Key points are not available for this paper at this time.
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. 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. L. Infect. Immun. PubMed Google Scholar, A. Microbiol. Rev. 2002; PubMed Scopus Google Scholar, M. M. Nature. 2001; PubMed Scopus Google Scholar). The of V. cholerae in the aquatic is by several of the known T2S is important for V. cholerae to and chitin and play a role in of chitin and egg masses as a of 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, M. H. M. Environ. Microbiol. 2003; 69: PubMed Scopus Google Scholar). in to human cells and in colonization of that a colonization in the aquatic environment and the human host T.J. Jude B.A. Taylor R.K. Nature. 2005; 438: 863-866Crossref PubMed Scopus (227) Google Scholar). has been to play a role in infection by the of V. cholerae through the of the gastrointestinal tract and by causing from the the of the M. P. Proc. Natl. Acad. Sci. U.S.A. PubMed Scopus Google Scholar, Camilli A. Infect. Immun. 2006; PubMed Scopus Google the of known T2S substrates on V. cholerae pathogenesis and comprehensive studies have not yet been undertaken to secreted In of the T2S of V. cholerae, we have applied proteomic approaches, including a coupled with multidimensional liquid chromatography and tandem mass identified 16 new putative T2S-dependent including three serine proteases VesA, VesB, and VesC. This on the characterization of these three identified is the first a comprehensive analysis of the V. cholerae have used a of proteomic to the protein of and and we the of T2S substrates from five to and that different proteomic is and complex protein are Although of the be for a of proteins, the from proteomic that V. cholerae a variety of including sialidase, cytolysin, two aminopeptidases, four three serine and a TagA-related protein in to cholera toxin, HAP, GbpA, and lipase and to other T2S in V. cholerae the T2S machinery is in transport of enzymes that play a role in the and and might bacterial fitness in different ecological including the human The T2S machinery is of as many as different substrates, as for the T2S system in L. N.P. Future Microbiol. 2009; 4: 797-805Crossref PubMed Scopus (63) Google Scholar, 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). proteomic studies on the analysis of the of V. cholerae under a growth and the of the proteins in V. cholerae was not cholera toxin and two identified T2S substrates, were not in this because the of are under and growth that from the used A.E. Lybarger S.R. Sandkvist M. J. Bacteriol. 2007; 189: 8484-8495Crossref PubMed Scopus (49) Google Scholar, M. K. J. Microbiol. PubMed Google Scholar). the other we that V. cholerae and several proteins to play a role in different steps of chitin and the were in the of This was not because has been shown that these in V. cholerae in R. A. J. Microbiol. 2007; 103: PubMed Scopus Google Scholar). Besides these proteins play an in V. cholerae by participating in processing of other or proteins discovered in the used to the mechanism of recognition by the T2S machinery and provide a new for the of secreted proteins in V. cholerae the of cholera toxin, HAP, and GbpA, the of secreted proteins on V. cholerae pathogenesis and is we on the initial characterization of the three identified VesA, VesB, and VesC, and activities a used for proteases. several of that VesA a role in the processing of the A subunit of cholera toxin in has been well that growth of V. cholerae in the of serine protease the processing and activation of we that the was that of VesA J. Biol. Chem. Full Text PDF PubMed Google with proteins have shown that proteases as HAP, and are of processing A B.A. M. Infect. Immun. PubMed Google Scholar, J. Biol. Chem. Full Text PDF PubMed Google Scholar). studies with human intestinal cell have that the of an host serine is to process A C. S. A. R.K. T.R. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). In of these is very that host proteases are responsible for A activation during intestinal colonization of V. be yet to be during which Vibrio proteases as VesA and play roles in the processing and activation of A. in this for the first a analysis that V. cholerae N16961 that VesA is the primary protease responsible for A processing in on the of the A was in the knock-out that is of A on by and the for proteins that are responsible for the processing of A is and B.A. M. Infect. Immun. PubMed Google Scholar). of the very of in V. cholerae strain is the primary protease responsible for the processing of A. This is by the that V. cholerae N16961 in the of the serine protease which VesA and VesB, A not that protease is of processing cholera toxin under and a very has shown that induces a into that play a role in pathogenesis A. S. H. D.R. M.K. R.K. A. PubMed Scopus Google Scholar). we have this not to be for intestinal colonization of that the T2S system of V. cholerae proteins that are with cholera toxin, including VesA, and a that is responsible for the to in the intestinal and of the for cholera toxin I. H. Taylor M. V. M.J. M. Taylor G. J. Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar, J.M. A. Richardson J.B. Infect. Immun. PubMed Google Scholar). VesA, and the and activation of the toxin the of the T2S system in V. cholerae 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. L. Infect. Immun. PubMed Google Scholar, A. Microbiol. Rev. 2002; PubMed Scopus Google Scholar, M. M. Nature. 2001; PubMed Scopus Google Scholar). The of V. cholerae in the aquatic is by several of the known T2S is important for V. cholerae to and chitin and play a role in of chitin and egg masses as a of 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, M. H. M. Environ. Microbiol. 2003; 69: PubMed Scopus Google Scholar). in to human cells and in colonization of that a colonization in the aquatic environment and the human host T.J. Jude B.A. Taylor R.K. Nature. 2005; 438: 863-866Crossref PubMed Scopus (227) Google Scholar). has been to play a role in infection by the of V. cholerae through the of the gastrointestinal tract and by causing from the the of the M. P. Proc. Natl. Acad. Sci. U.S.A. PubMed Scopus Google Scholar, Camilli A. Infect. Immun. 2006; PubMed Scopus Google the of known T2S substrates on V. cholerae pathogenesis and comprehensive studies have not yet been undertaken to secreted In of the T2S of V. cholerae, we have applied proteomic approaches, including a coupled with multidimensional liquid chromatography and tandem mass identified 16 new putative T2S-dependent including three serine proteases VesA, VesB, and VesC. This on the characterization of these three identified
Sikora et al. (Wed,) studied this question.