DsbA Is Required for Stable Expression of Outer Membrane Protein ...

JOURNAL OF BACTERIOLOGY, Aug. 1999, p. 5126–5130 0021-9193/99/$04.00⫹0 Copyright © 1999, American Society for Microbiology. All Rights Reserved.

Vol. 181, No. 16

DsbA Is Required for Stable Expression of Outer Membrane Protein YscC and for Efficient Yop Secretion in Yersinia pestis MICHAEL W. JACKSON

AND

GREGORY V. PLANO*

Department of Microbiology and Immunology, University of Miami School of Medicine, Miami, Florida 33176 Received 11 March 1999/Accepted 10 June 1999

The role of the periplasmic disulfide oxidoreductase DsbA in Yop secretion was investigated in Yersinia pestis. A Y. pestis dsbA mutant secreted reduced amounts of the V antigen and Yops and expressed reduced amounts of the full-sized YscC protein. Site-directed mutagenesis of the four cysteine residues present in the YscC protein resulted in defects similar to those found in the dsbA mutant. These results suggest that YscC contains at least one disulfide bond that is essential for the function of this protein in Yop secretion. Human-pathogenic Yersinia (Yersinia enterocolitica, Yersinia pseudotuberculosis, and Yersinia pestis) resist host defenses, in part through the expression and delivery of a set of plasmidencoded virulence proteins termed Yops (for reviews, see references 7 and 13). Secretion of Yops across the bacterial inner and outer membranes occurs via a type III secretion mechanism (19). Maximal expression and secretion of Yops in vitro occurs at 37°C in medium lacking calcium. The genes encoding the Y. pestis type III secretion apparatus (21) are clustered within several large transcriptional units which include yscBCDEFGHIJKL (12, 18, 23), yscNOPQRSTU (5, 9), yscW (virG) (2, 16), and yopNtyeAsycNyscXYV (lcrD) (6, 8, 10, 14, 15, 22). Many of the ysc gene products show significant similarities to components of other type III secretion systems (13). The yscC gene product shares sequence homology with a diverse family of outer membrane proteins termed secretins (13, 16). Members of this family are involved in the translocation of macromolecules across the bacterial outer membrane. Several of these proteins have been shown to form sodium dodecyl sulfate (SDS)-resistant multimers consisting of 12 to 14 monomers in the bacterial outer membrane (16, 17). It was recently demonstrated that the Y. enterocolitica YscC protein forms a ringshaped structure of approximately 20 nm with an apparent central pore (16). Mutational inactivation of yscC or of any of the other ysc genes (with the exception of yscB and yscH) prevents Yop secretion (2, 3, 5, 12–14, 18, 22). Assembly of a secretion apparatus, which spans both membranes and requires at least 21 distinct gene products for function, would likely require assistance from the cellular network of protein chaperones and disulfide oxidoreductases. The involvement of the periplasmic disulfide oxidoreductase DsbA in the secretion or release of virulence factors has previously been investigated in the enteropathogenic Escherichia coli (31), Klebsiella pneumoniae (24), Shigella flexneri (28, 30), and Erwinia chrysanthemi (25). Here, we identify the Y. pestis dsbA gene, construct a dsbA::phoA insertion mutant, and investigate the role of the dsbA gene product in Yop secretion. Isolation and nucleotide sequence of the Y. pestis dsbA gene. Degenerate PCR primers dsbA1 (5⬘-WSNTTYTWYTGYCC NCAYTG-3⬘) and dsbA2 (5⬘-TTNACRAANAYNGCNGG NAC-3⬘) consistently amplified a single 384-bp band from Y. pestis KIM10 (pCD1⫺, pPCP1⫺, pMT1⫹) chromosomal DNA.

The nucleotide sequence of the PCR fragment showed significant similarity to the E. coli dsbA gene (4). By using the dsbA PCR fragment as a probe, a 2.063-kb DraI fragment of Y. pestis KIM10 chromosomal DNA containing dsbA was identified and inserted into plasmids pBluescript SK(⫺) (Stratagene, La Jolla, Calif.) and pGEM-7zf (Promega, Madison, Wis.), generating plasmids pDSBA1 (Fig. 1) and pDSBA3, respectively. Determination of the nucleotide sequence of this fragment revealed a 621-nucleotide open reading frame coding for a predicted 207-amino-acid protein of 23,100 Da. The amino acid sequence of the predicted 207-amino-acid product of this open reading frame showed significant similarity (43 to 73% identity) to the DsbA proteins of other gram-negative bacteria. A predicted signal peptide with a Val-Thr-Ala-Ala putative cleavage site at residues 17 to 20 and a disulfide oxidoreductase active site Cys-Pro-His-Cys at residues 49 to 52 was identified (4). We concluded that the 207-codon open reading frame represents the coding sequence for the Y. pestis dsbA gene. Construction and characterization of a Y. pestis dsbA mutant. A Y. pestis dsbA mutant was constructed by phoA insertion mutagenesis and allelic exchange. The Y. pestis dsbA gene was disrupted by the insertion of an approximately 2.4-kb phoA cassette (11) into the unique KpnI site within dsbA of pDSBA3, generating plasmid pDSBA3-PhoA (Fig. 1). An approximately 4.5-kb ApaI-SacI fragment of pDSBA3-PhoA was inserted into the suicide vector pUK4134 (26), generating plasmid pUK4134.P10. The suicide plasmid was utilized to move the dsbA mutation into Y. pestis KIM5-3001.P22 [Smr, pCD1::Mu dI1734-22 (YopE⫺), pMT1, pPCP1] as previously described (26), generating the Y. pestis dsbA insertion mutant Y. pestis KIM5-3001.P23. KIM5-3001.P22 was generated by electroporation of pCD1::Mu dI1734-22 isolated from KIM5-3122 (27) [pCD1::Mu dI1734-22 (YopE⫺), pMT1, pPCP1] into KIM63001 (Smr, pMT1, pPCP1). The presence of the phoA cassette within the KIM5-3001.P23 dsbA locus was confirmed by PCR with both dsbA- and phoA-specific primers. The periplasmic ␤-lactamase resistance protein contains a single DsbA-dependent nonessential disulfide bond whose presence can be determined by comparing the SDS-polyacrylamide gel electrophoresis (PAGE) migration of the protein in the presence and absence of reducing agents (20). To test the ability of Y. pestis KIM5-3001.P23 to form this disulfide bond, Y. pestis KIM5 and KIM5-3001.P23 were transformed with pBluescript SK(⫺), and positive transformants were analyzed by SDS-PAGE and immunoblotting with antiserum specific for ␤-lactamase (5-prime, 3-prime, Inc., Boulder, Colo.). ␤-Lactamase from Y. pestis KIM5 migrated faster in the absence of 5%

* Corresponding author. Mailing address: Department of Microbiology and Immunology, University of Miami School of Medicine, Miami, FL 33176. Phone: (305) 243-6310. Fax: (305) 243-4623. E-mail: [email protected]. 5126

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FIG. 1. Physical and genetic maps of the Y. pestis dsbA region. The approximately 2-kb DraI fragment contains the dsbA gene and an unidentified open reading frame designated orf1. The approximate location of the degenerative oligonucleotide primers dsba1 and dsba2 used to amplify a portion of the Y. pestis dsbA gene are indicated by arrows. The unique KpnI restriction endonuclease site was used for insertion of the pPHO7 phoA cassette. Plasmids pDSBA1 and pDSBA2 were used in complementation studies.

␤-mercaptoethanol (␤ME) than in its presence, while the migration of ␤-lactamase from Y. pestis KIM5-3001.P23 migrated at the slower rate in both the presence and absence of reducing agents (Fig. 2A). These data indicate that Y. pestis KIM53001.P23 is defective in periplasmic disulfide bond formation, which is consistent with the phenotype of a dsbA mutant. Secretion of YopM, YopN, and Vantigen by the Y. pestis dsbA insertion mutant. Rates of secretion of YopM, YopN, and V antigen by Y. pestis KIM5 and the isogenic dsbA mutant KIM53001.P23 with and without plasmid pDSBA1 were determined by SDS-PAGE and immunoblot analysis as previously described (22). The parent Y. pestis KIM5 and the dsbA mutant Y. pestis KIM5-3001.P23 both secreted YopM, YopN, and V antigen in the absence of calcium; however, the secretion of these

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proteins was dramatically reduced in the dsbA mutant (Fig. 2B; YopN and V antigen data not shown). Complementation of Y. pestis KIM5-3001.P23 with plasmid pDSBA1 completely restored normal levels of YopM, YopN, and V antigen secretion, indicating that the defect in Yop secretion was specifically due to the disruption of the Y. pestis dsbA gene. YscC is unstable in the Y. pestis dsbA insertion mutant. SDS-PAGE and immunoblotting with antisera specific for YscB, -C, -D, -G, -J, -R, -W (VirG), and LcrD (9, 22, 23) were used to directly test for defects in the expression and stability of specific components of the Yop secretion apparatus. No defect in the expression of YscB, -D, -G, -J, -R, -W (VirG), or LcrD was detected (data not shown); however, the amount of YscC monomer detected in the Y. pestis dsbA mutant was specifically reduced (Fig. 2C). A unique anti-YscC reactive band migrating below the YscC monomer was also detected in the sample from the dsbA mutant. This peptide was most likely a proteolytic degradation product of YscC. In addition, the YscC complex (16, 23), which normally migrates as a discrete band at the top of the stacking gel, appeared as a diffuse, slightly lower-molecular-weight band in the dsbA mutant. Complementation of the dsbA mutant with either pDSBA1 or pDSBA2 restored the normal expression, stability, and migration of the YscC protein and of the YscC complex. Expression of the upstream yscB gene product and the downstream yscD and yscJ gene products was not significantly affected in the dsbA mutant, indicating that the reduced amount of YscC monomer was not due to decreased transcription of the yscABCDEFGHIJKL operon (data not shown). Together, these results suggest that YscC and the YscC complex are susceptible to proteolytic degradation in the Y. pestis dsbA mutant. The carboxyl-terminal region of YscC contains four cysteine residues required for stable expression of YscC in Y. pestis. Examination of the predicted YscC amino acid sequence (21,

FIG. 2. Effect of the dsbA mutation on periplasmic disulfide bond formation, Yop secretion, and YscC expression in Y. pestis. (A) Y. pestis KIM5-3001.P22 (parent) and the dsbA mutant Y. pestis KIM5-3001.P23 (dsbA::phoA) carrying plasmid pBluescript SK(⫺) (Apr) were grown at 37°C in the presence of calcium (2.5 mM). Bacterial cells were resuspended in SDS-PAGE solubilization buffer with (⫹␤ME) or without (⫺␤ME) 5% ␤ME, separated by SDS-PAGE, blotted to Immobilon-P membranes (Millipore Corp., Bedford, Mass.), and probed with antiserum specific for ␤-lactamase. The reduced (red.) and oxidized (ox.) forms of ␤-lactamase are shown by arrowheads. (B) Immunoblot analysis of culture supernatant (S) and cell pellet (P) fractions from the parent and dsbA mutant, with or without plasmid pDSBA1, grown at 37°C in the presence (⫹) or absence (⫺) of calcium. Antiserum specific for YopM was used to detect this protein (arrowheads). (C) Immunoblot analysis of cell pellet fractions from the parent and the dsbA mutant, with or without plasmid pDSBA1, a yscC deletion mutant KIM5-3001.12, and a lcrD deletion mutant KIM5-3001.3. Antiserum specific for YscC was utilized to detect the YscC monomer and the YscC-containing SDS-resistant complex (arrowheads).

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FIG. 3. Effect of cysteine-to-serine missense mutations in yscC on Yop secretion and YscC expression in Y. pestis. (A) Immunoblot analysis of culture supernatant (S) and cell pellet (P) fractions from the yscC deletion mutant KIM5-3001.12 and the yscC deletion mutant complemented with plasmids pYSCC1, pYSCC1 (C551S), pYSCC1 (C576S), pYSCC1 (C591S), and pYSCC1 (C598S) grown at 37°C in the presence (⫹) or absence (⫺) of calcium. Antiserum specific for YopM was used to detect this protein (arrowheads). (B) Immunoblot analysis of cell pellet fractions from the yscC deletion mutant KIM5-3001.12 and the yscC deletion mutant complemented with plasmids pYSCC1, pYSCC1 (C551S), pYSCC1 (C576S), pYSCC1 (C591S), and pYSCC1 (C598S) grown at 37°C in the absence of calcium. Antiserum specific for YscC was utilized to detect the YscC monomer and the YscC-containing SDS-resistant complex (arrowheads).

23) revealed that the 607-residue YscC protein contained four cysteine residues (Cys551, Cys576, Cys591, and Cys598). All four cysteines were found within a 48-amino-acid region located near the carboxyl-terminus of YscC (see Fig. 5). Interestingly, the location and spacing of the four cysteines were conserved between the Y. pestis YscC protein and the Pseudomonas aeruginosa PscC protein (see Fig. 5) (29). Because the DsbA proteins of E. coli and other gram-negative bacteria are periplasmic enzymes that catalyze disulfide bond formation, the effects on YscC expression and stability could be mediated directly through the four carboxyl-terminal cysteine residues of YscC. To test the role of these amino acids in YscC function and stability, we used site-directed mutagenesis to change the four yscC codons encoding cysteine to codons encoding serine. Point mutations within yscC were generated in plasmid pYSCC1 (23) by the PCR-ligation-PCR technique (1), resulting in plasmids pYSCC1 (C551S), pYSCC1 (C576S), pYSCC1 (C591S), and pYSCC1 (C598S). The ability of these plasmids to complement normal YscC expression and function was determined by inserting the plasmids into the Y. pestis yscC deletion mutant KIM5-3001.12 (20) and measuring Yop secretion and YscC expression as described for the experiments whose results are shown in Fig. 2. The yscC deletion mutant expressed intracellular YopM, YopN, and V antigen; however, as shown previously (23), these proteins were not exported to the culture supernatant in the presence or absence of calcium (Fig. 3A; YopN and V antigen data not shown). Complementation of the yscC mutant with pYSCC1, pYSCC1 (C591S), or pYSCC1 (C598S) completely restored calcium-regulated Yop and V

antigen secretion. However, the yscC deletion mutant complemented with plasmids pYSCC1 (C551S) or pYSCC1 (C576S) exported significantly reduced amounts of YopM, YopN, and V antigen. Thus, replacement of cysteine codon 551 or 576 with serine codons resulted in reduced secretion of Yops and V antigen, similar to the effects observed in the dsbA insertion mutant. The effect of the dsbA insertion mutation upon the function and proteolytic stability of YscC could be mediated directly through the four cysteines in YscC. If so, substitution of serine residues for these cysteines would result in effects on YscC stability similar to those seen in the dsbA insertion mutant. SDS-PAGE and immunoblotting with antiserum specific for YscC demonstrated that the yscC deletion mutant complemented with pYSCC1 expressed individual, discrete bands representing the YscC monomer and the high-molecular-weight YscC complex (Fig. 3B). However, the amount of YscC monomer was dramatically reduced in the yscC deletion mutant complemented with pYSCC1 (C551S) or pYSCC1 (C576S). An apparent YscC degradation product, similar in size to the degradation product found in the dsbA insertion mutant, was identified in each of the strains expressing a YscC product with a cysteine-to-serine substitution. In addition to the appearance of a specific YscC degradation product, the high-molecularweight YscC complex migrated as a lower-molecular-weight diffuse band in each of the four strains expressing a cysteineto-serine mutant YscC protein (Fig. 3B). To confirm that Cys551, Cys576, Cys591, and Cys598 are involved in the formation of intramolecular, not intermolecu-

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FIG. 4. Electrophoretic mobility of YscC and YscC (C598S) in the presence or absence of ␤ME. Proteins from Y. pestis KIM5-3001 (parent), the yscC deletion mutant (⌬yscC), and the yscC deletion mutant complemented with pYSCC1 or pYSCC1 (C598S) were denatured in SDS at 100°C in the presence (⫹) or absence (⫺) of 5% ␤ME, separated by SDS-PAGE in a 7.5% acrylamide gel, blotted to Immobilon-P membranes, and incubated with antiserum specific for YscC. The reduced (red.) and oxidized (ox.) forms of YscC are shown by arrowheads.

lar, disulfide bonds, cell lysates from Y. pestis KIM5, the yscC deletion mutant, and the yscC deletion mutant complemented with pYSCC1 or pYSCC1 (C598S) were solubilized (at 100°C) in the presence or absence of ␤ME and analyzed by SDSPAGE and immunoblotting (Fig. 4). YscC migrated as a

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monomer in both the presence and absence of ␤ME. The migration of YscC was slightly faster in the absence of ␤ME, suggesting the presence of at least one disulfide bond. Interestingly, the yscC deletion mutant carrying pYSCC1 (C591S) or pYSCC1 (C598S) expressed a YscC product that migrated at the approximate size of a YscC dimer in the absence of ␤ME [YscC (S591S)] (data not shown), suggesting that the nonpaired cysteine in these mutants is capable of forming an intermolecular disulfide bond with a neighboring YscC (C598S). These data are consistent with the hypothesis that wild-type YscC contains two intramolecular disulfide bonds. Strains expressing a YscC product with either the C551S or C576S substitution showed both a significant reduction in Yop secretion and a dramatic effect upon the stability of the YscC monomer and YscC complex. The C591S and C598S mutations had no detectable effect upon Yop secretion; however, these mutations still had a significant, but less pronounced, effect upon YscC stability. To account for both the effect of the dsbA insertion and the cysteine-to-serine mutations on the stability and function of YscC, we suggest that YscC contains two DsbA-dependent disulfide bonds, one between Cys551 and Cys576 and a second between Cys591 and Cys598 (Fig. 5). Both predicted disulfide bonds are required for stable production of YscC; however, the predicted Cys551-S-S-Cys576 disulfide bond appears to be more important in this regard. Insertion mutagenesis of Y. pestis dsbA prevents the proper formation of these bonds, as does mutagenesis of the codons encoding the specific cysteine residues. These data indicate that while both disulfide bonds are required for the proteolytic stability of YscC, only disruption of the Cys551-S-S-Cys576 disulfide bond affected the function of YscC in Yop secretion. Although the individual cysteine-to-serine substitutions in YscC reproduced all of the defects in Yop secretion and YscC stability seen in the dsbA insertion mutant, these results do not preclude a role for DsbA in disulfide bond formation in other Y. pestis proteins involved in Yop secretion. Disulfide bonds are critical to the folding, stability, and function of many bacterial proteins. For example, a DsbA-dependent disulfide bond in bundlin, the major structural subunit of the enteropathogenic E. coli bundle-forming pilus, is essential for the proteolytic stability of this protein (31). In addition, the S. flexneri dsbA gene is required for surface presentation of the Spa32 protein and for the subsequent release of Ipa proteins (28). We determined that the Y. pestis dsbA mutant exerts its effect on Yop secretion at least in part by reducing the level of stable, functional YscC protein, an essential component of the Yop secretion apparatus. DsbA appears to be required for the formation of two disulfide bonds in YscC itself. These disulfide bonds appear to be critical for maintaining YscC in a stable

FIG. 5. Amino acid sequence alignment of the carboxyl-terminal regions of Y. pestis YscC and P. aeruginosa PscC. The four conserved cysteine residues are shown in bold. Putative disulfide bonds between Cys551 and Cys576 and between Cys591 and Cys598 are shown by dashed lines. Identical amino acid residues (vertical lines) and similar amino acid residues (pluses) in the two proteins are indicated.

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