show that a soluble polyanion such as heparin can be used to rupture the EEV outer envelope in vitro, exposing the IMV and allowing neutralization by antiIMV monoclonal antibodies. The combined administration of polyanions and anti-IMV antibodies protected mice against disease with Vaccinia virus, demonstrating the therapeutic potential of this strategy. The EEV virion is the form of Vaccinia virus that spreads within the host and therefore an increased understanding of its entry mechanism and the identification of a strategy to target EEV entry are important advances in this field. Shannon Amoils ORIGINAL RESEARCH PAPER Law, M. et al. Ligand-induced and nonfusogenic dissolution of a viral membrane. Proc. Natl Acad. Sci. USA 11, 5989–5994 (2006)

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N E W S & A N A LY S I S DISEASE WATCH | IN THE NEWS Gut feeling Signalling through Toll-like receptors (TLRs) is a key component of the innate immune response, but the precise role of TLR-based innate recognition at mucosal surfaces such as the gastrointestinal tract is difficult to assess. In a recent paper in Journal of Experimental Medicine, Mathias Hornef and colleagues have begun to address this by investigating the role of lipopolysaccharide (LPS) signalling through the TLR4–MD2 complex, using an assay involving primary intestinal epithelial cells (IECs) from fetal, newborn and adult mice. They found that although all cells examined expressed the TLR4 complex, the ability to respond to agonists varied with the developmental stage. Only fetal IECs were able to activate the signalling pathways downstream of TLR4 when challenged with LPS, interleukin-1β or tumour necrosis factor; newborn IECs spontaneously released inflammatory cytokines, but this was not enhanced by the presence of an agonist, and adult IECs were non-responsive. Further investigations comparing vaginally born mice with mice born by caesarean section revealed that the spontaneous activation of newborn IECs and concomitant acquisition of LPS resistance requires vaginal delivery and oral exposure to LPS. The authors conclude that: “This adaptive processing might be crucial to facilitate postnatal microbial colonization and subsequent development of the astonishingly stable, lifelong symbiosis.” JEM

World health report card As part of the 2006 World Health Day, the WHO have published their annual World Health Report. The theme of this year’s report was ‘working together for health’ and it highlighted the fact that although the global population is growing, the number of healthcare workers is not increasing sufficiently to allow national healthcare systems to either provide essential medical interventions such as immunization and access to treatment for HIV/AIDS, malaria and tuberculosis, or to respond effectively to a healthcare crisis (such as SARS or avian influenza). In fact, there is a chronic shortage of healthcare workers — including doctors, nurses and midwives — in 57 countries worldwide, including 36 countries in sub-Saharan Africa. More than 4 million additional workers are needed to plug the gap, according to the report, which includes

a 10-year plan to address the crisis. WHO Assistant Director-General Timothy Evans also commented specifically on the impact of the ‘brain drain’ of qualified healthcare professionals from developing countries, warning that industrialized nations “are likely to attract even more foreign staff because of their aging populations, who will need more long-term care”. The report recommends that 50% of all new donor funds for health should be spent on strengthening healthcare systems. WHO

untreated, can lead to irreversible blindness. The WHO recommends that trachomaaffected countries implement a national strategy based on the SAFE principles to eliminate the disease; SAFE involves eyelid surgery; antibiotics; facial cleanliness and environmental changes. Last month’s In the News reported on the successful results of a clinical trial on the use of a single oral dose of azithromycin as a treatment for the disease, and the free availability of azithromycin forms a core part of the SAFE strategy. WHO

Contact lens warning Post exposure Marburg vaccine?

The US FDA and CDC have issued an alert to healthcare professionals and their patients about the risk of corneal fungal infections in wearers of soft contact lenses. As of the beginning of May, almost 200 cases of suspected Fusarium corneal infection were being investigated. US company Bausch & Lomb voluntarily suspended sales of its lens cleaner ReNu With MoistureLoc in April after it was linked with the outbreak, but no direct link has yet been proven. However, news that a Fusarium outbreak in Hong Kong at the end of 2005 had also been linked with the Bausch & Lomb solution prompted some criticism that the company had not acted quickly enough. As we went to press, news had begun to emerge that Fusarium infections had also been reported in Europe. CDC

More trachoma progress At their 10th meeting held recently in Geneva, The Alliance for the Global Elimination of Blinding Trachoma by the Year 2020 (GET2020) reviewed the encouraging progress that has been made towards their goal of eliminating trachoma by the year 2020. Trachoma is associated with Chlamydia trachomatis infection of the eye and, if left

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The results of further work by Heinz Feldmann and colleagues on their live attenuated vaccine against the filovirus Marburg virus were reported recently in the Lancet. The work suggests that the vaccine, which is based on recombinant vesicular stomatitis virus (rVSV), could be used prophylactically after exposure to the virus, in addition to providing protection before exposure. In an efficacy assessment study carried out in a rhesus-macaque model of Marburg haemorrhagic fever, eight animals received an intramuscular dose of 1,000 plaque-forming units (pfu) of Marburg virus. Twenty to thirty minutes later, five animals received 1 x 107 pfu of the rVSVbased vaccine, given as four intramuscular injections in different anatomical sites, and the three control animals were treated with the vector alone. All of the five animals that received the vaccine survived for at least 80 days; by contrast, the three animals in the control group all died by day 12. The authors suggest that the 20–30-minute time interval between exposure to the virus and administration of the vaccine would be sufficient to respond to accidental needlestick exposure in a laboratory or healthcare situation. Lancet

Two new kids on the block Chronic granulomatous disease (CGD) is an inherited primary immune deficiency that is caused by a defect in the NADPH-oxidase complex in phagocytic cells, and CGD patients are therefore more susceptible to infection with various catalase-producing microorganisms, including Staphylococcus aureus and Aspergillus spp. In addition, patients often present with symptoms, such as inflammation of the lymph nodes, for which the causative infectious


organism cannot be identified; in such cases, broad-spectrum antimicrobials are usually given to cover all options. Now, Greenberg et al. report in PLoS Pathogens on the identification of a new Gram-negative bacterium associated with lymph-node inflammation in a CGD patient. The organism — named Granulobacter bethesdensis — is a new genus and species within the Acetobacteraceae, and is the first member of this family to be associated with invasive disease. Since the paper was accepted for publication, the researchers have isolated G. bethesdensis from two additional CGD patients. In a separate publication in a recent issue of Journal of Clinical Microbiology, Archaea have been linked to infectious diseases for the first time, with the identification of a Methanobrevibacter oralis-like archaeal phylotype in human endodontic infection. Eurekalert

Genetic test for HCV prognosis Up to 20% of individuals infected with the hepatitis C virus (HCV) go on to develop cirrhosis, a chronic condition that can result in liver failure or hepatocellular carcinoma. It has long been known that the risk of developing cirrhosis is associated with clinical risk factors, such as being male, being over 40 years of age at the time of infection, and alcohol abuse, but genetic risk factors have been difficult to find. Now, Celera have presented data which indicate that a genetic test to predict which individuals infected with HCV will go on to develop cirrhosis could be available in the not-toodistant future. Speaking at an international conference for liver researchers in April, the company announced the results of a study to identify genetic predictors that involved ~1,500 HCV-infected individuals and has identified a total of seven single-nucleotide

polymorphisms that can predict the risk of developing cirrhosis more accurately than the existing clinical risk factors. Celera press release

Leishmaniasis vaccine update There are four main forms of leishmaniasis, ranging from the relatively mild cutaneous leishmaniasis to visceral leishmaniasis, which can be fatal if left untreated and which affects an estimated 500,000 individuals annually, with 60,000 deaths. All leishmaniases are caused by parasitic protozoa belonging to the genus Leishmania, which are transmitted by phlebotomine sandflies and, so far, there is no effective vaccine for any of the leishmaniases. Progress does, however, seem to have been made recently, according to a paper in ACS Chemical Biology that featured heavily on the newswires. Carbohydrate epitopes are becoming increasingly attractive for vaccine formulations, but require a suitable carrier and adjuvant. Immunostimulating reconstituted influenza virosomes (IRIVs) combine both these properties and have already been used successfully in vaccine construction. Now, IRIV technology has been used to create a leishmaniasis vaccine formulation with the cap tetrasaccharide from the Leishmania donovani lipophosphoglycan, and preliminary results in mice show that a strong protective response was elicited. Eurekalert

Microbicides in the news Microbicides moved higher up the news agenda towards the end of April, as reports came in from Microbicides 2006 in Cape Town — the first time that this biennial conference has been held in Africa. Stories highlighted included the results of Phase I and Phase II clinical trials of new microbicide products, and there was also a focus on the need for research into the acceptability of microbicide use.

Avian influenza Human cases of infection with the H5N1 virus continued to be reported throughout April, in countries including Egypt, China and Indonesia, and the total number of confirmed cases in 2006 by 5 May was 62, with 38 deaths. In terms of the general media however, these cases received


comparatively little attention and instead news items focused mainly on efforts to develop a vaccine to be used in the event of a pandemic, with the US Federal Government announcing it was awarding $1 billion in contracts to five pharmaceutical companies for vaccine development. Speaking at an avian influenza conference influenza expert Robert Webster commented that the H5N1 virus is the worst flu virus he’s seen. In the United Kingdom, there was initial alarm when avian influenza was detected on a poultry farm in Norfolk. The 35,000 chickens on the farm were slaughtered as a precaution, but tests have confirmed that the virus was a lowpathogenicity H7N3 strain. Promed Mail.

Outbreak news Mumps. A mumps outbreak in the Midwest of America has been hitting the headlines recently as it is the worst outbreak of mumps in the country for 20 years. The state of Iowa has been badly hit, with the number of recorded cases reaching more than 1,500. Mass immunization clinics for 18–22-year olds have been organized. Cholera. A cholera outbreak in Angola that began in February has been exacerbated by recent heavy rains, and more than 1,000 deaths had been reported by the beginning of May, with more than 20,000 infections, according to Médicins san Frontières (MSF). The head of the MSF mission in Angola commented that the outbreak had yet to reach its peak and that the number of deaths could double. In the News was compiled with the assistance of David Ojcius, University of California, Merced, USA. David’s links to infectious disease news stories can be accessed on Connotea (http://www. connotea.org), under the username ojcius.

VOLUME 4 | JUNE 2006 | 421 © 2006 Nature Publishing Group

REVIEWS Listeria monocytogenes: a multifaceted model Mélanie Hamon*‡§, Hélène Bierne*‡§ and Pascale Cossart*‡§

Abstract | The opportunistic intracellular pathogen Listeria monocytogenes has become a paradigm for the study of host–pathogen interactions and bacterial adaptation to mammalian hosts. Analysis of L. monocytogenes infection has provided considerable insight into how bacteria invade cells, move intracellularly, and disseminate in tissues, as well as tools to address fundamental processes in cell biology. Moreover, the vast amount of knowledge that has been gathered through in-depth comparative genomic analyses and in vivo studies makes L. monocytogenes one of the most well-studied bacterial pathogens.

*Institut Pasteur, Unité des Interactions Bactéries Cellules, Paris 75015, France. ‡ Institut National de la Santé et de la Recherche Médicale, U604, Paris 75015, France. § Institut National de la Recherche Agronomique, USC2020, Paris 75015, France. Correspondence to P.C. e-mail: [email protected] doi:10.1038/nrmicro1413

The Gram-positive bacterium Listeria monocytogenes is a ubiquitous pathogen that thrives in diverse environments such as soil, water, various food products, humans and animals. The disease caused by this bacterium, listeriosis, is acquired by ingesting contaminated food products and mainly affects immunocompromised individuals, pregnant women and newborns. Listeriosis manifests as gastroenteritis, meningitis, encephalitis, mother-to-fetus infections and septicaemia, resulting in death in 25–30% of cases. The diverse clinical manifestations of infection with L. monocytogenes reflect its ability to cross three tight barriers in the human host. Following ingestion, L. monocytogenes crosses the intestinal barrier by invading the intestinal epithelium, thereby gaining access to internal organs. During severe infections, crossing the blood–brain barrier results in infection of the meninges and the brain, and in pregnant women, crossing the fetoplacental barrier leads to infection of the fetus1. L. monocytogenes infection has been a useful model for evaluation of the cellular interactions that are crucial for the initiation of the host T-cell response. However, this aspect of listerial biology is well documented and has recently been reviewed elsewhere2. This Review highlights the many ways in which L. monocytogenes manipulates its mammalian host, and it focuses on the lessons this bacterium has taught us in cell biology, bacterial pathophysiology, virulence-factor regulation, and bacterial adaptation to the host cytosol. Indeed, the ability of L. monocytogenes to invade and replicate in different cell types has been extensively studied and has revealed the sophisticated relationship between the bacterium and its host. The breadth of information gathered on the elaborate mimicries that are used by L. monocytogenes to subvert host processes


has made it an exceptional tool for the study of cellular processes such as actin-based motility, growth-factormediated signalling, endocytosis and cellular adhesion. Furthermore, available animal models, genetic tools and genomics have facilitated the compilation of information on different aspects of L. monocytogenes biology and have made this bacterium one of the most useful model organisms for the study of bacterial pathogenesis and pathophysiology.

An intracellular bacterium and a cell biologist L. monocytogenes is a facultative intracellular bacterium. Its life cycle reflects its remarkable adaptation to intracellular survival and multiplication in macrophages and other cell types1,3,4 (FIG. 1). Similar to the situation for most pathogens, the invasion of macrophages by L. monocytogenes is a passive process, but entry into non-professional phagocytes is induced by binding of the bacterial surface proteins internalin A (InlA) and InlB to receptors on the host cell. Both of these invasins are necessary and sufficient for bacterial entry into cell types such as enterocytes, hepatocytes, fibroblasts, epithelial cells and endothelial cells, but InlAmediated entry is restricted to the smaller number of cell types that express its receptor. Entry of L. monocytogenes into mammalian cells is a dynamic process that requires actin polymerization and membrane remodelling, and is an excellent example of how a bacterium can manipulate host-cell signalling and endocytic pathways to its advantage. L. monocytogenes can also harness the actinpolymerization machinery in the cytoplasm to facilitate intracellular and intercellular movement. New mechanisms by which L. monocytogenes manipulates the host cell are emerging through the use of microarray analyses that aim to determine the genes that are specifically activated by bacterial entry into the host cell5,6. VOLUME 4 | JUNE 2006 | 423

© 2006 Nature Publishing Group

REVIEWS Listeria monocytogenes

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Figure 1 | Schematic representation and electron micrographs of the Listeria monocytogenes life cycle. a | L. monocytogenes induces its entry into a non-professional phagocyte. b | Bacteria are internalized in a vacuole (also known as a phagosome). c,d | The membrane of the vacuole is disrupted by the secretion of two phospholipases, PlcA and PlcB, and the pore-forming toxin listeriolysin O. Bacteria are released into the cytoplasm, where they multiply and start to polymerize actin, as observed by the presence of the characteristic actin tails (see Supplementary information S3 (figure)). e | Actin polymerization allows bacteria to pass into a neighbouring cell by forming protrusions in the plasma membrane. f | On entry into the neighbouring cell, bacteria are present in a double-membraned vacuole, from which they can escape to perpetuate the cycle. F-actin, filamentous actin. Electron micrographs a–c,e–f are reproduced with permission from REF. 113 © (1998) European Molecular Biology Organization, and d is reproduced with permission from REF. 30 © (1992) Elsevier.

InlB: subverting cellular-signalling and endocytic pathways. Binding of InlB to its cellular receptor Met results in the entry of L. monocytogenes into different cell types. Met is a protein tyrosine kinase, and the endogenous ligand of this receptor is hepatocyte growth factor (HGF) 7 (FIG. 2) . In vivo, Met is expressed mainly by cells of epithelial origin, whereas HGF is produced mainly by fibroblasts and stromal cells. The binding of HGF to Met activates cellular survival and proliferation signals, and it induces cytoskeletal rearrangements that function in cellular motility and differentiation. Binding of InlB activates

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the protein-tyrosine-kinase activity of Met, as well as the phosphatidylinositol 3-kinase (PI3K) and the Ras–mitogen-activated protein kinase (MAPK) pathways, all of which are required for the uptake process7–9. Interestingly, although InlB and HGF both bind and activate Met, InlB does not strictly mimic HGF. Indeed, the kinetics of InlB-induced signalling are different from those of HGF-induced signalling7, and at an equal concentration, InlB seems to induce a more potent activation of the Ras–MAPK pathway than does HGF10. Differences in signalling might be explained by the finding that InlB also binds gC1qR


REVIEWS a HGF-mediated signalling

b InlB-mediated signalling Listeria monocytogenes InlB

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Figure 2 | Met signalling induced by hepatocyte growth factor (HGF) and internalin B (InlB). a | Phosphorylation of Met leads to the recruitment and activation of many transducers, which in turn recruit cytosolic signalling proteins. Signalling mediated by HGF activates survival and proliferation signals, and it induces cytoskeletal rearrangements that are important for cellular motility and differentiation. On stimulation with HGF, the endocytosis of Met, similar to most signalling receptors, is an important regulatory mechanism that downregulates the cell-surface expression of the activated receptor. b | Met signalling mediated by the Listeria monocytogenes protein InlB induces cytoskeletal rearrangements that are important for bacterial entry into non-phagocytic cells. Clathrin components of the endocytic machinery are also recruited to the site of entry. The link between the cytoskeletal machinery (shown on the right) and the endocytic machinery (shown on the left) is still unclear. InlB, through the GW repeats at its C terminus, also binds gC1qR (the receptor for the globular part of complement component C1q) and glucosaminoglycans (GAGs), which are negatively charged polysaccharides that are present at cell surfaces. Both components might contribute to entry of L. monocytogenes by modulating the interaction of InlB with Met. ABI, Abl interactor 1; Arp, actin-related protein; CD2AP, CD2-associated protein; CIN85, Cbl-interacting protein of 85 kDa; EPS15, epidermal-growth-factor-receptor substrate 15; F-actin, filamentous actin; GAB1, GRB2-associated binding protein 1; GGA3, Golgi-localized, γ-ear-containing, ADP-ribosylation-factor-binding protein 3; GRB2, growth-factor-receptor-bound protein 2; HRS, HGF-regulated tyrosine-kinase substrate; N-WASP, neural Wiskott–Aldrich syndrome protein; PI3K, phosphatidylinositol 3-kinase; PLCγ, phospholipase C-γ; PtdIns(3,4,5)P3, phosphatidylinositol-3,4,5-trisphosphate; PtdIns(4,5)P2, phosphatidylinositol-4,5-bisphosphate; SHC, SRC-homology-2domain-containing transforming protein C; SOS, son of sevenless; Ub, ubiquitin; WAVE, Wiskott–Aldrich syndrome protein (WASP)-family verprolin homologous protein.

(the receptor for the globular part of complement component C1q), which might therefore function as a co-receptor for InlB11. Furthermore, InlB and HGF do not compete for binding to Met7, indicating that they might bind distinct sites on Met. These results are consistent with the fact that InlB and HGF have no sequence homology and are structurally unrelated. Crystal structures of InlB bound to Met could provide valuable information about the molecular mechanism that underlies the activation of Met by InlB. The signalling pathways that are activated by InlB ultimately lead to cytoskeletal rearrangements and entry of L. monocytogenes. How activation of the PI3K and the Ras–MAPK pathways leads to cytoskeletal rearrangements has been extensively studied, and many of the proteins that are crucial for invagination and internalization have been identified. Local actin remodelling at the site of InlB attachment is mediated by the recruitment and activation of the actin-nucleation complex, Arp2/3, which promotes actin nucleation and polymerization (discussed later). The mechanism of


Arp2/3 activation seems to be cell-type dependent, but it involves a combination of the small GTPases Rac and CDC42 and proteins of the Wiskott–Aldrich syndrome protein (WASP) family, which includes neural WASP (N-WASP) and WAVE 12. Proteins of the Ena/VASP family (enabled homologue/vasodilator-stimulatedphosphoprotein family), which promote actin-filament elongation, are also central to the process. In addition, cofilin, which is essential for depolymerization of actin filaments, functions successively as a stimulator and a downregulator of actin rearrangements that occur during the internalization process 12,13. All of these components that are recruited by the binding of InlB to Met also have a role in growth-factor-receptor activation. Therefore, although there are differences between the kinetics of signalling mediated by InlB and HGF, the machinery that is recruited to the site of activation seems to be the same for both molecules, showing the utility of L. monocytogenes as a tool to study cellular signalling by Met or other growth-factor receptors.


REVIEWS Recently, the study of InlB-induced internalization revealed an unexpected mechanism used by L. monocytogenes during host-cell entry. L. monocytogenes invades epithelial cells by subverting clathrin-mediated endocytosis14 (FIG. 2b; see Supplementary information S1 (figure)), a process that is used by mammalian cells to take up nutrients and to recycle membrane proteins. Owing to their size, which is usually 1–3 µm, bacteria were thought to enter cells through a mechanism related to phagocytosis (that is, an actin-dependent mechanism), which differs from ‘endocytosis’, a process that is thought to internalize particles no larger than 120 nm and that was considered to be actin independent until recently. Therefore, the finding that L. monocytogenes can induce its internalization by using clathrin-dependent machinery was surprising and indicated that clathrin-coated structures can engulf much larger particles than previously thought. Cell-surface expression of Met is downregulated by HGF, and similarly, soluble InlB induces the degradation of Met, through monoubiquitylation and clathrin-mediated endo cytosis 14. Furthermore, as shown using RNA-interferencemediated knockdown, important components of the endocytic machinery are required for internalization of L. monocytogenes. Although the underlying molecular mechanisms have not been defined, other invasive bacteria had previously been reported to use clathrinmediated entry, implying that this mechanism is not restricted to Listeria species and could be a commonly used mechanism for bacterial entry 15–17. Although the endocytic machinery is important for entry of L. monocytogenes, this bacterium might exploit other mechanisms, because inhibitors of endocytosis reduce bacterial entry but do not completely abolish it14. Plasma-membrane microdomains known as lipid rafts have also been shown to be important for the entry of L. monocytogenes18. Because lipid rafts are usually associated with clathrin-independent endocytic pathways, this indicates either that L. monocytogenes exploits more than one endocytic mechanism or that the separation among the classes of endocytosis is not as well demarcated as conventionally thought. Further work is necessary to decipher how lipid rafts, clathrin-mediated endocytosis and actin-mediated phagocytosis combine to enable listerial entry and cellular invasion.

Adherens junctions Together with tight junctions and desmosomes, these are specialized structures that allow epithelial cells to adhere to each other, and they have epithelial cadherin (E-cadherin) as a major component.

InlA: exploiting intercellular junctions. Similar to InlB, InlA induces local cytoskeletal rearrangements in the host cell to stimulate uptake of L. monocytogenes by epithelial cells. InlA is a covalently linked bacterial cell-wall protein that binds the host epithelial-cell protein E-cadherin19 (FIG. 3). E-cadherin is a transmembrane protein that belongs to a large family of cell–cell adhesion molecules that are required for the correct formation of adherens junctions between epithelial cells. E-cadherin is localized at these cellular junctions, where its intracellular domain forms a complex with the cytoskeleton through the catenins (which are cadherin-binding proteins), and its extracellular domain is in contact with E-cadherin molecules on neighbouring cells 20. InlA binds the extracellular

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domain of E-cadherin, but it is the intracellular domain of E-cadherin that is essential for the cytoskeletal rearrangements that are required for bacterial entry21. Because all of the components of the endogenous machinery of cell–cell junctions seem to be recruited on binding of InlA, L. monocytogenes is a good system for the study of cellular adhesion and identification of components that are involved in this process. Although it is known that assembly and attachment of the E-cadherin, α-catenin and β-catenin complex to the cytoskeleton is central to both intercellular adhesion and L. monocytogenes entry21, neither the mechanisms that hold cells together nor the molecular mechanisms that are required for InlA-dependent entry are as yet fully understood. Until recently, the accepted model of intercellular adhesion proposed that α-catenin anchors the E-cadherin–β-catenin complex to the actin cytoskeleton, providing a stable structure that maintains tissue integrity. However, it has become apparent that the dynamics of this process are more complex. As was recently shown, α-catenin cannot simultaneously interact with actin filaments and the E-cadherin–β-catenin complex, indicating that α-catenin is not an actin anchor but is, instead, an actin-filament organizer22,23. Further investigation is required to understand this mechanism fully; however, the dynamic interactions between the cadherin–catenin complex and the underlying actin cytoskeleton are consistent with the findings that L. monocytogenes can regulate and rearrange actin structures at intercellular junctions through adhesion to E-cadherin, and further emphasize the validity of the listerial model for analysing events at intercellular junctions. The validity of using L. monocytogenes as a tool to study cell–cell junction formation was shown by a recent study of InlA-dependent entry that identified the protein ARHGAP10 (Rho GTPase-activating protein 10) as a novel cellular component that is involved in the recruitment of α-catenin to cell–cell junctions. This study also showed that ARHGAP10 was essential for listerial entry (see Supplementary information S2 (figure))24. Overexpression of ARHGAP10 disrupted the cytoskeleton and increased the local concentration of α-catenin, indicating that ARHGAP10 has a direct role in regulation of the dynamics of cell–cell junction formation. Furthermore, ARHGAP10 was shown to control the activity of RhoA and CDC42, two proteins that regulate cell–cell junction formation. Components that generate the tension that is required to hold neighbouring cells together, that is, myosin VIIA and its ligand vezatin25, have been found to be important for L. monocytogenes entry, indicating that these components might generate the force that is necessary for engulfment of the bacterium through phagocytosis26. These findings also indicate that the tension that holds two cells together could be similar to the tension that is exerted during phagocytosis, as if each cell is attempting to engulf its neighbour. An analogous process known as ‘frustrated phagocytosis’ occurs when macrophages adhere to immune-complex-coated surfaces27.


REVIEWS a Intercellular junctions

b InlA-induced entry F-actin

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Figure 3 | Adherens junction and internalin A (InlA)-induced bacterial entry. a | Adherens junctions hold adjacent cells together through the transmembrane protein epithelial cadherin (E-cadherin). The intracellular domain of E-cadherin recruits α-catenin and β-catenin, and α-catenin bridges the actin cytoskeleton and E-cadherin. Formins, which interact directly with α-catenin, are also essential for forming actin cables at cell–cell junctions, although the mechanism by which they achieve this is not understood. b | The receptor for the Listeria monocytogenes protein InlA is E-cadherin. Many components that are important for adherens junctions are recruited to the site of bacterial entry, where the cytoskeletal rearrangements that are required for invasion occur. ARF6, ADP-ribosylation factor 6; ARHGAP10, Rho GTPase-activating protein 10; Arp, actin-related protein; F-actin, filamentous actin; G-actin, globular actin.

Harnessing the actin-polymerization machinery. Following internalization into a host-cell vacuole, L. monocytogenes lyses the membrane-bound phagosome (discussed later) and escapes into the cytoplasm, where it can polymerize host actin and propel itself through the cell and into neighbouring cells28. The ability to spread from cell to cell without coming in contact with the extracellular milieu allows the bacterium to propagate through tissues and avoid contact with circulating antibodies or other extracellular bactericidal compounds. At the leading edge of moving cells, the control of actin polymerization is a complex mechanism triggered by intricate signalling pathways that take place at the plasma membrane. L. monocytogenes bypasses these signalling pathways and directly nucleates actin constitutively, making it a simple and effective model for studying the dynamics of actin polymerization. L. monocytogenes polymerizes actin asymmetrically along its surface, producing an actin tail that propels the bacterium through the cytoplasm28,29 (see Supplementary


information S3 (figure)). Polymerization of host actin is mediated by the bacterial surface protein ActA, the first protein that was identified to have functions that promote actin nucleation30–32. It was later found that ActA mimics its eukaryotic counterparts, proteins of the WASP family (which includes N-WASP and WAVE)33. The N-terminal region of ActA, which is essential for actin-based motility 34, has homology to the C-terminal region of WASP, which binds the actin-nucleation complex Arp2/3 (REF. 35). Accordingly, both ActA and WASP-family proteins function as nucleation-promoting factors (NPFs) for the Arp2/3 complex and are involved in forming a ternary complex that is composed of Arp2/3, an NPF and actin. The Arp2/3 complex consists of seven proteins, including two proteins that are related to actin, Arp2 and Arp3, which (as a result of their structural similarity to actin) are thought to function as a template for polymerization. Discovery of the Arp2/3 complex ensued from studies of ActA partners, thus the role of Arp2/3 as a key actin-nucleating complex was consequently revealed.


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Met E-cadherin Guinea pig and rabbit

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Figure 4 | Host specificity of Listeria monocytogenes proteins internalin A (InlA) and InlB. a | InlA and InlB can bind and induce entry of L. monocytogenes into human cells that express the respective cell-surface receptors, epithelial cadherin (E-cadherin) or Met. However, a single amino-acid change in E-cadherin (at position 16; see b) prevents InlA from binding mouse E-cadherin, and for unknown reasons, InlB cannot recognize or activate guinea pig or rabbit Met. b | A diagrammatic representation of the crystal structure of the leucine-rich-repeat region of InlA (purple) bound to E-cadherin (green) is shown. The position of the crucial proline residue at position 16 of E-cadherin is indicated. It is this residue that determines intermolecular recognition and therefore host specificity. The crystal-structure representation is reproduced with permission from REF. 114 © (2002) Elsevier.

Filopodia Rod-like cell-surface projections that are composed of actin filaments. They are found on various cell types and have sensory or exploratory functions.

Lamellipodia Thin actin-rich structures that form protrusions at the edge of the cell and are essential for cellular motility.

The L. monocytogenes model has also been useful for understanding the importance and the function of VASP, the other main ligand of ActA. VASP binds the central proline-rich domain of ActA and promotes efficient actin-based motility34,36–38, highlighting the importance of this protein in actin polymerization. Only recently, however, have the complex molecular mechanisms of VASP activity begun to emerge. VASP seems to promote listerial motility by recruiting the actin-binding protein profilin, which promotes polymerization at actin-filament barbed ends39. VASP also seems to induce faster growth of the actin network at the bacterial surface by causing the release of Arp2/3 from ActA40. In addition, VASP seems to decrease Y-branch formation, thereby increasing parallel alignment of actin filaments41. Although their mechanistic role is not fully elucidated, proteins of the Ena/VASP family are important in the formation of actin fibres, filopodial tips and the lamellipodial leading edge42. Intracellular and intercellular movement using actin polymerization is not restricted to L. monocytogenes. A growing number of intracellular pathogens, including Rickettsia species, Shigella species, mycobacteria, Burkholderia pseudomallei and vaccinia virus, show this feature during infection (see Supplementary information S3 (figure), and for recent reviews, see REFS 43,44).

A paradigm in pathophysiology As a pathogen that displays such interesting features as strong T-cell activation, a sophisticated relationship with its host and crossing of protective barriers, L. monocytogenes has more recently also emerged as a model to study the pathophysiology of a complex bacterial infection. Findings from in vitro work have been

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used to bypass stringent host species specificity and generate relevant model systems to study infection in vivo. In this respect, L. monocytogenes is a good example of how in vitro studies can help to generate animal models that more closely reflect human infection. For many years, the animal model used to study L. monocytogenes infection was intravenous infection of mice. This model provides a dose-dependent infection with dissemination of the bacteria into organs and was crucial in the discovery of cell-mediated immunity 3. However, recent molecular evidence showed that the mouse model is inadequate to study the crossing of barriers that is characteristic of listeriosis. It had long been known that oral inoculation of mice (rather than intravenous infection), which most closely reflects the human mode of infection, is not efficient because only small numbers of L. monocytogenes cross the mouse intestinal barrier. The reason for this was uncovered by molecular in vitro studies that showed that a single amino-acid difference in the mouse cellular receptor for InlA, E-cadherin, prevented it from binding InlA, thereby showing the inadequacy of the mouse model for study of the invasive role of InlA45 (FIG. 4). Consequently, a novel animal system was developed to study the crossing of the intestinal barrier: a transgenic mouse that expresses human E-cadherin on the surface of enterocytes46. This model showed that InlA has a key role in disease, because it is essential for crossing of the intestinal barrier. In this model system, InlA could interact with E-cadherin, and a wild-type strain of L. monocytogenes was able to cause disease through oral inoculation. So far, E-cadherin has been found only at cell–cell junctions and on the basolateral face of epithelial cells, so the mechanism by which L. monocytogenes gains access to E-cadherin was enigmatic. Two hypotheses were put forward to explain how InlA could target E-cadherin 46. The first hypothesis proposed that L. monocytogenes could gain access to E-cadherin at the tips of intestinal microvilli, where apoptotic epithelial cells slough off. The second hypothesis proposed a synergy between InlA- and InlB-dependent internalization, because activation of Met by HGF has been shown to stimulate the disassembly of junctions between epithelial cells46. Recent results have shown that L. monocytogenes invades the intestinal epithelium at sites of cell extrusion at the tips of villi47 and that the contribution of InlB to crossing of the intestinal barrier is insignificant in vivo48. Whether the mechanism of intestinal invasion is used to cross other barriers is unknown. Synergy between InlA and InlB could still be important for crossing of the placental barrier49. Because the transgenic mice described here express human E-cadherin only on enterocytes, it is not possible to study the role of InlA in deeper tissues. The generation of transgenic mice that express E-cadherin on all cells is in progress. At present, the role of InlA can also be studied in guinea pigs or rabbits, because E-cadherin is recognized by InlA in these animals. However, recent studies show a species specificity for InlB: InlB does not recognize or activate guinea-pig


REVIEWS or rabbit Met. Therefore, the role of InlA and InlB in infection cannot be studied using these animal models48. A human model remains the best model system for studying listerial infection, and human explants have been successfully used to determine the mechanism by which L. monocytogenes crosses the maternofetal barrier49. Human placental villus explants, together with primary or immortalized trophoblastic cells, were used to show that InlA is a key bacterial protein that is required for crossing of the human maternofetal barrier, although InlA is not essential for this role in the pregnant guinea-pig model49,50. Crossing of the blood– brain barrier is still poorly understood. However, the optimization of animal models should help to decipher this crucial step. Other model systems are being developed to identify the host factors that are required for the intracellular survival of L. monocytogenes and possibly of other intracellular pathogens. Drosophila melanogaster has attracted attention as a model because of the many genetic and immunological studies that have been carried out using this organism, and it has been successfully used to test L. monocytogenes virulence51. Moreover, genome-wide RNA-interference screens in D. melanogaster S2 cells (which are macrophage-like cells) have revealed many new host factors that are important for entry into the host cell, escape from the vacuole and intracellular growth of L. monocytogenes52,53. Another noteworthy organism that has been shown to support a listerial infection is Caenorhabditis elegans54. a P

plcA

P1 and P2

prfA

b Ribosome subunits

SD prfA mRNA

ATG prfA

High temperature Low temperature

No PrfA

PrfA Virulence-gene expression

Figure 5 | The PrfA regulator. a | Schematic representation of the prfA region. During exponential phase, prfA is mostly transcribed as a bicistronic product from the promoter (P) upstream of plcA. By contrast, during stationary phase, prfA is mostly transcribed as a monocistronic product from P1 and/or P2. b | Mechanism that controls the thermoregulated expression of PrfA in the promoter upstream of prfA. At low temperatures (≤30°C), a secondary structure forms in the untranslated region of prfA, and this prevents ribosome binding and therefore expression of PrfA. At high temperatures (≥37°C), melting of the prfA untranslated region allows ribosomes to bind and PrfA expression to occur. SD, Shine–Dalgarno sequence. This figure is modified with permission from REF. 58 © (2002) Elsevier.


Novel regulatory mechanisms L. monocytogenes is a facultative intracellular pathogen that can live both inside and outside its host. This bacterium has therefore evolved sophisticated regulatory mechanisms to ensure that virulence factors are optimally expressed when they are required. These regulatory mechanisms might prove to be general mechanisms used by other bacteria. PrfA: a tightly regulated protein. Most of the virulence proteins that have been identified in L. monocytogenes are under the control of one transcriptional regulator, PrfA, which itself is tightly regulated by environmental conditions. During exponential growth, prfA is mainly transcribed as a bicistronic mRNA from the plcA promoter. By contrast, during stationary phase, a monocistronic mRNA is preferentially transcribed from a promoter upstream of prfA55–57 (FIG. 5a). Similar to many other pathogens, L. monocytogenes can sense conditions in the mammalian host and respond by expressing virulence genes. A novel regulation of PrfA by temperature, owing to the structure of the upstream untranslated region of the prfA mRNA, was recently discovered58 (FIG. 5b). At low temperature (30°C), the prfA leader transcript controls translation of the downstream mRNA by forming a secondary structure that masks the ribosome-binding site. At mammalian host temperature (37°C), this structure partially melts to expose the ribosome-binding site, thereby allowing translation to occur. Fusion of the prfA leader transcript to the gene that encodes green fluorescent protein (GFP) also resulted in thermoregulation of GFP. This mechanism might be used by other bacteria, as has previously been suggested for the Yersinia pestis activator protein LcrF59. Such post-transcriptional regulation of prfA allows rapid expression of the encoded transcription factor and therefore efficient transcription of virulence factors as soon as the bacterium enters the host. Other environmental conditions — such as osmolarity, iron concentrations, pH, the presence of fermentable sugars, stress (through σB), and conditions in the host-cell intracellular compartment — have been shown to regulate prfA and PrfA-controlled genes through mechanisms that are not completely understood60,61. Post-translational regulation of PrfA by a putative cofactor is suggested by its structure, which resembles that of the cyclic-AMP receptor62,63. The number of mechanisms that regulate PrfA is probably indicative of the importance of this crucial virulence factor during infection. Listeriolysin O: a pH-sensing protein. L. monocytogenes thrives in the cytoplasm of numerous cell types. Following internalization, bacteria escape from membrane-bound phagosomes by secreting two phospholipases, PlcA and PlcB, and the pore-forming toxin listeriolysin O (LLO), thereby gaining access to the cytoplasm (FIG. 1). Although LLO is a member of a large family of cholesterol-dependent cytolysins that are secreted by numerous Gram-positive bacteria, L. monocytogenes is the only pathogen that secretes this type of toxin inside the host cell. Therefore, secretion of LLO must


REVIEWS Premature unfolding

Neutral pH

Soluble monomer D1 Membrane-bound monomers D2 D3

D4

Pre-pore oligomer

Formation of the pre-insertion β-sheet Pore oligomer

Low pH

Out

In

Figure 6 | Listeriolysin O (LLO) pore-forming mechanism. At low (acidic) pH, the soluble LLO monomer interacts with the host-cell plasma membrane, presumably by binding cholesterol. On contact with the membrane, structural rearrangements in one monomer expose residues that can form hydrogen bonds with other monomers, thereby allowing oligomerization into a pre-pore complex. Following oligomerization, two α-helical bundles from each monomer extend to form transmembrane β-hairpins (red) that punch through the membrane. Pores formed by LLO and other cholesterol-binding proteins can be 250–300 Å in diameter. At neutral pH, domain 3 (D3) of the monomer prematurely unfolds, rendering the protein unable to form pores. This figure is modified with permission from REF. 115 © (2005) American Society for Microbiology.

Two-component regulatory system A two-protein signaltransduction system that is important for the bacterial response to environmental changes. It consists of a membrane-bound sensor protein kinase and a transcriptional-response regulator.

Elongation factor A protein that allows tRNAs to bind the ribosome and is essential for elongation of the polypeptide chain.

be tightly regulated, because the bacterium needs to balance efficient escape from the vacuole against prevention of host-cell damage to allow its intracellular survival. Unlike other pore-forming toxins, the activity of LLO is optimal at acidic pH (1,200 bp) from public databases (RDPII, GenBank, EMBL and DDBJ) up to May 2005 and from published reports of clones, strains or sequences described as ‘uncultured bacterium’ with previously determined phylogenetic affinity to the ε-proteobacteria. To construct a phylogenetic foundation for more detailed analyses, a Neighbour Joining (NJ) tree was constructed in PAUP* (REF.7), calculating distances under the general time-reversible model incorporating invariable sites and rate heterogeneity. The analyses revealed that a few previously affiliated ε-proteobacterial 16S rRNA gene sequences were chimeric or misidentified (see Supplementary information S1 (table)). The four clades that contain environmental sequences were then subjected to more rigorous maximum likelihood analyses using PHYML8 with the same model chosen for the NJ analysis. To estimate nodal supports, 100 bootstrap replicates were performed. The ε-proteobacterial sequences currently belong to two valid orders, the Nautiliales (genera Nautilia, Caminibacter and Lebetimonas) 2,9–11 and the Campylobacterales (families Campylobacteraceae, Helicobacteraceae and Hydrogenimonaceae)12,13. Excluding clinical systems (such as infectious associations with humans) affiliated with the Campylobacter and Helicobacter genera, the remaining ε-proteobacterial sequences are diagnosed www.nature.com/reviews/micro


REVIEWS Thermophile An organism that grows optimally at high temperatures, usually above 45°C.

Autotroph An organism that can use carbon dioxide as the sole source of carbon for growth.

Heterotroph An organism that uses organic compounds as nutrients to produce energy for growth.

Chemocline A chemical gradient from high to low concentrations, often consisting of a relatively small stratum where the concentration changes rapidly between the two endpoints.

Mesophile An organism that grows optimally at moderate temperatures, ranging between 20°C and 45°C.

into four robust phylogenetic clusters — classified here as the Nautiliales, Arcobacter, Sulfurospirillum and environmental sequence clusters — that consist of sequences retrieved from various marine systems (for example, deep-sea hydrothermal vents, vent fauna and deep-sea marine subsurfaces) and terrestrial systems (for example, groundwater, caves and springs) (FIG. 1). With few exceptions, ε-proteobacterial sequence affinities strongly correlate with ecotype for each of the phylogenetic clusters (denoted as coloured lines in FIG. 2 and coloured text in Supplementary information S2 (figure)) and metabolic capabilities (denoted as coloured symbols in Supplementary information S2 (figure)). Within the deeply branching group of the Nautiliales, sequences have been retrieved exclusively from hydrothermal systems, and cultured representatives of the family are thermophilic, autotrophic and can reduce elemental sulphur with molecular hydrogen (see Supplementary information S2 (figure), part a). Even within the Sulfurospirillum (FIG. 1; see Supplementary information S2 (figure), part b) and Arcobacter (FIG. 1; see Supplementary information S2 (figure), part c) clusters, nearly all of the sequences are grouped based on environmental setting and metabolism. For instance, although all characterized Sulfurospirillum spp. ferment

Ar co ba c

ic o ba cte r

lla line Wo

Campylobacter

te r

Candidatus A. sulfidicus Oilfield 'FWKO B'

Hel

m illu pir s o fur Sul sp. Am-N Hydrogenimonas Thioreductor Nitratiruptor Caminibacter Lebetimonas Nautilia Nautiliales

Sulfurovum

Nitratifractor

Thiovulum Thiomicrospira sp. CVO Thiomicrospira denitrificans

tal men Environ

Sulfuricurvum

Sulfurimonas

Figure 1 | Phylogeny of 1,037 near full-length (>1,200 bp) ε-proteobacterial sequences collected from public databases and published research. Sequences were aligned using Muscle v3.52 (REF. 116) followed by removal of highly divergent and ambiguous regions using Gblocks v0.91b (REF. 117). The phylogeny was reconstructed using Neighbour Joining under a general time-reversible model of evolution. Major taxonomic divisions, and all of the currently recognized genera, are indicated. Branches for environmental sequences are coloured to represent either marine (blue) or terrestrial (green) habitats.


using fumarate and can reduce nitrate to ammonia, with the exception of Sulfurospirillum multivorans14, one feature that phylogenetically distinguishes the cultured sulfurospirilla is their ability to respire using alternative electron acceptors under heterotrophic conditions15 (TABLE 1; see Supplementary information S2 (figure), part b). Sequences from different strains that respire using similar elements are more closely related to each other compared with other species within the family, despite strains originating from different geographical locations (for example, Sulfurospirillum carboxydovorans, Sulfurospirillum arcachonense and Sulfurospirillum sp. Am-N). Although arcobacters have been implicated in human and animal enteric diseases 16, few studies have combined isolation and molecular techniques to examine their habitat range17. The type species of the genus Arcobacter nitrofigilis was isolated from a salt-marsh plant root18, but there is still significant diversity among the arcobacters. Similar to the sulfurospirilla, Arcobacter sequences retrieved from marine and terrestrial habitats group together (FIG. 1) and with ecotype (see Supplementary information S2 (figure), part c). Although the metabolic capabilities of most arcobacters have not been studied in detail, many of the cultured representatives originate from marine environments with a well defined geochemical interface between dissolved oxygen and sulphide concentrations17. For example, ‘Candidatus Arcobacter sulfidicus’ was isolated from coastal marine sediments with an oxygen–sulphide chemocline19. This bacterium undergoes mesophilic, chemolithoautotrophic growth, and produces filamentous sulphur with sulphide and oxygen as the electron donor and acceptor, respectively. Based on radio- and stable-isotopic experiments of carbonfixation processes, Candidatus A. sulfidicus was the first ε-proteobacterium thought to assimilate inorganic carbon sources, not through the Calvin–Benson pathway, but by means of the reductive TCA cycle (rTCA cycle)19. The phylogenetic assignment of the remaining sequences is problematic. Based on the bootstrap supported phylogenetic topology, there is a large group that is distinct from the other major clusters (FIGS 1,2; see Supplementary information S2 (figure), part d). This cluster represents the largest increase in 16S rRNA genesequence diversity throughout the ε-proteobacteria and includes several recently described genera. Currently, this sequence cluster has no hierarchical taxonomic classification and future taxonomic revision is required to elucidate the possibility that the cluster might represent more than one hierarchical group. We have provisionally named this clade Thiovulgaceae fam. nov. for ease of reference throughout this review20–22. Thiovulgaceae is derived from thio meaning ‘sulphur’ and vulgar meaning ‘of, pertaining to, common’, forming Thiovulga meaning ‘pertaining to sulphur’;-aceae represents the ending to denote a family. The cultured genera that belong to the family are Gram-negative bacteria that have rod-, vibrio- or filamentous-shaped non-spore-forming cells. Organisms are found in mesophilic conditions, and cultured representatives are


REVIEWS gro

up I

ro up

I

Mari ne

Te rr es tr ia

lg

Sulfurovum

Nitratifractor

n ou Gr

Lower Kane Cave group VI

Sulfuricurvum

I up gro

Lower Kane Cave group I Lower Kane Cave group IV

r wate und Gro

Lower Kane Cave group II/V

group

II

Marine: deep-sea vents, sediments Marine: basinal sediments Marine: deep-sea vent metazoans Terrestrial: water, contaminated water Terrestrial: acid mine drainage, lakes, springs Terrestrial: hydrocarbon groundwater Terrestrial: cave microbial mats Marine: pelagic Marine: whale bone

r ate dw

Termite gut

Sulfurimonas Marine group II

Thiomicrospira

Thiovulum

Figure 2 | The provisional Thiovulgaceae fam. nov. clade. This figure is expanded from FIG. 1. Branches are coloured to represent ecotype. Based on additional, more rigorous maximum likelihood analysis, terrestrial group I is placed outside of the Thiovulgaceae fam. nov. as shown in Supplementary information S2 (figure). Chemolithoautotroph An organism that obtains energy from inorganic compounds and carbon from CO2.

Calvin–Benson pathway Also known as the Calvin– Benson cycle. A series of biochemical, enzyme-mediated reactions in which CO2 is reduced and incorporated into organic molecules.

Reductive TCA cycle (rTCA cycle). The TCA cycle in reverse, leading to the fixation of CO2. Represents a putatively ancient metabolic pathway in which autotrophic carbon fixation occurs under anaerobic conditions.

chemolithoautotrophic and can use molecular hydrogen and/or reduced sulphur compounds as electron donors. Members of the family have been isolated from both marine and freshwater habitats. The Thiovulgaceae fam. nov. family is a member of the order Campylobacterales and comprises the genera Thiovulum 23, Nitratifractor 24, Sulfurovum 25, Sulfuricurvum26, Thiomicrospira27,28 and Sulfurimonas29, which cluster into four main sequences groups, within which are two discrete ecological units — marine group (MG) and groundwater group (GG) (FIG. 2; see Supplementary information S2 (figure), part d). Ecotype groups are closely related to each other (for example MG I and MG II), but relatedness is not supported by bootstrap values, which indicates that significant diversity has yet to be uncovered within the cluster. Unlike the phylogenetic and ecotype patterns within the Sulfurospirillum and Arcobacter clusters, there are little recognizable

460 | JUNE 2006 | VOLUME 4

fine-scale ecotype clade associations. Both MG I and MG II are composed of sequences retrieved from deep-sea vents and sediments, or are associated with vent fauna; however, MG II contains a slightly broader ecotype diversity than MG I, as MG II contains a clade of terrestrial wastewater (sludge) organisms and Thiomicrospira spp. The newly described genera Sulfurovum 25 and Nitratifractor24 are affiliated with MG I, and the sulphuroxidizing genera Sulfurimonas 29, Thiovulum 23 and Thiomicrospira27,28 are affiliated with MG II. A large group of sequences retrieved from groundwater is separated into two clusters, GG I and GG II. Whereas GG I includes the genus Sulfuricurvum26, Lower Kane Cave groups I and IV30, and sequences isolated from wastewater, sludge or groundwater contaminated with petroleum, uranium or tricholoroethene, GG II consists of sequences only from Lower Kane Cave (FIG. 2; see Supplementary information S2 (figure), part d).


REVIEWS Table 1 | Physiological characteristics of ε-proteobacteria from deep-sea hydrothermal habitats and other selected environments Isolate/phylogenetic Isolation association site

Growth Carbon Electron temperature metabolism donor

Electron acceptor

Sulphur/nitrate reduction to:

Ref.

Order Nautiliales, Family Nautiliaceae Nautilia lithotrophica

Alvinella pompejana 53 ˚C tube, 13˚N EPR

Mixotroph

H2, formate

Sulphite, elemental sulphur

H2S

10

Nautilia sp. str. Am-H


Mixotroph

H2, formate

Elemental sulphur

H2S

44

Caminibacter hydrogeniphilus


Mixotroph

H2, complex organic compounds

Nitrate, elemental sulphur

H2S/ NH3

9

Caminibacter profundus

Vent cap, Rainbow Field, MAR

55 ˚C

Autotroph

H2

Nitrate, oxygen (microaerobic) elemental sulphur

H2S/ NH3

2

Caminibacter mediatlanticus

Chimney, Rainbow Field, MAR

55 ˚C

Autotroph

H2


H2S/ NH3

46

Lebetimonas acidiphila

In situ colonization system, TOTO, MA

50 ˚C

Autotroph

H2

Elemental sulphur

H2S

11

55 ˚C

Autotroph

H2

Nitrate, oxygen (microaerobic), elemental sulphur

H2S/ NH3

13

55 ˚C

Autotroph

H2


H2S /N2

24

32 ˚C

Autotroph

H2


H2S/ NH3

45

Order uncertain, Family Hydrogenimonaceae Hydrogenimonas thermophila

Chimney, Kairei Field, CIR

Order uncertain, Family Nitratiruptoraceae Nitratiruptor tergarcus

Chimney, Iheya North Field, OT

Order uncertain, Family Thioreductoraceae Thioreductor micantisoli

Sediment, Iheya North Field, OT

Order Campylobacterales, Family Campylobacteraceae Sulfurospirillum sp. str. Am-N

Alvinella pompejana, 13˚N EPR

41 ˚C

Heterotroph

Formate, fumarate

Elemental sulphur

H2S

44

Arcobacter sp. str. FWKO B

Production water, Coleville oil field

30 ˚C

Autotroph

H2, formate, sulphide


H2S/NO2–

28

Order uncertain, Family Thiovulgaceae Sulfurovum lithotrophicum

Sediment, Iheya North Field, OT

30 ˚C

Autotroph

Elemental sulphur, thiosulphate

Nitrate, oxygen (microaerobic)

N2

25

Nitratifractor salsuginis

Chimney, Iheya North Field, OT

37 ˚C

Autotroph

H2


N2

24

Sulfurimonas autotrophica

Sediment, Hatoma Knoll, OT

25 ˚C

Autotroph

Elemental sulphur, thiosulphate

Oxygen (microaerobic)

Sulfuricurvum kujiense

Groundwater, Japan oil storage cavity

25 ˚C

Autotroph

H2, sulphide, thiosulphate, elemental sulphur


NO2–

26

Thiomicrospira sp. str. CVO

Production water, Coleville oil field

30 ˚C

Mixotroph

Sulphide, elemental sulphur

Oxygen (microaerobic), nitrate, nitrite

N2, N2O

28

29

Location abbreviations: CIR, Central Indian Ridge; EPR, East Pacific Rise; MA, Mariana Volcanic Arc; MAR, Mid-Atlantic Ridge; OT, Okinawa Trough; TOTO, TOTO caldera deep-sea hydrothermal field. Chemical abbreviations: H2S, hydrogen sulphide; NH3, ammonia; N2O, nitrous oxide; NO2–, nitric oxide.

An additional sequence cluster, representing other terrestrial ecotypes (TG), is placed outside of the Thiovulgaceae fam. nov. and other families within the order Campylobacterales (see Supplementary information S2 (figure), part d). The TG I sequences from acid mine drainage, Lower Kane Cave, contaminated groundwater and termite guts might represent greater


taxonomic diversity than previously hypothesized. Moreover, other than these few termite-gut sequences, virtually nothing is known about the potential of ε-proteobacterial symbioses with terrestrial organisms. Future work in these poorly investigated or unexplored terrestrial systems should increase the known diversity of ε-proteobacterial groups. VOLUME 4 | JUNE 2006 | 461


REVIEWS Box 1 | Integrating ecology and biogeochemistry: hydrothermal vents as a case study The ε-proteobacteria have been found at, and sometimes dominate, four main deep-sea hydrothermal vent-specific habitats: mats on the surfaces of rocks, chimneys and animal surfaces (a in the figure); discharged vent fluids and subseafloor (b); within the hydrothermal vent plume (c); and symbiotic associations with vent animals such as Alvinella pompejana, Alviniconcha aff. hessleri, and Rimicaris spp.103 (d). The metabolically versatile ε-proteobacteria are uniquely suited to thrive in deep-sea habitats and other extreme settings. These hydrothermal-vent habitats are all dynamic suboxic to anaerobic environments, and the ε-proteobacteria use many metabolic processes, including sulphur oxidation, sulphur/ sulphite reduction, nitrate (NO3– ) or nitric oxide (NO2–) reduction to ammonium or nitrogen, and hydrogen and formate oxidation (see figure). In the figure, known electron donors are shown in the yellow shaded blocks and electron acceptors in the green shaded blocks. Some of the ε-proteobacteria might use complex sulphur species for sulphur oxidation or they might biotically influence the formation of iron-sulphur minerals, such as pyrite, at vents104–106. Carbon monoxide might also be used as an electron donor and metal(oid)s (iron, manganese, arsenic and selenium) might be used as electron acceptors, although these metabolisms have not been fully examined in vent habitats. These energy pathways can either be coupled with autotrophy (probably through the reductive TCA cycle), mixotrophy or heterotrophy (TABLE 1). The ε-proteobacterial groups establish themselves as the primary (and perhaps the first) colonizers in the dynamic diffuse flow vent environment because of their metabolic flexibility ( TABLE 1), specialized gene assemblages87, the possibility of special modes for attachment to surfaces43, rapid colonization at O2–H2S interfaces (possibly by formation of filamentous sulphur from hydrogen sulphide39) and phylotypic diversification over time as new habitats are colonized following eruptions or with titanium ring for Alvinella colonization (TRAC) deployment34,43,48,70. Ecological principles indicate that there is a tendency for the most productive species in an ecosystem to be the most dominant in a habitat, thereby pushing other species to comparatively lower densities. It is not surprising that the metabolically versatile ε-proteobacteria colonize extensive areas that are warmer (20–60oC) and have higher concentrations of sulphur species than locations where typical chemolithoautotrophic γ-proteobacteria are found. Hydrothermal fluids: Anaerobic, >350°C Substrates: H2, CO, CO2, H2S, As, FeSx, Mn, Se, organic acids

Seawater: Aerobic, 4°C Substrates: O2, NO3–

c Hydrothermal vent plume Caminibacter profundus Lebetimonas acidiphila

H2 S0, NO3–, O2 H2 S0

a Chimney structures Caminibacter mediatlanticus Hydrogenimonas thermophila Nitratifractor salsuginis Nitratiruptor tergarcus

H2 H2 H2 H2

NO3–, S0 NO3–, O2, S0 NO3–, O2 NO3–, O2, S0

–

–

a Vent fauna associations Caminibacter hydrogeniphilus Nautilia lithotrophica Nautilia sp. Am-H Sulfurospirillium sp. Am-N

H2, organics H2, formate H2, formate Formate, fumarate

NO3–, S0 S0, SO32– S0, SO32– S0

d Symbiotic associations No cultured representatives

b Hydrothermal sediments/subsurface Sulfurimonas autotrophica S0, S2O32– O2 Sulfurovum lithotrophicum S0, S2O32– NO3–, O2 NO3–, S0 Thioreductor micantisoli H2

Sea water seeping through crust and back to the vent system Heat from magma below

In the figure, the cultured species from each hydrothermal-vent-specific habitat are shown. Coloured arrows indicate the flow of either hot hydrothermal fluids (red) or cold sea water (blue). S0, elemental sulphur; SO32– sulphite; S2O3, thiosulphate.

Mixotroph An organism that can use both heterotrophic and autotrophic metabolic processes.

ε-Proteobacteria from marine systems Hydrothermal vents and vent-associated subsurfaces. To interpret the possible ecological and geological significance of the uncultured ε-proteobacterial groups, we look to the exemplary hydrothermal vent system (BOX 1). Since the discovery of hydrothermal vents in 1977, the importance of microorganisms as the prevailing biological feature in these environments has been clearly established. Whereas early studies focused on the endosymbiotic microbial assemblages of the vent tube worm, Riftia pachyptila31, the accumulated

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knowledge about the microbial diversity of vent sites now reveals that ε-proteobacteria are probably key players in the cycling of (at least) carbon, nitrogen and sulphur, and have important roles in symbiotic associations with vent metazoans (BOX 2). Moreover, deep-sea hydrothermal environments can be regarded as one of the largest reservoirs of diverse environmental ε-proteobacteria on Earth, ranging from the deeplybranching Nautiliales and Nitratiruptor groups to the Sulfurospirillum, Arcobacter, and the MG I and MG II of the Thiovulgaceae fam. nov.


REVIEWS Although PCR biases and differences in library construction and screening32 might skew interpretations, most 16S rRNA gene-based studies indicate an overwhelming dominance of ε-proteobacteria in the free-living populations in vent fluids or on, or near, the natural surfaces of vent chimney structures where the most intensive hydrogeochemical mixing occurs between ambient (1–4oC), oxygenated bottom sea water and high-temperature, anoxic and sulphide-enriched vent fluid diffusing from the interior portions of the chimney 33–37. The full-cycle rRNA approach (which includes 16S rRNA gene clone library construction and fluorescence in situ hybridization (FISH)) has unequivocally shown that up to 90% of the microbial communities found in these hydrothermal sites are composed of ε-proteobacteria, predominately associated with the Nautilia and Sulfurimonas genera33,37. Other lines of evidence also point to the importance of ε-proteobacteria at vents. Taylor and Wirsen38 showed that the flocculent discharge emanating from diffuse flow vents was similar to the filamentous sulphur production by chemolithoautotrophic sulphur-oxidizing bacteria, which were later shown to belong to the genus Arcobacter19. Filamentous sulphur mats composed of both vibrioid and filamentous sulphide-oxidizers, many of which were probably ε-proteobacteria, have also been found on titanium devices deployed at vents39. Several other research groups have retrieved ε-proteobacterial 16S rRNA gene sequences from vent caps or other in situ colonization devices34,40–43. For instance, ε-proteobacteria comprised ~81% of the total microbial community from an in situ colonization device deployed into diffuse flow vent emissions for 4 days in the Mid-Okinawa Trough34. In this study, water samples collected from 2 m and 10 m away from the chimney structure had dramatically different percentages of ε-proteobacteria, from 83% to 17.6%, respectively, indicating that the ε-proteobacteria tolerate the immediate and proximal vent conditions, probably owing to the increased availability of energy sources compared with more distal habitats or cold sea water (BOX 1).

Deep-sea hydrothermal-vent habitats have also been important for obtaining pure cultures of diverse phylogenetic groups. The first successful isolations were of hydrogen-oxidizing, sulphur-reducing, thermophilic chemolithoautotrophs from Alvinella pompejana symbiont-associated biomass and tube samples; these isolates belong to the Nautiliales10,44. Recently, several previously uncultivated, phylogenetically diverse ε-proteobacterial groups were isolated from various geologically and geographically distinct deepsea hydrothermal fields, all with a diverse range of physiological characteristics and utilization of electron donors (for example, hydrogen and sulphur) and acceptors (for example, sulphur and nitrogen) coupled to carbon fixation2,11,13,24,29,34,45–47 (FIG. 1; TABLE 1; see Supplementary information S2 (figure)). A sub-seafloor model proposed by Huber et al. indicates that ε-proteobacteria thrive in diffuse flow areas surrounding vent chimneys and heated crustal fields and sediments, where sea water mixes with hydrothermal fluids; ε-proteobacterial cells are discharged when eruptions at mid-ocean-ridge axes flush fluids and resident microorganisms within the seafloor crust and sediment to the seafloor surface and into sea water. Indeed, eruption plumes contain diverse microbial communities, but ε-proteobacteria make up ~20–60% of 16S rRNA gene clone libraries from these plumes, with more intergroup diversity occurring from particle-attached libraries than from free-living populations 48. Eventually, the particles and microorganisms fall to the ocean floor and again become part of the marine sediment and subsurface habitats. These hydrogeological processes have the potential to link microorganisms in all of the marine habitats, and might result in a homogenized genetic pool of microorganisms over time. This could be one explanation why members of ε-proteobacteria from the deep-sea hydrothermal vent and sediment systems are closely related to each other.

Box 2 | Episymbionts of Alvinella pompejana

Phylotype A group of sequences that show some threshold of sequence similarity, usually >97%, and that also form a monophyletic clade.

Epibiont An organism that lives attached to a host organism without apparent consequence (benefit or detriment) to the host.

At least two endemic hydrothermal vent fauna, Alvinella pompejana (East Pacific Rise) and Rimicaris exoculata (Mid-Atlantic Ridge), contain ε-proteobacterial episymbionts107,108. A. pompejana, also known as the Pompeii worm because of its heat tolerance, builds paper-like tube colonies attached to hydrothermal-vent chimneys along the East Pacific Rise. The hydrothermal vent shrimp, R. exoculata, forms large clusters on the warmer sections of vents along the Mid-Atlantic Ridge. A. pompejana, the biology of which has been extensively reviewed109, contains two closely related ε-proteobacterial phylotypes that comprise over 65% of a 16S rRNA gene library107,110 derived from episymbiont biomass. These two groups, both within Marine Group I, are filamentous and distinctly separated horizontally on individual dorsal expansions of A. pompejana, indicating niche specialization (M. T. Cottrell and S. C. Cary, unpublished data and REF. 110). More recently, the first endosymbiotic ε-proteobacterium, represented by a single ε-proteobacterial phylotype, was discovered in a deep-sea hydrothermal-vent-endemic gastropod Alvinoconcha spp.111,112 There have been few clues as to the role of the epibionts of A. pompejana because, despite many attempts, the filamentous ε-proteobacterial symbionts have not yet been cultured. Surveys of geochemical conditions within A. pompejana tubes revealed high temperatures (~20– 80oC) and anoxia, exceeding that of any known metazoan habitat, surprisingly low or trace free hydrogen sulphide (78% of the metabolically active fraction (RNA), with the ε-proteobacteria dominating the lower, sulphide-rich sediment fractions56. The strong relationship between δ- and ε-proteobacteria could be due to their respective roles in the sulphur cycle (that is, sulphate reduction for the δ-proteobacteria and sulphur oxidation for the ε-proteobacteria).

Push cores Soft sediment collected using a hollow plastic collection tube that is pushed into the sediment, after which the ends are closed.

Methane cold seeps Areas of the deep ocean floor where oil and methane gas bubble up from under seasediment layers at ambient temperatures, providing an energy source that can sustain deep-sea microbial communities.

ε-Proteobacteria in terrestrial systems According to some researchers, an immense subsurface microbial biosphere might exist that is not just associated with marine sediments or deep-sea hydrothermal vent systems57,58. On the basis of recent PCR-based investigations of terrestrial ecotypes, including naturally sulphur-rich environments such as oil-field brines15,27,28,59, hydrocarbon-contaminated groundwater15,59–61, uncontaminated groundwater62, sulphidic springs63–65 and limestone caves30,66–68, we are beginning to discover the importance of ε-proteobacteria in these habitats. Although there are terrestrial sequences belonging to the Sulfurospirillium and Arcobacter clusters, generally, most of the recently acquired ε-proteobacterial sequences from natural, uncontaminated terrestrial habitats are affiliated with the Thiovulgaceae fam. nov. (FIG. 2; see Supplementary information S2 (figure)). Several chemolithoautotrophic, nitrate-reducing, sulphur-oxidizing, microaerophilic ε-proteobacteria have been isolated from oil-field brines and oil-contaminated groundwater, including Arcobacter sp. strain FWKO B28, Thiomicrospira sp. strain CVO27,28 and Sulfuricurvum kujiense26,61 of GG I (FIGS 1,2; see Supplementary information S2 (figure)). So far, S. kujiense (isolated from hydrocarbon-contaminated Japanese groundwater60) is the only cultured terrestrial representative in the Thiovulgaceae fam. nov. Sulphidic caves (limestone caves with discharging hydrogen-sulphide-rich groundwater) allow easy access to the subsurface and are currently one of the beststudied natural terrestrial sites for ε-proteobacteria30,63,66–68. Lower Kane Cave serves as an ideal model system for understanding terrestrial ε-proteobacteria, particularly the terrestrial Thiovulgaceae fam. nov., because all of the sequences retrieved so far from Lower Kane Cave are affiliated with this evolutionary lineage (FIG. 2; see

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Supplementary information S2 (figure))30,68. Subsurface terrestrial habitats are geographically isolated from one another owing to geological structures, hydrostratigraphic connectivity and plate tectonics, and terrestrial organisms should have limited dispersal mechanisms and are not capable of atmospheric dispersal processes68,69. Therefore, high sequence similarity for the GG I sequences retrieved from around the world indicates that the ancestral population giving rise to the modern group might have originated from one geographical location. However, because GG II consists of only Lower-KaneCave-derived sequences, this clade might be endemic only to Lower Kane Cave. More research is needed to validate these relationships.

Ecological significance of ε-proteobacteria The ε-proteobacteria have significant roles in the habitats in which they thrive, as primary colonizers, primary producers or in symbiotic associations. Lopez-Garcia et al.70 suggest that, in deep-sea habitats, the ε-proteobacteria maximize overall ecosystem function owing to their high biomass and growth rates, rapid adaptations to changing geochemical conditions and metabolic versatility. These factors all facilitate the colonization of new substrates and habitats39,43. For Candidatus A. sulfidicus, the formation of filamentous sulphur might also stimulate colonization of surfaces in marine habitats35. Because nearly all of the microorganisms isolated from deep-sea hydrothermal-vent or marine-sediment ecotypes are chemolithoautotrophs (TABLE 1), these colonizers also serve as one of the crucial sources for organic carbon to the ecosystems (BOXES 1,2), especially at oxic–anoxic interfaces50,71,72. To assess the importance of chemolithoautotrophy in marine and terrestrial settings, however, in situ rates of carbon production or substrate use are needed. Carbon-fixation rates estimated for Candidatus A. sulfidicus were equal to, or exceeded, those of known sulphur-oxidizing bacteria that use the Calvin cycle19. Furthermore, based on previous work that describes the organic carbon-stable-isotope compositions for some marine-vent organisms73,74 and corresponding carbon-isotope fractionation patterns, organic carbon is probably supplied from primary producers that use the rTCA cycle74. In Lower Kane Cave, M. L. P. estimated that the rate of chemolithoautotrophic primary productivity by H14CO3 assimilation was 96.5 ± 6.0 mg carbon gram dry weight per hour for the ε-proteobacterial-dominated microbial mats24, which is comparable to rates of other autotrophic organisms75. Stable-carbon-isotope analyses in Lower Kane Cave corroborate that chemolithoautotrophically produced carbon supports the otherwise nutrient-poor system30. Although the autotrophic carbonfixation pathways were not evident from the study, the Calvin–Benson cycle was implicated in carbon fixation. In addition to cycling carbon, we know that ε-proteobacteria metabolically convert various forms of reduced and oxidized sulphur and nitrogen compounds (TABLE 1, which has important bearing on the speciation of sulphur/nitrogen within a habitat and on global sulphur/nitrogen cycling, as well as on geochemical and geological processes. Few studies have measured rates


REVIEWS NTPs, dNTPs

Wood–Ljungdahl pathway) or the rTCA (or Arnon) cycle,

Gluconeogenesis/glycolysis Amino acids PEP CO2

Pyruvate PS

Fdox 2[H]

CO2

LPS

Oxaloacetate

2[H] Fdred

Malate

Acetyl-CoA

Fumarate 2[H]

ATP-CL

ATP, CoA

ADP + P

Frd Succinate

Lipids

Citrate

ATP + CoASH

Isocitrate Succinyl CoA KGS

2-Oxoglutarate (α-Ketoglutarate)

CO2

CO2, 2[H]

Fdred 2[H]

Fdox

Ammonium assimilation

Figure 3 | The reductive or reverse TCA (rTCA) cycle of carbon fixation. The two ferredoxin-linked (Fd) CO2-fixation reactions (green) are oxygen sensitive; therefore, this cycle is generally found in anaerobic to microaerophilic microorganisms. The net product of the cycle is one molecule of acetyl-coenzyme A (CoA) synthesized from two molecules of CO2. Acetyl-CoA can be converted to pyruvate and phosphoenolpyruvate (PEP), which can either regenerate the intermediates of the cycle or be used for gluconeogenesis. Many rTCA-cycle intermediates are used in the generation of other cellular components, as indicated by the green arrows. Key enzymes ATP citrate lyase (ATP-CL), pyruvate synthase (PS, also known as pyruvate:ferredoxin oxidoreductase), ketoglutarate synthase (KGS, also known as 2-oxoglutarate:ferredoxin oxidoreductase) and fumarate reductase (Frd) are shown in blue ovals. ATP-CL, KGS and Frd allow the TCA cycle to operate in reverse (red arrow indicates reverse direction). A shared feature of the Calvin–Benson and rTCA cycles is their bidirectionality; in the presence of small organic compounds, microorganisms can use the rTCA cycle in the forward, oxidizing direction.

of sulphur/nitrogen oxidation/reduction in cultured organisms, much less in the environments in which they dominate. In Lower Kane Cave, an assessment of sulphide-consumption rates in the cave revealed that, under microaerophilic conditions, the chemolithoautotrophic ε-proteobacterial Lower Kane Cave group II (GG II of the Thiovulgaceae fam. nov.) consumed sulphide more rapidly than abiotic hydrogen sulphide loss mechanisms, and were consequently found to be responsible for sulphuric-acid dissolution of the cave host limestone76. These results not only linked the biogeochemical carbon and sulphur cycles but also provided evidence for the geological importance of the ε-proteobacteria to processes such as cave development76. Wood–Ljungdahl pathway Also known as the acetylcoenzyme A pathway. An ancient carbon-fixation pathway found in bacteria and archaea in which CO2 is converted to acetate; the key enzyme is acetyl-coenzyme A synthase/CO dehydrogenase.

ε-Proteobacteria and the rTCA cycle Many of the ε-proteobacteria studied so far are chemolithoautotrophs, and it is relevant to the evolutionary history of this group that chemolithoautotrophy is thought to be the first type of metabolic pathway to have evolved77,78. One of two extant autotrophic pathways, the acetyl-coenzyme A (CoA) pathway (also called the


most closely resembles the first known autotrophic pathway79–83 (FIG. 3). Until the latest studies on chemolithoautotrophic ε-proteobacteria, the rTCA cycle had been described in only a few microorganisms, including the green sulphur bacterium Chlorobium limicola (Chlorobiaceae), a few members of the δ-proteobacteria (for example Desulfobacter hydrogenophilus) and some members of the thermophilic Aquificales and archaeal Thermoproteaceae groups84–86. Within the ε-proteobacteria, the rTCA cycle was initially thought to be a potential CO2 fixation pathway in Candidatus A. sulfidicus 19 . Subsequently, two fosmids were sequenced from fosmid libraries linked to the dominant ε-proteobacterial episymbionts of A. pompejana87. Both fosmids contained the key indicator gene in the rTCA cycle, ATP citrate lyase (aclBA). Evidence for the potential presence and significance of the rTCA cycle for autotrophic carbon fixation at deep-sea vents has accumulated from phylogenetic analysis of rTCA genes amplified directly from hydrothermal vent chimney samples, from enzymatic expression analyses of aclB88–90, and from genetic analyses of the cultures of Candidatus A. sulfidicus and the chemolithoautotrophic Nautilia sp. strain AmH19,87. Phylogenetic evidence points to the close evolutionary relatedness among the acl gene-encoded ATP citrate lyases (ATP-CLs) of Persephonella marina (Aquificales), the ε-proteobacteria, and plants and animals 87,90. The plant and bacterial ATP-CLs are encoded by two subunits, aclB (the small subunit) and aclA (the large subunit). Sections of the acl subunits have significant homologies to the large subunit of succinyl-CoA synthetase and the small subunit of succinyl-CoA synthetase and citrate synthetase91. The acl gene of C. limicola is more distantly related and might be an ancestral form92. The phylogenetic relationships point to the possibility of transfer of the acl gene to the eukaryotic population somewhere between the Chlorobium and Aquificales split. However, there is evidence of two types of citrate-cleaving systems within the Aquificales themselves, with the citrate-cleaving citryl-CoA lyase/synthetase enzymes found in both Hydrogenobacter thermophilus and Aquifex aeolicus, and ATP citrate lyase in P. marina87,91.

ε-Proteobacteria throughout Earth’s history Although more data are needed to resolve the evolution of the citrate-cleaving system in relation to the evolution of the rTCA cycle and the early evolution of life, the presence and use of ATP-CL in some ε-proteobacteria clearly incite some interesting questions with respect to the overall evolutionary history of the ε-proteobacteria. How old is the subdivision? What role did these organisms have in Early Earth habitats? Because marine deep-sea vents are thought to be some of the most ancient colonized habitats on Earth93, it is not far-reaching to hypothesize that, as the ε-proteobacteria are dominant and important organisms in modern vents and similar extreme habitats, this group has been significant to ecological and biogeochemical processes throughout much of Earth’s history. VOLUME 4 | JUNE 2006 | 465


REVIEWS Aphotic Receiving no light or energy from the sun.

1.

2.

Research to reconstruct the evolutionary relationships among prokaryotic genomes using comparative 16S rRNA phylogeny and protein-sequence analyses has resulted in mixed information regarding the origin of non-photosynthetic sulphur-oxidizing bacteria and the ε-proteobacteria94–98. However, there is sufficient evidence to indicate that the ε-proteobacteria, along with the δ-proteobacteria, have closer genetic ancestry with the Chlorobiaceae and Aquificales than with the other Proteobacteria94,97. Sheridan et al. estimated that the divergence of 16S rRNA gene sequences of ε-proteobacteria dated to 1.37 billion years ago (Ga), which also corresponds to work by Brocks et al., who detected the presence of the Chlorobiaceae, along with the purple sulphur bacteria (Chromatiaceae) in rocks from 1.64 Ga based on unique hydrocarbon biomarkers (molecular fossils). However, much remains to be explored about the evolution of the ε-proteobacteria because, unlike the Chlorobiaceae and Chromatiaceae99, no molecular fossils have yet been identified in the rock record for the ε-proteobacteria. On the basis of biological and isotopic evidence98–102, the time period when ε-proteobacteria might have arisen on Early Earth, as far back as ~2 Ga, is marked by a shift from a reducing to an oxidizing ocean and global atmosphere owing to cyanobacterial photosynthesis98,100. Although considerable debate surrounds the nature of the geochemical conditions on Early Earth, and the relative timing of this transition is hotly deliberated101, recent sulphur-isotope data from ancient mineral deposits indicate that free oxygen was present in the atmosphere and surface environments as early as ~2.2 Ga102, which has implications for the evolution of oxygen-dependent sulphur-oxidation pathways. For present-day sulphuroxidizing bacteria that require aerobic or even microaerophilic conditions (for instance, those affiliated with the γ-proteobacteria), the availability of free oxygen would have been crucial for growth. However, oxygen is not essential for many of the isolated sulphur-reducing or sulphur-oxidizing ε-proteobacteria, especially for the deeply-branching Nautiliales, which are obligate anaerobes, or for other ε-proteobacteria that use various alternative electron acceptors (TABLE 1). Because the metabolic characteristics and ecotype preferences of the modern ε-proteobacteria, including thermophilic growth, anaerobic metabolism and autotrophy through the rTCA cycle, are similar to the Chlorobiaceae and Aquificales, the evolution and significance of the ε-proteobacteria throughout Earth’s history are provocative avenues to pursue in future research.

Garrity, G. M., Bell, J. A. & Lilburn, T. In Bergey’s Manual of Systematic Bacteriology Vol. 2 (eds Brenner, D. J., Krieg, N. R., Staley, J. T. & Garrity, G. M.) 1145 Part C (Springer, New York, 2005). Miroshnichenko, M. L. et al. Caminibacter profundus sp. nov., a novel thermophile of Nautiliales ord. nov within the class ‘ε-proteobacteria’, isolated from a deep-sea hydrothermal vent. Int. J. Syst. Evol. Microbiol. 54, 41–45 (2004). This paper erected the order Nautiliales, (containing the family Nautiliacea and the genera Nautilia and Caminibacter), which is only the second order to be described from the ε-proteobacteria.

3.

4.

5.

Future prospects The ε-proteobacteria represent a unique assemblage of microorganisms that, despite the attention given to the pathogenic members, have had little defined taxonomic or ecological consideration. Our taxonomic considerations of the ε-proteobacteria reveal that there is a large group of environmentally relevant ε-proteobacteria known mainly from phylogenetic studies of 16S rRNA genes, which we have provisionally termed Thiovulgaceae fam. nov. Our phylogenetic analysis indicates that there are several clades of ε-proteobacteria (for example, Thiovulgaceae fam. nov., Thioreductor and Nitratiruptor) that will most likely reveal more diversity in the future. In many sulphidic habitats, especially at oxic– anoxic interfaces, ε-proteobacteria are not only present in the microbial communities, but might be the dominant microorganisms involved in the cycling and recycling of carbon, nitrogen and sulphur compounds. Quantitative measurements of ε-proteobacteria (for example, using FISH) and biogeochemical cycling (by measuring uptake or consumption rates) are few, and more studies are needed to correlate the roles of ε-proteobacteria with their dominance in aphotic, sulphur-rich environments. Certainly, cave and terrestrial spring environments are more suited to these types of measurements than deep-sea hydrothermal vents or the deep subsurface because these are readily accessible sites where phototrophic productivity can be eliminated. Molecular methods, including FISH, metabolic gene presence/expression quantification and genome sequencing, hold promise for understanding the biogeochemical roles of ε-proteobacteria in remote extreme environments. At present, genomes from at least six environmentally relevant chemolithoautotrophic ε-proteobacteria from the Nautilia, Caminibacter, Arcobacter, Thiomicrospira, Sulfurovum and Nitratiruptor genera are being sequenced. More projects are needed, however, to understand the metabolic flexibility of this group, and to better characterize the pathogenic ε-proteobacteria. Genome projects will also further our understanding of ε-proteobacterial phylogenetic diversity and ecophysiology, and will undoubtedly allow for the identification of molecular markers to elucidate the evolutionary history of the entire class, including the Thiovulgaceae fam. nov. Another major challenge is to integrate this extensive molecular information and in situ biogeochemical culture-based strategies to improve our ability to isolate diverse metabolic groups.

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6.

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how to integrate these diverse methods into a unified taxonomic scheme. Gevers, D. et al. Re-evaluating prokaryotic species. Nature Rev. Microbiol. 3, 733–739 (2005). Swofford, D. PAUP*: Phylogenetic Analysis Using Parsimony (* and Other Methods) 4th edn (Sinauer Associates, Sunderland, 2003). Guindon, S., Lethiec, F., Duroux, P. & Gascuel, O. PHYML Online — a web server for fast maximum likelihood-based phylogenetic inference. Nucleic Acids Res. 33, W557–W559 (2005).


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Acknowledgements B.J.C was partially supported by the National Science Foundation. A.S.E was partially supported by the College of Basic Sciences at Louisiana State University, USA. The authors thank T.E. Hanson for his valuable input in to this review.

Competing interests statement The authors declare no competing financial interests.

DATABASES The following terms in this article are linked online to: Entrez Genome Project: http://www.ncbi.nlm.nih.gov/ entrez/query.fcgi?db=genomeprj Aquifex aeolicus | Chlorobium limicola | Helicobacter pylori | Nautilia sp. strain AmH | Persephonella marina

FURTHER INFORMATION Barbara J. Campbell’s homepage: http://www.ocean.udel.edu/cms/bcampbell Annette Summers Engel’s laboratory: http://www.geol.lsu.edu/Faculty/Engel/profile.html A metagenome of Alvinella pompejana symbiont database: http://ocean.dbi.udel.edu/index.php DDBJ: http://www.ddbj.nig.acjp EMBL: http://www.ebi.ac.uk/embl ε-Proteobacteria sequence data files and alignments: http://geol.lsu.edu/Faculty/Engel/epsilon.htm GenBank: http://www.ncbi.nlm.nih.gov/Genbank RDPII: http://wdcm.nig.ac.jp/RDP/html/index.html

SUPPLEMENTARY INFORMATION See online article: S1 (table) | S2 (figure) Access to this links box is available online.


PERSPECTIVES SCIENCE AND SOCIETY

Advances in tuberculosis vaccine strategies Yasir A. W. Skeiky and Jerald C. Sadoff

Abstract | Tuberculosis (TB), an ancient human scourge, is a growing health problem in the developing world. Approximately two million deaths each year are caused by TB, which is the leading cause of death in HIV-infected individuals. Clearly, an improved TB vaccine is desperately needed. Heterologous prime–boost regimens probably represent the best hope for an improved vaccine regimen to prevent TB. This first generation of new vaccines might also complement drug treatment regimens and be effective against reactivation of TB from the latent state, which would significantly enhance their usefulness. Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), is the second leading infectious cause of mortality worldwide — M. tuberculosis has infected approximately two billion individuals in total, which is one third of the world’s population1. Currently, TB is the leading cause of death in HIV-infected individuals owing to the high incidence of dual infections and the accompanying inhibition of the immune system by both HIV/ AIDS and TB. Ninety percent of the estimated deaths from TB and 95% of the estimated eight million new cases of TB each year occur in developing countries, which comprise 85% of the world’s population2. Incidence is increasing fastest in African countries that are affected by HIV, followed by Eastern Europe and the former Soviet Union, both of which are plagued by multi-drug resistant strains of TB3,4 that present an ominous global threat. The TB pandemic has continued despite widespread use of the only available licensed TB vaccine bacille Calmette–Guérin (BCG)5 under the WHO expanded program on immunization (EPI) and despite the use of directly observed chemotherapy programmes (DOTS) for those diagnosed with active disease. Failure or delay in diagnosis of active TB cases, relapse rates of up to 5% after DOTS6,7, and the lengthy treatment required to cure TB, which often results in poor compliance and the consequent emergence of multi-drug resistant M. tuberculosis strains8–10, present

serious challenges for the DOTS strategy. Although BCG seems to provide protection against disseminated disease in newborns and children, its efficacy against pulmonary TB is poor, which highlights the need for a better vaccine regimen. There are two potential vaccination strategies against TB; those given before (pre-exposure, prophylactic) or after (postexposure, therapeutic) exposure to the pathogen. Therapeutic vaccines can also be used in combination with drug therapy. Specific vaccines designed to prevent latent TB or reactivation from the latent state are in the early stages of development. Here, we review the development of crucial new prophylactic vaccines, the primary goal of which is to prevent infection itself or disease that occurs after infection. Such vaccines include recombinant BCG (rBCG), attenuated strains of M. tuberculosis, subunit vaccine approaches and live, non-replicating viral vector-based delivery systems used alone or in prime–boost regimens. A formidable challenge facing the development of these new vaccines is their safety, especially in populations afflicted with both HIV and TB. Infection with Mycobacterium tuberculosis The development of a delayed-type hypersensitivity response to tuberculin antigen (TST) is considered an indicator of M. tuberculosis infection. Only 20–50% of individuals


that are exposed to M. tuberculosis become infected, as evidenced by a positive TST in single case contact studies11. Around 5% of infected individuals develop pulmonary disease or some other clinical form of TB over 2–5 years, whereas the other 95% of infected TST positive individuals develop latent TB infection (LTBI). Individuals with LTBI have about a 5% lifetime chance of reactivating their latent infection, resulting in the development of clinical TB11. M. tuberculosis is transmitted by the aerosol route of infection. After phagocytosis, the organism successfully hides from immune-mediated destruction inside the endosome of the macrophage using various mechanisms, which include changing the endosomal pH, inhibiting apoptosis and destroying toxic superoxides that are secreted by immune cells. Infected alveolar macrophages and dendritic cells migrate to adjacent lymph nodes where mycobacterial antigens are presented and a T-helper-1 (TH1)-type response is initiated. This complex immune response ultimately results in the formation of the granuloma around infected macrophages. IFN-γ, which synergizes with tumour-necrosis factor-α (TNF-α), is central to the activation of macrophages and the isolation of M. tuberculosis inside the granuloma. The granuloma is initially composed of CD4+ and CD8+ T cells, but a complex array of T cells, including CD4+, CD8+, γδ T cells and CD1-restricted αβ T cells are also invoked to orchestrate immune responses and to contain the infection12–14. At later stages, the granuloma is surrounded by a fibrotic wall and lymphoid follicular structures. The granuloma can persist for decades and can contain the tubercle infection in a state of dormancy by depriving mycobacteria of oxygen and nutrients. However, failure to contain the infection can result in release of organisms, and caseation and necrosis with active clinical disease and transmission (FIG. 1). Requirements for TB vaccine development Scientific rationale. Most humans have complete or partial resistance to tuberculous disease following exposure or infection with M. tuberculosis. Further indication that resistance to TB is mediated in part by the


PERSPECTIVES a

TNF-α

d

CD8+ T cell

IFN-γ

Class I

TH1

Fibroblast

MHC class II

e M. tuberculosisinfected macrophage

Macrophage –

CD4+ T cell

γδ T cell

Nucleus

Perforin, granzyme

Early phagosome

c Cross-priming (weak)

UreC, SOD, nlaA gene product

Granuloma, containment TH1

Dendritic cell

b

CD1

Apoptosis

f rBCG–pfo + ∆SOD ∆nlaA rBCG∆UreC:Hly+

Cross-priming (strong)

Reactivation, dissemination

Figure 1 | The immune response to Mycobacterium tuberculosis infection or vaccination with BCG or recombinant modified BCG. a | M. tuberculosis subverts macrophage phagosome maturation through the production of UreC, which inhibits the acidification of the early phagosome. M. tuberculosis also produces factors such as superoxide dismutase (SOD) and the nlA gene product, which might inhibit host defences by interfering with host cell apoptosis. In the phagosome, M. tuberculosis antigens have access to the MHC class II machinery resulting in a predominant CD4+ T-cell response. However, apoptosis of infected macrophages produces extracellular vesicles carrying glycolipids and protein antigens. These vesicles are taken up by dendritic cells as a result of cross-priming and presented in the context of MHC class I (CD8+ T-cell responses), MHC class II (CD4+ T-cell responses) and CD1 molecules. b | Enhancement of MHC class I antigen presentation and CD8+ T-cell responses by modified BCG constructs. rBCG vaccines such as rBCG–pfo (with deletion of UreC and expression of the pH-independent perfringolysin) or rBCGΔUreC:Hly+ (with deletion of UreC and expression

immune system stems from the findings that certain groups of immunocompromised people have a high susceptibility to tuberculous disease. These include people with Mendelian inherited defects in immune mediators such as INF-γ 15,16, patients treated with TNF-α-antagonist drugs for rheumatoid arthritis17, and AIDS patients with decreased numbers of CD4+ T cells that are also functionally impaired18. Results from clinical trials with BCG, although not consistent, have nonetheless provided proofof-principle that vaccination can be effective

+

of the acidic pH-dependent Hly (listeriolysin)) facilitate the formation of pores in the membrane of the early phagosome, thereby allowing leakage of BCG mycobacteria and antigens into the cytoplasm. This leads to potent MHC class I presentation, enhancement of apoptosis and increased cross-priming and uptake by dendritic cells (c), resulting in enhanced CD8+ and CD4+ T-cell responses as well as γδ T-cell and CD1-restricted αβ T-cell activation. rBCG constructs with diminished SOD activity or deletion of nlaA can further enhance apoptosis. d | CD8+ T cells produce effector cytokines (IFN-γ and TNF-α) and lyse infected cells through the release of granules (perforin and granzyme) or by Fas-mediated lysis, whereas activated CD4+ TH1 cells produce IFN-γ, which activates macrophages to kill M. tuberculosis. e | Infected macrophages can also be contained within granulomas in a state of mycobacterial dormancy for decades. f | However, M. tuberculosis can reactivate as a result of a weakened immune system, with release of organisms from the granuloma and progression to active clinical disease.

against the consequences of infection with M. tuberculosis. Therefore, although resistance to TB is in part a property of the hostmediated immune response, in people not naturally resistant, vaccination could confer an additional degree of protection. Although animal-challenge models have not been proven to correlate with protection in humans, new rBCG vaccines or heterologous prime–boost regimens (TABLE 1) show better protection than BCG in such models. Such prime–boost regimens consist of a prime vaccine using BCG or rBCG,

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followed by a heterologous booster vaccine that contains antigens shared with the prime. Immunological correlates of protection. Rapid vaccine development and improvement requires reliable correlates of vaccineinduced human protection. In mouse TB challenge studies, IFN-γ-secreting CD4+ T cells are important mediators of protection19,20, and attempts to induce TB-antigenspecific IFN-γ-secreting CD4+ T cells has been the dominant theme of most TB vaccine research.


PERSPECTIVES Table 1 | Summary of new-generation tuberculosis vaccine work in progress

Vaccine

Description

Development stage

Researchers/sponsors

Live mycobacterial vaccines: modified BCG rBCG30

BCG Tice engineered to overexpress Ag85B

Phase I trial in US, completed in 2004; no M. Horwitz, UCLA; D. Hoft, St Louis serious adverse events University, US; T. Littlejohn, Winston, Salem, North Carolina, US Sponsor: Sequella, Aeras*

rBCG-Aeras 403

BCG Danish with endosome escape and overexpression of several proteins including Ag85A, Ag85B and TB10.4

Ongoing pre-clinical studies and GMP production; Phase I clinical trial planned for 2006

R. Sun, D. Hone, M. Stone, J. Sadoff , Aeras*

rBCGΔUre:CHly+

BCG Pasteur with endosome escape

Ongoing pre-clinical studies and GMP production; Phase I clinical trial planned for 2006

S. Kaufmann, Max Planck Institute for Infection Biology, Berlin, Germany

BCG::RDI

BCG Pasteur with reintroduction of RD-1 locus which contains protective Ags

Ongoing pre-clinical studies

S. Cole, Institute Pasteur, Paris, France

Pro-apoptotic BCG

BCG Tice with diminished superoxide dismutase activity


D. Kernodle, Vanderbilt, US Sponsor: NIH, VA, Aeras*

Live mycobacterial vaccines: modified Mycobacterium tuberculosis M. tuberculosis PhoP

Deletion of virulence associated gene phoP from Mtb MT103 strain

Ongoing pre-clinical studies and GLP production

B. Martin, University of Zaragoza, Spain; B. Gicquel, Institute Pasteur, Paris, France

M. tuberculosis mc26030

Mtb H37Rv with deletion of panCD and RD-1 locus

Ongoing pre-clinical studies and GMP production; Phase I clinical testing planned for 2006

W. Jacobs, Albert Einstein College of Medicine, New York Sponsor: NIH

M. tuberculosis mc26020

Mtb H37Rv with deletion of the lysA and the panCD locus

Ongoing pre-clinical studies and GMP production; Phase I clinical testing planned for 2006

W. Jacobs, Albert Einstein College of Medicine Sponsor: NIH

Mtb72F

Recombinant fusion protein (Mtb39 and Mtb32) in AS02A and AS01B adjuvants

Phase I trials completed in US and Europe; no serious adverse events; additional trials ongoing in Europe

Y. Skeiky, S. Reed, Corixa Corp. Seattle, US Sponsor: Glaxo Smith Kline, Aeras*

Hybrid-1 (85B–ESAT6)

Recombinant fusion of Ag85B–ESAT-6 in IC31 adjuvant

Phase I clinical trials commenced in 2005

P. Andersen, SSI, Denmark Sponsor: SSI, TBVAC

HyVac-4 (Ag85B–TB10.4)

Recombinant fusion of Ag85B–TB10.4 in IC31 adjuvant

Ongoing pre-clinical studies and GLP production; Phase I clinical trial planned for 2007

P. Andersen, SSI, Denmark Sponsor: SSI, Aeras*

Heatshock protein

Nascent BCG proteins associated with purified heat-shock proteins


C. Colaco, ImmunoBiology, UK Sponsor: ImmunoBiology, Aeras*

Subunit vaccines

Naked DNA and viral-vectored vaccines Hsp65 (GroEL) DNA

Conserved antigen from Mycobacterium leprae for immunotherapy


D. Lowrie, National Institute for Medical Research, London UK. Sponsor: Sequella Inc.

MVA85A

Attenuated strain of vaccinia (modified vaccinia virus) expressing Ag85A

Completed and ongoing Phase I clinical trials; immunogenic, no serious adverse events reported

A. Hill, H. McShane, Oxford University, UK Sponsor: Wellcome Trust

Aeras 402 (Ad35.TB-S)

Non-replicating Ad35 expressing multiple TB proteins including Ag85A, Ag85B and TB10.4

Ongoing pre-clinical studies; GMP production for Phase I clinical trials planned for 2006

Aeras*, Crucell Sponsor: Bill and Melinda Gates Foundation


J. Fulkerson, D. Hone, M. Stone, D. Onyabe, J. Sadoff Sponsor: Aeras*

Double-stranded RNA capsids Double-stranded RNA capsids

Double-stranded RNA capsids encoding TB antigens for oral delivery

*Supported by the Bill and Melinda Gates Foundation. Phase I clinical trial: trial that assesses the safety and immunogenicity of a drug or a vaccine in healthy volunteers. BCG, bacille Calmette–Guérin; GLP, good laboratory practice; GMP, good manufacturing practice; NIH, National Institutes of Health; SSI, Statens Serum Institute; TB, tuberculosis; VA, Medical Research Service, Department of Veterans Affairs.

MHC class I restricted, IFN-γ-secreting CD8+ T cells are also important in TB challenge models19,21 and experiments in knockout mice have reported that vaccines can induce significant protection in the

absence of CD4+ T cells22. CD8+ T cells might also be important in the control of LTBI23. The potential role of both vaccineinduced CD4+ T cells and CD8+ T cells is


not surprising as the granuloma is composed of CD4+ T cells with an outer ring of CD8+ T cells24,25. Potent new TB vaccines will probably need to induce a balanced antigen-specific CD4+ and CD8+ T-cell


PERSPECTIVES response, but the detailed phenotypes and numbers of such cells required for protection is unknown. Furthermore, antibody production against mycobacterial cell wall components might also be important26. At present, there are no proven human immunological correlates of vaccine-induced protection. The detailed phenotypic properties and levels of vaccine-induced T cells in protected versus non-protected volunteers in Phase IIb and Phase III efficacy studies are needed to generate such correlates. In an ongoing trial conducted by the South African TB Vaccine Initiative (SATVI) group (G. Hussey, T. Hawkridge, L. Geiter and W. Hanekom), cells from over 7,000 BCG-vaccinated 10-week-old infants have been collected and banked27,105 for the detailed analysis of vaccine-induced T cells. One correlate of protective immunity that could be explored using this approach is the activation of antigen-specific memory CD4+ and CD8+ T cells that secrete IFN-γ, IL-2 and TNF-α28. Combinations of these and other phenotypic markers in individual T cells — such as CD69, MIP1β, CD45RA, CD27, memory markers such as CD127 (IL-7 receptor)28,29, cytotoxic markers such as FAS ligand, CD107a/b and CD3/4/8 phenotype28 — show potential as correlates of immune protection. γδ T cells that recognize phospholigands, and CD1-restricted T cells that recognize the abundant glycolipids of the mycobacterial cell wall through CD1 molecules (reviewed in REFS 12–14), might also require examination for their potential as immune correlates. Strategies to improve BCG The current BCG vaccine. BCG, the attenuated strain of Mycobacterium bovis, was derived from the parental strain by passage on potato-derived media. Almost three billion doses have been used in various forms since 1921. Approximately 115 million doses are distributed each year30,31 providing almost 80% coverage of infants worldwide32. Neonatal vaccination with BCG has been reported as effective in reducing the incidence of childhood TB in endemic areas33 and in reducing the rate of complications from disseminated TB. However, the ongoing SATVI trial of over 11,000 newborns vaccinated with BCG showed an incidence of culture or smear-proven TB of around 3% by the age of 3 years27,105. The protective effect of BCG might wane over time resulting in a variable outcome that is insufficient to control TB in most endemic areas34. Although several older

Antigen overexpression. Antigen 85B (Ag85B) is a secretory and immunogenic protein of M. tuberculosis and BCG with mycolyl-transferase activity, which is required for mycobacterial cell wall synthesis. M. Horwitz’s group (UCLA) have produced a TB vaccine candidate, rBCG30 that overexpresses Ag85B and that induces increased protection compared with its parental BCG strain in animal challenge studies40,41. There were no significant safety issues in a Phase I clinical trial of rBCG30 in over 30 healthy adult volunteers. Increased immunogenicity compared with the parent BCG strain was reported42, although the study did not meet its predefined immunogenicity endpoints.

apoptosis-mediated CD8+ T-cell responses. Listeria monocytogenes Hly (listeriolysin) is a sulphydryl-activated cytolysin that forms pores in the membrane of the early phagosome. S. Kaufmann’s group (Max Planck Institute) have produced an rBCG vaccine (rBCGΔUreC:Hly+) that secretes Hly, which punches holes in the endosome and allows some leakage of BCG from the phagosome into the cytoplasm of infected host cells43, also resulting in apoptosis of these cells (FIG. 1). Because the activity of Hly is optimal in an acidic environment, the BCG urease gene (ureC), which enables mycobacteria to block the acidification of the early macrophage phagosome, was also deleted. Enhanced presence of BCG in the cytoplasm increases MHC class I presentation of BCG antigens to CD8+ T cells, thereby increasing the CD8+ T-cell immune response. Furthermore, translocation of BCG into the cytoplasm triggers apoptosis, which in turn kills the bacilli and releases antigens, a powerful signal for induction of cellular immunity44. The antigens released as apoptotic blebs (vesicles that carry mycobacterial antigens) are subsequently taken up and processed by dendritic cells, which activate T cells by a mechanism referred to as ‘cross-priming’ (reviewed in REF. 45). In preclinical mouse-protection studies, the rBCGΔUreC:Hly+ vaccine induced better efficacy than the BCG strain and was safer in immunocompromised SCID mice46. Also, unlike the parental BCG strain the ΔUreC:Hly+ rBCG was shown to protect significantly against the virulent M. tuberculosis strain, Beijing/W. This vaccine is scheduled for Phase I trials in early 2006 (REFS 47,48). R. Sun, M. Stone and D. Hone’s group at Aeras have constructed rBCG strains that escape from the endosome using the pH-independent perfringolysin (rBCG–pfo, (FIG. 1)), instead of Hly49, with overexpression of several immunodominant antigens including Ag85A, Ag85B and TB10.4. This strain combines the increased protection seen with rBCG30 with the increased protection and safety of the endosome-escape mutants. This vaccine is scheduled for Phase I trials in 2006 (REF. 27).

Endosome escape. Because protective immunity against M. tuberculosis infection seems to involve both CD4+ and CD8+ T cells, antigens must be presented by both MHC class I and II molecules. BCG that resides in the phagosome gains access to MHC class II molecules, thereby primarily activating CD4+ T cells. To be presented by MHC class I molecules and to activate CD8+ T cells, BCG must either escape from the endosome and gain access to the cytoplasm of the infected cells or induce

Reducing BCG interference with immunity. Usually, host superoxides produced by macrophages kill M. tuberculosis and can amplify host T-cell-recruiting signals. D. Kernodle’s group (Vanderbilt) has shown that secretion of superoxide dismutase (SOD) and other compounds that inactivate superoxides by M. tuberculosis promotes granuloma formation in which M. tuberculosis is secluded, and therefore contributes to inhibition of host defences and persistence of the organism50.

studies demonstrated efficacy as high as 80% in children, adolescents and young adults, with effects lasting up to 60 years in a study of native Americans35–37, and a recent study from Turkey claims that vaccination with two doses of BCG confers some protection in children against infection and disease38, a large-scale study from India over a wide age range did not show any overall efficacy of two different BCG vaccines compared with placebo39. The reasons for the varying efficacy of BCG in protection against pulmonary TB are not completely understood. Potential explanations that have been suggested (reviewed in REFS 30,32) include: interference with the immune response to BCG caused by previous exposure to environmental mycobacteria; differences among BCG vaccine sub-strains; and phenotypic changes in the vaccine during passage from the original cultures and during the manufacturing process; the deletion of protective antigens from BCG; failure of BCG to stimulate adequate, balanced anti-mycobacterial CD4+ and CD8+ T-cell responses; variability in dose, route of administration, age of administration and genetic differences among vaccinees; and lyophilization of the vaccine. Several strategies are being used to improve the efficacy of BCG and are described below.

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PERSPECTIVES Attenuated M. tuberculosis and rBCG with reduced secretion of functional SOD are more potent vaccines than their parental strains51. M. tuberculosis and BCG, similar to other successful human pathogens, also produce various other gene products that inhibit the human immune system. W. Jacobs (Albert Einstein College of Medicine) recently suggested that promoting a pro-apoptotic vaccine by specific deletion of genes such as nlaA (Rv3238c), which inhibit apoptosis of infected cells, might also lead to a superior vaccine52. Cells infected with rBCG or recombinant M. tuberculosis vaccines that cannot inhibit caspase-8-mediated apoptosis that usually accompanies infection consequently undergo apoptosis. This leads to cross priming and powerful cellular immune responses against these live vaccines, as caspase-8-mediated apoptosis is one of the most powerful known inducers of cellular immunity53,54. Reintroduction of deleted genes. S. Cole’s group (Institute Pasteur) have reintroduced selected genes into BCG that were deleted during BCG attenuation55. Examples include the RD1 locus56 (see TubercuList in Online links box), which encodes the immunodominant and protective ESAT-6 and CFP-10 proteins — reintroduction of this locus enhanced the protective efficacy of the recombinant BCG::RD1-2F9 vaccine compared with BCG57. However, this approach, although increasing the potency of BCG, might also increase its virulence58 and pose problems for human use. New attenuated live TB vaccines An alternative to BCG modification is to use molecularly attenuated M. tuberculosis that contains over 120 M. tuberculosis genes not found in BCG59–61. A desirable feature of effective and safe live M. tuberculosis vaccine strains is their ability to persist in the host for a limited duration, greater protective efficacy, as well as an improved safety profile compared with the currently used BCG vaccine. The obstacles facing live TB vaccines with respect to safety concerns, public perception and regulatory hurdles were discussed at a recent meeting, and the danger of reversion of live TB vaccines to a virulent state was considered a crucial issue62. At the minimum, two non-reverting independent mutations as well as proven safety in immunocompromised SCID mice are recommended for attenuated M. tuberculosis vaccines. Also, the potential release of antibiotic-resistance gene markers from live vaccines into the environment, with subsequent transfer to other organisms, is considered an important concern.

Inactivation of PhoP, which regulates expression of many M. tuberculosis proteins, by C. Soto’s group (University of Zaragoza, Spain)63 attenuated M. tuberculosis virulence while maintaining the pattern of immune responses associated with the parental M. tuberculosis strain64. This mutant has shown promising early animal protection but will probably require a second, independent, nonreverting mutation to satisfy human safety concerns62. Auxotrophic mutant vaccine candidates (requiring the addition of a nutrient(s) for survival) have been produced by transposon mutagenesis. These mutants maintain their infective ability, have a limited duration of replication in the host65,66 and protect against virulent TB challenge in the guinea pig model67. The extent of the attenuation and safety of these vaccines remains to be defined in immunocompromised SCID mice. The Jacobs and Bloom groups have targeted the deletion of proteins involved in M. tuberculosis lipid metabolism by disrupting both the panC and panD genes (which are involved in pantothenic acid synthesis). Further deletion of the lysA gene produced vaccine mc26020 (panC panD lysA), which does not replicate in vivo. Deletion of the RD1 region (implicated in the attenuation of BCG) from the panC, panD mutant produced vaccine mc26030 (panC panD ∆RD1)68–70. Both of these highly attenuated mutants were safer than BCG in immunocompromised animals and provided similar levels of protection in mouse challenge studies. The mc26030 is scheduled for Phase I trials in 2006 (REF. 52). Subunit vaccine strategies Vaccine antigen identification. Antigens of M. tuberculosis that activate T cells in previously infected humans and animals are considered good vaccine candidates. Biochemical approaches initially identified several of the most abundant proteins (Ag85 complex, MPT32, PhoS, DnaK, GroES, MPT46, MPT53, MPT63 and the 19-kDa lipoprotein) secreted by M. tuberculosis71–73. Subsequently, antigens in the low molecular mass range of 6–10 kDa (ESAT-6 family) and 26–34 kDa (Ag85B and MPT59)74,75 were defined. T-cell and serological expression cloning approaches76–79 identified several important dominant immunogenic antigens including Mtb32 (a secreted serine protease) and Mtb39 (a member of the PPE (proline-prolineglumatic acid) family of proteins presumably localized on the cell wall)80,81. Proteomic approaches82,83 have also identified several promising antigens such as Rv3407 (REF. 84).


Antigen vaccines have advanced towards clinical development using animal protection models and assessment of their potency in stimulating immune cells derived from peripheral blood mononuclear cells of healthy, previously infected donors to produce IFN-γ. Antigen vaccine candidates therfore include Ag85A and Ag85B (REFS 85,86), ESAT-6, TB10.4 and Mtb9.9 (REFS 75,79,87) and the PPE family of proteins, Mtb39a–e and Mtb41 (REFS 76,78,81). Recombinant fusion proteins. Y. Skeiky, S. Reed and P. Andersen’s groups have used a genetic fusion approach to generate hybrid protein vaccines consisting of Mtb39 and Mtb32 (Mtb72F)88, or Ag85B and ESAT-6 (Hybrid-1)89, respectively. These candidate vaccines were selected on the basis of their strong recognition by immune cells in infected individuals and experimentally vaccinated or infected animals. The generation of recombinant protein subunit vaccines comprising multiple open reading frames is both cost effective and simple, and the fusion vaccines can be more immunogenic than the individual components. Both fusion recombinant vaccine constructs (Mtb72F and Hybrid-1) in selected adjuvants protect as well as BCG in the mouse and guinea pig TB challenge models88,90–92. Multiple Phase I clinical trials of Mtb72F have been completed in the United States and Europe (results pending) and additional trials are being conducted in Europe. Phase I trials of Hybrid-I started in Europe in November 2005. ESAT-6 (the component of Hybrid-I that is absent from all BCG, but present in M. tuberculosis strains) is the basis of a new generation of diagnostic tests for M. tuberculosis infection. Therefore, if used as a vaccine, Hybrid-I could potentially compromise the diagnostic utility of ESAT-6 in distinguishing between infection and vaccination93,94. Therefore, P. Andersen’s group in collaboration with Aeras has developed a new fusion construct, HyVac-4, which consists of Ag85B–TB10.4, and which avoids diagnostic interference and should also boost BCG-primed individuals because TB10.4 is immunogenic in BCG-vaccinated subjects87,95. Prime–boost vaccine strategies. The attraction of the heterologous prime–boost approach is that it preferentially expands TB-specific pre-existing memory T cells against antigenic epitopes shared by both the prime and booster vaccines. This approach also minimizes the potential of


PERSPECTIVES generating antibody and T-cell neutralizing effects that would impact on the ‘take’ and effectiveness of a non-replicating viralvector boost (FIG. 2). An early study using a BCG prime–boost approach showed that mice boosted with an adjuvanted Ag85A protein when they had lost their BCG-induced capacity to resist an aerosol TB challenge were protected better than non-BCG-primed animals given adjuvanted protein96. This suggests that adjuvanted proteins including Mtb72F, Hybrid-1 and HyVac-4 could induce superior protection when given as boosters in primary regimens, or in adolescence. Such recombinant protein regimens, however, might induce primarily antigen-specific CD4+ T cells instead of the desired balanced CD4+ and CD8+ T-cell responses. A recent study in mice of a BCG prime followed by a booster with an M. tuberculosis DNA vaccine encoding the gene product Rv3407 (a 10-kDa protein of unknown function) also showed superior protection compared with BCG alone84. Protection that was superior to BCG was also demonstrated in guinea pigs immunized with a BCG prime–Mtb72F DNA boost regimen followed by aerosol challenge with virulent M. tuberculosis. Remarkably, histological examination of the lungs of these guinea pigs revealed minimal granulomatous lesions with evidence of lesion healing and airway remodelling at a time when animals immunized with BCG and then challenged were dying90. Whereas, so far, the success of human DNA vaccination in general has been disappointing, non-replicating recombinant poxviruses and adenoviruses are efficient at boosting previously primed T-cell responses and show great potential for use in heterologous prime–boost immunization strategies. Priming with BCG and boosting with a recombinant Modified Vaccinia Ankara virus (MVA) that expresses Ag85A induced increased levels of CD4+ and CD8+ T cells and enhanced protection in animals compared with a vaccination regimen using either BCG or MVA85A alone97. In a Phase I clinical trial recently reported by H. McShane and A. Hill’s group (Oxford University, UK), immunization of volunteers from 6 months to 38 years after BCG vaccination with MVA resulted in persistent, high levels of IFN-γ secreting CD4+ T cells, which were substantially higher than seen following vaccination with BCG or MVA85A alone, which only induced transient responses106. Adenoviral vectors are potent vaccinedelivery vehicles. A recent study showed that vaccination of mice by the intranasal

a Newborn

b 14 weeks

c 24 weeks and adolescence Recombinant protein with adjuvant

rBCG or BCG

Recombinant protein with adjuvant

Dendritic cell Viral vector

Nucleocaspid in bacteria

CD4+ T cell

CD8+ T cell

Expansion of Ag-specific T cells

Memory T cells

Amplification of Ag-specific T cells

Figure 2 | Prime-boost vaccination strategies. Boosting or augmenting BCG or a recombinant, modified BCG (rBCG), rather than trying to completely replace it, is a practical approach for future immunization against TB. Heterologous prime–boost regimens begin with vaccination of newborns using BCG or modified rBCG as the prime (a). This is followed by a booster vaccination in infancy (b) and later in childhood and adolescence (c) using different antigen-delivery systems (heterologous boosting) composed of selected antigens in common with the prime. The booster can include viral vectors such as recombinant adenovirus or poxvirus engineered to express immunogenic and protective mycobacterial candidate antigens (for example, Ag85A, Ag85B and TB10.4) or recombinant proteins (for example, HyVac-4, Hybrid-1 and Mtb72F) delivered with adjuvants (such as AS02A, AS01B, IC31 and LipoVac). Priming with BCG or rBCG followed by a booster with adenovirus and a subsequent boost with recombinant protein in adjuvant formulation is also being contemplated. Alternatively, Shigella spp. nucleocapsid vectors offer a possible alternative oral vaccine boost (b). Shigella vectors deliver double stranded RNA capsids into the cytoplasm where they produce mRNA that directs cellular production of M. tuberculosis antigens. The heterologous prime–boost approach preferentially expands BCG or TB-specific pre-existing memory T cells against antigenic epitopes shared by both the prime and booster vaccines. Administration of the booster vaccination in an alternative vector minimizes the generation of anti-vector immunity that is typically seen with repeated administration using the same vaccine (homologous boosting), thereby inducing a powerful synergistic immune response. Unlike BCG (which induces a predominant CD4+ T-cell response), rBCG that has access to the cytoplasm activates a broad repertoire of antigen-specific CD4+ and CD8+ T cells (a). Subsequent boosting results in the expansion of selected antigen-specific memory CD4+ and CD8+ T cells and the selective enrichment of high avidity T cells to combat exposure to the pathogen (b,c).

mucosal route with a recombinant adenoviral (Ad5 based) construct expressing the M. tuberculosis antigen Ag85A (AdAg85A) conferred protection (mediated by both CD4+ and CD8+ T cells) that was superior to that seen with BCG vaccination against a pulmonary M. tuberculosis challenge98. Aeras, in collaboration with Crucell, has constructed non-replicating Ad35 vectors that are less prevalent in the environment than Ad5 (REFS 99 and 100), and that express several

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M. tuberculosis proteins including Ag85A, Ag85B and TB10.4 (REFS 27,100). Vaccination of mice with a single dose of the rAd35–TB construct conferred protection against M. tuberculosis challenge107. BCG prime followed by a booster with a single dose of these adenovirus recombinant vaccines in mice have yielded significantly increased antigenspecific CD4+ and CD8+ T-cell responses compared with adenovectored TB vaccines or BCG administered alone. Aeras has


PERSPECTIVES constructed rBCG vaccines with over 50% escape from the endosome that overexpress these same antigens to function as a prime for this adenovector boost27 (R. Sun et al., unpublished observations). J. Fulkerson, D. Hone, D. Onyabe and M. Stone at Aeras have also constructed double stranded RNA capsids which can be orally delivered using engineered Shigella spp. vectors. Shigella delivers these capsids into the cytoplasm where they produce mRNA that directs cellular production of M. tuberculosis antigens101 (J. Fulkerson et al., unpublished observations) and induces cellular immunity. Such an oral vaccine boost to an rBCG prime might have the low cost and ease of deliverability desirable in a TB vaccine for the developing world. Conclusions A safe and effective heterologous prime– boost regimen, which boosts or augments BCG or rBCG, is perhaps the most realistic strategy for future TB control through immunization. Ideally the rBCG prime in such a regimen would include the overexpression of important antigens from different stages of the pathogen’s life cycle and would induce cross-priming and increased CD4+ and CD8+ T-cell responses, as well as increased safety in immunocompromised individuals. The ideal booster would comprise recombinant proteins delivered with adjuvants, or delivered by non-replicating viral or capsid vectors. Although there is a possibility that the current generation of new vaccines might protect against reactivation from the latent state, these vaccines are not generally focused on the ‘latency’ dosR regulated antigens recently identified by microarray and other techniques102,103. Furthermore, animal models of latency for preclinical vaccine evaluation are just being developed104. The best hope for these vaccines probably lies with overexpression of dosR regulated proteins by rBCG followed by a multivalent booster vaccine. Based on current clinical development timelines, first generation vaccine regimens for protection against infection and disease could be licensed and available in the next 7–9 years with vaccines against latency and reactivation 2–3 years later. Because of its great promise and practical usefulness, rBCG prime– heterologous boost strategies for malaria and HIV are also currently being explored. Yasir A. W. Skeiky and Jerald C. Sadoff are at the Aeras Global TB Vaccine Foundation, 1405 Research Blvd, Rockville, Maryland 20850, USA. e-mails: [email protected]; [email protected] doi10.1038/nrmicro1419

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Acknowledgements We are grateful to Dr. L. Barker for his input and critical reading of this article. The Aeras Global TB Vaccine Foundation is supported by a major grant from the Bill and Melinda Gates Foundation.


DATABASES The following terms in this article are linked online to: Entrez Gene: http://www.ncbi.nlm.nih.gov/entrez/query. fcgi?db=gene lysA | panC | panD | Rv3238c | ureC Entrez Genome Project: http://www.ncbi.nlm.nih.gov/ entrez/query.fcgi?db=genomeprj Listeria monocytogenes | Mycobacterium bovis | Mycobacterium tuberculosis UniProtKB: http://ca.expasy.org/sprot Ag85A | Ag85B | DnaK | GroES | Hly | IFN-γ | MPT32 | MPT46 | MPT53 | MPT63 | Mtb9.9 | Mtb32 | Phop | TB10.4

FURTHER INFORMATION Author’s homepage: www.aeras.org CDC HIV/AIDS Prevention: http://www.cdc.gov/hiv/dhap.htm South African TB Vaccine Initiative: http://www.satvi.uct.ac.za TubercuList: http://genolist.pasteur.fr./TubercuList Access to this links box is available online.


PERSPECTIVES OPINION

The trypanolytic factor of human serum Etienne Pays, Benoit Vanhollebeke, Luc Vanhamme, Françoise Paturiaux-Hanocq, Derek P. Nolan and David Pérez-Morga

Abstract | African trypanosomes (the prototype of which is Trypanosoma brucei brucei) are protozoan parasites that infect a wide range of mammals. Human blood, unlike the blood of other mammals, has efficient trypanolytic activity, and this needs to be counteracted by these parasites. Resistance to this activity has arisen in two subspecies of Trypanosoma brucei — Trypanosoma brucei rhodesiense and Trypanosoma brucei gambiense — allowing these parasites to infect humans, and this results in sleeping sickness in East Africa and West Africa, respectively. Study of the mechanism by which T. b. rhodesiense escapes lysis by human serum led to the identification of an ionic-pore-forming apolipoprotein — known as apolipoprotein L1 — that is associated with high-density-lipoprotein particles in human blood. In this Opinion article, we argue that apolipoprotein L1 is the factor that is responsible for the trypanolytic activity of human serum. Similar to other species of African trypanosome (such as Trypanosoma congolense and Trypanosoma vivax), Trypanosoma brucei brucei, Trypanosoma brucei rhodesiense and Trypanosoma brucei gambiense are protozoan parasites that are transmitted to various mammals by tsetse flies (Glossina palpalis and Glossina morsitans) (FIG. 1). During a blood meal by the tsetse fly, infective trypanosome forms, known as metacyclic forms, that are present in the salivary glands of the fly are inoculated into mammalian hosts, such as humans, cattle, antelopes, buffaloes and lions. This allows the transformation of the parasites into long, slender bloodstream forms, which actively divide and colonize the blood until a quorum-sensing signal triggers their differentiation into short, stumpy forms. These short, stumpy forms do not divide and are competent for transformation into procyclic forms as soon as they are ingested by a tsetse fly. The procyclic forms then proliferate in the mid-gut of the fly and eventually differentiate into metacyclic forms after a complex journey in the insect vector. The metacyclic forms are quiescent and are ready to be transferred back to mammals. Because they reside extracellularly in their mammalian hosts, the bloodstream forms of trypanosomes need to resist all components of host defence. The main protective feature of these parasites is a thick and dense surface coat, which covers the entire plasma membrane. This coat consists

of a monolayer of ∼107 molecules of a single glycoprotein known as variant surface glycoprotein (VSG). The VSG molecules are attached to the plasma membrane by glycosylphosphatidylinositol (GPI) anchors, and they form homodimers1. Close interactions between adjacent VSG homodimers prevent antibodies from reaching the inner antigenic determinants of the coat, and the elongated shape of the VSGs keeps toxins away from the plasma membrane. In addition to these protective functions, VSGs undergo continuous variation, which leads to the periodic change of the VSG loops that are exposed at the surface of the parasite. These loops contain the only antigenic determinants of living intact trypanosomes that are recognized by the immune system. Therefore, such variation is known as antigenic variation, and it allows the parasite to evade antibody-mediated clearance2–5. Only one VSG variant is produced at any one time, because only one VSG allele at a time can be transcribed from the repertoire of VSG genes, which consists of more than 1,000 sequences. This transcription occurs at one of several VSG gene expression sites (ESs), and antigenic variation can result from two distinct mechanisms: transcriptional switching between VSG ESs, and homologous recombination between the active VSG gene and another VSG gene from the repertoire. In addition to the defences that are usually encountered in mammals, African


trypanosomes need to defy a novel innate immune mechanism that evolved in humans and in some non-human primates — an efficient trypanolytic factor that is present in serum. Here, we summarize current knowledge of the trypanolytic activity of human serum and of how trypanosomes that infect humans resist this activity. Two serum proteins — haptoglobin-related protein (HPR) and apolipoprotein L1 (APOL1) — have been proposed as candidates for providing the trypanolytic activity. In this Opinion article, we argue that the characterization of the mechanism of resistance of T. b. rhodesiense, as well as the study of the phenotype of lysis induced by APOL1, indicates that APOL1 is the sole factor that is responsible for trypanolysis. Studies of trypanolytic activity Between 1902 and 1912, Laveran and Mesnil6 reported that sera from humans and other primates — such as various Papio (baboon), Cercocebus (mangabey) and

Procyclic form (mid-gut)

Metacyclic form (salivary glands)

Proliferative

Quiescent Insect host

Mammalian host Quiescent

Proliferative

Short, stumpy form (blood)

Long, slender form (blood)

Figure 1 | Life cycle of Trypanosoma brucei. The life cycle of T. brucei alternates between the insect host (the tsetse fly) and a mammalian host (such as humans, cattle, antelopes, buffaloes and lions). In both the insect vector and the mammalian host, colonization occurs through the proliferation of rapidly dividing trypanosome forms. These forms eventually transform into resting (quiescent) parasite cells that are preprogrammed for cellular differentiation after changes in the environment (that is, transfer from one host to the other). The long, slender bloodstream forms also adapt to their environment while present in the mammalian host, and this adaptation involves continuous variation of the main surface antigen, which is known as variant surface glycoprotein.


PERSPECTIVES Mandrillus (forest baboon) species — kill trypanosomes, although they did note differences in activity between these sera. Human serum did not affect the T. brucei subspecies that infect humans: that is, T. b. rhodesiense and T. b. gambiense. By contrast, sera from various Papio species lysed T. b. rhodesiense and showed 10–25-fold higher activity than human serum against human-serumsensitive trypanosomes6. Sera from Cercocebus and Mandrillus species were much less active against human-serumsensitive trypanosomes, and serum from Pan troglodytes (chimpanzees) was devoid of activity, despite the close evolutionary link between chimpanzees and humans6. These findings were confirmed in subsequent studies, which also showed that serum from Gorilla gorilla (gorillas) has trypanolytic activity7–9. Trypanolysis occurs through high-densitylipoprotein-particle-mediated endocytosis. Mainly as a result of the pioneering work of Mary Rifkin10,11, the trypanolytic activity was shown to be associated with high-density lipoprotein (HDL) particles (BOX 1), in particular with the densest HDL subfraction, HDL3 (REFS 12–15). Further studies indicated that receptor-mediated endocytosis of these HDL particles by the trypanosome was involved in lysis16–22. In this case, internalization of the lytic particles would involve their delivery to increasingly acidic compartments of the endocytic pathway. That these processes occur is supported by the findings that either neutralization of the pH of endosomes (with a membrane-permeable weak base, such as chloroquine)16,17 or inhibition of endocytosis itself (by RNA-interference-mediated knockdown of actin mRNA)22 results in inhibition of lysis. The identity of the trypanosome cell-surface receptor for HDL particles remains elusive, but it is probably a lipoprotein scavenger receptor, because HDL particles and low-density lipoprotein (LDL) particles have both been shown to compete with binding of the trypanolytic factor to

the trypanosome surface23. However, the lipoprotein scavenger receptor of trypanosomes must considerably differ from that of other eukaryotes, because sequence analysis did not uncover any candidate genes in the trypanosome genome. HPR as the trypanolytic factor. Purification of the trypanolytic HDL particles in the HDL3 subfraction yielded two types of complex, which differed in their lipid content but contained several of the same proteins, including APOA1 and the serine-proteaselike protein HPR24–27. The involvement of APOA1 in trypanolysis was quickly discounted because of its wide distribution among different types of HDL complex and because of its failure to show trypanolytic activity28–30. Several lines of evidence, however, suggested that HPR has a role in trypanolysis. First, haptoglobin-specific antibodies, which also recognize HPR, inhibited trypanolysis when added to lytic HDL particles24. Second, haptoglobin, which is similar to HPR, also prevented trypanolysis when added to these assays of trypanolysis, possibly as a result of competition30,31 (although a differential effect was observed for the two types of trypanolytic complex that had been purified32–34). Third, HPR is not expressed in chimpanzees, the serum of which lacks trypanolytic activity (as indicated earlier)6– 9,35. Last, HPR was also thought to be the specific component of the trypanolytic HDL particles that is recognized by the parasite cell-surface receptor36. The trypanolytic effect of the HPRcontaining HDL particles was thought to be a consequence of damage to the lysosomal membrane. According to Hajduk and colleagues16,24,37, HDL particles caused lipid peroxidation of the lysosomal membrane of the parasite, leading to disruption of this membrane, release of proteolytic enzymes into the cytoplasm and auto-digestion of the parasite cell. The peroxidase activity of HDL particles was initially ascribed to a putative HPR–haemoglobin dimer24 and then to the

Box 1 | High-density lipoprotein (HDL) particles In the blood, most lipids are contained in soluble complexes known as lipoproteins. HDL particles are spherical particles that comprise a hydrophobic lipid core (which mainly consists of triglycerides and cholesteryl esters) surrounded by a hydrophilic layer (which consists of phospholipids, unesterified cholesterol and several proteins that are collectively known as apolipoproteins). In terms of protein content, HDL particles mainly contain apolipoprotein A1 (APOA1), which specifically captures and solubilizes free cholesterol, thereby enabling HDL particles to function as cholesterol scavengers. Several HDL-particle subfractions can be separated on the basis of density. The subfraction known as HDL3, which contains both APOL1 and haptoglobin-related protein (HPR), is the densest. The high protein content of HDL particles renders them denser than other lipoprotein particles, including low-density lipoprotein (LDL) particles.

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Fenton reaction between hydrogen peroxide and iron (an iron-salt-dependent decomposition of hydrogen peroxide, which generates highly reactive hydroxyl radicals)37. Findings supporting these views included the inhibition of lysis by antioxidants and protease inhibitors (factors that were proposed to block lipid peroxidation and cellular autodigestion, respectively)16,24,37, the detection of end-products of lipid peroxidation during trypanolysis37 and the observation (using electron microscopy) of discontinuities in the lysosomal membrane of cells undergoing lysis16. By contrast, Raper, Tomlinson and colleagues27,38 could not detect binding of haemoglobin to HPR, found no evidence of lipid peroxidation, observed no effect of antioxidants and protease inhibitors on trypanolysis, and found no reactive oxygen intermediates associated with trypanolysis. As detailed later, one explanation for these conflicting findings could be that both the proposed identity and the proposed mode of action of the trypanolytic factor were incorrect. Studies of resistance to trypanolysis In addition to studies of the trypanolytic activity, research on the resistance of certain trypanosome subspecies to lysis provided another angle from which to approach identification of the trypanolytic factor. In contrast to T. b. brucei, the subspecies T. b. gambiense and T. b. rhodesiense escape the trypanolytic activity of human serum and cause sleeping sickness, a lethal disease in humans. T. b. gambiense is permanently resistant to human serum. By contrast, T. b. rhodesiense loses resistance after being isolated from humans and transferred to other animals: for example, after 30–70 passages in mice6,39. Following the injection of these mice with human serum, T. b. rhodesiense regains resistance to lysis, and this acquisition of resistance was shown to be associated with antigenic variation39. Specifically, for a given isolate of T. b. rhodesiense, such resistance arising on selection in human serum involved a change in expression of the VSG to a particular VSG variant known as ETat 1.10. This VSG, however, was not itself responsible for resistance to lysis, because other humanserum-resistant T. b. rhodesiense clones were shown to express different VSGs, including VSGs that are expressed by clones that are sensitive to lysis39. This paradox was resolved by the discovery that, in these trypanosomes, selection in human serum involved transcriptional switching to a particular VSG ES, which was named R-ES40. Although


PERSPECTIVES R-ES initially contained the gene encoding the VSG variant ETat 1.10, replacement of this gene by gene conversion resulted in conservation of the resistance phenotype, indicating that a component of R-ES, but not the VSG gene itself, conferred resistance. All VSG ESs have polycistronic transcription units that contain several genes (known as expression-site-associated genes, ESAGs) in addition to the VSG gene41, and these are under monoallelic control of expression, similar to the VSG genes. R-ES was found to contain a gene known as serum-resistance associated (SRA) (FIG. 2), which had previously been identified as being transcribed only in resistant clones42. The presence of this gene in a VSG ES that is systematically selected for transcription in human serum provided a straightforward explanation for its exclusive expression in human-serumresistant clones. Transfection of SRA alone into T. b. brucei (which is sensitive to lysis by human serum) conferred full resistance, thereby identifying SRA as necessary and sufficient for resistance of T. b. rhodesiense40. The finding that SRA is an ESAG present in a particular VSG ES explained the link between resistance to lysis by human serum and antigenic variation. SRA encodes a truncated VSG that is devoid of surface-exposed loops, owing to an in-frame deletion43–45. Together with the localization of this gene in a VSG ES, this finding indicates that SRA was generated by antigenic-variation-associated homologous recombination of VSG genes. Interestingly, another component that is involved in adaptation of the parasite to the host, the parasite surface receptor for host transferrin, was also found to originate from a VSG gene, and the genes that encode this heterodimeric receptor are also located in VSG ESs41. So, the VSG ESs are loci that result in the generation of various VSG-derived proteins that help the parasite to adapt to the host. SRA and R-ES seem to be conserved in most isolates of T. b. rhodesiense46–50. So, these genetic elements are clearly important for the propagation of this trypanosome subspecies, for which they are also excellent specific markers51. Given the high probability that SRA evolved only once, T. b. rhodesiense is likely to have arisen as a clone of T. b. brucei that differs mainly or solely by its ability to express SRA on selection in human serum. The genetic variability that is observed between T. b. rhodesiense isolates is probably a result of spreading of R-ES into various backgrounds, through genetic exchange48. Using reverse-transcription PCR, it has also been shown that R-ES

a

VSG repertoire

ES1

7

6

5

4

8

8

7

6

R-ES

6

7

5

4 SRA

5

ES transcription

b

VSGn

VSG

VSG

VSG Homologous recombination: gene conversion VSG 3 2 11 1

Homologous recombination: reciprocal exchange

Transcriptional switching ES2

VSG

8

8

3

2

11

1

VSG

VSG 70-Base-pair repeats

Telomeric repeats

Trypanosoma brucei rhodesiense metacyclic form

VSG variant expressed VSG

VSG

SRA

VSG

Human serum

Transcriptional switching Homologous recombination

Non-human mammalian serum ?

Figure 2 | Antigenic variation and resistance to human serum of Trypanosoma brucei rhodesiense. a | Genetic mechanisms of antigenic variation. The Trypanosoma brucei genome contains more than 1,000 variant surface glycoprotein (VSG) genes (shown as coloured boxes). Only one of these VSG variants is expressed at any one time. Gene expression occurs in a telomeric expression site (ES), where the VSG gene is co-transcribed with expression-site-associated genes (ESAGs; shown as numbered boxes).The T. brucei genome contains a set of 15–20 similar (but not identical) ESs, 3 examples of which are depicted. In T. b. rhodesiense, one particular ES, known as R-ES, contains the serum-resistance associated (SRA) gene, which confers resistance to lysis by human serum40. Antigenic variation (that is, the expression of a different VSG variant) can result from two mechanisms (indicated in red): transcriptional switching and homologous recombination. Transcriptional switching occurs by a process known as in situ activation, in which expression of the active ES is turned off, and expression of a previously silent ES is turned on. This process does not involve DNA rearrangement. By contrast, homologous recombination involves replacement of the active VSG gene. This can occur by one of two mechanisms: gene conversion, which involves replacement with a copy of a VSG gene from the repertoire; or reciprocal exchange, which involves replacement with a VSG gene from another ES (and thereby exchange of a VSG gene between two ESs). These replacements occur through recombination between homologous regions, such as 70-base-pair repeats. b | Gain and loss of resistance to human serum. Tsetse flies inject the mammalian host with T. b. rhodesiense in the metacyclic form, and these trypanosomes then transform into long, slender bloodstream forms (FIG. 1), in which different VSG ESs can be activated. Only trypanosomes in which the R-ES is active can resist lysis by human serum. Under these conditions, in situ activation (transcriptional switching) of other VSG ESs is counter-selected (indicated by a small red cross), owing to the requirement for SRA expression to resist lysis. Therefore, antigenic variation only occurs through homologous recombination targeted to the active VSG gene. In non-human hosts, expression of the R-ES seems to be counter-selected, although in situ inactivation of the R-ES does require many passages in mice6,39. This counter-selection might result from the absence of most ESAGs from the R-ES, because these ESAGs might not be completely dispensable.



PERSPECTIVES a APOL1 Met60

Trp235

Ala238

Pro304

Ala339

Leu398 C terminus

Pore-forming domain

N terminus

Membrane-addressing domain

b

c

d

pH ~7 N

C C

Asp249

SRA-interacting domain

N SRA (amino acids 31–79)

Arg287 Glu253 Glu256

Arg284 Ile58, Leu61 and Ile62

Glu260 APOL1 (amino acids 248–291)

C

APOL1 (amino acids 340–392)

N APOL1 (amino acids 90–235)

C

pH ~5 N

N

C

N

C N Colicin A (pore-forming domain)

Figure 3 | Models of the three domains of apolipoprotein L1 (APOL1). A diagrammatic representation of APOL1 and the location of each of its domains is shown in a. The structure of each domain is also shown in a ribbon-diagram format in b,c,d, where the protein backbone is shown as a coloured ribbon and the amino-acid side chains are shown in black (uncharged atoms), blue (positively charged atoms) and red (negatively charged atoms). a | APOL1 contains three domains: a pore-forming domain, a membrane-addressing domain and an SRA (serum-resistance associated)-interacting domain. b | The region of APOL1 that spans the methionine residue at position 60 and the tryptophan residue at position 235 contains a domain that is structurally and functionally similar to the ionic-pore-forming domain of bacterial colicins such as colicin A. Analogous helices of the two proteins are shown in the same colour. Colicin A diagram modified with permission from REF. 64 © (2005) American Association for the Advancement of Science. c | The region of APOL1 that spans the alanine residue at position 238 and the proline residue at position 304 contains a membrane-addressing domain. Computer modelling of this region predicts a pH-dependent structure. At neutral pH, such as in the blood, the two α-helices of the membrane-

is not active when the parasite is present in non-human sera52. This finding indicates that R-ES, which lacks several ESAGs that are usually found in other VSG ESs40 (FIG. 2a), is counter-selected when the parasite is not exposed to human serum (FIG. 2b). The data also confirm and explain the early observations of Laveran and Mesnil6, as well

addressing domain interact through two salt bridges (indicated by pink squares), which are formed by the side chains of the amino acids indicated. This hairpin structure shows a segregation between hydrophobic (orange) and hydrophilic (green) surfaces, as indicated by space-filling models. At acidic pH, such as in the lysosome (pH 5.3), the salt bridges are predicted to dissociate as a result of neutralization of the negatively charged residues, and this would lead to loss of the large hydrophobic surface. Experimental evidence indicates that this surface is required for the association of APOL1 with high-density lipoprotein (HDL) particles and that treatment with acid results in dissociation of APOL1 from HDL particles (B.V. and E.P., unpublished observations). Figure modified with permission from REF. 64 © (2005) American Association for the Advancement of Science. d | The region of APOL1 that spans the alanine residue at position 339 and the leucine residue at position 398 forms a long α-helix that strongly and specifically interacts with the N-terminal α-helix of the Trypanosoma brucei rhodesiense protein SRA. The residues of SRA that are important for this interaction, as determined by studies involving point mutations, are indicated. Figure modified with permission from Nature REF. 54 © (2003) Macmillan Publishers Ltd.

as more recent observations in the field47, that T. b. rhodesiense is sensitive to human serum when grown in non-primate animals. Therefore, when living in non-human hosts, T. b. rhodesiense probably does not differ from T. b. brucei, but the difference arises when T. b. rhodesiense is exposed to human serum, which triggers selection of R-ES

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and transcription of SRA. Interestingly, T. b. gambiense does not carry SRA, despite its constitutive resistance to human serum53. Therefore, the mechanism ensuring resistance of this subspecies to lysis must differ from that of T. b. rhodesiense, and this mechanism is under investigation at present.


PERSPECTIVES APOL1 Although SRA has a signal peptide, it is not targeted to the cell surface of the parasite (unlike the VSGs from which it is derived) but accumulates in the lysosome54. This could result from the absence of a GPI anchor (REFS 55,56, and D.P.N., F.P.H. and E.P., unpublished observations). Similar to VSGs, SRA is characterized by a long N-terminal hairpin that contains two amphipathic α-helices1,44. The analysis of various mutated forms of SRA expressed in T. b. brucei (which itself does not express SRA) showed that only the N-terminal amphipathic α-helix helix A is required to provide resistance54. In VSGs, this helix is usually involved in coil-coiling interactions with the other amphipathic α-helix, helix B, and with helices of adjacent VSGs, to form a dimer1. So, by analogy, it was envisaged that SRA interacts with the trypanolytic factor of human serum by coil-coiling. Chromatographic separation of human serum on SRA affinity columns showed that the protein APOL1 bound strongly and specifically to SRA. This binding was found to result from hydrophobic coil-coiling interactions between helix A of SRA and the C-terminal α-helix of APOL1 (REF. 54) (FIG. 3a,d). In addition, point mutations that disrupt helix A of SRA were shown to result in loss of binding to APOL1 (REF. 54). APOL1 is associated with the HDL3 subfraction of human serum57. Trypanosomes internalize APOL1containing particles, and APOL1 is then routed to the lysosome, where it colocalizes with SRA54. Under conditions that dissociate serum lipoprotein complexes (that is, in the presence of detergent), separation of human serum by affinity chromatography on either APOL1-specific antibodies or SRA selectively removed APOL1, and the only other detectable components that were subsequently eluted with APOL1 were serum albumin and antibodies54. This chromatographic step, followed by dialysis to remove the detergent, resulted in total loss of the trypanolytic activity of the serum54. Applying the same protocol but using affinity chromatography on an SRA molecule with a disrupted helix A did not result in the removal of APOL1 or in the loss of trypanolytic activity, showing that the depletion of APOL1 (and not the treatment with detergent) was responsible for the loss of trypanolytic activity54. Furthermore, addition of purified native or recombinant APOL1 to the depleted serum or to fetal calf serum (which naturally lacks APOL1) restored or conferred, respectively,

the ability to lyse human-serum-sensitive trypanosomes but not human-serumresistant trypanosomes54. The resistance of trypanosomes to lysis was associated with the C-terminal helix of APOL1 (which interacts with helix A of SRA), because removal of this helix from APOL1 conferred the ability to lyse both humanserum-sensitive and human-serumresistant trypanosomes54. Finally, the natural phenotype of this system can be entirely reconstituted with recombinant proteins: recombinant-SRA-expressing T. b. brucei were found to be resistant to lysis following exposure to recombinant APOL1, whereas wild-type T. b. brucei were readily lysed by APOL1 (REF. 54). This finding indicates that APOL1 mimics the natural killing activity of human serum, and it also proves that SRA blocks the activity of APOL1 on trypanosomes. From these studies, it was concluded that APOL1 is the trypanolytic factor of human serum and that, in T. b. rhodesiense, SRA neutralizes APOL1 through coil-coiling interactions with the C-terminal helix of APOL1. Accordingly, APOL1 was found to be absent from chimpanzee serum, which is non-trypanolytic (as discussed earlier)6,8,9, and sequencing of the chimpanzee genome provided a straightforward explanation for this finding: the APOL1 gene is absent from these animals58. Resistance to APOL1 during the T. b. rhodesiense life cycle. In long, slender bloodstream forms of T. b. rhodesiense (FIG. 1), the expression of SRA allows neutralization of APOL1 in the lysosome54. This mechanism of resistance considerably differs from the previously proposed mechanism, the selective inhibition of endocytosis of the trypanolytic factor19. Recent data obtained using T. b. brucei transfected with an epitope-tagged version of SRA indicate that neutralization of the trypanolytic factor by SRA can occur in endosomes, before the lytic factor reaches the lysosome59. These findings are consistent with the observation that the interaction between SRA and APOL1 can occur under both neutral and acidic conditions (that is, between pH 7.5 and 5.0)54. In accordance with the apparent lability of SRA40,46,59, the distribution of SRA between the endosomes and the lysosome might depend on the relative level of endocytic-protease activity, as indicated by studies in which the endo-lysosomal protease trypanopain was inhibited (L.V. and E.P., unpublished observations). Therefore, it can be speculated that SRA is routed to the


lysosome through the endocytic pathway and that the interaction of SRA with APOL1 can occur at different steps of this pathway and is followed by the proteolytic cleavage of this complex. Degradation of APOL1, however, is not required for the trypanosome to be resistant to lysis, because the presence of both APOL1 and SRA in the lysosome (in contrast to APOL1 alone) has been shown to prevent lysis from occurring54. That is, interaction with SRA seems to be sufficient for inactivation of APOL1. In procyclic forms of T. b. rhodesiense (FIG. 1), which are present in the tsetse fly, R-ES is inactive, so SRA is not expressed. However, procyclic forms can resist lysis by human serum in vitro60, and this is probably a consequence of the low level of endocytosis by trypanosomes at this stage22. Metacyclic forms of T. b. rhodesiense (FIG. 1), which are transferred from the tsetse fly to the mammalian host, are the first to be confronted with mammalian blood. Because R-ES is probably inactive in these forms of the parasite3, it is not clear how they resist APOL1 before their transformation into bloodstream forms. In vitro, metacyclic forms of T. b. rhodesiense were found to be mostly sensitive to lysis by human serum61. In the same assay, metacyclic forms of T. b. brucei and T. b. gambiense were always sensitive and resistant, respectively, to lysis by human serum. Therefore, metacyclic forms of T. b. rhodesiense are not constitutively resistant to human serum. After inoculation by the tsetse fly, trypanosomes can remain for several days in tissue spaces or lymphatic vessels, so it is possible that some metacyclic forms escape exposure to APOL1 before they begin to express SRA. Proposed mechanism of APOL1-mediated trypanolysis. The physiological function of APOL1 in humans remains elusive, although there is indirect evidence that it is involved in lipid metabolism62, as well as evidence of a link between overexpression in the brain and schizophrenia63. For this reason, the way in which APOL1 kills trypanosomes was not apparent. A weak similarity was, however, found between the N-terminal region of APOL1 and the pore-forming domain of colicins, which are bacterial toxins that kill competing bacteria by forming ionic pores in the inner cell membrane. This finding led to experiments that uncovered the capacity of APOL1 to generate ionic pores in biological membranes, both in vivo and in vitro64. Briefly, the region of APOL1 that spans amino acids 60 to 235, which was predicted by computer modelling to form a structure


PERSPECTIVES A Recombinant APOL1

Normal human serum

B a

0h

1h

2h

3h

4h

c

b

5h

Flagellar pocket

Fixation artefacts Lysosome

Lysosome

Nucleus

1 µm

1 µm

1 µm

Figure 4 | Phenotype of trypanosome lysis by human serum or by recombinant apolipoprotein L1 (APOL1). A | Micrographs show the expansion of the lysosome of Trypanosoma brucei brucei over time in the presence of 1 µg per ml recombinant APOL1 or 10% normal human serum. Lower panels are reproduced with permission from REF. 64 © (2005) American Association for the Advancement of Science. B | Transmission electron micrographs show fixed T. b. brucei that either were not exposed to human serum (a) or were exposed to human serum for 1.5 hours (b,c). In control cells (a), the lysosome is small and dispersed, and cannot be seen clearly. In treated cells (b,c), the swollen lysosome is indicated. Fixation artefacts, which have led to the suggestion that the lysosomal membrane is disrupted during lysis16, are also shown (c). Quantitative measurements of lysosomal surface area and cytoplasmic surface area over time are shown in Supplementary information S1 (figure)). Both the swelling of the lysosome and the absence of general cellular swelling can be observed in Supplementary information S2 (movie).

that resembles the pore-forming domain of colicins (FIG. 3a,b), showed bactericidal activity with a marked similarity to that of colicins and generated anionic pores in synthetic lipid-bilayer membranes. This pore-forming domain of APOL1 was found to be adjacent to a pH-sensitive membraneaddressing domain, which was predicted to bind HDL particles at neutral pH only (that is, in the blood but not in the late endosomes or the lysosome)64 (FIG. 3a,c). Both domains were required for the toxic activity of APOL1 in vivo, against bacteria and against trypanosomes. By contrast, the C-terminal α-helix of APOL1 (which interacts with SRA and is required for resistance to lysis) was dispensable for toxic activity in both cases. In trypanosomes, recombinant APOL1 was found to mimic completely the lytic activity of normal human serum, causing

depolarization of the lysosomal membrane (that is, loss of the differential charge between the two faces of the membrane), followed by continuous swelling of the lysosome until the parasite cell lysed64 (FIG. 4). In addition, APOL1 was detected at the periphery of the lysosome but not in the lumen64. Given the predicted structure of its membraneaddressing domain (FIG. 3c), APOL1 is thought to undergo a conformational change that allows its dissociation from HDL particles at low pH (such as in the late endosomes and the lysosome) and its subsequent insertion into the lysosomal membrane, where it forms a pore. On exposure to recombinant APOL1, similar to normal human serum, lysosomal swelling was found to be the first detectable morphological change, and as this process continued, there were no marked alterations in other intracellular structures

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(FIG. 4; see Supplementary information S1,S2 (figure and movie)), in particular no general vacuolization and cellular swelling (although this is in contrast to findings reported in REF. 65; discussed later). Lysosomal swelling was found to be associated with the influx and intracellular accumulation of chloride ions (Cl–). Both of these processes were blocked by either depletion of extracellular Cl– or addition of the anionchannel blocker DIDS (4,4′-diisothiocyano2,2′-disulphonate stilbene)64. Although DIDS completely inhibited the influx of Cl– into trypanosomes and the subsequent lysis, it had no direct effect on the pore-forming activity of APOL1 in vitro, indicating that additional Cl– channels are involved in trypanolysis. So, it was concluded that APOL1 triggers the lysosomal influx of Cl– from the cytoplasm, where the concentration of this anion is high (106 mM)66. This movement would then reduce the cytoplasmic Cl– concentration, activating the compensatory entry of extracellular Cl– through DIDS-sensitive channels in the plasma membrane64. This model of the trypanolytic process is shown in FIG. 5. The cascade of Cl– movement would be accompanied by the influx of water into the lysosome and osmotic swelling of this compartment. This accounts for the observed inhibition of trypanolysis by osmotically active molecules such as sucrose (the presence of which in the cytosol reduces the influx of water into the lysosome)67. The internal pressure resulting from the continuous enlargement of the lysosome would be responsible for the lysis of the parasite, because this pressure is expected to irreversibly damage the plasma membrane of the parasite. This model also explains the observed fraying of the parasite surface coat and the leakage of ions that occur before lysis67. The finding that trypanosomes with considerably swollen lysosomes can survive for a period of time clearly conflicts with the idea that lysis involves disruption of the lysosomal membrane following lipid peroxidation, a mechanism that is still proposed by some researchers16,20,24. Indeed, the extreme swelling of the lysosome seems to be incompatible with a damaged lysosomal membrane. The main argument for lysosomal-membrane disruption is that discontinuities can be detected in the membrane, using electron microscopy16; however, these are probably the result of common fixation artefacts68 (FIG. 4Bc). A recent study proposed an alternative mechanism for trypanolysis: that lytic HDL particles generate cation-selective pores that are active at the plasma membrane after they have been recycled from the lysosome69.


PERSPECTIVES Given the cation concentration gradients at the plasma membrane (that is, extracellular to cytoplasmic concentration ratios of 140 mM/14 mM and 6 mM/116 mM for sodium ions (Na+) and potassium ions (K+), respectively 66), these pores would allow an influx of Na+ into the cytoplasm. However, in this study 69, the electrophysiological measurements were carried out on a crude population of lipoproteins that contained various pore-forming proteins and peptides, and this population generated complex ion currents, possibly owing to the formation of irrelevant (non-physiological) pores or the influx of ions through nonspecific channels. In addition, although pore-forming activity was monitored under conditions that mimic the interface between the lysosomal and cytoplasmic compartments, its involvement in trypanolysis was entirely attributed to ionic fluxes produced at the plasma membrane, and we think that this conclusion is questionable for two main reasons. First, it is unlikely that plasma-membrane fluxes are relevant to the trypanolytic process that is induced by normal human serum, because we have found that this process is characterized by swelling of the lysosome but not of the cytoplasm64 (FIG. 4; see Supplementary information S1, S2 (figure and movie)). This phenotype is in contrast to that observed using purified lytic HDL particles65, indicating that the lysis that occurs in the presence of purified lytic HDL particles is likely to involve nonspecific toxicity. Second, the specificity of the pores for particular cations was not measured in the study reported in REF. 69. However, if the pores conduct Na+ to a similar extent to which they seem to conduct K+, then (because the Na+ and K+ gradients across the plasma membrane are approximately equal in magnitude but opposite in direction66) the most likely initial outcome would be the electroneutral exchange of the two cations, which would have no osmotic consequences. Therefore, in our opinion, the hypothesis that the trypanolytic factor generates cationic pores is not proven by the data in REF. 69, and pore formation at the plasma membrane does not account for the phenotype of trypanolysis by normal human serum. Identification of the trypanolytic factor of human serum: APOL1 or HPR? Initially, HPR was considered to be the trypanolytic factor of human serum, and recent papers present evidence for why this protein, in addition to APOL1, is a trypanolytic factor9,69,70. This view is based, in part, on the observation that serum from baboons is

Blood pH ~7 HDL particle

DIDS Flagellum

APOL1

Lysosome Cl–

Flagellar pocket

Cl– and H2O

SRA pH 5.3 Swollen lysosome pH ~7 Late endosome or lysosome Plasma membrane Early endosome

Cytosol

Figure 5 | Model of the mechanism of trypanolysis by apolipoprotein L1 (APOL1). APOL1 contains a pore-forming domain (red) and a membrane-addressing domain (blue). It is associated with highdensity lipoprotein (HDL) particles (green) that are present in the blood. These particles are internalized by bloodstream-form trypanosomes through HDL-receptor-mediated endocytosis that is initiated in the flagellar pocket. Following internalization, HDL particles progress through the endocytic pathway, from early endosomes (which have neutral pH) to late endosomes (which are acidic) and then to the lysosome (which is also acidic). We think that most data indicate that trypanolysis occurs by the following process. The lysosomal pH induces a conformational change in the membrane-addressing domain of APOL1, leading to dissociation from the HDL particle and binding to the lysosomal membrane. The pore-forming domain of APOL1 enables it to form a pore in the membrane, and this leads to the flux of chloride ions (Cl–) from the cytoplasm to the lumen of the lysosome. Cytoplasmic Cl– deprivation is compensated by the activity of a DIDS (4,4′-diisothiocyano-2,2′-disulphonate stilbene)sensitive Cl– transporter in the plasma membrane. This ionic flux is presumably accompanied by secondary cationic movements64,67,69, and it triggers the movement of water (H2O) into the lysosome and osmotic swelling of this compartment. The resultant uncontrolled swelling of the lysosome increases intracellular pressure, probably leading to damage to the plasma membrane (dashed line), which is ultimately the cause of cell death. The Trypanosoma brucei rhodesiense protein serum-resistance associated (SRA) interacts with APOL1 in late endosomes and the lysosome, and this prevents APOL1 from forming pores, possibly through sequestration of APOL1 followed by proteolytic degradation.

trypanolytic and contains HPR but not APOL1 (REFS 8,9). However, serum from these primates kills both human-serumsensitive and human-serum-resistant T. b. rhodesiense clones, and it seems to have considerably more lytic activity than does human serum6,8,9. In our opinion, these observations indicate that the trypanolytic activity of baboon serum differs from the trypanolytic activity of human serum and that HPR cannot be the common trypanolytic factor in both cases, because HPR does not lyse human-serum-resistant T. b. rhodesiense. It is also argued that HPR is a trypanolytic factor because HPR-specific antibodies inhibit the activity of lytic HDL particles9,24,70. These experiments, however, involved lipoprotein complexes that were not dissociated with detergent. In addition, it has been reported that antibodies specific


for APOA1 (a protein that does not have trypanolytic activity28–30) inhibit lysis70. So, one possible explanation for the inhibitory activity of HPR-specific antibodies is that antibodies specific for any exposed constituent of HDL particles can inhibit lysis through a nonspecific mechanism, perhaps by aggregation of HDL particles or by interference in the endocytic process. In their recent study, Hajduk and colleagues70 propose that both HPR and APOL1 are trypanolytic but that these proteins need to be associated in the same HDL particle for maximal activity. This proposal is based on trypanolytic assays carried out using purified HPR and APOL1 or using human HDL particles fractionated into particles that contain both HPR and APOL1, either protein alone or neither protein. In the first case (using purified proteins), toxicity was


PERSPECTIVES Box 2 | The function of apolipoproteins (APOLs) In evolutionary terms, APOL1 appeared recently, as it has been identified only in humans and gorillas8,75. It belongs to a multigene family that has six members76,77, and homologous sequences are present in other eukaryotes (particularly in other mammals). Among APOL1-like proteins, the sequence of the pore-forming domain is similar, whereas that of the membrane-addressing domain is more variable; the C-terminal helix is the most conserved region (see Supplementary information S3 (figure)). Unlike other APOL1-family members, APOL1 is secreted (probably because of the presence of a signal peptide), but sequestration in high-density lipoprotein (HDL) particles presumably neutralizes its activity in serum. Other APOL-family members are most probably intracellular proteins. Given the pore-forming activity of APOL1 (REF. 64), intracellular APOLs could be ion channels in organelles, such as the lysosome, and might thereby control the volume of these organelles. The potential of the C-terminal helix of APOL1 to interact with the Trypanosoma brucei rhodesiense protein serum -resistance associated (SRA) and thereby neutralize APOL1 activity, together with the evidence that this helix is dispensable for trypanolytic activity despite its high level of sequence conservation54,64, raises the interesting possibility that this region controls the ionic-pore-forming activity of APOLs through interaction with proteins that have SRA-like helices. It is tempting to propose that the generation of a secreted version of these pore-forming proteins in the great apes of Africa was linked to the presence of trypanosomes in this region and that it was selected because of its trypanolytic activity. However, the conservation of APOL1 in humans (despite the wide geographical dispersion of humans) does not support this idea but indicates that APOL1 fulfils additional functions. In this regard, it is interesting that the expression of APOL1, APOL2 and APOL3 is strongly induced by the pro-inflammatory cytokine tumour-necrosis factor75,78 and that overexpression of APOL6 triggers apoptosis, presumably through a BH3 domain (B-cell lymphoma 2 (BCL-2)-homology domain 3)79. So, APOLs might be involved in various cellular responses to danger signals.

Pore-forming proteins and cell death The ability of pore-forming proteins to trigger cell death can be illustrated by several well-known examples — bacterial colicins, diphtheria toxin and apoptotic proteins from the BCL-2 family — although different mechanisms are involved in the cell death that is mediated by these three types of protein (reviewed in REF. 80). Despite a low level of sequence homology, these proteins all have poreforming domains with a similar structure: that is, several α-helices organized around a conserved hydrophobic hairpin. Computer modelling and experimental data indicate that the pore-forming domain of APOL1 also belongs to this category64 (FIG. 3). As is the case for BCL-2-family members, the pore-forming domain of APOL1 might have been inherited from bacteria. HDL particles and innate immunity The association of APOL1 with HDL particles is likely to occur through hydrophobic interactions that are mediated by the membrane-addressing domain at neutral pH64 (FIG. 3). The presence of pore-forming microbicidal components in HDL particles is not an unprecedented finding. Various APOLs that are associated with HDL particles can show bactericidal activity81. In addition, defensins and cathelicidins, which are pore-forming antibacterial peptides, also seem to be associated with lipoproteins in serum82. The anchoring of microbicidal proteins in lipoprotein particles would ensure the sequestration of these proteins when their activity is not required, thereby preventing cytotoxic effects while maintaining high serum concentrations. Therefore, lipoproteins could have a role as carriers for components of the innate immune system21,70.

detected following direct incubation of trypanosomes with high concentrations of either protein. However, we argue that these data cannot be fully interpreted without information relating to the phenotype of lysis. For example, when the antimicrobial peptides cathelicidins are purified from lipoprotein complexes, they can kill trypanosomes in vitro through disruption of the plasma membrane71; however, there is no evidence that these cathelicidins are active against trypanosomes in vivo. In the second case (using fractionated HDL particles), almost 100% of the trypanolytic activity was recovered in the HDL-particle fraction that contained both HPR and APOL1, whereas less than 0.4% of the trypanolytic activity was detected in fractions that contained

either APOL1 or HPR. We think that the small trypanolytic effect of these HDL particles that contain undefined amounts of either HPR or APOL1 could be a result of nonspecific toxicity conferred by the experimental treatment. Indeed, fractionation does seem to confer non-physiological toxicity, because the phenotype of trypanolysis by fractionated HDLs differed from that of human serum and involved generalized vacuolization and cellular swelling65. Furthermore, fractionation resulted in a higher trypanolytic activity than that naturally present in serum31,33, and it rendered chimpanzee HDL particles trypanolytic9. One possibility is that HDLparticle fractionation activates microbicidal peptides, including cathelicidins71. A definitive assessment of this idea could be made by

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determining the effect of fractionated HDL particles on trypanosomes that are naturally resistant to human serum. In summary, we outline the following five arguments in support of our contention that APOL1 is the only trypanolytic factor of human serum. First, the addition of recombinant APOL1 to fetal calf serum, which lacks HPR, rendered the serum trypanolytic, and the phenotype of lysis was identical to that observed for human serum54,64. It should be noted that free APOL1 (that is, APOL1 not associated with HDL particles), whether native or recombinant protein, seems to have less trypanolytic activity than human serum64,70, and this finding could be interpreted in two ways. The reduced activity of free APOL1 might result from a lack of synergy with HPR69,70: that is, both APOL1 and HPR are required for maximal trypanolytic activity. Alternatively, it is our contention that the reduced activity of free APOL1 reflects a requirement for APOL1 to be associated with HDL particles for efficient binding and uptake by the HDL receptor of the parasite11,23. Indeed, kinetic studies indicated that free recombinant APOL1 seemed to be as effective as human serum at lysis of trypanosomes, except that lysis was delayed64. Accordingly, trypanolysis by recombinant APOL1 was strongly accelerated on reconstitution of APOL1 in lipoproteins54,64. Therefore, we think that the activity of APOL1 does not require synergy with HPR. APOL1-transgenic mice would be useful for evaluating the in vivo efficiency of trypanosome killing by APOL1 alone, but such mice are not available at present. Injection of recombinant APOL1 into wildtype mice, however, was shown to be necessary and sufficient for complete inhibition of infection with T. b. brucei 72. It remains to be determined whether similar experiments with HPR-transgenic mice would show increased parasite killing by APOL1. Second, the presence of HPR in APOL1free serum did not trigger trypanolysis and did not affect trypanosome growth. This was observed in vivo for transgenic mice that expressed HPR at a level similar to that of humans73, as well as for human serum that was specifically depleted of APOL1 (through separation of the proteins present in detergent-dissociated HDL particles by affinity chromatography on SRA)54,64. Third, depletion of APOL1 from human serum without removal of HPR (as above) led to a complete loss of trypanolytic activity54,64. The converse experiment — selective depletion of HPR without removal of APOL1— has not yet been carried out.


PERSPECTIVES Fourth, trypanolysis did not occur when recombinant APOL1 that was mutated in either the pore-forming domain or the membrane-addressing domain was added to serum specifically depleted of APOL1, even if HPR was present64. Conversely, recombinant APOL1 that lacked the C-terminal (SRA-interacting) helix was necessary and sufficient to kill T. b. rhodesiense independent of HPR54,72. Fifth, expression of SRA alone conferred complete resistance to either human serum or recombinant APOL1. This finding can be explained by the interaction between SRA and APOL1 (REF. 54), and there is no evidence that SRA could interfere with HPR. For example, no trace of HPR has been found in the APOL1-containing serum fraction that binds SRA affinity columns in the presence of detergent (discussed earlier)54. We propose, therefore, that APOL1 is responsible for most, if not all, of the trypanolytic activity that is associated with human serum. In our opinion, the idea that APOL1 operates synergistically with HPR for maximal trypanolytic activity in the context of a subclass of HDL particles that contains APOL1, HPR and APOA1 is not supported by the available evidence. Conclusions and future directions Despite considerable progress, many questions that relate to the trypanolytic activity of human serum remain to be answered. In addition, the physiological function of APOL1 and other APOLs remains unclear (BOX 2; see Supplementary information S3 (figure)). At present, the most important points that require further investigation are the identity of the trypanosome receptor for the trypanolytic HDL particles, the basis of resistance of T. b. gambiense to APOL1 and the putative involvement of HPR in the mechanism of trypanosome killing by APOL1. To resolve the last issue, a crucial experiment will be to compare the trypanosome-infection characteristics of mice that are transgenic for HPR, APOL1 or both genes. So far, however, attempts to generate APOL1-transgenic mice have not been successful. Recent advances in this field of research include a breakthrough in the diagnosis of sleeping sickness, for which the presence of SRA has proved to be a reliable marker of infection with T. b. rhodesiense47–51, and the use of APOL1 as a treatment that might cure the disease. As discussed in this article, when APOL1 is not bound to HDL particles, it kills trypanosomes but with delayed kinetics compared with human serum64. So, to convert APOL1 into a drug that is effective against

trypanosomes that infect humans, two modifications have been carried out. First, the C-terminal region, which is recognized by SRA, has been removed. Second, this truncated APOL1 molecule has been fused to an antibody module (known as a nanobody) that is derived from single-chain camel antibodies, thereby targeting the toxin (APOL1) to invariant determinants (high-mannose side chains) of the trypanosome surface74. The efficacy of trypanolysis by this protein construct was shown in mice: injection of the modified APOL1 resulted in inhibition of parasitaemia caused by either T. b. brucei or T. b. rhodesiense72. APOL1 might also be useful for solving the problem of nagana, a lethal disease that is caused by infection of cattle with T. b. brucei. Transgenic cattle expressing APOL1 derivatives such as the truncated-APOL1–nanobody conjugate would be expected to resist infection by T. b. brucei and T. b. rhodesiense, because this truncated trypanolytic factor cannot be neutralized by the T. b. rhodesiense ‘antidote’ SRA. As well as allowing healthy animal production in areas in which T. b. rhodesiense is endemic, the use of such ‘trypanolytic cattle’ should lead to a marked reduction in the main reservoir of T. b. rhodesiense, thereby contributing to the prevention of epidemics of sleeping sickness. Etienne Pays, Benoit Vanhollebeke, Luc Vanhamme, Françoise Paturiaux-Hanocq and David Pérez-Morga are at the Laboratory of Molecular Parasitology, Institute of Molecular Biology and Medicine (IBMM), Université Libre de Bruxelles, 12 rue des Professeurs Jeener et Brachet, B-6041 Gosselies, Belgium.

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Derek P. Nolan is at the Department of Biochemistry, Trinity College, Dublin 2, Ireland.

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Correspondence to E.P. e-mail: [email protected]

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Acknowledgements This paper is dedicated to the memory of M. Steinert. This work was supported by the Fonds National de la Recherche Scientifique (FNRS), the United Nations Children’s Fund, United Nations Development Programme, World Bank, and World Health Organization Special Programme for Research and Training in Tropical Diseases (TDR), and the Interuniversity Attraction Poles Programme (Belgian Science Policy).


DATABASES The following terms in this article are linked online to: Entrez Genome Project: http://www.ncbi.nlm.nih.gov/ entrez/query.fcgi?db=genomeprj T. brucei UniProtKB: http://ca.expasy.org/sprot APOA1 | APOL1 | HPR

FURTHER INFORMATION Etienne Pays’s laboratory: http://www.ulb.ac.be/ibmm/homeuk_13.html

SUPPLEMENTARY INFORMATION See online article: S1 (figure) | S2 (movie) | S3 (figure) Access to this links box is available online.


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