Calcium Is Essential for the Major Pseudopilin in the Type 2 Secretion System

The pseudopilus is a key feature of the type 2 secretion system (T2SS) and is made up of multiple pseudopilins that are similar in fold to the type 4 pilins. However, pilins have disulfide bridges, whereas the major pseudopilins of T2SS do not. A key question is therefore how the pseudopilins, and in particular, the most abundant major pseudopilin, GspG, obtain sufficient stability to perform their function. Crystal structures of Vibrio cholerae, Vibrio vulnificus, and enterohemorrhagic Escherichia coli (EHEC) GspG were elucidated, and all show a calcium ion bound at the same site. Conservation of the calcium ligands fully supports the suggestion that calcium ion binding by the major pseudopilin is essential for the T2SS. Functional studies of GspG with mutated calcium ion-coordinating ligands were performed to investigate this hypothesis and show that in vivo protease secretion by the T2SS is severely impaired. Taking all evidence together, this allows the conclusion that, in complete contrast to the situation in the type 4 pili system homologs, in the T2SS, the major protein component of the central pseudopilus is dependent on calcium ions for activity. The pseudopilus is a key feature of the type 2 secretion system (T2SS) and is made up of multiple pseudopilins that are similar in fold to the type 4 pilins. However, pilins have disulfide bridges, whereas the major pseudopilins of T2SS do not. A key question is therefore how the pseudopilins, and in particular, the most abundant major pseudopilin, GspG, obtain sufficient stability to perform their function. Crystal structures of Vibrio cholerae, Vibrio vulnificus, and enterohemorrhagic Escherichia coli (EHEC) GspG were elucidated, and all show a calcium ion bound at the same site. Conservation of the calcium ligands fully supports the suggestion that calcium ion binding by the major pseudopilin is essential for the T2SS. Functional studies of GspG with mutated calcium ion-coordinating ligands were performed to investigate this hypothesis and show that in vivo protease secretion by the T2SS is severely impaired. Taking all evidence together, this allows the conclusion that, in complete contrast to the situation in the type 4 pili system homologs, in the T2SS, the major protein component of the central pseudopilus is dependent on calcium ions for activity. In Gram-negative bacteria, the type 2 secretion system (T2SS) 2The abbreviations used are: T2SStype 2 secretion systemEHECenterohemorrhagic E. coliEpsextracellular protein secretionGspgeneral secretory pathwayASUasymmetric unitr.m.s.d.root mean square deviationBis-Tris2-(bis(2-hydroxyethyl)amino)-2-(hydroxymethyl)propane-1,3-diolCHES2-(cyclohexylamino)ethanesulfonic acidTLStranslation/libration/ screw. is used for the secretion of several important proteins across the outer membrane (1Cianciotto N.P. Trends Microbiol. 2005; 13: 581-588Abstract Full Text Full Text PDF PubMed Scopus (264) Google Scholar). The T2SS is also called the terminal branch of the general secretory pathway (Gsp) (2Pugsley A.P. Possot O. Mol. Microbiol. 1993; 10: 665-674Crossref PubMed Scopus (49) Google Scholar) and, in Vibrio species, the extracellular protein secretion (Eps) apparatus (3Sandkvist M. Michel L.O. Hough L.P. Morales V.M. Bagdasarian M. Koomey M. DiRita V.J. Bagdasarian M. J. Bacteriol. 1997; 179: 6994-7003Crossref PubMed Google Scholar). This sophisticated multiprotein machinery spans both the inner and the outer membrane of Gram-negative bacteria and contains 11–15 different proteins. The T2SS consists of three major subassemblies (4Johnson T.L. Abendroth J. Hol W.G. Sandkvist M. FEMS Microbiol. Lett. 2006; 255: 175-186Crossref PubMed Scopus (181) Google Scholar, 5Filloux A. Biochim. Biophys. Acta. 2004; 1694: 163-179Crossref PubMed Scopus (220) Google Scholar, 6Peabody C.R. Chung Y.J. Yen M.R. Vidal-Ingigliardi D. Pugsley A.P. Saier Jr., M.H. Microbiology. 2003; 149: 3051-3072Crossref PubMed Scopus (283) Google Scholar, 7Keizer D.W. Slupsky C.M. Kalisiak M. Campbell A.P. Crump M.P. Sastry P.A. Hazes B. Irvin R.T. Sykes B.D. J. Biol. Chem. 2001; 276: 24186-24193Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar, 8Sandkvist M. Infect. Immun. 2001; 69: 3523-3535Crossref PubMed Scopus (266) Google Scholar, 9Sauvonnet N. Vignon G. Pugsley A.P. Gounon P. EMBO J. 2000; 19: 2221-2228Crossref PubMed Scopus (186) Google Scholar): (i) the outer membrane complex comprising mainly the crucial multisubunit secretin GspD; (ii) the pseudopilus, which consists of one major and several minor pseudopilins; and (iii) an inner membrane platform, containing the cytoplasmic secretion ATPase GspE and the membrane proteins GspL, GspM, GspC, and GspF. type 2 secretion system enterohemorrhagic E. coli extracellular protein secretion general secretory pathway asymmetric unit root mean square deviation 2-(bis(2-hydroxyethyl)amino)-2-(hydroxymethyl)propane-1,3-diol 2-(cyclohexylamino)ethanesulfonic acid translation/libration/ screw. The pseudopilus is a key element of the T2SS that forms a helical fiber spanning the periplasm. The fiber is assembled from multiple subunits of the major pseudopilin GspG (4Johnson T.L. Abendroth J. Hol W.G. Sandkvist M. FEMS Microbiol. Lett. 2006; 255: 175-186Crossref PubMed Scopus (181) Google Scholar, 5Filloux A. Biochim. Biophys. Acta. 2004; 1694: 163-179Crossref PubMed Scopus (220) Google Scholar, 10Nunn D.N. Lory S. Proc. Natl. Acad. Sci. U.S.A. 1991; 88: 3281-3285Crossref PubMed Scopus (178) Google Scholar, 11Nunn D.N. Lory S. J. Bacteriol. 1993; 175: 4375-4382Crossref PubMed Google Scholar, 12Sandkvist M. Mol. Microbiol. 2001; 40: 271-283Crossref PubMed Scopus (325) Google Scholar, 13Köhler R. Schäfer K. Müller S. Vignon G. Diederichs K. Philippsen A. Ringler P. Pugsley A.P. Engel A. Welte W. Mol. Microbiol. 2004; 54: 647-664Crossref PubMed Scopus (92) Google Scholar, 14Durand E. Bernadac A. Ball G. Lazdunski A. Sturgis J.N. Filloux A. J. Bacteriol. 2003; 185: 2749-2758Crossref PubMed Scopus (131) Google Scholar). The pseudopilus is thought to form a plug of the secretin pore in the outer membrane and/or to function as a piston during protein secretion. In recent years, studies of the T2SS pseudopilins led to structure determinations of all individual pseudopilins (13Köhler R. Schäfer K. Müller S. Vignon G. Diederichs K. Philippsen A. Ringler P. Pugsley A.P. Engel A. Welte W. Mol. Microbiol. 2004; 54: 647-664Crossref PubMed Scopus (92) Google Scholar, 15Yanez M.E. Korotkov K.V. Abendroth J. Hol W.G. J. Mol. Biol. 2008; 375: 471-486Crossref PubMed Scopus (39) Google Scholar, 16Yanez M.E. Korotkov K.V. Abendroth J. Hol W.G. J. Mol. Biol. 2008; 377: 91-103Crossref PubMed Scopus (41) Google Scholar, 17Korotkov K.V. Hol W.G. Nat. Struct. Mol. Biol. 2008; 15: 462-468Crossref PubMed Scopus (109) Google Scholar). The recent structure of the helical ternary complex of GspK-GspI-GspJ suggested that these three minor pseudopilins form the tip of the pseudopilus (17Korotkov K.V. Hol W.G. Nat. Struct. Mol. Biol. 2008; 15: 462-468Crossref PubMed Scopus (109) Google Scholar). A crystal structure of GspG from Klebsiella oxytoca was in a previous study combined with electron microscopy data to arrive at a helical arrangement, with no evidence for special features, such as disulfide bridges, other covalent links, or metal-binding sites, for stabilizing this major pseudopilin or the pseudopilus (13Köhler R. Schäfer K. Müller S. Vignon G. Diederichs K. Philippsen A. Ringler P. Pugsley A.P. Engel A. Welte W. Mol. Microbiol. 2004; 54: 647-664Crossref PubMed Scopus (92) Google Scholar). The pseudopilins of the T2SS share a common fold with the type 4 pilins (15Yanez M.E. Korotkov K.V. Abendroth J. Hol W.G. J. Mol. Biol. 2008; 375: 471-486Crossref PubMed Scopus (39) Google Scholar, 16Yanez M.E. Korotkov K.V. Abendroth J. Hol W.G. J. Mol. Biol. 2008; 377: 91-103Crossref PubMed Scopus (41) Google Scholar, 17Korotkov K.V. Hol W.G. Nat. Struct. Mol. Biol. 2008; 15: 462-468Crossref PubMed Scopus (109) Google Scholar, 18Parge H.E. Forest K.T. Hickey M.J. Christensen D.A. Getzoff E.D. Tainer J.A. Nature. 1995; 378: 32-38Crossref PubMed Scopus (400) Google Scholar, 19Craig L. Pique M.E. Tainer J.A. Nat. Rev. Microbiol. 2004; 2: 363-378Crossref PubMed Scopus (571) Google Scholar, 20Craig L. Volkmann N. Arvai A.S. Pique M.E. Yeager M. Egelman E.H. Tainer J.A. Mol. Cell. 2006; 23: 651-662Abstract Full Text Full Text PDF PubMed Scopus (321) Google Scholar, 21Hansen J.K. Forest K.T. J. Mol. Microbiol. Biotechnol. 2006; 11: 192-207Crossref PubMed Scopus (66) Google Scholar). Pilins are proteins incorporated into pili, long appendages on the surface of bacteria forming thin, strong fibers with multiple functions (19Craig L. Pique M.E. Tainer J.A. Nat. Rev. Microbiol. 2004; 2: 363-378Crossref PubMed Scopus (571) Google Scholar, 21Hansen J.K. Forest K.T. J. Mol. Microbiol. Biotechnol. 2006; 11: 192-207Crossref PubMed Scopus (66) Google Scholar). Type 4 pilins and pseudopilins contain a prepilin leader sequence that is cleaved off by a prepilin peptidase, yielding mature protein (10Nunn D.N. Lory S. Proc. Natl. Acad. Sci. U.S.A. 1991; 88: 3281-3285Crossref PubMed Scopus (178) Google Scholar, 11Nunn D.N. Lory S. J. Bacteriol. 1993; 175: 4375-4382Crossref PubMed Google Scholar, 22Nunn D. Trends Cell Biol. 1999; 9: 402-408Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar). A distinct feature of the type 4 pilins is the occurrence of a disulfide bridge connecting β4 to a Cys in the so-called “D-region” near the C terminus (21Hansen J.K. Forest K.T. J. Mol. Microbiol. Biotechnol. 2006; 11: 192-207Crossref PubMed Scopus (66) Google Scholar). In a recent study (23Kang H.J. Coulibaly F. Clow F. Proft T. Baker E.N. Science. 2007; 318: 1625-1628Crossref PubMed Scopus (271) Google Scholar) on the thin fibers of Gram-positive bacteria, isopeptide units appeared to be essential for providing these filaments sufficient cohesion and stability. A key question was therefore whether the major pseudopilin GspG also requires a special feature to obtain sufficient stability to perform its function. The gene fragment corresponding to the soluble domain of V. cholerae GspG (residues 26–137) was cloned into a pCDFDuet-1-based vector (Novagen) for expression with an N-terminal hexahistidine tag followed by a tobacco etch virus protease cleavage site. BL21(DE3) Escherichia coli cells were grown in Luria broth at 37 °C and induced for 3 h with 0.5 mm isopropyl-1-thio-β-d-galactopyranoside at 30 °C. GspG was purified from the soluble fraction of the lysed cells using nickel-nitrilotriacetic acid (Qiagen) followed by His tag cleavage with tobacco etch virus protease, an ion-exchange purification step using a 30Q column (GE Healthcare), and a final size exclusion on a Superdex 75 column (GE Healthcare) for both ion-exchange peaks. After dialysis against 10 mm sodium acetate, pH 5.0, crystals were obtained only for the first ion-exchange peak. The optimized crystals were grown using the vapor diffusion method in 22.5% polyethylene glycol 3350, 0.1 m sodium acetate, pH 5.0, 0.04 m zinc acetate. The crystals diffracted to ∼4 Å initially. After two cycles of crystal annealing by blocking the cryo-stream, the resolution limit improved to 2.5 Å, and a preliminary data set was collected in-house (supplemental Table 1). Data were processed using HKL2000 (24Otwinowski Z. Minor W. Methods Enzymol. 1997; 276: 307-326Crossref PubMed Scopus (38572) Google Scholar). The structure was solved by molecular replacement with Phaser (25McCoy A.J. Grosse-Kunstleve R.W. Adams P.D. Winn M.D. Storoni L.C. Read R.J. J. Appl. Cryst. 2007; 40: 658-674Crossref PubMed Scopus (14559) Google Scholar) using the structure of K. oxytoca GspG (13Köhler R. Schäfer K. Müller S. Vignon G. Diederichs K. Philippsen A. Ringler P. Pugsley A.P. Engel A. Welte W. Mol. Microbiol. 2004; 54: 647-664Crossref PubMed Scopus (92) Google Scholar) as a model. In the search model, the non-equivalent residues were truncated to Ala/Gly, and the swapped β4-strand was removed. After density modification with Resolve (26Terwilliger T. J Synchrotron Radiat. 2004; 11: 49-52Crossref PubMed Scopus (361) Google Scholar), it was apparent that there are several metal ions in the structure. Those ions were initially thought to be Zn2+ ions present in the crystallization solution. Therefore, the data were collected at zinc remote and inflection wavelengths at beamline BL9-2 at the Stanford Synchrotron Radiation Laboratory (SSRL). Using anomalous data from either zinc remote or a combination of zinc remote/inflection data sets, SHELXD (27Sheldrick G.M. Acta Crystallogr. A. 2008; 64: 112-122Crossref PubMed Scopus (80754) Google Scholar) found three zinc positions, two with full occupancy and one with ∼50% occupancy. The initial model was improved using ARP/wARP (28Perrakis A. Harkiolaki M. Wilson K.S. Lamzin V.S. Acta Crystallogr. D Biol. Crystallogr. 2001; 57: 1445-1450Crossref PubMed Scopus (460) Google Scholar) and finalized using manual rebuilding in Coot (29Emsley P. Cowtan K. Acta Crystallogr. D Biol. Crystallogr. 2004; 60: 2126-2132Crossref PubMed Scopus (23384) Google Scholar) alternating with refinement by REFMAC5 (30Murshudov G.N. Vagin A.A. Dodson E.J. Acta Crystallogr. D. 1997; 53: 240-255Crossref PubMed Scopus (13870) Google Scholar) using one TLS group per chain. From subsequent tests, it appeared that the structure could have been solved ab initio using the zinc anomalous signal. The two monomers in the asymmetric unit (ASU) form an antiparallel dimer with two Zn2+ ions in the interface and a third Zn2+ ion participating in crystal contact with a symmetry-related molecule (Fig. 1). The two monomers superimpose with an r.m.s.d. of 0.4 Å over 112 Cα atoms. The gene fragments corresponding to the soluble domains of V. vulnificus GspG (residues 26–137) and EHEC GspG (residues 17–136) were cloned into a pCDFDuet-1-based vector similar to V. cholerae GspG. The expression was performed as for V. cholerae GspG, except that Luria broth was supplemented with 1 mm calcium chloride, but no calcium chloride was added during subsequent purification and crystallization. The purification followed the procedure outlined for V. cholerae GspG with the first ion-exchange peak used for crystallization. Crystals of V. vulnificus GspG were grown using the vapor diffusion method in 1.95 mdl-malic acid, pH 5.0. A data set was collected at beamline BL9-2 at SSRL (supplemental Table 1). Data were processed using XDS (31Kabsch W. J. Appl. Cryst. 1993; 26: 795-800Crossref Scopus (3232) Google Scholar). The structure was solved by molecular replacement with Phaser (25McCoy A.J. Grosse-Kunstleve R.W. Adams P.D. Winn M.D. Storoni L.C. Read R.J. J. Appl. Cryst. 2007; 40: 658-674Crossref PubMed Scopus (14559) Google Scholar) using the V. cholerae GspG structure as a model. After manual rebuilding in Coot (29Emsley P. Cowtan K. Acta Crystallogr. D Biol. Crystallogr. 2004; 60: 2126-2132Crossref PubMed Scopus (23384) Google Scholar), the three monomers in the ASU were refined with REFMAC5 (30Murshudov G.N. Vagin A.A. Dodson E.J. Acta Crystallogr. D. 1997; 53: 240-255Crossref PubMed Scopus (13870) Google Scholar) using three TLS groups per chain as defined by the TLSMD server (32Painter J. Merritt E.A. Acta Crystallogr D Biol Crystallogr. 2006; 62: 439-450Crossref PubMed Scopus (1106) Google Scholar). The three subunits in the ASU superimpose pairwise with an r.m.s.d. of 0.2–0.4 Å over 111 Cα atoms. The V. vulnificus GspG structures superimpose onto the V. cholerae GspG structures with an r.m.s.d. of 0.6–0.8 Å over 111 Cα atoms and 91% sequence identity in the superimposed region. A cluster of crystals was obtained during screening in a condition containing 1.0 m sodium citrate, 0.1 m CHES, pH 9.5. An individual crystal was separated from the cluster, frozen, and used for data collection. Data were collected at beamline BL9-2 at SSRL and processed using XDS (31Kabsch W. J. Appl. Cryst. 1993; 26: 795-800Crossref Scopus (3232) Google Scholar). The structure was solved by molecular replacement with Phaser (25McCoy A.J. Grosse-Kunstleve R.W. Adams P.D. Winn M.D. Storoni L.C. Read R.J. J. Appl. Cryst. 2007; 40: 658-674Crossref PubMed Scopus (14559) Google Scholar) using the V. cholerae GspG structure, with the Ca2+-binding loop and C-terminal α-helix removed, as a search model. The two monomers in the ASU were rebuilt using ARP/wARP (28Perrakis A. Harkiolaki M. Wilson K.S. Lamzin V.S. Acta Crystallogr. D Biol. Crystallogr. 2001; 57: 1445-1450Crossref PubMed Scopus (460) Google Scholar) and finalized using Coot (29Emsley P. Cowtan K. Acta Crystallogr. D Biol. Crystallogr. 2004; 60: 2126-2132Crossref PubMed Scopus (23384) Google Scholar). The structure was refined with REFMAC5 (30Murshudov G.N. Vagin A.A. Dodson E.J. Acta Crystallogr. D. 1997; 53: 240-255Crossref PubMed Scopus (13870) Google Scholar) using three TLS groups per chain as defined by the TLSMD server (32Painter J. Merritt E.A. Acta Crystallogr D Biol Crystallogr. 2006; 62: 439-450Crossref PubMed Scopus (1106) Google Scholar). The two subunits in the ASU superimpose with an r.m.s.d. of 2.0 Å over 114 Cα atoms. Large only in the N-terminal The exclusion of residues from the the r.m.s.d. to 0.5 onto the V. cholerae GspG structure an r.m.s.d. of Å over Cα atoms with sequence identity in the superimposed region. The and the were as Sandkvist M. J. Bacteriol. 2007; PubMed Scopus Google Scholar). were in gene with using as a used for the in and were and was using as a and the for the were by were grown in Luria broth supplemented with and and to the and The were and the protease was as (4Johnson T.L. Abendroth J. Hol W.G. Sandkvist M. FEMS Microbiol. Lett. 2006; 255: 175-186Crossref PubMed Scopus (181) Google Scholar). were grown to were by cells in with mm and for 10 of 1.0 was onto and GspG was with against V. cholerae GspG, by and (GE system and were used for The soluble domain of the V. cholerae GspG (residues 26–137) was and purified similar to the A peak was obtained the ion-exchange The stability of proteins was by using as as A. G. R. J. M. G. 2004; PubMed Scopus Google Scholar), in the of 1 mm calcium chloride or in the of 1 mm were for The of GspG was and 0.4 °C for no calcium chloride, and for the were and 0.4 °C for no calcium chloride, and sequence were made using R. T. R. 2003; PubMed Scopus Google Scholar). P. E. 2003; PubMed Scopus Google Scholar) was to the The were using The Scholar). solved the crystal structures of V. cholerae, V. vulnificus, and EHEC all to 2.0 Å yielding a of of these three GspG (supplemental Table 1 and and these structures with the structure of K. oxytoca GspG and show the fold of an N-terminal a and a C-terminal However, found two that have been the C-terminal residues in all GspG structures a helical 2 and in contrast with a in K. oxytoca GspG. In the structure, a the two pseudopilin in the asymmetric unit (13Köhler R. Schäfer K. Müller S. Vignon G. Diederichs K. Philippsen A. Ringler P. Pugsley A.P. Engel A. Welte W. Mol. Microbiol. 2004; 54: 647-664Crossref PubMed Scopus (92) Google Scholar). a distinct electron density is present in of the two subunits of the V. cholerae GspG structure, by the of and and by chain and in an The density of the peak (Fig. and the of ligands initial evidence that the ion bound is a calcium evidence that the two ions in the loop are calcium ions (i) the anomalous using the Å data and (Fig. at the Ca2+-binding sites, which is to the and at the zinc with full the for and the for Zn2+ at this (ii) the anomalous in the Å data set show and at the two Ca2+-binding with the and at the zinc with full which with the for and the for Zn2+ at this (iii) refinement as calcium ions at full occupancy for the of in chain A and in chain which are to the of and for the atoms. The structure determinations of V. vulnificus and EHEC GspG were to the of ions across the GspG In the V. vulnificus structure, the of the three subunits have the of the structure, the evidence of calcium binding is The anomalous (supplemental using the Å and at the Ca2+-binding in and corresponding with the of for at this The chain of in chain C of the Ca2+-binding to in a crystal on the of anomalous refined the ion in chain C at occupancy. of the calcium ions of in chain in chain and in chain which are to the of and for the atoms. The acid of from EHEC and K. oxytoca but distinct sequence in the Ca2+-binding with of V. cholerae and V. vulnificus The proteins have an of three forming in the Vibrio GspG the two (Fig. and 1). in the EHEC GspG structure, both subunits show a electron density with binding in the same as in V. cholerae and V. vulnificus GspG. The for the metal ions with full occupancy are in chain A and in chain which are to the of and for the atoms. The ligands of the EHEC calcium are and which are to and of V. cholerae GspG, with as the of and with the of and the of a (Fig. The of the ion is in with The GspG sequence (supplemental that the two are are a and only in is one an also how the in the Ca2+-binding with the of three forming the V. cholerae GspG in the therefore to be two of major is a with chain ligands two ligands and a the two The is the with two chain one chain two and no the two is most that K. oxytoca GspG to the and that the in the crystal structure (13Köhler R. Schäfer K. Müller S. Vignon G. Diederichs K. Philippsen A. Ringler P. Pugsley A.P. Engel A. Welte W. Mol. Microbiol. 2004; 54: 647-664Crossref PubMed Scopus (92) Google Scholar) the site. The of by GspG was by the residues and in V. cholerae GspG with and the on extracellular secretion of protease by V. protease secretion was was in a GspG (Fig. that the of the two T2SS function by The and (Fig. either in with the chain from and is of of from the that the and the proteins were at similar to that of GspG (Fig. also stability of the and the GspG using a A. G. R. J. M. G. 2004; PubMed Scopus Google Scholar, D. J.K. M.J. 2005; PubMed Scopus Google Scholar, V.S. J. E. T. E. P. J. 2001; PubMed Google Scholar) and found that the is GspG, with a of °C °C. the stability of the was by or whereas the stability of the protein and studies that the of the in V. cholerae GspG the of the T2SS. The of calcium binding by GspG could protein secretion by the T2SS in a of for by a of the C-terminal for in the pseudopilus (supplemental that three major used by secretion are dependent on different stabilizing disulfide in the type 4 pilins (21Hansen J.K. Forest K.T. J. Mol. Microbiol. Biotechnol. 2006; 11: 192-207Crossref PubMed Scopus (66) Google Scholar), isopeptide in the thin fibers of Gram-positive bacteria (23Kang H.J. Coulibaly F. Clow F. Proft T. Baker E.N. Science. 2007; 318: 1625-1628Crossref PubMed Scopus (271) Google Scholar), and calcium ions in the pseudopilus of the T2SS an calcium is and in the tip pseudopilin (17Korotkov K.V. Hol W.G. Nat. Struct. Mol. Biol. 2008; 15: 462-468Crossref PubMed Scopus (109) Google Scholar). the T2SS is dependent on calcium binding in multiple from the of of for providing the EHEC for the and the SSRL for with