Life Stage-specific Proteomes of Legionella pneumophila Reveal a Highly Differential Abundance of Virulence-associated Dot/Icm effectors
2015; Elsevier BV; Volume: 15; Issue: 1 Linguagem: Inglês
10.1074/mcp.m115.053579
ISSN1535-9484
AutoresPhilipp Auraß, Thomas Gerlach, Dörte Becher, Birgit Voigt, Susanne Karste, Jörg Bernhardt, Katharina Riedel, Michael Hecker, Antje Flieger,
Tópico(s)Vibrio bacteria research studies
ResumoMajor differences in the transcriptional program underlying the phenotypic switch between exponential and post-exponential growth of Legionella pneumophila were formerly described characterizing important alterations in infection capacity. Additionally, a third state is known where the bacteria transform in a viable but nonculturable state under stress, such as starvation. We here describe phase-related proteomic changes in exponential phase (E), postexponential phase (PE) bacteria, and unculturable microcosms (UNC) containing viable but nonculturable state cells, and identify phase-specific proteins. We present data on different bacterial subproteomes of E and PE, such as soluble whole cell proteins, outer membrane-associated proteins, and extracellular proteins. In total, 1368 different proteins were identified, 922 were quantified and 397 showed differential abundance in E/PE. The quantified subproteomes of soluble whole cell proteins, outer membrane-associated proteins, and extracellular proteins; 841, 55, and 77 proteins, respectively, were visualized in Voronoi treemaps. 95 proteins were quantified exclusively in E, such as cell division proteins MreC, FtsN, FtsA, and ZipA; 33 exclusively in PE, such as motility-related proteins of flagellum biogenesis FlgE, FlgK, and FliA; and 9 exclusively in unculturable microcosms soluble whole cell proteins, such as hypothetical, as well as transport/binding-, and metabolism-related proteins. A high frequency of differentially abundant or phase-exclusive proteins was observed among the 91 quantified effectors of the major virulence-associated protein secretion system Dot/Icm (> 60%). 24 were E-exclusive, such as LepA/B, YlfA, MavG, Lpg2271, and 13 were PE-exclusive, such as RalF, VipD, Lem10. The growth phase-related specific abundance of a subset of Dot/Icm virulence effectors was confirmed by means of Western blotting. We therefore conclude that many effectors are predominantly abundant at either E or PE which suggests their phase specific function. The distinct temporal or spatial presence of such proteins might have important implications for functional assignments in the future or for use as life-stage specific markers for pathogen analysis. Major differences in the transcriptional program underlying the phenotypic switch between exponential and post-exponential growth of Legionella pneumophila were formerly described characterizing important alterations in infection capacity. Additionally, a third state is known where the bacteria transform in a viable but nonculturable state under stress, such as starvation. We here describe phase-related proteomic changes in exponential phase (E), postexponential phase (PE) bacteria, and unculturable microcosms (UNC) containing viable but nonculturable state cells, and identify phase-specific proteins. We present data on different bacterial subproteomes of E and PE, such as soluble whole cell proteins, outer membrane-associated proteins, and extracellular proteins. In total, 1368 different proteins were identified, 922 were quantified and 397 showed differential abundance in E/PE. The quantified subproteomes of soluble whole cell proteins, outer membrane-associated proteins, and extracellular proteins; 841, 55, and 77 proteins, respectively, were visualized in Voronoi treemaps. 95 proteins were quantified exclusively in E, such as cell division proteins MreC, FtsN, FtsA, and ZipA; 33 exclusively in PE, such as motility-related proteins of flagellum biogenesis FlgE, FlgK, and FliA; and 9 exclusively in unculturable microcosms soluble whole cell proteins, such as hypothetical, as well as transport/binding-, and metabolism-related proteins. A high frequency of differentially abundant or phase-exclusive proteins was observed among the 91 quantified effectors of the major virulence-associated protein secretion system Dot/Icm (> 60%). 24 were E-exclusive, such as LepA/B, YlfA, MavG, Lpg2271, and 13 were PE-exclusive, such as RalF, VipD, Lem10. The growth phase-related specific abundance of a subset of Dot/Icm virulence effectors was confirmed by means of Western blotting. We therefore conclude that many effectors are predominantly abundant at either E or PE which suggests their phase specific function. The distinct temporal or spatial presence of such proteins might have important implications for functional assignments in the future or for use as life-stage specific markers for pathogen analysis. Legionella pneumophila is a Gram-negative bacterium ubiquitously found in water and soil. In their natural environment, the bacteria infect, counteract host defense mechanisms, and intracellularly replicate in protozoa, especially amoebae. When inhaled by humans, L. pneumophila uses similar strategies to propagate in alveolar macrophages leading to Legionnaires disease, a severe pneumonia (1.Fraser D.W. Tsai T.R. Orenstein W. Parkin W.E. Beecham H.J. Sharrar R.G. Harris J. Mallison G.F. Martin S.M. McDade J.E. Shepard C.C. Brachman P.S. Legionnaires' disease: description of an epidemic of pneumonia.N. Engl. J. Med. 1977; 297: 1189-1197Crossref PubMed Google Scholar, 2.McDade J.E. Shepard C.C. Fraser D.W. Tsai T.R. Redus M.A. Dowdle W.R. Legionnaires' disease: isolation of a bacterium and demonstration of its role in other respiratory disease.N. Engl. J. Med. 1977; 297: 1197-1203Crossref PubMed Google Scholar, 3.Horwitz M.A. Silverstein S.C. Legionnaires' disease bacterium (Legionella pneumophila) multiples intracellularly in human monocytes.J. Clin. Invest. 1980; 66: 441-450Crossref PubMed Google Scholar, 4.Rowbotham T.J. Preliminary report on the pathogenicity of Legionella pneumophila for freshwater and soil amoebae.J. Clin. Pathol. 1980; 33: 1179-1183Crossref PubMed Scopus (678) Google Scholar). Upon infection of amoebae and human cells, the intracellular life cycle of L. pneumophila includes at least two distinct stages, a replicative phase, where the bacteria efficiently proliferate, and a transmissive phase, where bacterial replication halts as nutrients become scarce. During transmissive phase, the expression of transmission traits, such as motility, cytotoxicity, accumulation of the storage lipid polyhydroxybutyrate, increased osmotic robustness, production of virulence traits, is triggered and the bacteria are highly infectious (5.Byrne B. Swanson M.S. Expression of Legionella pneumophila virulence traits in response to growth conditions.Infect. Immun. 1998; 66: 3029-3034Crossref PubMed Google Scholar, 6.James B.W. Mauchline W.S. Dennis P.J. Keevil C.W. Wait R. Poly-3-hydroxybutyrate in Legionella pneumophila, an energy source for survival in low-nutrient environments.Appl. Environ. Microbiol. 1999; 65: 822-827Crossref PubMed Google Scholar, 7.Cirillo J.D. Falkow S. Tompkins L.S. Growth of Legionella pneumophila in Acanthamoeba castellanii enhances invasion.Infect. Immun. 1994; 62: 3254-3261Crossref PubMed Google Scholar). The phenotypic switch is regulated in a complex fashion and involves the action of various regulatory proteins, such as CsrA, RpoS, LetA/S, FliA, RelA, and the small ncRNAs RsmY/Z (8.Molofsky A.B. Swanson M.S. Differentiate to thrive: lessons from the Legionella pneumophila life cycle.Mol. Microbiol. 2004; 53: 29-40Crossref PubMed Scopus (243) Google Scholar, 9.Rasis M. Segal G. The LetA-RsmYZ-CsrA regulatory cascade, together with RpoS and PmrA, post-transcriptionally regulates stationary phase activation of Legionella pneumophila Icm/Dot effectors.Mol. Microbiol. 2009; 72: 995-1010Crossref PubMed Scopus (0) Google Scholar, 10.Sahr T. Bruggemann H. Jules M. Lomma M. Albert-Weissenberger C. Cazalet C. Buchrieser C. Two small ncRNAs jointly govern virulence and transmission in Legionella pneumophila.Mol. Microbiol. 2009; 72: 741-762Crossref PubMed Scopus (0) Google Scholar). Accordingly, it has been shown that phase transition goes along with significant transcriptomic changes (11.Faucher S.P. Mueller C.A. Shuman H.A. Legionella Pneumophila transcriptome during intracellular multiplication in human macrophages.Front. Microbiol. 2011; 2: 60Crossref PubMed Scopus (78) Google Scholar, 12.Bruggemann H. Hagman A. Jules M. Sismeiro O. Dillies M.A. Gouyette C. Kunst F. Steinert M. Heuner K. Coppee J.Y. Buchrieser C. Virulence strategies for infecting phagocytes deduced from the in vivo transcriptional program of Legionella pneumophila 2.Cell Microbiol. 2006; 8: 1228-1240Crossref PubMed Scopus (0) Google Scholar, 13.Hovel-Miner G. Pampou S. Faucher S.P. Clarke M. Morozova I. Morozov P. Russo J.J. Shuman H.A. Kalachikov S. SigmaS controls multiple pathways associated with intracellular multiplication of Legionella pneumophila.J. Bacteriol. 2009; 191: 2461-2473Crossref PubMed Scopus (75) Google Scholar, 14.Weissenmayer B.A. Prendergast J.G. Lohan A.J. Loftus B.J. Sequencing illustrates the transcriptional response of Legionella pneumophila during infection and identifies seventy novel small non-coding RNAs.PLoS ONE. 2011; 6: e17570Crossref PubMed Scopus (69) Google Scholar) which must impact the life stage-specific proteomes. Remarkably, broth-grown L. pneumophila show a comparable phenotypic switch by expressing a phenotype similar to the replicative phase during exponential phase (E) 1The abbreviations used are:Eexponential phase2DE2D gel electrophoresisBCYEbuffered charcoal yeast extractBYEbuffered yeast extractextrextracellular proteinsHAhaemagglutininLCVLegionella-containing vacuoleNSAFnormalized spectral abundance factoromapouter membrane-associated proteinsPEpost-exponential phaseSPIsignal peptidase I signal peptideswcpsoluble whole cell proteinT2SStype II secretion systemT4BSStype IVB secretion systemTatTwin-arginine translocation systemUNCunculturable microcosmsVBNCviable but nonculturable. and to transmissive phase in post-exponential phase (PE) (8.Molofsky A.B. Swanson M.S. Differentiate to thrive: lessons from the Legionella pneumophila life cycle.Mol. Microbiol. 2004; 53: 29-40Crossref PubMed Scopus (243) Google Scholar, 12.Bruggemann H. Hagman A. Jules M. Sismeiro O. Dillies M.A. Gouyette C. Kunst F. Steinert M. Heuner K. Coppee J.Y. Buchrieser C. Virulence strategies for infecting phagocytes deduced from the in vivo transcriptional program of Legionella pneumophila 2.Cell Microbiol. 2006; 8: 1228-1240Crossref PubMed Scopus (0) Google Scholar, 14.Weissenmayer B.A. Prendergast J.G. Lohan A.J. Loftus B.J. Sequencing illustrates the transcriptional response of Legionella pneumophila during infection and identifies seventy novel small non-coding RNAs.PLoS ONE. 2011; 6: e17570Crossref PubMed Scopus (69) Google Scholar). exponential phase 2D gel electrophoresis buffered charcoal yeast extract buffered yeast extract extracellular proteins haemagglutinin Legionella-containing vacuole normalized spectral abundance factor outer membrane-associated proteins post-exponential phase signal peptidase I signal peptide soluble whole cell protein type II secretion system type IVB secretion system Twin-arginine translocation system unculturable microcosms viable but nonculturable. Currently, several proteomic studies are available for L. pneumophila. One of the first proteomic maps characterizing L. pneumophila grown on agar media was published by Lebeau et al. One hundred ten different proteins of bacterial cell lysates were identified by means of 2D gel electrophoresis (2DE), tryptic digest, and mass spectrometry (MS) (15.Lebeau I. Lammertyn E. De Buck E. Maes L. Geukens N. Van Mellaert L. Arckens L. Anne J. Clerens S. First proteomic analysis of Legionella pneumophila based on its developing genome sequence.Res. Microbiol. 2005; 156: 119-129Crossref PubMed Scopus (15) Google Scholar). Hayashi et al. conducted a similar approach and analyzed the growth phase-dependent L. pneumophila cell extract proteome after growth in broth. They determined four proteins overabundant in E and 64 proteins accumulating in PE. About 60% of the differentially abundant proteins were defined as enzymes and were categorized to: carbohydrate metabolism, amino acid metabolism, and lipid metabolism (16.Hayashi T. Nakamichi M. Naitou H. Ohashi N. Imai Y. Miyake M. Proteomic analysis of growth phase-dependent expression of Legionella pneumophila proteins which involves regulation of bacterial virulence traits.PLoS ONE. 2010; 5: e11718Crossref PubMed Scopus (16) Google Scholar). In the context of bacterial pathogenicity, exported proteins designed to directly interact with host cells are of particular interest. Several protein secretion systems of L. pneumophila are associated with bacterial virulence, such as the Defect in organelle trafficking/Intracellular multiplication (Dot/Icm) type IVB secretion system (T4BSS), the Legionella type II secretion pathway (Lsp) (T2SS), the type I (Lss), and the Twin-arginine translocation (Tat) systems (15.Lebeau I. Lammertyn E. De Buck E. Maes L. Geukens N. Van Mellaert L. Arckens L. Anne J. Clerens S. First proteomic analysis of Legionella pneumophila based on its developing genome sequence.Res. Microbiol. 2005; 156: 119-129Crossref PubMed Scopus (15) Google Scholar, 17.Segal G. Purcell M. Shuman H.A. Host cell killing and bacterial conjugation require overlapping sets of genes within a 22-kb region of the Legionella pneumophila genome.Proc. Natl. Acad. Sci. U.S.A. 1998; 95: 1669-1674Crossref PubMed Scopus (434) Google Scholar, 18.Isaac D.T. Isberg R. Master manipulators: an update on Legionella pneumophila Icm/Dot translocated substrates and their host targets.Future Microbiol. 2014; 9: 343-359Crossref PubMed Scopus (63) Google Scholar, 19.Rossier O. Cianciotto N.P. Type II protein secretion is a subset of the PilD-dependent processes that facilitate intracellular infection by Legionella pneumophila 1.Infect. Immun. 2001; 69: 2092-2098Crossref PubMed Scopus (0) Google Scholar, 20.Cianciotto N.P. Many substrates and functions of type II secretion: lessons learned from Legionella pneumophila.Future Microbiol. 2009; 4: 797-805Crossref PubMed Scopus (61) Google Scholar, 21.Fuche F. Vianney A. Andrea C. Doublet P. Gilbert C. Functional type 1 secretion system involved in Legionella pneumophila virulence.J. Bacteriol. 2015; 197: 563-571Crossref PubMed Scopus (23) Google Scholar, 22.De Buck E. Lebeau I. Maes L. Geukens N. Meyen E. Van Mellaert L. Anne J. Lammertyn E. A putative twin-arginine translocation pathway in Legionella pneumophila.Biochem. Biophys. Res. Commun. 2004; 317: 654-661Crossref PubMed Scopus (0) Google Scholar, 23.Rossier O. Cianciotto N.P. The Legionella pneumophila tatB gene facilitates secretion of phospholipase C, growth under iron-limiting conditions, and intracellular infection.Infect. Immun. 2005; 73: 2020-2032Crossref PubMed Scopus (75) Google Scholar, 24.Jacobi S. Heuner K. Description of a putative type I secretion system in Legionella pneumophila.Int. J. Med. Microbiol. 2003; 293: 349-358Crossref PubMed Scopus (30) Google Scholar). More than 300 and 25 proteins are translocated into the host cell by the T4BSS or are exported by the T2SS, respectively (18.Isaac D.T. Isberg R. Master manipulators: an update on Legionella pneumophila Icm/Dot translocated substrates and their host targets.Future Microbiol. 2014; 9: 343-359Crossref PubMed Scopus (63) Google Scholar, 25.Cianciotto N.P. Type II secretion and Legionella virulence.Curr. Top. Microbiol. Immunol. 2013; 376: 81-102PubMed Google Scholar). Previous proteomics studies gave an insight into the variety of L. pneumophila proteins present in the culture supernatant and on their dependence on type II secretion, Tat secretion, or export via outer membrane vesicles. In those studies, 2DE/MS analysis was used and identified at least 20 type II-secreted proteins, 20 proteins with differential abundance in wild type and a Tat mutant as well as 181 culture supernatant proteins of which 33 specifically were associated with outer membrane vesicles (20.Cianciotto N.P. Many substrates and functions of type II secretion: lessons learned from Legionella pneumophila.Future Microbiol. 2009; 4: 797-805Crossref PubMed Scopus (61) Google Scholar, 25.Cianciotto N.P. Type II secretion and Legionella virulence.Curr. Top. Microbiol. Immunol. 2013; 376: 81-102PubMed Google Scholar, 26.DebRoy S. Dao J. Soderberg M. Rossier O. Cianciotto N.P. Legionella pneumophila type II secretome reveals unique exoproteins and a chitinase that promotes bacterial persistence in the lung.Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 19146-19151Crossref PubMed Scopus (155) Google Scholar, 27.De Buck E. Hoper D. Lammertyn E. Hecker M. Anne J. Differential 2-D protein gel electrophoresis analysis of Legionella pneumophila wild type and Tat secretion mutants.Int. J. Med. Microbiol. 2008; 298: 449-461Crossref PubMed Scopus (17) Google Scholar, 28.Galka F. Wai S.N. Kusch H. Engelmann S. Hecker M. Schmeck B. Hippenstiel S. Uhlin B.E. Steinert M. Proteomic characterization of the whole secretome of Legionella pneumophila and functional analysis of outer membrane vesicles.Infect. Immun. 2008; 76: 1825-1836Crossref PubMed Scopus (122) Google Scholar). Furthermore, in a recent study by Hoffmann et al., the proteome of intracellular L. pneumophila 1h post infection was analyzed by means of 1D gel electrophoresis and subsequent liquid chromatography-MS/MS (29.Hoffmann C. Finsel I. Otto A. Pfaffinger G. Rothmeier E. Hecker M. Becher D. Hilbi H. Functional analysis of novel Rab GTPases identified in the proteome of purified Legionella-containing vacuoles from macrophages.Cell. Microbiol. 2014; 16: 1034-1052Crossref PubMed Scopus (70) Google Scholar). Four hundred thirty-four Legionella proteins were detected in Legionella-containing vacuoles (LCV) purified from RAW 264.7 macrophages, or 944 obtained from D. discoideum including 29 or 60 Dot/Icm effectors, respectively (29.Hoffmann C. Finsel I. Otto A. Pfaffinger G. Rothmeier E. Hecker M. Becher D. Hilbi H. Functional analysis of novel Rab GTPases identified in the proteome of purified Legionella-containing vacuoles from macrophages.Cell. Microbiol. 2014; 16: 1034-1052Crossref PubMed Scopus (70) Google Scholar). In addition to E and PE, various studies reported on L. pneumophila in a viable but nonculturable (VBNC) state which might be considered as a third life stage devoted to the survival of unfavorable conditions (30.Steinert M. Emody L. Amann R. Hacker J. Resuscitation of viable but nonculturable Legionella pneumophila Philadelphia JR32 by Acanthamoeba castellanii.Appl. Environ. Microbiol. 1997; 63: 2047-2053Crossref PubMed Google Scholar, 31.Alleron L. Merlet N. Lacombe C. Frere J. Long-term survival of Legionella pneumophila in the viable but nonculturable state after monochloramine treatment.Curr. Microbiol. 2008; 57: 497-502Crossref PubMed Scopus (89) Google Scholar, 32.Alleron L. Khemiri A. Koubar M. Lacombe C. Coquet L. Cosette P. Jouenne T. Frere J. VBNC Legionella pneumophila cells are still able to produce virulence proteins.Water Res. 2013; 47: 6606-6617Crossref PubMed Scopus (0) Google Scholar, 33.Garcia M.T. Jones S. Pelaz C. Millar R.D. Abu Kwaik Y. Acanthamoeba polyphaga resuscitates viable non-culturable Legionella pneumophila after disinfection.Environ. Microbiol. 2007; 9: 1267-1277Crossref PubMed Scopus (107) Google Scholar, 34.Al-Bana B.H. Haddad M.T. Garduno R.A. Stationary phase and mature infectious forms of Legionella pneumophila produce distinct viable but non-culturable cells.Environ. Microbiol. 2014; 16: 382-395Crossref PubMed Scopus (30) Google Scholar). Bacteria in the VBNC state fail to grow on routine laboratory media on which they would normally grow, but retain insignia of life, such as intact membranes, metabolic activity, transcription, respiration, and some attributes of virulence (35.Oliver J.D. Recent findings on the viable but nonculturable state in pathogenic bacteria.FEMS Microbiol. Rev. 2010; 34: 415-425Crossref PubMed Scopus (706) Google Scholar). It has been shown that unculturable L. pneumophila may resuscitate to a fully virulent form by passage through an amoebal host (30.Steinert M. Emody L. Amann R. Hacker J. Resuscitation of viable but nonculturable Legionella pneumophila Philadelphia JR32 by Acanthamoeba castellanii.Appl. Environ. Microbiol. 1997; 63: 2047-2053Crossref PubMed Google Scholar, 33.Garcia M.T. Jones S. Pelaz C. Millar R.D. Abu Kwaik Y. Acanthamoeba polyphaga resuscitates viable non-culturable Legionella pneumophila after disinfection.Environ. Microbiol. 2007; 9: 1267-1277Crossref PubMed Scopus (107) Google Scholar, 34.Al-Bana B.H. Haddad M.T. Garduno R.A. Stationary phase and mature infectious forms of Legionella pneumophila produce distinct viable but non-culturable cells.Environ. Microbiol. 2014; 16: 382-395Crossref PubMed Scopus (30) Google Scholar, 36.Ohno A. Kato N. Yamada K. Yamaguchi K. Factors influencing survival of Legionella pneumophila serotype 1 in hot spring water and tap water.Appl. Environ. Microbiol. 2003; 69: 2540-2547Crossref PubMed Scopus (82) Google Scholar) and therefore provide a largely undetected source of infections. Unculturable Legionellae have been observed for example after prolonged incubation in laboratory tap water microcosms at different temperatures, including elevated temperature at 42 °C (36.Ohno A. Kato N. Yamada K. Yamaguchi K. Factors influencing survival of Legionella pneumophila serotype 1 in hot spring water and tap water.Appl. Environ. Microbiol. 2003; 69: 2540-2547Crossref PubMed Scopus (82) Google Scholar), heat shock (50–70 °C) (37.Epalle T. Girardot F. Allegra S. Maurice-Blanc C. Garraud O. Riffard S. Viable but Not Culturable Forms of Legionella pneumophila Generated After Heat Shock Treatment Are Infectious for Macrophage-Like and Alveolar Epithelial Cells After Resuscitation on Acanthamoeba polyphaga.Microb. Ecol. 2015; 69: 215-224Crossref PubMed Scopus (32) Google Scholar), following disinfection (31.Alleron L. Merlet N. Lacombe C. Frere J. Long-term survival of Legionella pneumophila in the viable but nonculturable state after monochloramine treatment.Curr. Microbiol. 2008; 57: 497-502Crossref PubMed Scopus (89) Google Scholar, 32.Alleron L. Khemiri A. Koubar M. Lacombe C. Coquet L. Cosette P. Jouenne T. Frere J. VBNC Legionella pneumophila cells are still able to produce virulence proteins.Water Res. 2013; 47: 6606-6617Crossref PubMed Scopus (0) Google Scholar, 33.Garcia M.T. Jones S. Pelaz C. Millar R.D. Abu Kwaik Y. Acanthamoeba polyphaga resuscitates viable non-culturable Legionella pneumophila after disinfection.Environ. Microbiol. 2007; 9: 1267-1277Crossref PubMed Scopus (107) Google Scholar), and after treatment with heavy metals (38.Hwang M.G. Katayama H. Ohgaki S. Effect of intracellular resuscitation of Legionella pneumophila in Acanthamoeba polyphage cells on the antimicrobial properties of silver and copper.Environ. Sci. Technol. 2006; 40: 7434-7439Crossref PubMed Scopus (29) Google Scholar). Major differences in the transcriptional program underlying the phenotypic switch between E and PE in L. pneumophila, which are important in understanding bacterial virulence, were formerly described (11.Faucher S.P. Mueller C.A. Shuman H.A. Legionella Pneumophila transcriptome during intracellular multiplication in human macrophages.Front. Microbiol. 2011; 2: 60Crossref PubMed Scopus (78) Google Scholar, 12.Bruggemann H. Hagman A. Jules M. Sismeiro O. Dillies M.A. Gouyette C. Kunst F. Steinert M. Heuner K. Coppee J.Y. Buchrieser C. Virulence strategies for infecting phagocytes deduced from the in vivo transcriptional program of Legionella pneumophila 2.Cell Microbiol. 2006; 8: 1228-1240Crossref PubMed Scopus (0) Google Scholar, 13.Hovel-Miner G. Pampou S. Faucher S.P. Clarke M. Morozova I. Morozov P. Russo J.J. Shuman H.A. Kalachikov S. SigmaS controls multiple pathways associated with intracellular multiplication of Legionella pneumophila.J. Bacteriol. 2009; 191: 2461-2473Crossref PubMed Scopus (75) Google Scholar, 14.Weissenmayer B.A. Prendergast J.G. Lohan A.J. Loftus B.J. Sequencing illustrates the transcriptional response of Legionella pneumophila during infection and identifies seventy novel small non-coding RNAs.PLoS ONE. 2011; 6: e17570Crossref PubMed Scopus (69) Google Scholar). This and the study by Hayashi et al. indicated that cell-associated proteomes of E and PE L. pneumophila indeed reveal differences (16.Hayashi T. Nakamichi M. Naitou H. Ohashi N. Imai Y. Miyake M. Proteomic analysis of growth phase-dependent expression of Legionella pneumophila proteins which involves regulation of bacterial virulence traits.PLoS ONE. 2010; 5: e11718Crossref PubMed Scopus (16) Google Scholar). However, the big variety and differential abundance of proteins present at the three L. pneumophila life stages and distinct subproteomes (soluble whole cell, outer membrane-associated, and extracellular) have so far not been comprehensively described. Here, we present a global proteomic analysis of E, PE, and UNC of L. pneumophila and visualize the protein composition and occurring changes in Voronoi treemaps. Our results reveal (1) different life stage- and accordingly virulence-related protein patterns, (2) phase-dependent differential protein abundance including many Dot/Icm effectors, (3) life stage-exclusive proteins, and (4) protein localization of a wide array of proteins. To delineate growth phase-related proteomic changes of L. pneumophila, E/PE bacteria and UNC were generated and analyzed by mass spectrometry. E and PE bacteria were assessed for swcp, extr, and omap. Each sample was analyzed in biological triplicates to allow for statistical tests and to improve consistency. Protein identification and quality criteria were very strict throughout the study (see section Proteome Analysis). Only proteins identified in each of three biological replicates were quantified by means of normalized spectral abundance factors (NSAFs) (see section Data Analysis). In order to reduce mainly cytosolic contaminants, quantified extr and omap were assessed for quantitative enrichment of individual proteins compared with swcp, and only enriched proteins were further analyzed (see section Data Analysis of omap and extr). To test for phase related differential abundance of proteins, a t test as implemented in Scaffold (v.4.4.5) was used (p < 0.05). Ratios of quantity of significantly different proteins were log2 transformed and only those were approved who exceeded 1 or fell below −1 (49.Hempel K. Herbst F.A. Moche M. Hecker M. Becher D. Quantitative proteomic view on secreted, cell surface-associated, and cytoplasmic proteins of the methicillin-resistant human pathogen Staphylococcus aureus under iron-limited conditions.J. Proteome Res. 2011; 10: 1657-1666Crossref PubMed Scopus (52) Google Scholar). Within proteins with an apparent phase specific abundance, subfractions termed "on/off proteins" and "phase exclusive proteins" were defined (see section Data Analysis). L. pneumophila was routinely grown on buffered charcoal yeast extract (BCYE) agar for 2–3 days at 37 °C (39.Edelstein P.H. Improved semiselective medium for isolation of Legionella pneumophila from contaminated clinical and environmental specimens.J. Clin. Microbiol. 1981; 14: 298-303Crossref PubMed Google Scholar). For growth in liquid laboratory medium, L. pneumophila was inoculated at an OD660 = 0.2–0.3 and was cultured in buffered yeast extract (BYE) broth at 37 °C with continuous shaking at 250 rpm. Bacterial growth was checked by determining the optical density of the culture at wavelength 660 nm (OD660) using a Beckman spectrophotometer DU520 (Beckman Coulter, Brea, CA). E and PE L. pneumophila for proteomics analysis were obtained from liquid cultures. E starter cultures were diluted in BYE broth to an OD660 = 0.2. E samples were removed after ∼4.5 h shaking at 37 °C. PE samples were obtained at 13.5 h when the bacteria entered stationary growth phase. To confirm that the bacteria reached PE, samples were subjected to Western blotting with antibodies directed against flagellin (kindly provided by Klaus Heuner, Robert Koch-Institut Berlin (40.Heuner K. Bender-Beck L. Brand B.C. Luck P.C. Mann K.H. Marre R. Ott M. Hacker J. Cloning and genetic characterization of the flagellum subunit gene (flaA) of Legionella pneumophila serogroup 1.Infect. Immun. 1995; 63: 2499-2507Crossref PubMed Google Scholar)), a growth phase-related marker of PE L. pneumophila (5.Byrne B. Swanson M.S. Expression of Legionella pneumophila virulence traits in response to growth conditions.Infect. Immun. 1998; 66: 3029-3034Crossref PubMed Google Scholar). For the generation of L. pneumophila UNC, PE cells were washed two times in sterilized tap water and subsequently inoculated in sterilized tap water to an OD660 of 0.3, corresponding to ∼2 × 108 CFU/ml. The water microcosms were statically incubated at 42 °C to induce VBNC bacteria as described elsewhere (41.Ohno A. Kato N. Sakamoto R. Kimura S. Yamaguchi K. Temperature-dependent parasitic relationship between Legionella pneumophila and a free-living amoeba (Acanthamoeba castellanii).Appl. Environ. Microbiol. 2008; 74: 4585-4588Crossref PubMed Scopus (45) Google Scholar). To study the effect of temperature for efficient generation of VBNC L. pneumophila, samples were incubated at 4 °C, 21 °C, 42 °C, and only the latter condition lead to loss of culturability and maintenance of viability in a reasonable time (∼110 days). Samples were considered UNC when repeated plating of 1 ml cultures containing 108 bacteria on BCYE yielded zero CFU after 5 days of incubation at 37 °C. To determine the amount of VBNC and dead bacteria, BacLight Live/Dead staining and the cFDA assay (both purchased from Invitrogen) were used. The BacLight Live/Dead staining kit was used according to the manufacturer's protocol and the stained cells were imaged by epifluorescence microscopy. Additionally, the quantity of esterase-positive bacteria was determined after treatment with 6-carboxyfluoresceindia
Referência(s)