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Identification of Redox Partners of the Thiol-Disulfide Oxidoreductase SdbA in Streptococcus gordonii.

ABSTRACT: We previously identified a novel thiol-disulfide oxidoreductase, SdbA, in Streptococcus gordonii that formed disulfide bonds in substrate proteins and played a role in multiple phenotypes. In this study, we used mutational, phenotypic, and biochemical approaches to identify and characterize the redox partners of SdbA. Unexpectedly, the results showed that SdbA has multiple redox partners, forming a complex oxidative protein-folding pathway. The primary redox partners of SdbA that maintain its active site in an oxidized state are a surface-exposed thioredoxin family lipoprotein called SdbB (Sgo_1171) and an integral membrane protein annotated as CcdA2. Inactivation of sdbB and ccdA2 simultaneously, but not individually, recapitulated the sdbA mutant phenotype. The sdbB-ccdA2 mutant had defects in a range of cellular processes, including autolysis, bacteriocin production, genetic competence, and extracellular DNA (eDNA) release. AtlS, the natural substrate of SdbA produced by the sdbB-ccdA2 mutant lacked activity and an intramolecular disulfide bond. The redox state of SdbA in the sdbB-ccdA2 mutant was found to be in a reduced form and was restored when sdbB and ccdA2 were knocked back into the mutant. In addition, we showed that SdbB formed a disulfide-linked complex with SdbA in the cell. Recombinant SdbB and CcdA2 exhibited oxidase activity and reoxidized reduced SdbA in vitro Collectively, our results demonstrate that S. gordonii uses multiple redox partners for oxidative protein folding.IMPORTANCE Streptococcus gordonii is a commensal bacterium of the human dental plaque. Previously, we identified an enzyme, SdbA, that forms disulfide bonds in substrate proteins and plays a role in a number of cellular processes in S. gordonii Here, we identified the redox partners of SdbA. We showed that SdbA has multiple redox partners, SdbB and CcdA2, forming a complex oxidative protein-folding pathway. This pathway is essential for autolysis, bacteriocin production, genetic competence, and extracellular DNA (eDNA) release in S. gordonii These cellular processes are considered to be important for the success of S. gordonii as a dental plaque organism. This is the first example of an oxidative protein-folding pathway in Gram-positive bacteria that consists of an enzyme that uses multiple redox partners to function.

SUBMITTER: Jalal NA

PROVIDER: S-EPMC6482928 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:UnlabelledStreptococcus gordonii is a commensal inhabitant of the human oral cavity. To maintain its presence as a major component of oral biofilms, S. gordonii secretes inhibitory molecules such as hydrogen peroxide and bacteriocins to inhibit competitors. S. gordonii produces two nonmodified bacteriocins (i.e., Sth1 and Sth2) that are regulated by the Com two-component regulatory system, which also regulates genetic competence. Previously we found that the thiol-disulfide oxidoreductase SdbA was required for bacteriocin activity; however, the role of SdbA in Com signaling was not clear. Here we demonstrate that ΔsdbA mutants lacked bacteriocin activity because the bacteriocin gene sthA was strongly repressed and the peptides were not secreted. Addition of synthetic competence-stimulating peptide to the medium reversed the phenotype, indicating that the Com pathway was functional but was not activated in the ΔsdbA mutant. Repression of bacteriocin production was mediated by the CiaRH two-component system, which was strongly upregulated in the ΔsdbA mutant, and inactivation of CiaRH restored bacteriocin production. The CiaRH-induced protease DegP was also upregulated in the ΔsdbA mutant, although it was not required for inhibition of bacteriocin production. This establishes CiaRH as a regulator of Sth bacteriocin activity and links the CiaRH and Com systems in S. gordonii. It also suggests that either SdbA or one of its substrates is an important factor in regulating activation of the CiaRH system.ImportanceStreptococcus gordonii is a noncariogenic colonizer of the human oral cavity. To be competitive in the oral biofilm, S. gordonii secretes antimicrobial peptides called bacteriocins, which inhibit closely related species. Our previous data showed that mutation of the disulfide oxidoreductase SdbA abolished bacteriocin production. In this study, we show that mutation of SdbA generates a signal that upregulates the CiaRH two-component system, which in turn downregulates a second two-component system, Com, which regulates bacteriocin expression. Our data show that these systems are also linked in S. gordonii, and the data reveal that the cell's ability to form disulfide bonds is sensed by the CiaRH system.

Project description:The actinobacterium Corynebacterium matruchotii has been implicated in nucleation of oral microbial consortia leading to biofilm formation. Due to the lack of genetic tools, little is known about basic cellular processes, including protein secretion and folding, in this organism. We report here a survey of the C. matruchotii genome, which encodes a large number of exported proteins containing paired cysteine residues, and identified an oxidoreductase that is highly homologous to the Corynebacterium diphtheriae thiol-disulfide oxidoreductase MdbA (MdbACd). Crystallization studies uncovered that the 1.2-Å resolution structure of C. matruchotii MdbA (MdbACm) possesses two conserved features found in actinobacterial MdbA enzymes, a thioredoxin-like fold and an extended ?-helical domain. By reconstituting the disulfide bond-forming machine in vitro, we demonstrated that MdbACm catalyzes disulfide bond formation within the actinobacterial pilin FimA. A new gene deletion method supported that mdbA is essential in C. matruchotii Remarkably, heterologous expression of MdbACm in the C. diphtheriae ?mdbA mutant rescued its known defects in cell growth and morphology, toxin production, and pilus assembly, and this thiol-disulfide oxidoreductase activity required the catalytic motif CXXC. Altogether, the results suggest that MdbACm is a major thiol-disulfide oxidoreductase, which likely mediates posttranslocational protein folding in C. matruchotii by a mechanism that is conserved in ActinobacteriaIMPORTANCE The actinobacterium Corynebacterium matruchotii has been implicated in the development of oral biofilms or dental plaque; however, little is known about the basic cellular processes in this organism. We report here a high-resolution structure of a C. matruchotii oxidoreductase that is highly homologous to the Corynebacterium diphtheriae thiol-disulfide oxidoreductase MdbA. By biochemical analysis, we demonstrated that C. matruchotii MdbA catalyzes disulfide bond formation in vitro Furthermore, a new gene deletion method revealed that deletion of mdbA is lethal in C. matruchotii Remarkably, C. matruchotii MdbA can replace C. diphtheriae MdbA to maintain normal cell growth and morphology, toxin production, and pilus assembly. Overall, our studies support the hypothesis that C. matruchotii utilizes MdbA as a major oxidoreductase to catalyze oxidative protein folding.

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Identification of Redox Partners of the Thiol-Disulfide Oxidoreductase SdbA in Streptococcus gordonii.

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