Legionella pneumophila Strain 130b Possesses a Unique Combination of Type IV Secretion Systems and Novel Dot/Icm Secretion System Effector Proteins

ABSTRACT Legionella pneumophila is a ubiquitous inhabitant of environmental water reservoirs. The bacteria infect a wide variety of protozoa and, after accidental inhalation, human alveolar macrophages, which can lead to severe pneumonia. The capability to thrive in phagocytic hosts is dependent on the Dot/Icm type IV secretion system (T4SS), which translocates multiple effector proteins into the host cell. In this study, we determined the draft genome sequence of L. pneumophila strain 130b (Wadsworth). We found that the 130b genome encodes a unique set of T4SSs, namely, the Dot/Icm T4SS, a Trb-1-like T4SS, and two Lvh T4SS gene clusters. Sequence analysis substantiated that a core set of 107 Dot/Icm T4SS effectors was conserved among the sequenced L. pneumophila strains Philadelphia-1, Lens, Paris, Corby, Alcoy, and 130b. We also identified new effector candidates and validated the translocation of 10 novel Dot/Icm T4SS effectors that are not present in L. pneumophila strain Philadelphia-1. We examined the prevalence of the new effector genes among 87 environmental and clinical L. pneumophila isolates. Five of the new effectors were identified in 34 to 62% of the isolates, while less than 15% of the strains tested positive for the other five genes. Collectively, our data show that the core set of conserved Dot/Icm T4SS effector proteins is supplemented by a variable repertoire of accessory effectors that may partly account for differences in the virulences and prevalences of particular L. pneumophila strains.

[1]  J. Graham,et al.  Role for the Ankyrin eukaryotic-like genes of Legionella pneumophila in parasitism of protozoan hosts and human macrophages. , 2008, Environmental microbiology.

[2]  W. Dowdle,et al.  Legionnaires' disease: isolation of a bacterium and demonstration of its role in other respiratory disease. , 1977, The New England journal of medicine.

[3]  M. Barbacid,et al.  Genetic analysis of Ras signalling pathways in cell proliferation, migration and survival , 2010, The EMBO journal.

[4]  F. Maxfield,et al.  Legionella pneumophila inhibits acidification of its phagosome in human monocytes , 1984, The Journal of cell biology.

[5]  C. Pericone,et al.  Evidence for Acquisition of Legionella Type IV Secretion Substrates via Interdomain Horizontal Gene Transfer , 2005, Journal of bacteriology.

[6]  M. Swanson,et al.  Association of Legionella pneumophila with the macrophage endoplasmic reticulum , 1995, Infection and immunity.

[7]  R. ffrench-Constant,et al.  New plasmids and putative virulence factors from the draft genome of an Australian clinical isolate of Photorhabdus asymbiotica. , 2010, FEMS microbiology letters.

[8]  Matthew Berriman,et al.  ACT: the Artemis comparison tool , 2005, Bioinform..

[9]  Lisa N Kinch,et al.  AMPylation of Rho GTPases by Vibrio VopS Disrupts Effector Binding and Downstream Signaling , 2009, Science.

[10]  C. Roy,et al.  Legionella phagosomes intercept vesicular traffic from endoplasmic reticulum exit sites , 2002, Nature Cell Biology.

[11]  D. Schneider,et al.  Growth-phase-dependent mobility of the lvh-encoding region in Legionella pneumophila strain Paris. , 2006, Microbiology.

[12]  N. Grishin,et al.  Fido, a Novel AMPylation Domain Common to Fic, Doc, and AvrB , 2009, PloS one.

[13]  P. Edelstein.  Control of Legionella in hospitals. , 1986, The Journal of hospital infection.

[14]  D. Philpott,et al.  Nod-like proteins in immunity, inflammation and disease , 2006, Nature Immunology.

[15]  M. Yarmolinsky,et al.  Plasmid addiction genes of bacteriophage P1: doc, which causes cell death on curing of prophage, and phd, which prevents host death when prophage is retained. , 1993, Journal of molecular biology.

[16]  Zhao-Qing Luo,et al.  Multiple substrates of the Legionella pneumophila Dot/Icm system identified by interbacterial protein transfer. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[17]  M. Frace,et al.  Virulence Factors Encoded by Legionella longbeachae Identified on the Basis of the Genome Sequence Analysis of Clinical Isolate D-4968 , 2009, Journal of bacteriology.

[18]  N. Cianciotto,et al.  A bacterial ecto‐triphosphate diphosphohydrolase similar to human CD39 is essential for intracellular multiplication of Legionella pneumophila , 2007, Cellular microbiology.

[19]  B. Finlay,et al.  Type III Secretion Systems and Disease , 2007, Clinical Microbiology Reviews.

[20]  C. Buchrieser,et al.  Analysis of the Legionella longbeachae Genome and Transcriptome Uncovers Unique Strategies to Cause Legionnaires' Disease , 2010, PLoS genetics.

[21]  O. Gascuel,et al.  A simple, fast, and accurate algorithm to estimate large phylogenies by maximum likelihood. , 2003, Systematic biology.

[22]  C. Roy,et al.  A yeast genetic system for the identification and characterization of substrate proteins transferred into host cells by the Legionella pneumophila Dot/Icm system , 2005, Molecular microbiology.

[23]  Gilbert GREUB,et al.  Amoebal pathogens as emerging causal agents of pneumonia. , 2010, FEMS microbiology reviews.

[24]  S. Zink,et al.  Comparative assessment of virulence traits in Legionella spp. , 2003, Microbiology.

[25]  C. Buchrieser,et al.  Multigenome analysis identifies a worldwide distributed epidemic Legionella pneumophila clone that emerged within a highly diverse species. , 2008, Genome research.

[26]  H. Hilbi,et al.  Legionella pneumophila Exploits PI(4)P to Anchor Secreted Effector Proteins to the Replicative Vacuole , 2006, PLoS pathogens.

[27]  Rodrigo Lopez,et al.  Clustal W and Clustal X version 2.0 , 2007, Bioinform..

[28]  N. Cianciotto,et al.  Type II Protein Secretion Is a Subset of the PilD-Dependent Processes That Facilitate Intracellular Infection byLegionella pneumophila , 2001, Infection and Immunity.

[29]  Hiroki Nagai,et al.  Legionella translocates an E3 ubiquitin ligase that has multiple U‐boxes with distinct functions , 2008, Molecular microbiology.

[30]  Matthew Berriman,et al.  DNAPlotter: circular and linear interactive genome visualization , 2008, Bioinform..

[31]  M. Struelens,et al.  Pan-European Study on Culture-Proven Legionnaires' Disease: Distribution of Legionella pneumophila Serogroups and Monoclonal Subgroups , 2002, European Journal of Clinical Microbiology and Infectious Diseases.

[32]  E. Cascales,et al.  Biogenesis, architecture, and function of bacterial type IV secretion systems. , 2005, Annual review of microbiology.

[33]  J. Russo,et al.  Comparative sequence analysis of the icm/dot genes in Legionella. , 2004, Plasmid.

[34]  R. Isberg,et al.  Conjugative transfer by the virulence system of Legionella pneumophila. , 1998, Science.

[35]  G. Waksman,et al.  The structural biology of type IV secretion systems , 2009, Nature Reviews Microbiology.

[36]  S. Emr,et al.  Pathogen effector protein screening in yeast identifies Legionella factors that interfere with membrane trafficking. , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[37]  Shuqing Liu,et al.  Environmental Mimics and the Lvh Type IVA Secretion System Contribute to Virulence-Related Phenotypes of Legionella pneumophila , 2006, Infection and Immunity.

[38]  C. Buchrieser,et al.  Legionella pneumophila: population genetics, phylogeny and genomics. , 2009, Infection, genetics and evolution : journal of molecular epidemiology and evolutionary genetics in infectious diseases.

[39]  C. Worby,et al.  The fic domain: regulation of cell signaling by adenylylation. , 2009, Molecular cell.

[40]  M. Endrizzi,et al.  Naip5 Affects Host Susceptibility to the Intracellular Pathogen Legionella pneumophila , 2003, Current Biology.

[41]  C. Buchrieser,et al.  Adaptation of Legionella pneumophila to the host environment: role of protein secretion, effectors and eukaryotic-like proteins. , 2006, Current opinion in microbiology.

[42]  Derrick W. Crook,et al.  Transferable Antibiotic Resistance Elements in Haemophilus influenzae Share a Common Evolutionary Origin with a Diverse Family of Syntenic Genomic Islands , 2004, Journal of bacteriology.

[43]  S. Mukherjee,et al.  Bacterial FIC Proteins AMP Up Infection , 2009, Science Signaling.

[44]  R. Utsumi,et al.  Functional analysis of the fic gene involved in regulation of cell division. , 1991, Research in microbiology.

[45]  Michael Wagner,et al.  Amoebae as Training Grounds for Intracellular Bacterial Pathogens , 2005, Applied and Environmental Microbiology.

[46]  J. V. Lee,et al.  Distribution of Legionella pneumophila serogroups, monoclonal antibody subgroups and DNA sequence types in recent clinical and environmental isolates from England and Wales (2000–2008) , 2009, European Journal of Clinical Microbiology & Infectious Diseases.

[47]  D. Ferguson,et al.  Novel Type IV Secretion System Involved in Propagation of Genomic Islands , 2006, Journal of bacteriology.

[48]  Peer Bork,et al.  SMART 6: recent updates and new developments , 2008, Nucleic Acids Res..

[49]  Mario Juhas,et al.  Type IV secretion systems: tools of bacterial horizontal gene transfer and virulence , 2008, Cellular microbiology.

[50]  Narmada Thanki,et al.  CDD: specific functional annotation with the Conserved Domain Database , 2008, Nucleic Acids Res..

[51]  M. Heidtman,et al.  The Legionella pneumophila replication vacuole: making a cosy niche inside host cells , 2009, Nature Reviews Microbiology.

[52]  J. Russo,et al.  Relationships between a new type IV secretion system and the icm/dot virulence system of Legionella pneumophila , 1999, Molecular microbiology.

[53]  J. Cirillo,et al.  Identification of novel loci involved in entry by Legionella pneumophila. , 2000, Microbiology.

[54]  P. Green,et al.  Consed: a graphical tool for sequence finishing. , 1998, Genome research.

[55]  I. V. van Driel,et al.  Molecular Pathogenesis of Infections Caused by Legionella pneumophila , 2010, Clinical Microbiology Reviews.

[56]  C. Buchrieser,et al.  Legionella pathogenicity: genome structure, regulatory networks and the host cell response. , 2007, International journal of medical microbiology : IJMM.

[57]  P. Gros,et al.  Birc1e is the gene within the Lgn1 locus associated with resistance to Legionella pneumophila , 2003, Nature Genetics.

[58]  K. Heuner,et al.  Identification and characterization of a new conjugation/type IVA secretion system (trb/tra) of Legionella pneumophila Corby localized on two mobile genomic islands. , 2008, International journal of medical microbiology : IJMM.

[59]  H. Shuman,et al.  Host cell killing and bacterial conjugation require overlapping sets of genes within a 22-kb region of the Legionella pneumophila genome. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[60]  C. D. Price,et al.  Genetic Susceptibility and Caspase Activation in Mouse and Human Macrophages Are Distinct for Legionella longbeachae and L. pneumophila , 2007, Infection and Immunity.

[61]  T. Rowbotham,et al.  Preliminary report on the pathogenicity of Legionella pneumophila for freshwater and soil amoebae. , 1980, Journal of clinical pathology.

[62]  E. D. Cambronne,et al.  Recognition and Delivery of Effector Proteins into Eukaryotic Cells by Bacterial Secretion Systems , 2006, Traffic.

[63]  L. Holm,et al.  The Pfam protein families database , 2005, Nucleic Acids Res..

[64]  Tal Pupko,et al.  Genome-Scale Identification of Legionella pneumophila Effectors Using a Machine Learning Approach , 2009, PLoS pathogens.

[65]  David Posada,et al.  MODELTEST: testing the model of DNA substitution , 1998, Bioinform..

[66]  M. Jules,et al.  Legionella pneumophila adaptation to intracellular life and the host response: Clues from genomics and transcriptomics , 2007, FEBS letters.

[67]  J. Eisen,et al.  A simple, fast, and accurate method of phylogenomic inference , 2008, Genome Biology.

[68]  C. Bryant,et al.  Molecular mechanisms involved in inflammasome activation. , 2009, Trends in cell biology.

[69]  Andrés Moya,et al.  Legionella pneumophila pangenome reveals strain-specific virulence factors , 2010, BMC Genomics.

[70]  C. Buchrieser,et al.  Evidence in the Legionella pneumophila genome for exploitation of host cell functions and high genome plasticity , 2004, Nature Genetics.

[71]  N. Engleberg,et al.  Cloning and expression of Legionella pneumophila antigens in Escherichia coli , 1984, Infection and immunity.

[72]  M. Barbacid,et al.  RAS oncogenes: the first 30 years , 2003, Nature Reviews Cancer.

[73]  J. Celli,et al.  A Legionella pneumophila Effector Protein Encoded in a Region of Genomic Plasticity Binds to Dot/Icm-Modified Vacuoles , 2009, PLoS pathogens.

[74]  Thomas F Meyer,et al.  Type IV secretion systems and their effectors in bacterial pathogenesis. , 2006, Current opinion in microbiology.

[75]  I. Chou,et al.  The Genomic Sequence of the Accidental Pathogen Legionella pneumophila , 2004, Science.

[76]  L. Bermudez,et al.  Genetic and Phenotypic Differences between Legionella pneumophila Strains , 2002, Journal of Clinical Microbiology.

[77]  Dennis A. Ridenour,et al.  Identification of a Gene That Affects the Efficiency of Host Cell Infection by Legionella pneumophila in a Temperature-Dependent Fashion , 2003, Infection and Immunity.

[78]  Kim Rutherford,et al.  Artemis: sequence visualization and annotation , 2000, Bioinform..

[79]  X. Charpentier,et al.  The perplexing functions and surprising origins of Legionella pneumophila type IV secretion effectors , 2009, Cellular microbiology.

[80]  G F Mallison,et al.  Legionnaires' disease: description of an epidemic of pneumonia. , 1977, The New England journal of medicine.

[81]  M. Horwitz.  Formation of a novel phagosome by the Legionnaires' disease bacterium (Legionella pneumophila) in human monocytes , 1983, The Journal of experimental medicine.

[82]  Y. Kwaik,et al.  Molecular Mimicry by an F-Box Effector of Legionella pneumophila Hijacks a Conserved Polyubiquitination Machinery within Macrophages and Protozoa , 2009, PLoS pathogens.

[83]  N. Fry,et al.  Addition of neuA, the Gene Encoding N-Acylneuraminate Cytidylyl Transferase, Increases the Discriminatory Ability of the Consensus Sequence-Based Scheme for Typing Legionella pneumophila Serogroup 1 Strains , 2007, Journal of Clinical Microbiology.

[84]  S. Pukatzki,et al.  The type VI secretion system: translocation of effectors and effector-domains. , 2009, Current opinion in microbiology.

[85]  V. Bennett-Wood,et al.  Identification of Legionella pneumophila-Specific Genes by Genomic Subtractive Hybridization with Legionella micdadei and Identification of lpnE , a Gene Required for Efficient Host Cell Entry † , 2005 .

[86]  P. Christie,et al.  Biological Diversity of Prokaryotic Type IV Secretion Systems , 2009, Microbiology and Molecular Biology Reviews.

[87]  N. Cianciotto.  Many substrates and functions of type II secretion: lessons learned from Legionella pneumophila. , 2009, Future microbiology.

[88]  Georgios S. Vernikos,et al.  Comparative genome analysis of Salmonella Enteritidis PT4 and Salmonella Gallinarum 287/91 provides insights into evolutionary and host adaptation pathways. , 2008, Genome research.

[89]  C. Roy,et al.  Ankyrin Repeat Proteins Comprise a Diverse Family of Bacterial Type IV Effectors , 2008, Science.

[90]  T. Zusman,et al.  Identification of a Hypervariable Region Containing New Legionella pneumophila Icm/Dot Translocated Substrates by Using the Conserved icmQ Regulatory Signature , 2008, Infection and Immunity.

[91]  H. Hilbi,et al.  Icm/Dot‐dependent upregulation of phagocytosis by Legionella pneumophila , 2001, Molecular microbiology.

[92]  H. Hilbi,et al.  Environmental predators as models for bacterial pathogenesis. , 2007, Environmental microbiology.

[93]  E. Myers,et al.  Basic local alignment search tool. , 1990, Journal of molecular biology.

[94]  R. Isberg,et al.  Legionella pneumophila Dot/Icm translocated substrates: a sum of parts. , 2009, Current opinion in microbiology.

[95]  A. Weiss,et al.  Chemical Genetics Reveals Bacterial and Host Cell Functions Critical for Type IV Effector Translocation by Legionella pneumophila , 2009, PLoS pathogens.

[96]  O. Anderson,et al.  Legionella Eukaryotic-Like Type IV Substrates Interfere with Organelle Trafficking , 2008, PLoS pathogens.

[97]  K. Heuner,et al.  Description of a putative type I secretion system in Legionella pneumophila. , 2003, International journal of medical microbiology : IJMM.

[98]  M. Jules,et al.  The Legionella pneumophila F‐box protein Lpp2082 (AnkB) modulates ubiquitination of the host protein parvin B and promotes intracellular replication , 2010, Cellular microbiology.