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Two Periplasmic Disulfide Oxidoreductases, DsbA and SrgA, Target Outer Membrane Protein SpiA, a Component of the Salmonella Pathogenicity Island 2 Type III Secretion System

2004; Elsevier BV; Volume: 279; Issue: 33 Linguagem: Inglês

10.1074/jbc.m402760200

1083-351X

Tsuyoshi Miki, Nobuhiko Okada, Hirofumi Danbara,

Escherichia coli research studies

The formation of disulfide is essential for the folding, activity, and stability of many proteins secreted by Gram-negative bacteria. The disulfide oxidoreductase, DsbA, introduces disulfide bonds into proteins exported from the cytoplasm to periplasm. In pathogenic bacteria, DsbA is required to process virulence determinants for their folding and assembly. In this study, we examined the role of the Dsb enzymes in Salmonella pathogenesis, and we demonstrated that DsbA, but not DsbC, is required for the full expression of virulence in a mouse infection model of Salmonella enterica serovar Typhimurium. Salmonella strains carrying a dsbA mutation showed reduced function mediated by type III secretion systems (TTSSs) encoded on Salmonella pathogenicity islands 1 and 2 (SPI-1 and SPI-2). To obtain a more detailed understanding of the contribution of DsbA to both SPI-1 and SPI-2 TTSS function, we identified a protein component of the SPI-2 TTSS apparatus affected by DsbA. Although we found no substrate protein for DsbA in the SPI-1 TTSS apparatus, we identified SpiA (SsaC), an outer membrane protein of SPI-2 TTSS, as a DsbA substrate. Site-directed mutagenesis of the two cysteine residues present in the SpiA protein resulted in the loss of SPI-2 function in vitro and in vivo. Furthermore, we provided evidence that a second disulfide oxidoreductase, SrgA, also oxidizes SpiA. Analysis of in vivo mixed infections demonstrated that a Salmonella dsbA srgA double mutant strain was more attenuated than either single mutant, suggesting that DsbA acts in concert with SrgA in vivo. The formation of disulfide is essential for the folding, activity, and stability of many proteins secreted by Gram-negative bacteria. The disulfide oxidoreductase, DsbA, introduces disulfide bonds into proteins exported from the cytoplasm to periplasm. In pathogenic bacteria, DsbA is required to process virulence determinants for their folding and assembly. In this study, we examined the role of the Dsb enzymes in Salmonella pathogenesis, and we demonstrated that DsbA, but not DsbC, is required for the full expression of virulence in a mouse infection model of Salmonella enterica serovar Typhimurium. Salmonella strains carrying a dsbA mutation showed reduced function mediated by type III secretion systems (TTSSs) encoded on Salmonella pathogenicity islands 1 and 2 (SPI-1 and SPI-2). To obtain a more detailed understanding of the contribution of DsbA to both SPI-1 and SPI-2 TTSS function, we identified a protein component of the SPI-2 TTSS apparatus affected by DsbA. Although we found no substrate protein for DsbA in the SPI-1 TTSS apparatus, we identified SpiA (SsaC), an outer membrane protein of SPI-2 TTSS, as a DsbA substrate. Site-directed mutagenesis of the two cysteine residues present in the SpiA protein resulted in the loss of SPI-2 function in vitro and in vivo. Furthermore, we provided evidence that a second disulfide oxidoreductase, SrgA, also oxidizes SpiA. Analysis of in vivo mixed infections demonstrated that a Salmonella dsbA srgA double mutant strain was more attenuated than either single mutant, suggesting that DsbA acts in concert with SrgA in vivo. The secretion of proteins is a prerequisite for interactions between pathogenic bacteria and their hosts. In Gram-negative bacteria, several types of secretion pathways for proteins that are important for such bacterial interactions with host cells have been identified. The type I secretion mechanism requires three accessory proteins to form a transmembrane structure, which includes a channel spanning the inner and outer membranes. Proteins secreted by this pathway have an uncleaved C-terminal secretion signal responsible for directing the secretion of protein (1Koronakis V. Sharff A. Koronakis E. Luisi B. Hughes C. Nature. 2000; 405: 914-919Crossref PubMed Scopus (870) Google Scholar, 2Koronakis V. Andersen C. Hughes C. Curr. Opin. Struct. Biol. 2001; 11: 403-407Crossref PubMed Scopus (26) Google Scholar, 3Delepelaire P. Wandersman C. J. Biol. Chem. 1990; 265: 17118-17125Abstract Full Text PDF PubMed Google Scholar). The secretion of proteins by the type II secretion pathway (also known as the "general secretion pathway") involves two different steps. Proteins are first translocated through the inner membrane via signal peptides that interact with the Sec-dependent pathway, and the proteins are then transported across the outer membrane by either of several different terminal branches of the process (4Sandkvist M. Mol. Microbiol. 2001; 40: 271-283Crossref PubMed Scopus (327) Google Scholar, 5Pugsley A.P. Microbiol. Rev. 1993; 57: 50-108Crossref PubMed Google Scholar). The type III secretion system (TTSS) 1The abbreviations used are: TTSS, type III secretion system; CI, competitive index; HA, hemagglutinin; cfu, colony-forming units; PBS, phosphate-buffered saline; FBS, fetal bovine serum; DTT, dithiothreitol; SIF, Salmonella-induced filament. functions as a pathway for secretion across bacterial membranes and for the translocation of secreted proteins across the plasma membrane of eukaryotic cells (6Hueck C.J. Microbiol. Mol. Biol. Rev. 1998; 62: 379-433Crossref PubMed Google Scholar). The type IV secretion system also translocates proteins in a single step from the cytoplasm to the cytosol of a host cell (7Christie P.J. Vogel J.P. Trends Microbiol. 2000; 8: 354-360Abstract Full Text Full Text PDF PubMed Scopus (387) Google Scholar). Proteins secreted via the type V secretion system are autotransporter proteins, which are characterized by a unifying structure possessing an N-terminal signal sequence and a pore-forming C-terminal domain (8Henderson I.R. Cappello R. Nataro J.P. Trends Microbiol. 2000; 8: 529-532Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar, 9Henderson I.R. Nataro J.P. Infect. Immun. 2001; 69: 1231-1243Crossref PubMed Scopus (350) Google Scholar). In all of these secretion pathways, many of the proteins residing in or transiting through the periplasmic space acquire disulfide bonds after their translocation across the inner membrane. The formation of disulfide bonds is a key step in the folding of many secreted and membrane proteins. In Escherichia coli, disulfide bond formation is catalyzed by the Dsb proteins (10Kadokura H. Katzen F. Beckwith J. Annu. Rev. Biochem. 2003; 72: 111-135Crossref PubMed Scopus (448) Google Scholar). DsbA is a 21-kDa periplasmic protein with a CXXC motif in its active site, and it interacts with reduced substrate proteins, catalyzing the oxidation of their cysteine residues to disulfide bonds (11Bardwell J.C. McGovern K. Beckwith J. Cell. 1991; 67: 581-589Abstract Full Text PDF PubMed Scopus (830) Google Scholar). The inner membrane protein DsbB oxidizes DsbA (12Bardwell J.C. Lee J.O. Jander G. Martin N. Belin D. Beckwith J. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 1038-1042Crossref PubMed Scopus (361) Google Scholar, 13Guilhot C. Jander G. Martin N.L. Beckwith J. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 9895-9899Crossref PubMed Scopus (128) Google Scholar) and is re-oxidized directly by membrane-bound ubiquinones (14Kobayashi T. Kishigami S. Sone M. Inokuchi H. Mogi T. Ito K. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 11857-11862Crossref PubMed Scopus (208) Google Scholar, 15Bader M. Muse W. Ballou D.P. Gassner C. Bardwell J.C. 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A. 1996; 93: 13048-13053Crossref PubMed Scopus (243) Google Scholar, 19Missiakas D. Schwager F. Raina S. EMBO J. 1995; 14: 3415-3424Crossref PubMed Scopus (169) Google Scholar, 20Rietsch A. Bessette P. Georgiou G. Beckwith J. J. Bacteriol. 1997; 179: 6602-6608Crossref PubMed Scopus (194) Google Scholar, 21Katzen F. Beckwith J. Cell. 2000; 103: 769-779Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar). DsbA plays a central role in periplasmic protein folding. A dsbA mutant of an E. coli strain exhibits numerous in vivo phenotypes, including a loss of motility, an absence of alkaline phosphatase activity, sensitivity to benzylpenicillin and dithiothreitol, and resistance to phage M13, because of severe defects in disulfide bond formation in proteins requiring these processes (11Bardwell J.C. McGovern K. Beckwith J. Cell. 1991; 67: 581-589Abstract Full Text PDF PubMed Scopus (830) Google Scholar, 22Missiakas D. Georgopoulos C. Raina S. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 7084-7088Crossref PubMed Scopus (200) Google Scholar). In addition, dsbA-null mutants have a decreased growth rate in minimal media, as compared with the wild-type strain, and show mucoid colonies when grown on plates in minimal media (11Bardwell J.C. McGovern K. Beckwith J. Cell. 1991; 67: 581-589Abstract Full Text PDF PubMed Scopus (830) Google Scholar). Although DsbC mutants are defective at disulfide bond formation, a mutation in dsbC does not display any obvious phenotype, except for a defect in the expression of dsbC substrates that contain multiple disulfide bonds (17Rietsch A. Belin D. Martin N. Beckwith J. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 13048-13053Crossref PubMed Scopus (243) Google Scholar). In many pathogenic bacteria, DsbA is involved in pathogenicity through the catalysis of oxidative protein folding in virulence determinants. These virulence factors include the following: the cholera toxin of Vibrio cholerae (23Yu J. Webb H. Hirst T.R. Mol. Microbiol. 1992; 6: 1949-1958Crossref PubMed Scopus (112) Google Scholar, 24Peek J.A. Taylor R.K. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 6210-6214Crossref PubMed Scopus (184) Google Scholar); the heat-stable toxin of enterotoxigenic E. coli (25Yamanaka H. Kameyama M. Baba T. Fujii Y. Okamoto K. J. Bacteriol. 1994; 176: 2906-2913Crossref PubMed Google Scholar, 26Okamoto K. Baba T. Yamanaka H. Akashi N. Fujii Y. J. Bacteriol. 1995; 177: 4579-4586Crossref PubMed Google Scholar); a molecular chaperone, PapD, of P pili of uropathogenic E. coli (27Jacob-Dubuisson F. Pinkner J. Xu Z. Striker R. Padmanhaban A. Hultgren S.J. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 11552-11556Crossref PubMed Scopus (128) Google Scholar); bundle-forming pili and Intimin of enteropathogenic E. coli (28Zhang H.Z. Donnenberg M.S. Mol. Microbiol. 1996; 21: 787-797Crossref PubMed Scopus (119) Google Scholar, 29Hicks S. Frankel G. Kaper J.B. Dougan G. Phillips A.D. Infect. Immun. 1998; 66: 1570-1578Crossref PubMed Google Scholar); and Invasin of Yersinia pseudotuberculosis (30Leong J.M. Morrissey P.E. Isberg R.R. J. Biol. Chem. 1993; 268: 20524-20532Abstract Full Text PDF PubMed Google Scholar). DsbA is also required for the proper function of the TTSS in Yersinia pestis (31Jackson M.W. Plano G.V. J. Bacteriol. 1999; 181: 5126-5130Crossref PubMed Google Scholar), Shigella flexneri (32Watarai M. Tobe T. Yoshikawa M. Sasakawa C. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 4927-4931Crossref PubMed Scopus (100) Google Scholar), and Pseudomonas aeruginosa (33Ha U.H. Wang Y. Jin S. Infect. Immun. 2003; 71: 1590-1595Crossref PubMed Scopus (88) Google Scholar). In Y. pestis, a mutation in dsbA results in the unstable expression of an outer membrane protein, YscC, that constitutes the TTSS apparatus, which leads to the decreased translocation of Yop proteins. Substitution of cysteine residues in YscC reproduced all of the phenotypes seen in a dsbA mutation, suggesting that DsbA catalyzes the YscC as a substrate (31Jackson M.W. Plano G.V. J. Bacteriol. 1999; 181: 5126-5130Crossref PubMed Google Scholar). Salmonella enterica is a Gram-negative and facultative intracellular bacterium that is pathogenic to humans and animals; this pathogen is known to cause a broad spectrum of diseases such as gastroenteritis and bacteremia, as well as typhoid fever. The nature and severity of infection by Salmonella is generally dependent upon both the serovar and the host species. Typhoid and paratyphoid fevers result from systemic infection with human-adapted serovars such as S. enterica serovar Typhi and S. enterica serovar Paratyphi A. In contrast, infection with the broad host range-adapted serovar S. enterica serovar Typhimurium usually causes gastroenteritis in humans but produces a systemic infection similar to typhoid fever in susceptible mice. S. enterica utilize two different virulence-associated TTSS, encoded in Salmonella pathogenicity islands 1 and 2 (SPI-1 and SPI-2, respectively), for different stages of pathogenesis. The SPI-1 TTSS is required for the invasion of intestinal epithelial cells and the induction of the inflammatory response in the intestinal mucosa (6Hueck C.J. Microbiol. Mol. Biol. Rev. 1998; 62: 379-433Crossref PubMed Google Scholar, 34Galan J.E. Annu. Rev. Cell Dev. Biol. 2001; 17: 53-86Crossref PubMed Scopus (600) Google Scholar, 35Kaniga K. Tucker S. Trollinger D. Galan J.E. J. Bacteriol. 1995; 177: 3965-3971Crossref PubMed Scopus (257) Google Scholar). In contrast, SPI-2 TTSS is required for intracellular survival in macrophages and for systemic infection in the mouse model (36Ochman H. Soncini F.C. Solomon F. Groisman E.A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 7800-7804Crossref PubMed Scopus (530) Google Scholar, 37Shea J.E. Hensel M. Gleeson C. Holden D.W. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 2593-2597Crossref PubMed Scopus (646) Google Scholar, 38Hensel M. Shea J.E. Waterman S.R. Mundy R. Nikolaus T. Banks G. Vazquez-Torres A. Gleeson C. Fang F.C. Holden D.W. Mol. Microbiol. 1998; 30: 163-174Crossref PubMed Scopus (500) Google Scholar). The complete genome sequence of S. enterica serovar Typhimurium strain LT2 has revealed the presence of Dsb protein homologues (39McClelland M. Sanderson K.E. Spieth J. Clifton S.W. Latreille P. Courtney L. Porwollik S. Ali J. Dante M. Du F. Hou S. Layman D. Leonard S. Nguyen C. Scott K. Holmes A. Grewal N. Mulvaney E. Ryan E. Sun H. Florea L. Miller W. Stoneking T. Nhan M. Waterston R. Wilson R.K. Nature. 2001; 413: 852-856Crossref PubMed Scopus (1522) Google Scholar). Southern hybridization analysis has also demonstrated the wide distribution of a dsbA gene among Salmonella serovars (40Turcot I. Ponnampalam T.V. Bouwman C.W. Martin N.L. Can. J. Microbiol. 2001; 47: 711-721Crossref PubMed Google Scholar). A dsbA gene cloned from the S. enterica serovar Typhimurium can restore the dsbA– phenotype in an E. coli strain (40Turcot I. Ponnampalam T.V. Bouwman C.W. Martin N.L. Can. J. Microbiol. 2001; 47: 711-721Crossref PubMed Google Scholar), demonstrating that Salmonella disulfide oxidoreductase DsbA is functional, although the enzymatic activity of Salmonella DsbA seems to be different from that of E. coli (40Turcot I. Ponnampalam T.V. Bouwman C.W. Martin N.L. Can. J. Microbiol. 2001; 47: 711-721Crossref PubMed Google Scholar). Recently, a number of proteins affected by the dsbA mutation have been identified using two-dimensional gel electrophoresis with comparison of periplasmic proteins expressed in the wild-type and dsbA mutant strains in S. enterica serovar Typhi (41Agudo D. Mendoza M.T. Castanares C. Nombela C. Rotger R. Proteomics. 2004; 4: 355-363Crossref PubMed Scopus (22) Google Scholar). However, the presence of disulfide bonds in these proteins has not been determined. Thus, the membrane and secreted proteins, the folding of which is affected by DsbA in Salmonella, remain still obscure. In the S. enterica serovar Typhimurium, the Salmonella DsbA paralogue SrgA, encoded on the 94-kb virulence plasmid, functions as a disulfide oxidoreductase, whereas the enzymatic activity of SrgA is less efficient than that of DsbA when E. coli alkaline phosphatase is used as a substrate (42Bouwman C.W. Kohli M. Killoran A. Touchie G.A. Kadner R.J. Martin N.L. J. Bacteriol. 2003; 185: 991-1000Crossref PubMed Scopus (68) Google Scholar). SrgA specifically oxidizes the disulfide bond of PefA, the major structural subunit of the plasmid-encoded fimbriae Pef, and thus the disulfide oxidoreductase activity of SrgA is required for the assembly of Pef on the bacterial surface (42Bouwman C.W. Kohli M. Killoran A. Touchie G.A. Kadner R.J. Martin N.L. J. Bacteriol. 2003; 185: 991-1000Crossref PubMed Scopus (68) Google Scholar). Moreover, SrgA activity is dependent upon the presence of functional DsbB (42Bouwman C.W. Kohli M. Killoran A. Touchie G.A. Kadner R.J. Martin N.L. J. Bacteriol. 2003; 185: 991-1000Crossref PubMed Scopus (68) Google Scholar), suggesting that, similar to DsbA, SrgA is recycled to an active oxidized form by DsbB. Recently, it has been shown that a Salmonella strain that contains a mutation in dsbA is highly attenuated in a systemic infection of mice (43Ellermeier C.D. Slauch J.M. J. Bacteriol. 2004; 186: 68-79Crossref PubMed Scopus (43) Google Scholar). A Salmonella dsbA mutant strain has also shown decreased SPI-1 and SPI-2 TTSS function in terms of the translocation of effector proteins into mouse macrophage-like RAW264.7 cells (43Ellermeier C.D. Slauch J.M. J. Bacteriol. 2004; 186: 68-79Crossref PubMed Scopus (43) Google Scholar). However, the interaction between DsbA and the component proteins of SPI-1 and SPI-2 TTSS has not yet been determined. Thus, in order to obtain a more detailed understanding of the role of DsbA in Salmonella pathogenesis, we characterized the effect of DsbA on the activity of both SPI-1 and SPI-2 TTSS, and we identified a protein component of the SPI-2 TTSS apparatus affected by DsbA. Although we found no substrate for DsbA in the SPI-1 TTSS apparatus, we identified SpiA (also referred as SsaC), an outer membrane protein of SPI-2 TTSS, as a DsbA substrate. Furthermore, we demonstrated that a second disulfide oxidoreductase, SrgA, also oxidizes SpiA in vitro. An analysis of in vivo mixed infections showed that a dsbA srgA double mutant strain was more attenuated than either single mutant strain, suggesting that DsbA acts in concert with SrgA in vivo. Bacterial Strains, Plasmids, Primers, and Growth Conditions—The bacterial strains and plasmids used in this study are listed in Table I. The oligonucleotide primers used in this study are listed in Table II. E. coli and Salmonella strains were grown in Luria-Bertani (LB) broth or on LB agar under selection for resistance to ampicillin (100 μg/ml), chloramphenicol (25 μg/ml for plasmid-containing strains, 5 μg/ml for chromosomal integrants), kanamycin (25 μg/ml), nalidixic acid (50 μg/ml), or streptomycin (50 μg/ml), as required. A previously described intracellular salts-based minimal medium with limiting magnesium (MgM, pH 5.8) was used to induce SPI-2 gene expression (44Beuzon C.R. Banks G. Deiwick J. Hensel M. Holden D.W. Mol. Microbiol. 1999; 33: 806-816Crossref PubMed Scopus (174) Google Scholar). Phage P22-mediated transductions for Salmonella have been described previously (45Sternberg N.L. Maurer R. Methods Enzymol. 1991; 204: 18-43Crossref PubMed Scopus (181) Google Scholar). The dsbA::Tn5 mutation of E. coli strain SK101 (46Kamitani S. Akiyama Y. Ito K. EMBO J. 1992; 11: 57-62Crossref PubMed Scopus (225) Google Scholar) was introduced into strain MC1061 by using P1 phages.Table IBacterial strains and plasmids used in this studyNameRelevant characteristicsSource/Ref.Salmonella strainsSL1344Serovar Typhimurium, wild-type70Hoiseth S.K. Stocker B.A. Nature. 1981; 291: 238-239Crossref PubMed Scopus (1595) Google ScholarSB136SL1344 invA::kan, Km71Galan J.E. Ginocchio C. Costeas P. J. Bacteriol. 1992; 174: 4338-4349Crossref PubMed Scopus (444) Google ScholarTM100SL1344 dsbA::kan, KmrThis studyTM202SL1344 dsbC::kan, KmrThis studyTM133SL1344 spiAC133S, Cys-133 to Ser substitutionThis studyTM152SL1344 spiAC152S, Cys-152 to Ser substitutionThis studyTM114SL1344 ΔssaV::cat, CmrThis studyTM233SL1344 ssrA::kan, transductant, KmrThis studyTM171SL1344 ΔsrgA::kan, KmrThis studyTM101TM100 containing pMW-dsbAThis studyTM502SL1344 dsbA::cat, CmrThis studyTM505SL1344 dsbA::cat, ΔsrgA::kanThis studyE. coli strainsDH5αK-12 recA1 endA1 gyrA96 thi-1 hsdR17InvitrogensupE44 Δ(lacXYA-argR)U169 deoR (ϕ80 dlacΔ(lacZ)M15)SM10λpirthi-1 thr leu tonA lacY supE recA::RP4-2-Tc::Mu λpirSK101K-12 dsbA::Tn5, Kmr46Kamitani S. Akiyama Y. Ito K. EMBO J. 1992; 11: 57-62Crossref PubMed Scopus (225) Google ScholarMC1061K-12 araD139 Δ(ara, leu)7697 Δlac(IZY)X74 galE15 galK16 rpsL hsdR2 (rK- mK+) mcrA mcrB172Casadaban M.J. Cohen S.N. J. Mol. Biol. 1980; 138: 179-207Crossref PubMed Scopus (1753) Google ScholarTM161MC1061 dsbA::Tn5, KmrThis studyXL10-GoldK-12 Δ(mcrA)183 Δ(mcrCB-hsdSMR-mrr)173 endA1supE44 thi-1 recA1 gyrA96 relA1 lac HteStratagene[F′ proAB lacIqZΔM15 Tn10(Tetr) Amy Cmr]PlasmidspGEM-T EasyTA cloning vector, AmprPromegapMW118pSC101-based low copy number plasmid, AmprNippon GenepMW119pSC101-based low copy number plasmid, AmprNew England BiolabspACYC184p15A-based low copy number plasmid, Cmr, TetrNew England BiolabspFLAG-CTCFLAG tag expression vector, AmprSigmapUC18KPlasmid coding Kmr-encoding gene cassette48Menard R. Sansonetti P.J. Parsot C. J. Bacteriol. 1993; 175: 5899-5906Crossref PubMed Scopus (616) Google ScholarpCACTUSSuicide vector mobilized by RP4 and sucrose-sensitive, repts, Cmr47Morona R. van den Bosch L. Manning P.A. J. Bacteriol. 1995; 177: 1059-1068Crossref PubMed Scopus (159) Google ScholarpKD46Plasmid expressing λ Red recombinase, Ampr49Datsenko K.A. Wanner B.L. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 6640-6645Crossref PubMed Scopus (11299) Google ScholarpLD-lacZΩIntegrational plasmid with promoterless lacZ gene, SperThis studypMW-dsbAdsbA in pMW118, AmprThis studypAC-dsbAdsbA in pACYC184, CmrThis studypMW-srgAsrgA in pMW119, AmprThis studypAC-srgAsrgA in pACYC184, CmrThis studypMW-spiAspiA in pMW118, AmprThis studypTM9invG in pFLAG-CTC, AmprThis studypTM10spiA in pFLAG-CTC, AmprThis studypTM11spiAC133S in pFLAG-CTC, AmprThis studypTM12spiAC152S in pFLAG-CTC, AmprThis studypTM22sseJ-HA under control of native promoter in pMW118, AmprThis studypLD-hilAZhilA::lacZ operon fusion in pLD-lacZΩThis studypLD-invFZinvF::lacZ operon fusion in pLD-lacZΩThis studypLD-ssrAZssrA::lacZ operon fusion in pLD-lacZΩThis study Open table in a new tab Table IIPrimers used in this studyPrimerSequence (restriction sites underlined)dsbAST-15′-GATTACTGGCCTCGAGAGACAACG-3′ (XhoI)dsbAST-25′-CTGTGGGATCCGAAAGATATACAG-3′ (BamHI)dsbC-FW5′-CAACTTCATCAACTGCACCATCCGC-3′dsbC-RV5′-ATGGAGGAAGATCGGCGGGGAGTTC-3′srgA-FW5′-CTGGTCAGCAGCAGGAGAATCAGTG-3′srgA-RV5′-ACGCATAACCGGAATATTCCGGCTG-3′spiA-FW15′-CACAAGTAGTAGCTCTGAGCTTATTGC-3′spiA-RV15′-GGTTACCTTCATTCAGCCATACTTCCC-3′C133S-15′-CCTTTCATCACCGGGATCCGAGGTTAAAGAAATTACC-3′C133S-25′-GGTAATTTCTTTAACCTCGGATCCCGGTGATGAAAGG-3′C152S-15′-GTGAGCGGTGTTCCCAGCTCCCTGACTCGTATTAGTC-3′C152S-25′-GACTAATACGAGTCAGGGAGCTGGGAACACCGCTCAC-3′sipB-FW5′-AGGCTCGAGAATGACGCAAGTAGCATTAGC-3′ (XhoI)sipB-RV5′-AAAGGATCCTGCGCGACTCTGGCGCAGAAT-3′ (BamHI)sipC-FW5′-GGGCTCGAGATTAGTAATGTGGGAATAAAAT-3′ (XhoI)sipC-RV5′-AAAGGATCCAGCGCGAATATTGCCTGCGAT-3′ (BamHI)sseB-FW5′-GGGAAGCTTTCAGGAAACATCTTATGGGGA-3′ (HindIII)sseB-RV5′- CTCGAGGTACGTTTTCTGCGCTATCATA-3′ (XhoI)invG-FW5′-ACCGGACTCGAGACACATATTCTTTTGGCC-3′ (XhoI)invG-RV5′-CAAGGATCCTTTAATTGCCTCCTGACCTCT-3′ (BamHI)spiA-FW25′-GGGCTCGAGGTAAATAAACGTTTAATCTTA-3′ (XhoI)spiA-RV25′-CCCGGATCCACCATGAGATATGCCATTATT-3′ (BamHI)sseJ-Pro5′-GGGCTCGAGTCACATAAAACACTAGCACTT-3′ (XhoI)sseJ-RV5′-CCCGGATCCTTCAGTGGAATAATGATGAGC-3′ (BamHI)HA-R15′-GGGAGGCCTTATCCGTATGATGTGCCGGATTATGCGT AGGACTACAAGGACGACGATGACAAG-3′ (StuI)HA-R25′-GGGAGGCCTGACAGATCCTTCAGTGGAATA-3′ (StuI)ssaV-red-FW5′-ATTATATCGTTTGTCACTCACAATCAGCACATCACG GCTGGTGTAGGCTGGAGCTGCTTC-3′ssaV-red-RV5′-TCTGCGTCTTATGACGAGACGACAGCGCAGTATAGG TCCCCATATGAATATCCTCCTTAG-3′hilA-Pro5′-GTAAGTCGACGTCCAGATGACACTA-3′ (SalI)hilA-Rv5′-AACCGGATCCTGCATCTGAAAAGGA-3′ (BamHI)invF-Pro5′-ACCGGTCGACTCACGGAGGAAGGGA-3′ (SalI)invF-RV5′-TCCTGGATCCGGCAATTTTCATTGT-3′ (BamHI)ssrA-Pro5′-GGGGTCGACCTGTGAAATTCGCTCACAACC-3′ (SalI)ssrA-RV5′-GGGGGATCCTCAGTCGCTAATGAGCATTGA-3′ (BamHI) Open table in a new tab Construction of Plasmids—The dsbA and dsbC genes were amplified by PCR with the following primers: dsbAST-1 and dsbAST-2 for dsbA, and dsbC-FW and dsbC-RV for dsbC; strain SL1344 genomic DNA was used as the template. The PCR products were cloned into TA cloning vector pGEM-T Easy (Promega) in order to produce pGEM-dsbA and pGEM-dsbC, respectively. Plasmids pMW-dsbA and pAC-dsbA, used to complement the dsbA mutant strain, were constructed by inserting the BamHI-XhoI fragment containing the dsbA gene from pGEM-dsbA into the BamHI-SalI sites of pMW118 (Nippon Gene) and pACYC184 (New England Biolabs), respectively. The srgA gene was amplified from the 94-kb virulence plasmid of strain SL1344 using the primers srgA-FW and srgA-RV, and the PCR product was cloned into pGEM-T Easy. The fragment was then subcloned into pMW119 (Nippon Gene) at the SacI and SphI sites in order to generate pMW-srgA. The spiA gene was amplified by PCR with the primers spiA-FW1 and spiA-RV1, and strain SL1344 genomic DNA was used as the template. The PCR fragment was cloned into pGEM-T Easy, and the fragments were subcloned into pMW118 at the SacI and SphI sites in order to generate pMW-spiA. The SpiA point mutants were constructed by site-directed mutagenesis using the plasmid pGEM-sipA as a template and the respective oligonucleotides using the Quikchange XL site-directed mutagenesis kit (Stratagene) according to the manufacturer's instructions. The oligonucleotides C133S-1 and C133S-2, and C152S-1 and C152S-2 were used to replace cysteine residues with serine residues at positions 133 and 152 in the SpiA protein, respectively. The mutated plasmids were transformed into E. coli XL10-Gold supercompetent cells (Stratagene), and the presence of a respective mutation was confirmed by DNA sequencing. The resulting plasmids obtained were designated as pGEM-spiAC133S and pGEM-spiAC152S, respectively. To construct FLAG-tagged fusion proteins, the target genes were amplified by PCR using the following primers: sipB-FW and sipB-RV for sipB; sipC-FW and sipC-RV for sipC; sseB-FW and sseB-RV for sseB; invG-FW and invG-RV for invG; and spiA-FW2 and spiA-RV2 for spiA, spiAC133S, and spiAC152S. The PCR products were digested with XhoI and BamHI and were cloned into the XhoI-BglII site of pFLAG-CTC (Sigma). To construct the hemagglutinin (HA) epitope-tagged SseJ fusion protein, DNA fragment containing sseJ and its promoter region was amplified by PCR using sseJ-Pro and sseJ-RV. The PCR product was digested with XhoI and BamHI and was ligated to the same site of pMW118, yielding pTM21. To insert the HA epitope into the C-terminal SseJ, pTM21 was amplified by reverse PCR using the primers HA-R1 and HA-R2. The PCR product was digested with StuI and then self-ligated, yielding plasmid pTM22, which encodes the SseJ-HA fusion protein. Construction of Mutant Strains—Nonpolar mutants of dsbA and dsbC were constructed by allele exchange using the temperature- and sucrose-sensitive suicide vector pCACTUS (47Morona R. van den Bosch L. Manning P.A. J. Bacteriol. 1995; 177: 1059-1068Crossref PubMed Scopus (159) Google Scholar). A disruption mutation was created by the insertion of the SmaI-digested Kmr-encoding gene (kan) cassette from pUC18K (48Menard R. Sansonetti P.J. Parsot C. J. Bacteriol. 1993; 175: 5899-5906Crossref PubMed Scopus (616) Google Scholar) or promoterless cat gene into a unique EcoRV site in the coding region of dsbA and dsbC on pGEM-dsbA and pGEM-dsbC, respectively. The disrupted gene was then subcloned using SalI and SphI into similarly digested pCACTUS, and the resulting plasmid was introduced into strain SL1344 by electroporation for allele exchange mutagenesis, which was carried out as described previously (47Morona R. van den Bosch L. Manning P.A. J. Bacteriol. 1995; 177: 1059-1068Crossref PubMed Scopus (159) Google Scholar). Chromosomal mutations were verified by PCR and DNA sequencing analyses. For the construction of mutant strains expressing point-mutated SpiA proteins, the SacI-SphI fragments from pGEM-spiAC133S and pGEM-spiAC152S were subcloned into similarly digested pCACTUS, and each resulting plasmid was introduced into strain SL1344 by electroporation. The gene replacement of spiA to a point-mutated spiA was confirmed by DNA sequencing and by restriction enzyme digestion of the PCR-amplified segments with BamHI for spiAC135S and PvuII for spiAC152S. The ssaV mutant was constructed by the Red disruption system (49Datsenko K.A. Wanner B.L. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 6640-6645Crossref PubMed Scopus (11299) Google Scholar). The primers used for this series were ssaV-red-FW and ssaV-red-RV. Strain SH100 carrying pKD46 was used for gene disruption, as described previously (49Datsenko K.A. Wanner B.L. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 6640-6645Crossref PubMed Scopus (11299) Google Scholar). The disrupted genes were transferred by phage P22 transduction into strain SL1344. To construct lacZ transcriptional fusions, the DNA fragments containing the hilA, invF, and ssrA promoter regions were amplified by PCR using the primers hilA-Pro and