A proteomic approach to study Salmonella typhi periplasmic proteins altered by a lack of the DsbA thiol: Disulfide isomerase

Abstract

Two-dimensional electrophoresis (2-DE) was used to analyze the pleiotropic effects of a deficiency in DsbA, a periplasmic disulfide-bond oxidoreductase, in Salmonella typhi. With this aim, the dsbA gene was cloned and assayed for activity in a dsbA-null mutant of Escherichia coli. A dsbA/chloramphenicol acetylase construct was then used to disrupt the wild-type gene of S. typhi. The resultant dsbA-null mutant of S. typhi, like the E. coli mutant, exhibited a lack of flagellation and of glucose-1-phosphatase activity. Periplasmic extracts from the parental and mutant strains were analyzed by 2-DE using standard denaturing and nondenaturing conditions. Differences in protein expression were more marked in nondenaturing conditions. Ninety-nine protein spots were analyzed by peptide mass fingerprinting, and 65 spots were identified by searching a S. typhi database. Twenty-five spots were exclusively detected in the wild-type strain, 10 were found only in the mutant strain, and 21 were common to both strains. We observed a lack of DsbA, glucose-1-phosphatase and flagellin in the dsbA-null mutant, which explains two of the observed phenotypes. The AI-2 autoinducer-producing protein LuxS, which is involved in quorum-sensing signalling was also absent.

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

“…A dsbA gene cloned from the S. enterica serovar Typhimurium can restore the dsbA Ϫ 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.…”

Characterization of Two Homologous Disulfide Bond Systems Involved in Virulence Factor Biogenesis in Uropathogenic Escherichia coli CFT073

“…Several studies have demonstrated a direct role for both enzymes, particularly DsbA, in the biogenesis of virulence factors utilized by bacterial pathogens in various stages of the infection process (19). The protein forming the P-ring of E. coli flagella, FlgI, was one of the first DsbA substrates identified (10) and flagellum-mediated motility was subsequently demonstrated to require the presence of functional DsbA in several gram-negative pathogens, including Salmonella enterica (1), Proteus mirabilis (8), Erwinia carotovora subsp. atroseptica (9), Burkholderia cepacia (17), and Campylobacter jejuni (42).…”

The CydDC Family of Transporters and Their Roles in Oxidase Assembly and Homeostasis

“…Motility in bacteria relies heavily upon flagella, supramolecular structures that extend from the inside of the cytoplasm to outside of the cell. The importance of redox homeostasis in motility is emphasised by the finding that deletion of dsbA or dsbB leaves many bacteria from different genera non-motile, as functional flagella can no longer be made (Agudo, Mendoza, Castanares, Nombela, & Rotger, 2004;Bringer, Rolhion, Glasser, & DarfeuilleMichaud, 2007;Burall et al, 2004;Coulthurst et al, 2008;Dailey & Berg, 1993;Hayashi, Abe, Kimoto, Furukawa, & Nakazawa, 2000;Turcot, Ponnampalam, Bouwman, & Martin, 2001). It has been shown in E. coli that DsbA introduces a disulphide bond within FlgI, a component of the P-ring protein of the flagella motor found in the periplasm (Dailey & Berg, 1993).…”