Characterization of SrgA, a Salmonella enterica Serovar Typhimurium Virulence Plasmid-Encoded Paralogue of the Disulfide Oxidoreductase DsbA, Essential for Biogenesis of Plasmid-Encoded Fimbriae

Abstract

Disulfide oxidoreductases are viewed as foldases that help to maintain proteins on productive folding pathways by enhancing the rate of protein folding through the catalytic incorporation of disulfide bonds. SrgA, encoded on the virulence plasmid pStSR100 of Salmonella enterica serovar Typhimurium and located downstream of the plasmid-borne fimbrial operon, is a disulfide oxidoreductase. Sequence analysis indicates that SrgA is similar to DsbA from, for example, Escherichia coli, but not as highly conserved as most of the chromosomally encoded disulfide oxidoreductases from members of the family Enterobacteriaceae. SrgA is localized to the periplasm, and its disulfide oxidoreductase activity is dependent upon the presence of functional DsbB, the protein that is also responsible for reoxidation of the major disulfide oxidoreductase, DsbA. A quantitative analysis of the disulfide oxidoreductase activity of SrgA showed that SrgA was less efficient than DsbA at introducing disulfide bonds into the substrate alkaline phosphatase, suggesting that SrgA is more substrate specific than DsbA. It was also demonstrated that the disulfide oxidoreductase activity of SrgA is necessary for the production of plasmid-encoded fimbriae. The major structural subunit of the plasmidencoded fimbriae, PefA, contains a disulfide bond that must be oxidized in order for PefA stability to be maintained and for plasmid-encoded fimbriae to be assembled. SrgA efficiently oxidizes the disulfide bond of PefA, while the S. enterica serovar Typhimurium chromosomally encoded disulfide oxidoreductase DsbA does not. pefA and srgA were also specifically expressed at pH 5.1 but not at pH 7.0, suggesting that the regulatory mechanisms involved in pef gene expression are also involved in srgA expression. SrgA therefore appears to be a substrate-specific disulfide oxidoreductase, thus explaining the requirement for an additional catalyst of disulfide bond formation in addition to DsbA of S. enterica serovar Typhimurium.The ability of the bacterial cell to synthesize and secrete specific proteins is dependent upon several aspects of protein secretory pathways. While the passage of proteins through the secretory apparatus in the inner membrane has been relatively well studied (reviewed in references 18, 42, and 60), the events occurring once proteins reach the periplasm of gram-negative cells are less well understood. Even though the periplasm may not be their final destination, proteins begin the process of conformational folding once they reach the periplasm (74). This folding is accompanied by proline isomerization and disulfide bond formation, both necessary to attaining and maintaining native structure (23). These processes are mediated by peptidyl-prolyl isomerases (45) and disulfide oxidoreductases (2, 50, 56) that are present in the periplasm.Many proteinaceous structures, such as fimbriae (39, 78), flagella (14), and several bacterial toxins (46,53,65,77), either contain disulfide bonds or require disulfide bonds in some component of their assembly...

F-Like Type IV Secretion Systems Encode Proteins with Thioredoxin Folds That Are Putative DsbC Homologues

“…TrbB F and homologues encoded by the three H plasmids contain the thioredoxin C-X-X-C active-site motif, as well as the essential proline residue that is adjacent to the active-site loop. They also contain a hydrophobic or aromatic residue at the third position, which is consistent with a possible role in disulfide bond isomerization (13) Proteins that promote disulfide bond formation have previously been shown to be essential for the proper assembly of a number of periplasmic protein complexes, including the E. coli flagellar apparatus, the enteropathogenic E. coli bundle-forming pilus, the E. coli pap pili, the Klebsiella oxytoca type II secreton, and Salmonella enterica serovar Typhimurium plasmid-encoded fimbriae (15,58). In addition to previous studies on the effect of a dsbA mutation on F piliation as measured by filamentous phage sensitivity and electron microscopy (10), we observed that the mating efficiency of the F plasmid is also affected by the presence of a dsbA mutation in the donor cell and that the addition of DTT further decreases mating ability to undetectable levels.…”

Salmonella enterica Serovar Typhimurium Can Detect Acyl Homoserine Lactone Production by Yersinia enterocolitica in Mice

“…2). There was also a correlation between resolution and the number of Yersinia CFU recovered from the sample, with resolution requiring approximately 10 6 Yersinia CFU (Fig. 3).…”

“…Two genes in the rck operon, pefI and srgA (sdiA-regulated gene), appear to affect the expression and function of the pef operon. The pefI gene encodes a transcriptional regulator of the pef operon, and the srgA gene is a dsbA paralog that specifically catalyzes the oxidation of a disulfide bond in the PefA fimbrial subunit (6,30). Therefore, sdiA may indirectly affect the expression and assembly of plasmid-encoded fimbriae on the surface of the bacterium.…”

The Different Strategies Used by Salmonella to Invade Host Cells

“…Two genes in this operon, pefI (plasmid encoded fimbriae) and srgA (sdiA-regulated gene), affect the expression and function of the pef operon located upstream of the rck operon and involved in the biosynthesis of the Pef fimbriae. pefI encodes a transcriptional regulator of the pef operon, and SrgA is a DsbA paralog that efficiently oxidizes the disulfide bond of PefA, the major structural subunit of the Pef fimbriae (Bouwman et al, 2003). These fimbriae are involved in biofilm formation, adhesion to murine small intestine and fluid accumulation in the infant mouse (Baumler et al, 1996;Ledeboer et al, 2006).…”