Dataset Information

Identification of a novel Salmonella invasion locus homologous to Shigella ipgDE.

ABSTRACT: Genes essential for Salmonella typhimurium invasion have been localized to Salmonella pathogenicity island 1 (SPI1) on the chromosome. However, it is clear that other genes are required for the invasion process. Mutations that abolish the SPI1 invasion type III secretion system do not significantly reduce invasion into Chinese hamster ovary tissue culture cells. Two invasion defective mutants were isolated by screening 2,500 Tn10dTc insertion mutants of S. typhimurium in the tissue culture invasion assay. One of the invasion mutants, SVM167, has an insertion between centisomes 24.5 and 25.5 in an operon homologous to the ipgDEF operon of the Shigella flexneri and Shigella sonnei virulence plasmid. A second mutant, SVM168, has an insertion in an IS3-type element with homology to the Salmonella enteritidis IS1351 element and Yersinia enterocolitica IS1400 element from a high-pathogenicity island. Further characterization of SVM167 showed that culture supernatants from this mutant lack a previously uncharacterized protein that is also missing from culture supernatants of a SPI1 mutant, suggesting it can be secreted by the SPI1 type III secretion system. In addition, transcription of this operon, sigDE (Salmonella invasion gene), is dependent on the presence of sirA, an activator of hilA expression. HilA activates transcription of several of the SPI1 genes but does not appear to have a major role in activation of transcription from the sigDE promoter.

SUBMITTER: Hong KH

PROVIDER: S-EPMC107092 | biostudies-literature | 1998 Apr

REPOSITORIES: biostudies-literature

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Identification of a novel Salmonella invasion locus homologous to Shigella ipgDE.

Hong K H KH Miller V L VL

Journal of bacteriology 19980401 7

Genes essential for Salmonella typhimurium invasion have been localized to Salmonella pathogenicity island 1 (SPI1) on the chromosome. However, it is clear that other genes are required for the invasion process. Mutations that abolish the SPI1 invasion type III secretion system do not significantly reduce invasion into Chinese hamster ovary tissue culture cells. Two invasion defective mutants were isolated by screening 2,500 Tn10dTc insertion mutants of S. typhimurium in the tissue culture invas ...[more]

PMID: 9537377

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Project description:An avirulent, invasion plasmid insertion mutant of Shigella flexneri 5 (pHS1059) was restored to the virulence phenotype by transformation with a partial HindIII library of the wild-type invasion plasmid constructed in pBR322. Western immunoblot analysis of pHS1059 whole-cell lysates revealed that the synthesis of the invasion plasmid antigens VirG, IpaA, IpaB, IpaC, and IpaD was similar to that seen in the corresponding isogenic S. flexneri 5 virulent strain, M90T. IpaB and IpaC, however, were not present on the surface of pHS1059 as was found in M90T, suggesting that the transport or presentation of the IpaB and IpaC proteins onto the bacterial surface was defective in the mutant. pHS1059 was complemented by pWR266, which carried contiguous 1.2- and 4.1-kb HindIII fragments of the invasion plasmid. pHS1059(pWR266) cells were positive in the HeLa cell invasion assay as well as colony immunoblot and enzyme-linked immunosorbent assays, using monoclonal antibodies to IpaB and IpaC. These studies established that the antigens were expressed on the surface of the transformed bacteria. In addition, water extraction of pHS1059 and pHS1059(pWR266) whole cells, which can be used to remove IpaB and IpaC antigens from the surface of wild-type M90T bacteria, yielded significant amounts of these antigens from pHS1059(pWR266) but not from pHS1059. Minicell and DNA sequence analysis indicated that several proteins were encoded by pWR266, comprising the spa loci, which were mapped to a region approximately 18 kb upstream of the ipaBCDAR gene cluster. Subcloning and deletion analysis revealed that more than one protein was involved in complementing the Spa- phenotype in pHS1059. One of these proteins, Spa47, showed striking homology to ORF4 of the Bacillus subtilis flaA locus and the fliI gene sequence of Salmonella typhimurium, both of which bear strong resemblance to the alpha and beta subunits of bacterial, mitochondrial, and chloroplast proton-translocating F0F1 ATPases.

Project description:BackgroundSalmonella is a major bacterial pathogen associated with a large number of outbreaks of foodborne diseases. Many highly virulent serovars that cause human illness belong to Salmonella serogroup C1, and Salmonella ser. Choleraesuis is a prominent cause of invasive infections in Asia. Comparative genomic analysis in our previous study showed that two homologous genes, SC0368 and SC0595 in Salmonella ser. Choleraesuis were unique to serogroup C1. In this study, two single-deletion mutants (Δ0368 and Δ0595) and one double-deletion mutant (Δ0368Δ0595) were constructed based on the genome. All these mutants and the wild-type strain were subjected to RNA-Seq analysis to reveal functional relationships of the two serogroup C1-specific genes.ResultsData from RNA-Seq indicated that deletion of SC0368 resulted in defects in motility through repression of σ28 in flagellar regulation Class 3. Consistent with RNA-Seq data, results from transmission electron microcopy (TEM) showed that flagella were not present in △0368 and △0368△0595 mutants resulting in both swimming and swarming defects. Interestingly, the growth rates of two non-motile mutants △0368 and △0368△0595 were significantly greater than the wild-type, which may be associated with up-regulation of genes encoding cytochromes, enhancing bacterial proliferation. Moreover, the △0595 mutant was significantly more invasive in Caco-2 cells as shown by bacterial enumeration assays, and the expression of lipopolysaccharide (LPS) core synthesis-related genes (rfaB, rfaI, rfaQ, rfaY, rfaK, rfaZ) was down-regulated only in the △0368△0595 mutant. In addition, this study also speculated that these two genes might be contributing to serotype conversion for Salmonella C1 serogroup based on their apparent roles in biosynthesis of LPS and the flagella.ConclusionA combination of biological and transcriptomic (RNA-Seq) analyses has shown that the SC0368 and SC0595 genes are involved in biosynthesis of flagella and complete LPS, as well as in bacterial growth and virulence. Such information will aid to revealing the role of these specific genes in bacterial physiology and evolution within the serogroup C1.

Dataset Information

Identification of a novel Salmonella invasion locus homologous to Shigella ipgDE.

Publications

Identification of a novel Salmonella invasion locus homologous to Shigella ipgDE.

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