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Originally published In Press as doi:10.1074/jbc.M402760200 on May 28, 2004

J. Biol. Chem., Vol. 279, Issue 33, 34631-34642, August 13, 2004
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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*

Tsuyoshi Miki, Nobuhiko Okada{ddagger}, and Hirofumi Danbara

From the Department of Microbiology, School of Pharmaceutical Sciences, Kitasato University, Tokyo 108-8641, Japan

Received for publication, March 11, 2004 , and in revised form, May 28, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
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.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
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 (1–3). 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 (4, 5). The type III secretion system (TTSS)1 functions as a pathway for secretion across bacterial membranes and for the translocation of secreted proteins across the plasma membrane of eukaryotic cells (6). The type IV secretion system also translocates proteins in a single step from the cytoplasm to the cytosol of a host cell (7). 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 (8, 9). 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 (10). 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 (11). The inner membrane protein DsbB oxidizes DsbA (12, 13) and is re-oxidized directly by membrane-bound ubiquinones (14–16). DsbC and DsbG are the periplasmic components of the isomerization pathway. These proteins reshuffle misfolded multiple disulfide bonds (17, 18). The active sites of DsbC and DsbG are maintained in the reduced form by the inner membrane protein DsbD, which transfers electrons from the cytoplasmic protein thioredoxin onto DsbC and DsbG (17, 19–21).

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 (11, 22). 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 (11). 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 (17).

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 (23, 24); the heat-stable toxin of enterotoxigenic E. coli (25, 26); a molecular chaperone, PapD, of P pili of uropathogenic E. coli (27); bundle-forming pili and Intimin of enteropathogenic E. coli (28, 29); and Invasin of Yersinia pseudotuberculosis (30). DsbA is also required for the proper function of the TTSS in Yersinia pestis (31), Shigella flexneri (32), and Pseudomonas aeruginosa (33). 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 (31).

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 (6, 34, 35). In contrast, SPI-2 TTSS is required for intracellular survival in macrophages and for systemic infection in the mouse model (36–38).

The complete genome sequence of S. enterica serovar Typhimurium strain LT2 has revealed the presence of Dsb protein homologues (39). Southern hybridization analysis has also demonstrated the wide distribution of a dsbA gene among Salmonella serovars (40). A dsbA gene cloned from the S. enterica serovar Typhimurium can restore the dsbA– phenotype in an E. coli strain (40), demonstrating that Salmonella disulfide oxidoreductase DsbA is functional, although the enzymatic activity of Salmonella DsbA seems to be different from that of E. coli (40). 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 (41). 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 (42). 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 (42). Moreover, SrgA activity is dependent upon the presence of functional DsbB (42), 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 (43). 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 (43). 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.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
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 (44). Phage P22-mediated transductions for Salmonella have been described previously (45). The dsbA::Tn5 mutation of E. coli strain SK101 (46) was introduced into strain MC1061 by using P1 phages.


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  		TABLE I
Bacterial strains and plasmids used in this study

 


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  		TABLE II
Primers used in this study

 
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 (47). A disruption mutation was created by the insertion of the SmaI-digested Kmr-encoding gene (kan) cassette from pUC18K (48) 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 (47). 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 (49). 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 (49). 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 hilA-RV, invF-Pro and invF-RV, and ssrA-Pro and ssrA-RV. The PCR products digested with SalI and BamHI were ligated into the same sites of pLD-lacZ{Omega}, a derivative of suicide vector pGP704 (50) containing a promoterless lacZ gene and replacing the bla gene with the {Omega} fragment, producing pLD-hilAZ, pLD-invFZ, and pLD-ssrAZ. The resulting plasmids were transferred from E. coli SM10{lambda}pir to S. enterica serovar Typhimurium strain SH100 by conjugation. All fusion genes were introduced into the wild-type SL1344 and dsbA mutant TM100 strains by phage P22-mediated transduction. {beta}-Galactosidase activities of reporter gene fusions were determined according to the standard procedures (51) with the substrate o-nitrophenyl-{beta}-D-galactoside.

Mouse Virulence Assays—Female BALB/c mice (5–6 weeks old) were used for the mouse infection studies and were housed at Kitasato University according to the standard Laboratory Animal Care Advisory Committee guidelines. To prepare the inocula, bacteria were grown overnight at 37 °C in LB broth under conditions of shaking, and then the bacteria were used to inoculate fresh medium (1:100). The bacteria were grown to an A600 of 0.5 to 0.6 under the same conditions. For oral infection, 2 x 106 bacteria diluted in PBS were inoculated at a volume of 20 µl into groups of five mice. For intraperitoneal infection, 1 x 105 bacteria were injected at a volume of 100 µl into groups of five mice. The cfu were determined by plating serial dilutions of the inoculum. The survival of infected mice was observed daily for 4 weeks. In the cases involving mixed infections, the wild-type and mutant strains were grown separately and then were mixed prior to inoculation. The precise ratio of the two strains was determined retrospectively by a comparison of colony counts obtained on LB agar and LB agar with the appropriate antibiotics. Mice were sacrificed at 48 h after inoculation by carbon dioxide inhalation. The spleens were removed, placed in PBS, and homogenized by mechanical disruption. The number of wild-type and mutant bacteria in the spleen was then determined by plating a dilution series of the lysate onto LB agar alone and LB agar containing the appropriate antibiotics. Each competitive index (CI) value indicates the mean of at least three independent infections ± S.E. CI data were analyzed by Student's t test for statistical significance. p values of 0.05 or less were considered as significant.

Antibodies—Anti-SipB, anti-SipC, and anti-SseB antisera were generated by the immunization of mice with the SipB-, SipC-, and SseB-FLAG fusion proteins overexpressed in E. coli DH5{alpha} and purified on anti-FLAG M2 affinity gel (Sigma). Anti-{beta}-lactamase antiserum (dilution of 1:2,000) was kindly provided by Dr. Matsuhisa Inoue (Kitasato University, Kanagawa, Japan). The following antibodies were obtained from commercial sources: anti-CD107a H4A3 (LAMP-1, dilution of 1:1000, BD Pharmingen), anti-HA epitope tag HA.11 (dilution of 1:1000, Covance), anti-Salmonella lipopolysaccharide O4 group antigen (dilution of 1:1000, Denka Seiken), anti-FLAG M2 (dilution of 1:20,000, Sigma), and anti-DnaK (dilution of 1:1000, Calbiochem). Alexa 488-conjugated goat anti-mouse IgG and Alexa 594 goat anti rabbit IgG secondary antibodies (dilution of 1:500) were obtained from Molecular Probes. Alkaline phosphatase-conjugated goat anti-mouse IgG antibody was purchased from Sigma and was used at dilution of 1:10,000.

Cell Culture—HeLa cells (ATCC CCL-2) were grown in minimal essential medium (Sigma) supplemented with 10% fetal bovine serum (FBS), and HEp-2 cells (ATCC CCL-23) were grown in Dulbecco's modified Eagle's medium (Sigma) supplemented with 10% FBS. All of the cell lines used here were cultured in the presence of gentamicin and kanamycin and were maintained in a humidified atmosphere containing 5% CO2 at 37 °C.

Gentamicin Protection Assay—Bacteria were grown overnight at 37 °C in LB broth with aeration, diluted at 1:33 into fresh LB broth containing 0.3 M NaCl, and grown for another 3 h to obtain an A600 of 0.6 to 0.8. The bacteria were added to HEp-2 cells (2 x 105 cells/well) in 24-well plates at a multiplicity of infection of 100. The plates were centrifuged for 5 min at 500 x g to contact bacteria and HEp-2 cells, and then the plates were incubated for 1 h at 37 °C in the presence of 5% CO2. To remove extracellular bacteria, the cells were washed three times with Hanks' balanced salt solution, and were incubated in Dulbecco's modified Eagle's medium containing 10% FBS and gentamicin (100 µg/ml) for 1 h at 37 °C in the presence of 5% CO2. The cells were washed three times with Hanks' balanced salt solution and were lysed with 0.2 ml of 1% Triton X-100. After incubation for 15 min at 4 °C, the samples were vigorously mixed with 0.8 ml of PBS. The number of intracellular bacteria was determined by plating the cells on LB agar.

Bacterial Infection of HeLa Cells—HeLa cells were seeded onto glass coverslips (12-mm diameter) in 24-well plates at a density of 1 x 105 cells/well. Bacteria were grown at 37 °C overnight with aeration and then were subcultured in LB broth for 3 h. The cultures were diluted in minimal essential medium and added to the HeLa cells at a multiplicity of infection of 100. The cells were incubated for 10 min at 37 °Cina5% CO2. For the dsbA mutant strain, infection was carried out for 1 h to compensate for the invasion deficiency of the strain. Monolayers were washed three times with Hanks' balanced salt solution, and then the samples were incubated for 2 h in minimal essential medium containing 10% FBS and gentamicin (100 µg/ml) in order to kill the extracellular bacteria, after which the concentration of gentamicin was decreased to 5 µg/ml.

Immunofluorescence Microscopy—For the immunofluorescence analysis, the cells were fixed in 4% formaldehyde in PBS for 10 min at 4 °C. After being washed three times in PBS, the fixed cells were permeabilized in 0.1% Triton X-100 in PBS for 5 min. The samples were then probed with various primary and secondary antibodies, mounted using Vectashield solution (Vector Laboratories, Inc.), and viewed at x63 magnification on a Zeiss confocal laser scanning microscope (LSM510 META).

Preparation of Secreted Proteins—For the preparation of secreted proteins that are dependent on SPI-1 TTSS, the bacteria were grown in LB broth containing 0.3 M NaCl overnight at 37 °C without aeration. For the preparation of secreted proteins that are dependent on SPI-2 TTSS, the bacteria were grown in MgM minimal medium containing 0.1% casamino acids overnight at 37 °C with aeration. For the isolation of proteins released into the culture supernatants, the supernatants from bacterial cultures were filtered, and trichloroacetic acid was added to the samples at a final concentration of 10%. After incubation on ice for 3 h, the samples were centrifuged at 16,000 x g for 45 min, and the resulting precipitated proteins were dissolved in SDS-PAGE sample buffer. Proteins detached from the bacterial surface (detached fraction) were prepared as described previously (52). Briefly, bacteria grown in 100 ml of MgM minimal medium containing 0.1% casamino acids were pelleted by centrifugation at 6,000 x g for 10 min and resuspended in 10 ml of PBS. The bacterial suspension was then agitated on a Vortex mixer (Vortex Genie 2, Scientific Industries, Inc) at maximum speed for 1 min. Bacterial cells were pelleted by centrifugation at 10,000 x g for 10 min, and the supernatant was pass through a filter (0.2 µm-pore size) to remove residual bacteria. Protein in the detached fraction was recovered by trichloroacetic acid precipitation. After incubation on ice for 3 h, the samples were centrifuged at 16,000 x g for 45 min. The resulting precipitated proteins were air-dried and dissolved in SDS-PAGE sample buffer.

SDS-PAGE and Western Blot Analysis—The protein samples were normalized according to bacterial cfu, separated by SDS-PAGE, and transferred to polyvinylidene difluoride membranes (Immobilon, Millipore) for immunoblotting. Western blot analysis was carried out as described previously (53). Oxidized and reduced states of the proteins were examined by comparing the gel mobilities of the proteins in samples with and without DTT.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Characterization of Salmonella dsbA Mutation—A nonpolar dsbA mutant strain of S. enterica serovar Typhimurium, TM100, was examined for its pleiotropic phenotype, as reported previously (11, 40). The mutant strain exhibited mucoidy when grown on agar plates, as well as reduced motility and a high sensitivity to DTT; the mutant strain was unable to grow in LB medium in the presence of 8 mM DTT, as compared with the wild-type strain, which did grow in the same concentration of DTT. The growth rate of the dsbA mutant was similar to that of the wild-type strain when grown in rich media. To further confirm the effect of the dsbA mutation on the disulfide bond formation of {beta}-lactamase, a periplasmic protein with a single disulfide bond, we determined the mobility of {beta}-lactamase by SDS-PAGE under nonreducing conditions. Bacterial cell extracts from the wild-type strain SL1344 carrying plasmid pBlueScriptII SK(+), which expresses {beta}-lactamase, and a dsbA mutant strain TM100 carrying the same plasmid, were subjected to SDS-PAGE in the absence of DTT. {beta}-Lactamase was detected by immunoblotting with antiserum specific for {beta}-lactamase. As expected, the oxidized form of {beta}-lactamase was found in the wild-type strain but not in the dsbA mutant strain, confirming the defects in periplasmic disulfide bond formation (Fig. 1). These results were consistent with the phenotype of a dsbA mutant in E. coli (11). The dsbA mutant phenotype was completely restored by introducing the wild-type dsbA allele on the plasmid into S. enterica serovar Typhimurium strain TM100 (data not shown).



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  		FIG. 1.
Disulfide bond formation is defective in the S. enterica serovar Typhimurium dsbA::kan mutant strain. S. enterica serovar Typhimurium SL1344 (wild type) and TM100 (dsbA::kan) mutant strains were transformed with pBlueScript II SK(+)(pSK) carrying bla and were grown at 37 °C. Proteins of the whole-cell lysate were separated by SDS-PAGE under nonreducing conditions, and {beta}-lactamase was detected by Western blot analysis with antibody to {beta}-lactamase. The positions of the oxidized (ox.) and reduced (red.) forms of the protein are indicated.

 
Virulence of the dsbA::kan, but Not dsbC::kan, Mutant Is Attenuated in Vivo—To elucidate the role of dsbA in virulence, we examined lethality in mice after oral administration. A mutant strain, TM100 (dsbA::kan), did not lead to lethal infection when BALB/c were inoculated orally with 2 x 106 cfu/mouse, the dose at which the wild-type strain was 100% lethal (~50-fold higher than the reported LD50 for SL1344) (Fig. 2). Complementation with pMW-dsbA resulted in restoration of the virulence defect of the mutant strain TM100 in mice. In contrast, a strain with a mutation in dsbC was shown to have no changes in virulence when the bacteria were administered orally at 3 x 106 cfu/mouse (Fig. 2). These results demonstrated that DsbA, but not DsbC, is required for the full expression of pathogenesis in mice by infection with Salmonella.



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  		FIG. 2.
Virulence of S. enterica serovar Typhimurium dsbA::kan and dsbC::kan mutant strains. S. enterica serovar Typhimurium SL1344 (wild type) and TM100 (dsbA::kan) and TM202 (dsbC::kan) mutant strains were administered orally with 106 cfu to female BALB/c mice. The virulence of S. enterica serovar Typhimurium carrying a mutation in dsbC was not altered, but the loss of dsbA had a marked effect on virulence. Introduction of a cloned dsbA gene on the low copy number plasmid into the dsbA::kan mutant strain was able to restore virulence to the wild-type level.

 
Effects of dsbA Mutation on SPI-1 TTSS Function—It has been demonstrated that DsbA is essential for TTSS-mediated protein secretion in several pathogenic bacteria, including Y. pestis (31), S. flexneri (32), and P. aeruginosa (33). Therefore, to investigate the effects of dsbA mutation on SPI-1 TTSS function, secretion of Sip proteins by the wild-type and the isogenic dsbA mutant strain was determined by SDS-PAGE and immunoblotting analysis. The dsbA mutant strain TM100 secreted SipA, SipB, and SipC proteins at lower levels into the culture medium as compared with the corresponding wild-type levels (Fig. 3A). In addition, Western blot analysis using anti-SipB antiserum showed that the amount of these proteins in the whole-cell extracts was similar to that of the wild-type level (Fig. 3B). We next examined the interaction of the S. enterica serovar Typhimurium dsbA mutant strain with cultured HEp-2 cells using an assay for the examination of bacterial internalization and actin cytoskeleton reorganization. The inactivation of the dsbA gene in strain SL1344 resulted in an 85% reduction in the invasive capacity into HEp-2 cells compared with that of the wild-type strain (Fig. 3C). Consistent with these results, the mutant strain was also found to be less effective than the wild-type strain at inducing actin cytoskeleton rearrangement (Fig. 3D). Complementation of a mutant strain TM100 with plasmid pMW-dsbA completely restored these SPI-1 TTSS-dependent phenotypes, indicating that the disruption of the dsbA gene in S. enterica serovar Typhimurium was responsible for the observed reduced SPI-1 TTSS function.



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  		FIG. 3.
Effect of the dsbA::kan mutation on SPI-1 TTSS function. A, SDS-PAGE profiles of supernatant proteins from S. enterica serovar Typhimurium SL1344 (wild type), SB136 (invA::kan), TM100 (dsbA::kan), and TM101 (TM100 carrying pMW-dsbA) cultures. Proteins from the culture supernatants corresponding to 1 x 109 bacteria were precipitated as described under "Experimental Procedures," and the proteins were separated by SDS-PAGE and were visualized by Coomassie Blue staining. The arrows indicate previously identified proteins that were present in the culture supernatant from the wild-type strain but that were reduced in the case of the dsbA mutant strain (69). B, the amount of SipB in the culture supernatants and in the total cell fractions corresponding to 1 x 109 and 8 x 107 cells, respectively, from S. enterica serovar Typhimurium SL1344 (wild type), SB136 (invA::kan), TM100 (dsbA::kan), and TM101 (TM100 with pMW-dsbA) strains was analyzed by Western blotting with antibody raised against SipB-FLAG fusion protein. The same results were obtained when mouse anti-SipC antiserum was used against the same preparations. C, the invasiveness of S. enterica serovar Typhimurium SL1344 (wild type), SB136 (invA::kan), TM100 (dsbA::kan), and TM101 (TM100 with pMW-dsbA) into HEp-2 cells was determined by gentamicin protection assays. The data represent the percentage of the wild-type intracellular cfu and are presented as the means ± S.E. from triplicate experiments. D, the induction of localized actin cytoskeleton rearrangements in HeLa cells infected with SL1344 (wild type) (a), SB136 (invA::kan) (b), TM100 (dsbA::kan) (c), or TM101 (TM100 complemented with pMW-dsbA) (d). The actin cytoskeleton was visualized by phalloidin staining (green). Bacteria (red) were stained with anti-Salmonella antiserum. The exposure times for all of the images are identical.

 
Effects of dsbA Mutation on SPI-2 TTSS Function—S. enterica serovar Typhimurium contains two distinct TTSS encoded by SPI-1 and SPI-2. Thus, we next determined the effects of dsbA mutation on the second TTSS encoded by SPI-2. First, we compared the levels of the major SPI-2 TTSS-secreted proteins, SseB, SseC, and SseD, in fractions of the bacterial cell surface obtained from dsbA mutant and the wild-type strains. Bacteria were grown under conditions that induced the secretion of SPI-2 TTSS, and the cultures were subjected to vigorous mixing. The bacterial surface proteins (i.e. the detached fraction) were then analyzed by SDS-PAGE. Proteins with apparent molecular masses of about 20 (SseD), 21 (SseB), and 52 kDa (SseC) secreted by the wild-type strain were reduced in the detached fraction of the dsbA mutant strain (Fig. 4A). These proteins were absent in the detached fraction obtained from a mutant strain, TM114, defective in ssaV, a structural component of SPI-2 TTSS. Furthermore, immunoblot analysis using mouse anti-SseB antiserum showed that the secretion of SseB in the detached fraction of the dsbA mutant strain was decreased to ~20% of the wild-type level, whereas the total amount of SseB obtained from the whole-cell fraction of the mutant strain amounted to more than 90% of that obtained from the wild-type strain (Fig. 4B).



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  		FIG. 4.
Effect of dsbA::kan mutation on SPI-2 TTSS function. A, S. enterica serovar Typhimurium SL1344 (wild type), TM114 (ssaV::cat), and TM100 (dsbA::kan) strains were grown overnight in MgM medium at pH 5.8. Proteins secreted from equal amounts of bacterial cells corresponding to 1 x 1010 bacteria were recovered by vigorous mixing and concentrated by acetone precipitation, as described under "Experimental Procedures." Protein extracts were subjected to SDS-PAGE and were visualized by Coomassie Blue staining. The positions of the SPI-2 effector proteins SseB, SseC, and SseD are indicated. B, the amount of SseB in the detached fractions and in the total cell fractions corresponding to 1 x 1010 and 8 x 107 bacteria, respectively, from S. enterica serovar Typhimurium SL1344 (wild type), TM114 (ssaV::cat), and TM100 (dsbA::kan) strains was analyzed by Western blotting with antibody raised against SseB-FLAG fusion protein. C, confocal immunofluorescence micrographs of HeLa cells infected with S. enterica serovar Typhimurium SL1344 (wild type), TM114 (ssaV::cat), or TM100 (dsbA::kan) strains. Infected cells were fixed 22 h after bacterial invasion, and they were labeled for Salmonella (red) and LAMP-1, a marker of the SCV membrane (green).

 
Because SPI-2 TTSS activates and functions when bacteria reside within host cells, we next examined the ability of the dsbA mutant to form Salmonella-induced filaments (SIFs). SIF formation is an intracellular phenotype that is dependent on the presence of functional SPI-2 TTSS (54). HeLa cells were infected with the wild-type strain SL1344 and the dsbA mutant strain TM100. Twenty-two hours after infection, infected cells were fixed, permeabilized, and stained for SIFs and bacteria using antibodies to LAMP-1 and Salmonella, respectively. SIFs were clearly detected in HeLa cells infected with the wild-type strain, whereas no SIFs were observed in cells infected with the dsbA mutant. This result was similar to that obtained with the phenotype of the SPI-2 TTSS mutant strain TM114 (ssaV::cat) (Fig. 4C). These findings confirmed that intracellular SPI-2 TTSS function is dependent on the activity of DsbA.

A dsbA Mutation Does Not Affect the Expression of Either SPI-1 or SPI-2—In the above experiments, the defect in DsbA function appeared to render the phenotype that resembled that of strains deficient in the SPI-1 and SPI-2 secretion apparatus; however, there was no effect on the expression of the effector molecules secreted by SPI-1 and SPI-2 TTSS. Therefore, to further determine the role of dsbA in the expression of SPI-1 and SPI-2 TTSS, we constructed mutant strains containing hilA::lacZ and invF::lacZ transcriptional fusions for SPI-1 expression and ssrA::lacZ transcriptional fusion for SPI-2 expression on the chromosome both with a dsbA+ and a dsbA– background. Salmonella strains were grown under SPI-1- or SPI-2inducing conditions, and {beta}-galactosidase activity was measured in the wild-type and dsbA mutant strains. As shown in Table III, transcription of hilA and invF, which are regulatory genes for SPI-1 (55), was not affected by the loss of DsbA, whereas the transcription of ssrA, a regulatory gene for SPI-2 (38), was slightly increased in the dsbA mutant strain for reasons that remain unclear. Thus, disulfide bond formation may be essential for SPI-1 and SPI-2 TTSS apparatus assembly. These data were consistent with results showing that the amount of effector proteins of SPI-1 and SPI-2 in the total cell fractions was at the same level in the dsbA mutant strain as in the wild-type strain (see Figs. 3B and 4B).


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  		TABLE III
{beta}-Galactosidase activity from lacZ fusions to hilA, invF, and ssrA in S. enterica serovar Typhimurium SL1344 (wild-type) and TM100 (dsbA::kan) mutant strains

 
SPI-2 TTSS Outer Membrane Protein SpiA Requires DsbA to Function—It has been reported that the outer membrane protein YscC, a component of the Y. pestis TTSS apparatus, is oxidized by DsbA (31). Therefore, Salmonella DsbA may target YscC homologues, InvG and SpiA, which are major outer membrane proteins of SPI-1 and SPI-2 TTSS, as substrates. Thus, the ability of Salmonella DsbA to catalyze the outer membrane proteins, InvG of SPI-1 TTSS and SpiA of SPI-2 TTSS, was examined. The plasmids pTM9 and pTM10 expressing C-terminal FLAG-tagged InvG fusion protein (InvG-FLAG) and C-terminal FLAG-tagged SpiA fusion protein (SpiA-FLAG), respectively, were introduced into an S. enterica serovar Typhimurium strain that carried either the dsbA+ or dsbA– allele. The folding of these fusion proteins was investigated by SDS-PAGE in the presence or absence of 1 mM DTT, and by immunoblotting with anti-FLAG-M2 monoclonal antibody for the detection of the proteins. The mobility of the InvG-FLAG fusion protein was not affected by the absence or presence of DTT in the wild-type and dsbA mutant strains (Fig. 5A). In contrast, SpiA-FLAG fusion protein was found as a single band in all strains tested under reducing conditions, whereas different forms of the SpiA-FLAG fusion protein corresponding to the reduced and oxidized forms were observed in the wild-type and TM101 (dsbA–/dsbA+) strains, but not in the TM100 strain (dsbA::kan) under nonreducing conditions (Fig. 5B). These results indicated that an intramolecular disulfide bond was formed in SpiA. Consistent with these results, examination of the predicted amino acid sequences of InvG and SpiA revealed that SpiA contained two cysteine residues at positions 133 and 152, but no cysteine was present in the InvG protein.



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  		FIG. 5.
In vivo redox state of InvG (A), SpiA (B), and SpiA point mutant proteins SpiAC133S and SpiAC152S (C). A, the whole-cell lysates corresponding to 2 x 107 bacteria isolated from S. enterica serovar Typhimurium SL1344 (wild type) and TM100 (dsbA::kan) carrying plasmid pTM9 and expressing InvG-FLAG fusion protein were subjected to SDS-PAGE and were visualized by Western blot analysis with anti-FLAG antibody. The positions of the precursor (pre-InvG) and mature (InvG) forms of InvG-FLAG fusion proteins are indicated. B, whole-cell lysates corresponding to 2 x 107 bacteria isolated from S. enterica serovar Typhimurium SL1344 (wild type), TM100 (dsbA::kan), and TM101 (dsbA::kan/pMW-dsbA) carrying plasmid pTM10 and expressing SpiA-FLAG fusion protein were subjected to SDS-PAGE and were visualized by Western blot analysis with anti-FLAG antibody. The positions of the oxidized (ox.), reduced (red.), and precursor (pre-SpiA) forms of the SpiA-FLAG fusion proteins are indicated. C, the induction of cysteine to serine mutations in SpiA. S. enterica serovar Typhimurium SL1344 (wild type) was transformed with pTM10 for SpiA, pMT11 for SpiAC133S, and pTM12 for SpiAC152S. The whole-cell lysates corresponding to 2 x 107 bacteria isolated from these Salmonella strains were subjected to SDS-PAGE and were visualized by Western blot analysis with anti-FLAG antibody. The positions of the oxidized (ox.), reduced (red.), and precursor (pre-SpiA) forms of SpiA-FLAG fusion proteins are indicated. +DTT and –DTT indicate the presence or absence of DTT, respectively. The relative amount of protein loaded was determined by blotting samples with antibody against DnaK, a bacterial heat shock protein.

 
To further examine whether the folding of SpiA could be mediated directly by the two cysteines in SpiA, we carried out site-directed mutagenesis by using a plasmid pTM10 containing spiA-FLAG fusion gene with Cys-133 or Cys-152 replaced with serine residues. The plasmids pTM11 and pTM12 expressing point-mutated SpiA fusion proteins, SpiAC133S-FLAG and SpiAC152S-FLAG, respectively, were introduced into S. enterica serovar Typhimurium wild-type strain SL1344. SDS-PAGE and immunoblotting analysis of the point-mutated SpiAC133S and SpiAC152S proteins expressed by the wild-type strain showed that the mobility of these mutant proteins was not affected by either the presence or the absence of a reducing agent (Fig. 5C) in a manner that was consistent with the redox status of the SpiA protein. These results demonstrated that Cys-133 and Cys-152 in SpiA are involved in the formation of an intramolecular disulfide bond.

Two Cysteine Residues in SpiA Are Essential for SPI-2 TTSS Function—To investigate SPI-2 TTSS function in the SpiA point-mutated strains, TM133 (spiAC133S) and TM152 (spiAC152S), we examined the level of SseB protein expressed on the cell surface as well as the level of that released into the extracellular medium. The wild-type strain SL1344 was able to translocate SseB protein on the cell surface and release it into the culture supernatant. However, less SseB was released from the fractions of both mutant strains TM133 and TM152, as was the case with the SPI-2 TTSS mutant strain TM114 (ssaV::cat) (Fig. 6). In the dsbA mutant strain TM100, a reduced amount of SseB was expressed on the cell surface, and no SseB was found in the supernatant fraction (Fig. 6).



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  		FIG. 6.
Disulfide formation of SpiA is required for the efficient secretion of SPI-2 effector proteins. S. enterica serovar Typhimurium SL1344 (wild type) and TM114 (ssaV::cat), TM100 (dsbA::kan), TM133 (spiAC133S), and TM152 (spiAC152S) strains were grown in MgM at pH 5.8 to induce expression and secretion of SPI-2 substrate proteins. The cell-surface (detached) fraction, the culture supernatant, and the total cell fraction were prepared as described under "Experimental Procedures." Equal amounts of bacterial cells (total cell fraction) and proteins prepared from equal amounts of the detached fraction and the culture supernatants corresponding to 1 x 1010 and 3 x 1010 bacteria, respectively, were subjected to Western blot analysis with a polyclonal antiserum raised against SseB-FLAG fusion protein.

 
To analyze whether these Salmonella mutant strains could assemble functional SPI-2 TTSS in an intracellular manner, we examined the SPI-2 TTSS-dependent translocation of an effector protein, SseJ, a protein that is translocated by intracellular Salmonella, and is associated with Salmonella-containing vacuoles (SCVs) and SIFs after translocation (56, 57). HeLa cells were infected with Salmonella strains carrying the plasmid pTM22 that expresses the HA-tagged sseJ under the control of its native promoter. The cells were fixed at 22 h after infection and then were stained for immunofluorescence using anti-HA and anti-Salmonella lipopolysaccharide antibodies. SseJ-HA fusion protein was detected in the HeLa cells infected with the wild-type strain but was not found in the cells infected with the SpiA point-mutated strains equivalent to the SPI-2 TTSS mutant strain (Fig. 7A). Although a dsbA mutant strain failed to induce LAMP-1-positive SIFs in infected cells (see Fig. 4C), the same mutant strain harboring pTM22 was able to translocate the SseJ-HA fusion protein into the infected cell cytoplasm and form SIF structures (Fig. 7A). This phenotypic difference was probably due to limitations in the sensitivity of the antibodies used for immunostaining and/or the difference may have been due to the induction of SIF formation by the plasmid-based expression of sseJ.



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  		FIG. 7.
Disulfide formation of SpiA is required for the efficient SPI-2-dependent translocation of SseJ into host cells. A, confocal immunofluorescence micrographs of HeLa cells infected with S. enterica serovar Typhimurium SL1344 (wild type), TM114 (ssaV::cat), TM133 (spiAC133S), TM152 (spiAC152S), or TM100 (dsbA::kan) strains, all carrying plasmid pTM22 expressing an HA-tagged SseJ fusion protein. The cells were fixed 22 h after bacterial entry and were examined by confocal immunofluorescence microscopy. HA-tagged SseJ was detected by anti-HA monoclonal antibody (green), and whole bacteria were detected with anti-Salmonella antiserum (red). B, the expression of HA-tagged SseJ was dependent on the SPI-2 transcriptional activator SsrA. S. enterica serovar Typhimurium SL1344 (wild type), TM114 (ssaV::cat), TM133 (spiAC133S), TM152 (spiAC152S), TM100 (dsbA::kan), or TM233 (ssrA::kan) strains, all carrying plasmid pTM22, were grown in MgM at pH 5.8, and equal amounts of bacterial lysates corresponding to 8 x 107 cfu and were analyzed by Western blotting with anti-HA antibody. The relative amount of protein loaded was determined by blotting the samples with antibody against DnaK.

 
To investigate whether the reduced secretion of these mutant strains resulted from the decreased expression of the sseJ gene, the level of SseJ-HA fusion protein present in the whole-cell lysate was analyzed by Western blotting using anti-HA monoclonal antibody. The fusion protein was detected in the strains carrying mutations in spiAC133S, spiAC152S, and ssaV, and the levels were similar to those detected in the wild-type strain; however, the strain carrying a mutation in ssrA, a transcriptional regulatory gene of SPI-2 TTSS, did not produce similar results (Fig. 7B). These findings thus suggest that the lack of disulfide bond formation in the SpiA protein will affect the translocation of effector proteins through SPI-2 TTSS in infected host cells.

Because Salmonella strains carrying mutations in SPI-2 TTSS including spiA are highly attenuated (36, 38), we examined the virulence of strains carrying point mutations in spiA in mice to confirm their in vivo virulence phenotype. The results with control mice infected intraperitoneally with the wild-type strain SL1344 demonstrated that the strains were 100% lethal. In contrast, all mice infected with the spiAC133S or spiAC152S mutant strain survived, and this defect was complemented by introduction of the wild-type spiA allele on the plasmid (Table IV). Furthermore, attenuation of in vivo virulence in strains containing point mutation in spiA was analyzed by a competition index (CI) in mixed infection. As expected, the CI of strains carrying spiAC133S and spiAC152S against the wild-type strain was similar to that of SPI-2 ssrA mutant strain (Table IV). All of these results suggest that a deficiency in the disulfide bond formation in the SpiA protein will result in a virulence phenotype equivalent to that resulting from the loss of SPI-2 TTSS function.


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  		TABLE IV
Mouse virulence for S. enterica serovar Typhimurium strains after peritoneal infection

 
The Functional Relationship between the DsbA and SPI-2 TTSS in Vivo—To investigate further the interaction between DsbA and SPI-2 TTSS in vivo,a dsbA mutation was introduced to the ssaV mutant strain, and single and double mutant strains were analyzed by a CI in mixed infections. The results demonstrated that the CI of a strain carrying both dsbA and ssaV mutations against a single dsbA mutant strain was not significantly different from the CI of the ssaV mutant strain against the wild-type strain. Moreover, a dsbA ssaV double mutant strain was found to be no more attenuated than an ssaV single mutant (Table V). Therefore, DsbA appears to contribute to S. enterica serovar Typhimurium virulence through SPI-2 TTSS function in cases of systemic infection.


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  		TABLE V
CI value for mixed infection

 
Expression of SrgA, a Paralogue of DsbA, Can Be Restored in the dsbA Phenotype in Vitro and in Vivo—Based on the data that SPI-2 TTSS activity was retained in a dsbA mutant strain in contrast to strains carrying a point mutation in spiA, we considered the possibility that S. enterica serovar Typhimurium might possess a redox protein that functions as a disulfide oxidoreductase, in addition to DsbA. Recently, SrgA of S. enterica serovar Typhimurium was identified as the virulence plasmid-encoded paralogue of DsbA and was shown to oxidize PefA, the major structural subunit of plasmid-encoded fimbriae (42). To investigate the role of srgA in Salmonella virulence, we constructed a mutant strain carrying a mutation in srgA, and we compared the results with those obtained with the wild-type strain. A mutation in srgA showed no attenuation of virulence. An srgA mutation was then introduced to the dsbA mutant strain, and the wild-type and double mutant strains were analyzed by mixed infection of mice. The double mutant strain was found to be strongly attenuated than either single mutant (Table V), and the level of attenuation in the double mutant strain was similar to that of the SPI-2 mutant strains (see also Table IV) (58).

The ability of SrgA to oxidize the SpiA protein was then examined in an E. coli strain, TM161, a derivative of MC1061 containing a dsbA::Tn5 mutation. The oxidized form of SpiA was detected by the introduction of plasmids carrying dsbA and srgA into strain TM161, although a small amount of SpiA was catalyzed in a strain carrying the plasmid pAC-srgA (srgA+) in contrast to a strain carrying the plasmid pAC-dsbA (dsbA+) (Fig. 8). The relative oxidizing capacity of the SpiA protein between DsbA and SrgA may be required for further characterization, because srgA is Salmonella-specific and expression of srgA in the E. coli-based assay system used here was not determined. To further examine whether SrgA would be able to restore the defective phenotypes in a Salmonella dsbA mutant strain, a plasmid encoding srgA was introduced into TM100, a strain carrying a dsbA mutant allele. An SrgA-complemented strain (dsbA–/srgA+) showed motility and epithelial cell invasiveness as did a DsbA-complemented strain (dsbA–/dsbA+) (data not shown). Furthermore, introduction of the plasmid encoding srgA into a dsbA mutant strain restored its virulence in infected mice; moreover, this virulence was found to be equal to that of the wild-type strain (Table V), indicating that SrgA functions in a manner similar to DsbA in Salmonella. These results strongly suggest that S. enterica serovar Typhimurium maintains two functional disulfide oxidoreductases, DsbA and SrgA, and both target an SPI-2 TTSS outer membrane protein SpiA as a substrate in vivo.



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  		FIG. 8.
Two distinct periplasmic disulfide oxidoreductases, DsbA and SrgA, target SpiA. E. coli strains TM161 (dsbA::Tn5) carrying pTM10 expressing SpiA-FLAG fusion protein were transformed with pACYC184 (vector only), pAC-dsbA (cloned dsbA), or pAC-srgA (cloned srgA), and protein extracts corresponding 2 x 107 cells were subjected to SDS-PAGE under nonreduced conditions and visualized by Western blot analysis with anti-FLAG antibody. The positions of the oxidized (ox.), reduced (red.), and precursor (pre-SpiA) forms of SpiA-FLAG fusion proteins are indicated.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Disulfide bonds are crucial for the folding, stability, and function of many extracytoplasmic proteins (59, 60). There are two complementary pathways that catalyze the formation of disulfide bonds in the periplasmic space of E. coli. In the disulfide bond formation pathway, DsbA and DsbB add new disulfide bonds to folding proteins. The DsbC and DsbD proteins in the disulfide isomerization pathway rearrange existing bonds between incorrectly paired cysteines. In this study, we selectively mutated the dsbA and dsbC genes in S. enterica serovar Typhimurium, and we analyzed their virulence properties. By using a mouse infection model, we provided evidence that a mutation in dsbA, but not in dsbC, successfully attenuated the virulence of Salmonella. Because DsbA is a key protein-folding catalyst, inactivation of dsbA might block the folding of various secreted proteins, which include important virulence determinants of systemic infection by Salmonella, and such inactivation may thus have conferred a reduction of virulence in this mouse infection model.

It is known that DsbA is involved in the biogenesis of bacterial toxins and organelles on the bacterial surface, e.g. fimbriae and other adhesive molecules (27–30). In addition to these virulence factors, the dsbA gene is required for the translocation of effector proteins by TTSS in Y. pestis (31), S. flexneri (32), and P. aeruginosa (33). In Y. pestis, DsbA contributes to the stable expression of YscC, an outer membrane protein of the Yersinia TTSS apparatus (31). Therefore, a mutation in dsbA results in unstable YscC, leading to the reduced secretion of Yop proteins. An additional effect on the expression of the TTSS apparatus has also been reported in P. aeruginosa (33). To elucidate the mechanism of virulence attenuation achieved by dsbA mutation in S. enterica serovar Typhimurium, we characterized various virulence phenotypes dependent on both SPI-1 and SPI-2 TTSS in a dsbA mutant strain. Our data demonstrated that the translocation of effector proteins by both SPI-1 and SPI-2 TTSS was deficient in the dsbA mutant strain, whereas in contrast to P. aeruginosa, the expression of SPI-1 and SPI-2 TTSS was not affected by the loss of DsbA. Because effector proteins are directly translocated from the cytoplasm to the extracellular space of the bacterium via the TTSS apparatus, the DsbA protein of S. enterica serovar Typhimurium is required for the formation of the TTSS apparatus.

Assembly of the TTSS apparatus is thought to proceed in a manner similar to that of a flagella basal body-hook assembly (61). For example, the first step in the assembly process of the SPI-1 TTSS is the sec-dependent export of the proteins PrgH, PrgK, and InvG, which form the stable base substructure of the TTSS machinery. Therefore, the secretion of these proteins does not require a functional TTSS apparatus. This incomplete base structure is required for the transport of the main subunit of the needle substructure, PrgI, and other exported secretion components including InvJ. The integration of inner membrane components into base substructure is completed prior to secretion of PrgI and needle assembly. Thus, it is likely that DsbA could catalyze Salmonella TTSS component proteins that are exported to the periplasmic space in a sec-dependent manner. In the case of the SPI-1 TTSS apparatus, the base structure component proteins PrgH, PrgK, and InvG lack multiple cysteine residues after the cleavage of the putative signal sequence, and they are therefore not directly affected by mutations of DsbA. However, InvG forms a ring-like pore structure essential for the secretion of SPI-1 effector proteins from bacterial cells and requires an accessory lipoprotein, InvH, for its proper location (61). Although not essential for needle complex assembly, InvH facilitates the efficient outer membrane insertion of InvG (62, 63). Additional evidence has shown reduced virulence associated with invH mutation, and InvH is known to have two cysteine residues (64). Such evidence suggests the possibility that a mutation in dsbA causes a failure to form intramolecular disulfide bonds in InvH, resulting in the reduced stability of InvH, which leads to the less efficient secretion of SPI-1 effectors. The molecular characterization of the role played by DsbA in InvH is now in progress.

To provide a possible explanation for the role of DsbA in efficient SPI-1 TTSS function, the pleiotropic effect caused by the dsbA mutation should also be considered. Many proteins that fail to form proper disulfide bonds are degraded rapidly in bacterial cells (59). Additionally, it is known that abnormal states on the bacterial surface due to an aberrant redox balance in the periplasm occur as a result of dsbA mutation (11). Therefore, dsbA mutant strains are hypersensitive to the reductant DTT, benzylpenicillin, and some metal ions including Hg2+ and Cd2+ (22, 65). These structural changes occurring in bacterial membranes may affect the proper localization of SPI-1 TTSS secretory components such as the InvG outer membrane protein.

In contrast to the InvG protein of the SPI-1 TTSS apparatus, SpiA, an outer membrane component of SPI-2 TTSS, bears the sec-dependent signal sequence for secretion in its N-terminal region and possesses two cysteine residues after the cleavage of a putative signal sequence. Thus, we investigated whether SpiA is a disulfide bond-containing protein, the folded protein structure of which is required for SPI-2 TTSS function. Our results demonstrated that the SpiA protein produced by the wild-type strain, but not that produced by a dsbA mutant strain, exhibited different structures corresponding to reduced and oxidized forms of the protein, as shown by SDS-PAGE conducted under both reducing and nonreducing conditions. The replacement of the cysteine with a serine at position 133 or 152 in SpiA by site-directed mutagenesis has also revealed that these mutated proteins produced by the wild-type strain could not change their oxidized structure under nonreducing conditions. In addition, similar to the SPI-2 TTSS-deficient mutant strains, a Salmonella strain expressing the mutated SpiAC133S or SpiAC152S protein had less efficient SPI-2 TTSS function, as shown by both in vitro and in vivo studies. These results thus suggested that DsbA plays an important role in the assembly of the SPI-2 TTSS apparatus through the disulfide bond formation in the outer membrane protein SpiA.

A mutation of dsbA usually leads to phenotypic effects that resemble those of a mutation in the gene encoding the virulence factor per se. Our data showed that S. enterica serovar Typhimurium strains carrying a mutation in spiA completely lost SPI-2 TTSS function, but a dsbA mutant strain was shown to have retained reduced activity of SPI-2 TTSS, indicating the existence of a DsbA-like protein that was able to catalyze the folding of SpiA. In this study, we demonstrated that a virulence plasmid-encoded SrgA efficiently oxidized the disulfide bond of SpiA in an E. coli dsbA– background. Furthermore, because a combination of mutations in both dsbA and srgA genes conferred a greater reduction in virulence in mice than did single mutations in either the dsbA or srgA alone, both the DsbA and SrgA proteins appear to be functionally important for the activity of SPI-2 TTSS in Salmonella.

The distribution of the srgA allele among Salmonella serovars revealed that srgA is only carried by limited strains (42). An SrgA homologue, Dlp, encoded on the virulence plasmid of S. enterica serovar Enteritidis, and another homologue, Dlt, encoded on the chromosome of S. enterica serovar Typhi, were also shown to have disulfide oxidoreductase enzymatic activity (66). These data suggest that a second gene encoding disulfide oxidoreductase may be an additional horizontally acquired determinant of some of the Salmonella serovars. Most interesting, the enzymatic activity of SrgA has been shown to be substrate-specific (42). Thus, the maintenance of two different types of disulfide oxidoreductase may play an important role in the enhanced virulence of these Salmonella serovars.

Based on our data and previous studies showing that several environmental factors are required to induce the functional assembly of the SPI-2 TTSS apparatus (67), we propose here a model for the role of Salmonella periplasmic disulfide oxidoreductases in SPI-2 TTSS function. The expression of spiA is induced when Salmonella reside in phagosomes, where bacteria impose nutrient limitations. The product of the spiA gene is transported across the inner membrane via a sec-dependent pathway; this product is in the form of a precursor protein with an N-terminal signal peptide. The signal peptide is then removed, and the mature SpiA protein undergoes folding that is assisted by two distinct periplasmic disulfide oxidoreductases, DsbA and SrgA, and is released into the periplasmic compartment. During maturation of the SCV, the pH of the phagosomal lumen may change to acidic levels by fusion with vacuolar ATPase (68). This acidification could induce the oligomerization of SpiA subunits, resulting in the formation of functional pores in the outer membrane that are required for the secretion and translocation of effector proteins by SPI-2 TTSS (67). The translocation of effector proteins into the host cytosol allows the bacteria to form SIFs and to maintain SCV where Salmonella can then replicate and escape from intracellular killing caused by host defense mechanisms.


   FOOTNOTES
 
* This work was supported in part by a Grant-in-aid for Exploratory Research 15659105 and by a 21st Century Center of Excellence program grant from the Japanese Ministry of Education, Culture, Sports, Sciences, and Technology. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger} To whom correspondence should be addressed: Dept. of Microbiology, School of Pharmaceutical Sciences, Kitasato University, 5-9-1 Shirokane, Minato-ku, Tokyo 108-8641, Japan. Tel.: 81-3-5791-6256; Fax: 81-3-3444-4831; E-mail: okadan{at}pharm.kitasato-u.ac.jp.

1 The 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. Back


   ACKNOWLEDGMENTS
 
We thank Koreaki Ito for providing the E. coli strain SK101 and Matsuhisa Inoue for the gift of rabbit anti-{beta}-lactamase antibody.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

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