title: Two periplasmic disulfide oxidoreductases, DsbA and SrgA, target outer membrane protein SpiA, a component of the Salmonella pathogenicity island 2 type III secretion system.
abstract: 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.
authors.affiliation: Department of Microbiology, School of Pharmaceutical Sciences, Kitasato University, 5-9-1 Shirokane, Minato-ku, Tokyo 108-8641, Japan.
authors.forename: Tsuyoshi
authors.initials: T
authors.lastname: Miki
authors.forename: Nobuhiko
authors.initials: N
authors.lastname: Okada
authors.forename: Hirofumi
authors.initials: H
authors.lastname: Danbara
mesh_terms: D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism*; D015153:Blotting, Western; D002455:Cell Division; D017931:DNA Primers / Q000737:chemistry; D004220:Disulfides; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D044404:Genomic Islands; D005839:Gentamicins / Q000494:pharmacology; D006367:HeLa Cells; D006801:Humans; D007763:Lac Operon; D051379:Mice; D008807:Mice, Inbred BALB C; D008840:Microfilament Proteins / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D009154:Mutation; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000737:chemistry* / Q000378:metabolism / Q000502:physiology*; D050862:Oxidoreductases Acting on Sulfur Group Donors; D019897:Periplasm / Q000201:enzymology / Q000378:metabolism; D033903:Periplasmic Proteins / Q000737:chemistry* / Q000378:metabolism; D010957:Plasmids / Q000378:metabolism; D019704:Protein Disulfide-Isomerases / Q000378:metabolism / Q000502:physiology*; D021381:Protein Transport; D012475:Salmonella / Q000378:metabolism; D019779:Salmonella enterica / Q000378:metabolism*; D014774:Virulence
chemical_list: D001426:Bacterial Proteins; D017931:DNA Primers; D004220:Disulfides; D005839:Gentamicins; D008840:Microfilament Proteins; D033903:Periplasmic Proteins; C118287:SipA protein, Salmonella; D010088:Oxidoreductases; D050862:Oxidoreductases Acting on Sulfur Group Donors; C490374:SrgA protein, Salmonella typhimurium; C077913:periplasmic protein disulfide oxidoreductase; D019704:Protein Disulfide-Isomerases