Proteomic Analysis of the Vibrio cholerae Type II Secretome Reveals New Proteins, Including Three Related Serine Proteases
2011; Elsevier BV; Volume: 286; Issue: 19 Linguagem: Inglês
10.1074/jbc.m110.211078
ISSN1083-351X
AutoresAleksandra E. Sikora, Ryszard A. Zielke, Daniel A. Lawrence, Philip Andrews, Maria Sandkvist,
Tópico(s)Bacterial biofilms and quorum sensing
ResumoThe type II secretion (T2S) system is responsible for extracellular secretion of a broad range of proteins, including toxins and degradative enzymes that play important roles in the pathogenesis and life cycle of many Gram-negative bacteria. In Vibrio cholerae, the etiological agent of cholera, the T2S machinery transports cholera toxin, which induces profuse watery diarrhea, a hallmark of this life-threatening disease. Besides cholera toxin, four other proteins have been shown to be transported by the T2S machinery, including hemagglutinin protease, chitinase, GbpA, and lipase. Here, for the first time, we have applied proteomic approaches, including isotope tagging for relative and absolute quantification coupled with multidimensional liquid chromatography and tandem mass spectrometry, to perform an unbiased and comprehensive analysis of proteins secreted by the T2S apparatus of the V. cholerae El Tor strain N16961 under standard laboratory growth conditions. This analysis identified 16 new putative T2S substrates, including sialidase, several proteins participating in chitin utilization, two aminopeptidases, TagA-related protein, cytolysin, RbmC, three hypothetical proteins encoded by VCA0583, VCA0738, and VC2298, and three serine proteases VesA, VesB, and VesC. Focusing on the initial characterization of VesA, VesB, and VesC, we have confirmed enzymatic activities and T2S-dependent transport for each of these proteases. In addition, analysis of single, double, and triple protease knock-out strains indicated that VesA is the primary protease responsible for processing the A subunit of cholera toxin during in vitro growth of the V. cholerae strain N16961. The type II secretion (T2S) system is responsible for extracellular secretion of a broad range of proteins, including toxins and degradative enzymes that play important roles in the pathogenesis and life cycle of many Gram-negative bacteria. In Vibrio cholerae, the etiological agent of cholera, the T2S machinery transports cholera toxin, which induces profuse watery diarrhea, a hallmark of this life-threatening disease. Besides cholera toxin, four other proteins have been shown to be transported by the T2S machinery, including hemagglutinin protease, chitinase, GbpA, and lipase. Here, for the first time, we have applied proteomic approaches, including isotope tagging for relative and absolute quantification coupled with multidimensional liquid chromatography and tandem mass spectrometry, to perform an unbiased and comprehensive analysis of proteins secreted by the T2S apparatus of the V. cholerae El Tor strain N16961 under standard laboratory growth conditions. This analysis identified 16 new putative T2S substrates, including sialidase, several proteins participating in chitin utilization, two aminopeptidases, TagA-related protein, cytolysin, RbmC, three hypothetical proteins encoded by VCA0583, VCA0738, and VC2298, and three serine proteases VesA, VesB, and VesC. Focusing on the initial characterization of VesA, VesB, and VesC, we have confirmed enzymatic activities and T2S-dependent transport for each of these proteases. In addition, analysis of single, double, and triple protease knock-out strains indicated that VesA is the primary protease responsible for processing the A subunit of cholera toxin during in vitro growth of the V. cholerae strain N16961. IntroductionGram-negative bacteria have evolved at least six secretion pathways devoted to the transport of proteins through the cell envelope into either the extracellular environment or directly into host cells (1Pukatzki S. McAuley S.B. Miyata S.T. Curr. Opin. Microbiol. 2009; 12: 11-17Crossref PubMed Scopus (243) Google Scholar, 2Saier Jr., M.H. J. Membr. Biol. 2006; 214: 75-90Crossref PubMed Scopus (91) Google Scholar). The type II secretion (T2S) 2The abbreviations used are: T2S, type II secretion; iTRAQ, isotope tagging for relative and absolute quantification; AEBSF, 4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride; IPTG, isopropyl 1-thio-β-d-galactopyranoside; CFU, colony-forming unit; HAP, hemagglutinin protease; Fwd, forward; Rev, reverse; CT, cholera toxin; Boc, t-butoxycarbonyl; AMC, aminomethylcoumarin. system was first discovered in Klebsiella oxytoca and has been shown to be widely distributed among γ-proteobacteria (3d'Enfert C. Reyss I. Wandersman C. Pugsley A.P. J. Biol. Chem. 1989; 264: 17462-17468Abstract Full Text PDF PubMed Google Scholar, 4d'Enfert C. Ryter A. Pugsley A.P. EMBO J. 1987; 6: 3531-3538Crossref PubMed Scopus (153) Google Scholar, 5Sandkvist M. Infect. Immun. 2001; 69: 3523-3535Crossref PubMed Scopus (262) Google Scholar, 6Cianciotto N.P. Trends Microbiol. 2005; 13: 581-588Abstract Full Text Full Text PDF PubMed Scopus (263) Google Scholar). Depending on the bacterial species, the T2S complex consists of 12–16 different constituents that form a multiprotein apparatus spanning the entire cell envelope (7Sandkvist M. Mol. Microbiol. 2001; 40: 271-283Crossref PubMed Scopus (322) Google Scholar, 8Filloux A. Biochim. Biophys. Acta. 2004; 1694: 163-179Crossref PubMed Scopus (218) Google Scholar). The conserved components of the T2S machinery include the cytoplasmic ATPase (T2S E), the inner membrane platform (T2S C, F, L, and M), a pilus-like structure (T2S G–K), a protein responsible for the processing of pseudopilins (T2S O), and the secretion pore (T2S D) embedded in the outer membrane (9Johnson T.L. Abendroth J. Hol W.G. Sandkvist M. FEMS Microbiol. Lett. 2006; 255: 175-186Crossref PubMed Scopus (181) Google Scholar). The exoprotein precursors are synthesized with N-terminal signal peptides that direct them into the periplasmic space via either the Sec or Tat transport systems (10Pugsley A.P. Microbiol. Rev. 1993; 57: 50-108Crossref PubMed Google Scholar, 11Voulhoux R. Ball G. Ize B. Vasil M.L. Lazdunski A. Wu L.F. Filloux A. EMBO J. 2001; 20: 6735-6741Crossref PubMed Scopus (211) Google Scholar). After obtaining tertiary conformation, the exoproteins enter the T2S machinery and are subsequently translocated into the extracellular milieu (12Hirst T.R. Holmgren J. Proc. Natl. Acad. Sci. U.S.A. 1987; 84: 7418-7422Crossref PubMed Scopus (113) Google Scholar, 13Lory S. Curr. Opin. Microbiol. 1998; 1: 27-35Crossref PubMed Scopus (70) Google Scholar). Many key steps in the secretion process are still not well understood, including how the exoproteins are recognized by the T2S system, and a specific secretion signal common to known substrates has not yet been identified.The T2S system is devoted to secretion of a variety of substrates, including toxins, surface-associated virulence factors, a broad range of enzymes that hydrolyze macromolecules (such as lipids, polysaccharides, and proteins), surfactant(s) important for motility, and certain cytochromes (5Sandkvist M. Infect. Immun. 2001; 69: 3523-3535Crossref PubMed Scopus (262) Google Scholar, 14Zalewska-Piatek B. Bury K. Piatek R. Bruzdziak P. Kur J. J. Bacteriol. 2008; 190: 5044-5056Crossref PubMed Scopus (18) Google Scholar, 15Horstman A.L. Kuehn M.J. J. Biol. Chem. 2002; 277: 32538-32545Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar, 16Stewart C.R. Rossier O. Cianciotto N.P. J. Bacteriol. 2009; 191: 1537-1546Crossref PubMed Scopus (53) Google Scholar, 17Shi L. Deng S. Marshall M.J. Wang Z. Kennedy D.W. Dohnalkova A.C. Mottaz H.M. Hill E.A. Gorby Y.A. Beliaev A.S. Richardson D.J. Zachara J.M. Fredrickson J.K. J. Bacteriol. 2008; 190: 5512-5516Crossref PubMed Scopus (100) Google Scholar). The T2S-dependent proteins are of great interest because many of them play important roles in pathogenesis and/or contribute to bacterial fitness in different ecological niches (6Cianciotto N.P. Trends Microbiol. 2005; 13: 581-588Abstract Full Text Full Text PDF PubMed Scopus (263) Google Scholar, 18Cianciotto N.P. Future Microbiol. 2009; 4: 797-805Crossref PubMed Scopus (63) Google Scholar, 19Evans F.F. Egan S. Kjelleberg S. Environ. Microbiol. 2008; 10: 1101-1107Crossref PubMed Scopus (27) Google Scholar, 20Jha G. Rajeshwari R. Sonti R.V. Mol. Plant Microbe Interact. 2005; 18: 891-898Crossref PubMed Scopus (64) Google Scholar). Many of the T2S exoproteins were originally identified based on the loss of specific enzymatic activities in culture supernatants of the T2S mutants, and to date only a few comprehensive studies have been undertaken to define a broader array of secreted proteins (21DebRoy S. Dao J. Söderberg M. Rossier O. Cianciotto N.P. Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 19146-19151Crossref PubMed Scopus (168) Google Scholar, 22Evans F.F. Raftery M.J. Egan S. Kjelleberg S. J. Proteome Res. 2007; 6: 967-975Crossref PubMed Scopus (42) Google Scholar, 23Coulthurst S.J. Lilley K.S. Hedley P.E. Liu H. Toth I.K. Salmond G.P. J. Biol. Chem. 2008; 283: 23739-23753Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). Identification of the T2S substrates from different bacterial species might help to elucidate the mechanism of exoprotein recognition by the T2S system and provide a better understanding of the general role of T2S in pathogenesis and environmental survival.Our laboratory studies the T2S system in the causative agent of cholera, Vibrio cholerae. Cholera is a life-threatening diarrheal disease that predominantly occurs in developing countries of Asia, Africa, and South America (24Sack D.A. Sack R.B. Chaignat C.L. N. Engl. J. Med. 2006; 355: 649-651Crossref PubMed Scopus (75) Google Scholar). The T2S system, extracellular protein secretion (Eps), of V. cholerae is responsible for secretion of five known proteins, including cholera toxin, chitinase (ChiA-1), chitin-binding protein (GbpA), hemagglutinin protease (HAP), and lipase (25Sandkvist M. Morales V. Bagdasarian M. Gene. 1993; 123: 81-86Crossref PubMed Scopus (65) Google Scholar, 26Overbye L.J. Sandkvist M. Bagdasarian M. Gene. 1993; 132: 101-106Crossref PubMed Scopus (78) Google Scholar, 27Kirn T.J. Jude B.A. Taylor R.K. Nature. 2005; 438: 863-866Crossref PubMed Scopus (227) Google Scholar, 28Connell T.D. Metzger D.J. Lynch J. Folster J.P. J. Bacteriol. 1998; 180: 5591-5600Crossref PubMed Google Scholar, 29Sikora A.E. Lybarger S.R. Sandkvist M. J. Bacteriol. 2007; 189: 8484-8495Crossref PubMed Scopus (49) Google Scholar, 30Davis B.M. Lawson E.H. Sandkvist M. Ali A. Sozhamannan S. Waldor M.K. Science. 2000; 288: 333-335Crossref PubMed Scopus (87) Google Scholar).V. cholerae circulates between two very diverse environments, aquatic reservoirs and the gastrointestinal tract of the human body (31Cottingham K.L. Chiavelli D.A. Taylor R.K. Front. Ecol. Environ. 2003; 1: 80-86Crossref Google Scholar). The human host acquires V. cholerae through an oral route of infection with contaminated water or food. Following colonization of the small intestine, the bacteria produce and secrete cholera toxin. Although the disease is multifaceted, cholera toxin is the major virulence factor. It stimulates constitutive activation of cellular adenylate cyclase causing severe intestinal fluid loss and watery diarrhea. The rice-water like stools from cholera patients contain a large number of V. cholerae cells that are often shed back to the environment (32Nelson E.J. Harris J.B. Morris Jr., J.G. Calderwood S.B. Camilli A. Nat. Rev. Microbiol. 2009; 7: 693-702Crossref PubMed Scopus (378) Google Scholar). In the aquatic environment, V. cholerae associates with chitin particles, phyto- and zooplankton, and Chironomidae (nonbiting midges) egg masses (33Nalin D.R. Daya V. Reid A. Levine M.M. Cisneros L. Infect. Immun. 1979; 25: 768-770Crossref PubMed Google Scholar, 34Lipp E.K. Huq A. Colwell R.R. Clin. Microbiol. Rev. 2002; 15: 757-770Crossref PubMed Scopus (527) Google Scholar, 35Broza M. Halpern M. Nature. 2001; 412: 40Crossref PubMed Scopus (105) Google Scholar). The persistence of V. cholerae in the aquatic niche is likely facilitated by several of the known T2S substrates. GbpA is important for V. cholerae attachment to biotic and abiotic chitin surfaces; ChiA1 and HAP play a role in utilization of chitin and insect egg masses as a source of nutrients, respectively (27Kirn T.J. Jude B.A. Taylor R.K. Nature. 2005; 438: 863-866Crossref PubMed Scopus (227) Google Scholar, 28Connell T.D. Metzger D.J. Lynch J. Folster J.P. J. Bacteriol. 1998; 180: 5591-5600Crossref PubMed Google Scholar, 36Halpern M. Gancz H. Broza M. Kashi Y. Appl. Environ. Microbiol. 2003; 69: 4200-4204Crossref PubMed Scopus (69) Google Scholar). Moreover, gbpA mutants display defects in adherence to human epithelial cells and in colonization of infant mice, suggesting that GbpA represents a dual colonization factor functioning both in the aquatic environment and the human host (27Kirn T.J. Jude B.A. Taylor R.K. Nature. 2005; 438: 863-866Crossref PubMed Scopus (227) Google Scholar). Similarly, HAP has been suggested to play a role in infection by facilitating the penetration of V. cholerae through the mucus barrier of the gastrointestinal tract and by causing its detachment from the epithelium following the induction of the secretory diarrhea (37Finkelstein R.A. Boesman-Finkelstein M. Holt P. Proc. Natl. Acad. Sci. U.S.A. 1983; 80: 1092-1095Crossref PubMed Scopus (121) Google Scholar, 38Silva A.J. Leitch G.J. Camilli A. Benitez J.A. Infect. Immun. 2006; 74: 2072-2079Crossref PubMed Scopus (62) Google Scholar).Despite the significant impact of known T2S substrates on V. cholerae pathogenesis and physiology, comprehensive studies have not yet been undertaken to identify additional secreted factors. In pursuit of identifying the T2S secretome of V. cholerae, here we have applied global proteomic approaches, including a high throughput technique (iTRAQ) coupled with multidimensional liquid chromatography (LC) and tandem mass spectrometry (MS/MS). We identified 16 new putative T2S-dependent exoproteins, including three related serine proteases VesA, VesB, and VesC. This study focuses on the characterization of these three newly identified proteins.DISCUSSIONThis is the first study describing a comprehensive analysis of the V. cholerae T2 secretome. We have used a combination of proteomic approaches to compare the protein profiles of wild-type and T2S-deficient strains, and we expanded the list of T2S substrates from five to 21 (Tables 1 and 2). Our study shows that applying different proteomic approaches is beneficial and complementary, especially when complex protein samples are analyzed. Although further verification of the results may be necessary for a subset of proteins, together the results from our proteomic experiments indicate that V. cholerae secretes a variety of hydrolytic enzymes, including sialidase, cytolysin, two aminopeptidases, four chitinases, three trypsin-like serine proteases, and a TagA-related protein in addition to cholera toxin, HAP, ChiA, GbpA, and lipase (Tables 1 and 2). Similarly to other T2S systems, in V. cholerae the T2S machinery is primarily engaged in transport of hydrolytic enzymes that likely play a role in modifying the surroundings and generating nutrients and thus might support bacterial fitness in different ecological niches, including the human host. The T2S machinery is capable of secreting as many as 25 different substrates, as demonstrated for the T2S system in L. pneumophila (18Cianciotto N.P. Future Microbiol. 2009; 4: 797-805Crossref PubMed Scopus (63) Google Scholar, 21DebRoy S. Dao J. Söderberg M. Rossier O. Cianciotto N.P. Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 19146-19151Crossref PubMed Scopus (168) Google Scholar). Our proteomic studies focused on the analysis of the T2 secretome of V. cholerae cultured under a single growth condition, and therefore the complete repertoire of the T2-secreted proteins in V. cholerae was not revealed. For example, cholera toxin and lipase, two previously identified T2S substrates, were not detected in this study because the expression of their respective genes are under strict regulatory controls and require growth conditions that differ from the one used here (29Sikora A.E. Lybarger S.R. Sandkvist M. J. Bacteriol. 2007; 189: 8484-8495Crossref PubMed Scopus (49) Google Scholar, 39Iwanaga M. Yamamoto K. J. Clin. Microbiol. 1985; 22: 405-408Crossref PubMed Google Scholar). On the other hand, we did establish that V. cholerae produces and secretes several proteins predicted to play a role in different steps of chitin degradation and utilization even though the cultures were grown in the absence of chitin. This was not surprising, however, because it has been shown previously that these genes remain expressed in V. cholerae grown in LB medium (84Bhowmick R. Ghosal A. Chatterjee N.S. J. Appl. Microbiol. 2007; 103: 97-108Crossref PubMed Scopus (21) Google Scholar). Besides degrading chitin, these proteins may play an additional function in V. cholerae physiology by participating in processing of other GlcNAc containing carbohydrates or proteins.The proteins discovered in our study expand the tools used to investigate the molecular mechanism of substrate recognition by the T2S machinery and provide a new reference for elucidating the function of secreted proteins in V. cholerae physiology. With the exception of cholera toxin, HAP, and GbpA, the impact of individual secreted proteins on V. cholerae pathogenesis and metabolism is understudied. Hence, we focused on the initial characterization of the three newly identified proteases, VesA, VesB, and VesC, and investigated their hydrolytic activities against a fluorogenic peptide commonly used for trypsin-like proteases. We also established several lines of evidence that VesA plays a pivotal role in the processing of the A subunit of cholera toxin in vitro. Because it has been well documented that growth of V. cholerae in the presence of serine protease inhibitors markedly reduced the processing and activation of CT A, we assume that the activity inhibited was primarily that of VesA (81Mekalanos J.J. Collier R.J. Romig W.R. J. Biol. Chem. 1979; 254: 5855-5861Abstract Full Text PDF PubMed Google Scholar).Biochemical assays with purified proteins have shown that proteases such as HAP, trypsin, and elastase are capable of processing CT A (79Booth B.A. Boesman-Finkelstein M. Finkelstein R.A. Infect. Immun. 1984; 45: 558-560Crossref PubMed Google Scholar, 81Mekalanos J.J. Collier R.J. Romig W.R. J. Biol. Chem. 1979; 254: 5855-5861Abstract Full Text PDF PubMed Google Scholar). Moreover studies with human intestinal T84 cell lines have demonstrated that the presence of an unidentified host epithelial serine protease(s) is sufficient to process CT A (85Lencer W.I. Constable C. Moe S. Rufo P.A. Wolf A. Jobling M.G. Ruston S.P. Madara J.L. Holmes R.K. Hirst T.R. J. Biol. Chem. 1997; 272: 15562-15568Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). In light of these findings, it is very likely that host proteases are responsible for CT A activation during intestinal colonization of V. cholerae; however, there may be conditions, yet to be determined, during which Vibrio proteases such as VesA and HapA play significant roles in the processing and activation of CT A. Nevertheless, in this study for the first time a genetic analysis demonstrated that V. cholerae N16961 lacking vesA secretes mostly unprocessed CT A, suggesting that VesA is the primary protease responsible for CT A processing in vitro. Based on the immunoblot analysis, about 30% of the CT A was processed in the vesA knock-out strain, suggesting that additional factor(s) is also capable of CT A cleavage (Fig. 5A). Based on observations made by us and others, the most likely candidate(s) for additional endogenous proteins that are responsible for the processing of CT A is VesB and HAP (79Booth B.A. Boesman-Finkelstein M. Finkelstein R.A. Infect. Immun. 1984; 45: 558-560Crossref PubMed Google Scholar). Because of the very low level of HAP in V. cholerae strain N16961, VesB is likely the primary protease responsible for the residual processing of CT A. This conclusion is supported by the finding that V. cholerae N16961 grown in the presence of the serine protease inhibitor leupeptin, which inhibits both VesA and VesB, secretes intact CT A (supplemental Fig. 4).We did not find that VesC protease is capable of processing cholera toxin under tested conditions (Figs. 5 and 6). However, a very recent study has shown that VesC induces a hemorrhagic response when injected into rabbit ileal loops suggesting that it may play a role in pathogenesis (73Syngkon A. Elluri S. Koley H. Rompikuntal P.K. Saha D.R. Chakrabarti M.K. Bhadra R.K. Wai S.N. Pal A. PLoS ONE. 2010; 5: e13122Crossref PubMed Scopus (39) Google Scholar). As we have showed here; however, this activity does not seem to be required for intestinal colonization of mice.Importantly, our study revealed that the T2S system of V. cholerae secretes proteins that are functionally associated with cholera toxin, including VesA, Hap, and sialidase. Sialidase contains a sialic acid binding domain that is responsible for localizing the enzyme to higher organized sialic gangliosides in the intestinal mucus and cleavage of sialic acid groups revealing GM1, the receptor for cholera toxin (86Moustafa I. Connaris H. Taylor M. Zaitsev V. Wilson J.C. Kiefel M.J. von Itzstein M. Taylor G. J. Biol. Chem. 2004; 279: 40819-40826Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar, 87Galen J.E. Ketley J.M. Fasano A. Richardson S.H. Wasserman S.S. Kaper J.B. Infect. Immun. 1992; 60: 406-415Crossref PubMed Google Scholar). VesA, Hap, and sialidase likely work synergistically, ensuring the proper localization and activation of the toxin immediately after its secretion, thus further underscoring the importance of the T2S system in V. cholerae pathogenesis. IntroductionGram-negative bacteria have evolved at least six secretion pathways devoted to the transport of proteins through the cell envelope into either the extracellular environment or directly into host cells (1Pukatzki S. McAuley S.B. Miyata S.T. Curr. Opin. Microbiol. 2009; 12: 11-17Crossref PubMed Scopus (243) Google Scholar, 2Saier Jr., M.H. J. Membr. Biol. 2006; 214: 75-90Crossref PubMed Scopus (91) Google Scholar). The type II secretion (T2S) 2The abbreviations used are: T2S, type II secretion; iTRAQ, isotope tagging for relative and absolute quantification; AEBSF, 4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride; IPTG, isopropyl 1-thio-β-d-galactopyranoside; CFU, colony-forming unit; HAP, hemagglutinin protease; Fwd, forward; Rev, reverse; CT, cholera toxin; Boc, t-butoxycarbonyl; AMC, aminomethylcoumarin. system was first discovered in Klebsiella oxytoca and has been shown to be widely distributed among γ-proteobacteria (3d'Enfert C. Reyss I. Wandersman C. Pugsley A.P. J. Biol. Chem. 1989; 264: 17462-17468Abstract Full Text PDF PubMed Google Scholar, 4d'Enfert C. Ryter A. Pugsley A.P. EMBO J. 1987; 6: 3531-3538Crossref PubMed Scopus (153) Google Scholar, 5Sandkvist M. Infect. Immun. 2001; 69: 3523-3535Crossref PubMed Scopus (262) Google Scholar, 6Cianciotto N.P. Trends Microbiol. 2005; 13: 581-588Abstract Full Text Full Text PDF PubMed Scopus (263) Google Scholar). Depending on the bacterial species, the T2S complex consists of 12–16 different constituents that form a multiprotein apparatus spanning the entire cell envelope (7Sandkvist M. Mol. Microbiol. 2001; 40: 271-283Crossref PubMed Scopus (322) Google Scholar, 8Filloux A. Biochim. Biophys. Acta. 2004; 1694: 163-179Crossref PubMed Scopus (218) Google Scholar). The conserved components of the T2S machinery include the cytoplasmic ATPase (T2S E), the inner membrane platform (T2S C, F, L, and M), a pilus-like structure (T2S G–K), a protein responsible for the processing of pseudopilins (T2S O), and the secretion pore (T2S D) embedded in the outer membrane (9Johnson T.L. Abendroth J. Hol W.G. Sandkvist M. FEMS Microbiol. Lett. 2006; 255: 175-186Crossref PubMed Scopus (181) Google Scholar). The exoprotein precursors are synthesized with N-terminal signal peptides that direct them into the periplasmic space via either the Sec or Tat transport systems (10Pugsley A.P. Microbiol. Rev. 1993; 57: 50-108Crossref PubMed Google Scholar, 11Voulhoux R. Ball G. Ize B. Vasil M.L. Lazdunski A. Wu L.F. Filloux A. EMBO J. 2001; 20: 6735-6741Crossref PubMed Scopus (211) Google Scholar). After obtaining tertiary conformation, the exoproteins enter the T2S machinery and are subsequently translocated into the extracellular milieu (12Hirst T.R. Holmgren J. Proc. Natl. Acad. Sci. U.S.A. 1987; 84: 7418-7422Crossref PubMed Scopus (113) Google Scholar, 13Lory S. Curr. Opin. Microbiol. 1998; 1: 27-35Crossref PubMed Scopus (70) Google Scholar). Many key steps in the secretion process are still not well understood, including how the exoproteins are recognized by the T2S system, and a specific secretion signal common to known substrates has not yet been identified.The T2S system is devoted to secretion of a variety of substrates, including toxins, surface-associated virulence factors, a broad range of enzymes that hydrolyze macromolecules (such as lipids, polysaccharides, and proteins), surfactant(s) important for motility, and certain cytochromes (5Sandkvist M. Infect. Immun. 2001; 69: 3523-3535Crossref PubMed Scopus (262) Google Scholar, 14Zalewska-Piatek B. Bury K. Piatek R. Bruzdziak P. Kur J. J. Bacteriol. 2008; 190: 5044-5056Crossref PubMed Scopus (18) Google Scholar, 15Horstman A.L. Kuehn M.J. J. Biol. Chem. 2002; 277: 32538-32545Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar, 16Stewart C.R. Rossier O. Cianciotto N.P. J. Bacteriol. 2009; 191: 1537-1546Crossref PubMed Scopus (53) Google Scholar, 17Shi L. Deng S. Marshall M.J. Wang Z. Kennedy D.W. Dohnalkova A.C. Mottaz H.M. Hill E.A. Gorby Y.A. Beliaev A.S. Richardson D.J. Zachara J.M. Fredrickson J.K. J. Bacteriol. 2008; 190: 5512-5516Crossref PubMed Scopus (100) Google Scholar). The T2S-dependent proteins are of great interest because many of them play important roles in pathogenesis and/or contribute to bacterial fitness in different ecological niches (6Cianciotto N.P. Trends Microbiol. 2005; 13: 581-588Abstract Full Text Full Text PDF PubMed Scopus (263) Google Scholar, 18Cianciotto N.P. Future Microbiol. 2009; 4: 797-805Crossref PubMed Scopus (63) Google Scholar, 19Evans F.F. Egan S. Kjelleberg S. Environ. Microbiol. 2008; 10: 1101-1107Crossref PubMed Scopus (27) Google Scholar, 20Jha G. Rajeshwari R. Sonti R.V. Mol. Plant Microbe Interact. 2005; 18: 891-898Crossref PubMed Scopus (64) Google Scholar). Many of the T2S exoproteins were originally identified based on the loss of specific enzymatic activities in culture supernatants of the T2S mutants, and to date only a few comprehensive studies have been undertaken to define a broader array of secreted proteins (21DebRoy S. Dao J. Söderberg M. Rossier O. Cianciotto N.P. Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 19146-19151Crossref PubMed Scopus (168) Google Scholar, 22Evans F.F. Raftery M.J. Egan S. Kjelleberg S. J. Proteome Res. 2007; 6: 967-975Crossref PubMed Scopus (42) Google Scholar, 23Coulthurst S.J. Lilley K.S. Hedley P.E. Liu H. Toth I.K. Salmond G.P. J. Biol. Chem. 2008; 283: 23739-23753Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). Identification of the T2S substrates from different bacterial species might help to elucidate the mechanism of exoprotein recognition by the T2S system and provide a better understanding of the general role of T2S in pathogenesis and environmental survival.Our laboratory studies the T2S system in the causative agent of cholera, Vibrio cholerae. Cholera is a life-threatening diarrheal disease that predominantly occurs in developing countries of Asia, Africa, and South America (24Sack D.A. Sack R.B. Chaignat C.L. N. Engl. J. Med. 2006; 355: 649-651Crossref PubMed Scopus (75) Google Scholar). The T2S system, extracellular protein secretion (Eps), of V. cholerae is responsible for secretion of five known proteins, including cholera toxin, chitinase (ChiA-1), chitin-binding protein (GbpA), hemagglutinin protease (HAP), and lipase (25Sandkvist M. Morales V. Bagdasarian M. Gene. 1993; 123: 81-86Crossref PubMed Scopus (65) Google Scholar, 26Overbye L.J. Sandkvist M. Bagdasarian M. Gene. 1993; 132: 101-106Crossref PubMed Scopus (78) Google Scholar, 27Kirn T.J. Jude B.A. Taylor R.K. Nature. 2005; 438: 863-866Crossref PubMed Scopus (227) Google Scholar, 28Connell T.D. Metzger D.J. Lynch J. Folster J.P. J. Bacteriol. 1998; 180: 5591-5600Crossref PubMed Google Scholar, 29Sikora A.E. Lybarger S.R. Sandkvist M. J. Bacteriol. 2007; 189: 8484-8495Crossref PubMed Scopus (49) Google Scholar, 30Davis B.M. Lawson E.H. Sandkvist M. Ali A. Sozhamannan S. Waldor M.K. Science. 2000; 288: 333-335Crossref PubMed Scopus (87) Google Scholar).V. cholerae circulates between two very diverse environments, aquatic reservoirs and the gastrointestinal tract of the human body (31Cottingham K.L. Chiavelli D.A. Taylor R.K. Front. Ecol. Environ. 2003; 1: 80-86Crossref Google Scholar). The human host acquires V. cholerae through an oral route of infection with contaminated water or food. Following colonization of the small intestine, the bacteria produce and secrete cholera toxin. Although the disease is multifaceted, cholera toxin is the major virulence factor. It stimulates constitutive activation of cellular adenylate cyclase causing severe intestinal fluid loss and watery diarrhea. The rice-water like stools from cholera patients contain a large number of V. cholerae cells that are often shed back to the environment (32Nelson E.J. Harris J.B. Morris Jr., J.G. Calderwood S.B. Camilli A. Nat. Rev. Microbiol. 2009; 7: 693-702Crossref PubMed Scopus (378) Google Scholar). In the aquatic environment, V. cholerae associates with chitin particles, phyto- and zooplankton, and Chironomidae (nonbiting midges) egg masses (33Nalin D.R. Daya V. Reid A. Levine M.M. Cisneros L. Infect. Immun. 1979; 25: 768-770Crossref PubMed Google Scholar, 34Lipp E.K. Huq A. Colwell R.R. Clin. Microbiol. Rev. 2002; 15: 757-770Crossref PubMed Scopus (527) Google Scholar, 35Broza M. Halpern M. Nature. 2001; 412: 40Crossref PubMed Scopus (105) Google Scholar). The persistence of V. cholerae in the aquatic niche is likely facilitated by several of the known T2S substrates. GbpA is important for V. cholerae attachment to biotic and abiotic chitin surfaces; ChiA1 and HAP play a role in utilization of chitin and insect egg masses as a source of nutrients, respectively (27Kirn T.J. Jude B.A. Taylor R.K. Nature. 2005; 438: 863-866Crossref PubMed Scopus (227) Google Scholar, 28Connell T.D. Metzger D.J. Lynch J. Folster J.P. J. Bacteriol. 1998; 180: 5591-5600Crossref PubMed Google Scholar, 36Halpern M. Gancz H. Broza M. Kashi Y. Appl. Environ. Microbiol. 2003; 69: 4200-4204Crossref PubMed Scopus (69) Google Scholar). Moreover, gbpA mutants display defects in adherence to human epithelial cells and in colonization of infant mice, suggesting that GbpA represents a dual colonization factor functioning both in the aquatic environment and the human host (27Kirn T.J. Jude B.A. Taylor R.K. Nature. 2005; 438: 863-866Crossref PubMed Scopus (227) Google Scholar). Similarly, HAP has been suggested to play a role in infection by facilitating the penetration of V. cholerae through the mucus barrier of the gastrointestinal tract and by causing its detachment from the epithelium following the induction of the secretory diarrhea (37Finkelstein R.A. Boesman-Finkelstein M. Holt P. Proc. Natl. Acad. Sci. U.S.A. 1983; 80: 1092-1095Crossref PubMed Scopus (121) Google Scholar, 38Silva A.J. Leitch G.J. Camilli A. Benitez J.A. Infect. Immun. 2006; 74: 2072-2079Crossref PubMed Scopus (62) Google Scholar).Despite the significant impact of known T2S substrates on V. cholerae pathogenesis and physiology, comprehensive studies have not yet been undertaken to identify additional secreted factors. In pursuit of identifying the T2S secretome of V. cholerae, here we have applied global proteomic approaches, including a high throughput technique (iTRAQ) coupled with multidimensional liquid chromatography (LC) and tandem mass spectrometry (MS/MS). We identified 16 new putative T2S-dependent exoproteins, including three related serine proteases VesA, VesB, and VesC. This study focuses on the characterization of these three newly identified proteins.
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