The flagellar sigma factor FliA (sigma(28)) regulates the expression of Salmonella genes associated with the centisome 63 type III secretion system - PubMed (original) (raw)

The flagellar sigma factor FliA (sigma(28)) regulates the expression of Salmonella genes associated with the centisome 63 type III secretion system

K Eichelberg et al. Infect Immun. 2000 May.

Abstract

One of the essential features of all pathogenic strains of Salmonella enterica is the ability to enter into nonphagocytic cells. This pathogenic property is mediated by the Salmonella pathogenicity island 1 (SPI-1)-encoded type III secretion system. Expression of components and substrates of this system is subject to complex regulatory mechanisms. These mechanisms include a number of specific and global transcriptional regulatory proteins. In this study we have compared in S. enterica serovars Typhimurium and Typhi the effect of mutations in flagellar genes on the phenotypes associated with the SPI-1 type III protein secretion system. We found that serovar Typhi strains carrying a null mutation in either of the flagellar regulatory genes flhDC or fliA were severely deficient in entry into cultured epithelial cells and macrophage cytotoxicity. This defect could not be reversed by applying a mild centrifugal force, suggesting that the effects of the mutations were not due to the absence of motility. In contrast, the same mutations had no significant effect on the ability of serovar Typhimurium to enter into cultured Henle-407 cells or to induce macrophage cell death. Consistent with these observations, we found that the mutations in the flagellar regulatory proteins significantly reduced the expression of components of the SPI-1-encoded type III system in serovar Typhi but had a marginal effect in serovar Typhimurium. Our results therefore indicate that there is an overlap between regulatory mechanisms that control flagellar and type III secretion gene expression in Salmonella serovar Typhi.

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Figures

FIG. 1

Culture supernatant protein profiles of wild-type S. enterica serovars Typhi and Typhimurium. Culture supernatant proteins from wild-type serovar Typhi (ISP1820) and serovar Typhimurium (SL1344) were obtained as indicated in Materials and Methods, separated on an SDS–10% polyacrylamide gel, and stained with Coomassie blue. The arrows on the right indicate the positions of a subset of secreted proteins.

FIG. 2

Effect of flagellar gene mutations on the ability of Salmonella strains to enter into Henle-407 cells in the presence or absence of a mild centrifugal force. Transposon insertions in the three regulatory classes of flagellar genes were introduced into S. enterica serovars Typhimurium (A) and Typhi (B), and the strains were tested for their ability to enter cultured epithelial cells in the presence or absence of a mild centrifugal force. Values represent the percentage of the bacterial inoculum that survived the gentamicin treatment and have been standardized to the level of internalization of the wild-type (w.t.) strain in each category, which was considered 100%. The values represent the means ± standard deviations from one representative experiment performed with triplicate samples. Equivalent results were obtained in several repetitions of this experiment. The actual percentage of the wild-type serovar Typhimurium and Typhi inocula that resisted the gentamicin treatment under these assay conditions ranged between 40 and 60% in different experiments.

FIG. 3

Effect of flagellar mutations on Salmonella macrophage cytotoxicity. Transposon insertions were introduced in three flagellar genes (fliA, flgM, and flgK) in S. enterica serovars Typhimurium (A) and Typhi (B) and tested for cytotoxicity in cultured J774.A1 macrophages as described in Materials and Methods. The percentage of dead cells was determined after 3 h of infection by staining with the membrane-impermeant dye ethidium homodimer-1. During the first 10 min of infection, bacteria were spun onto the macrophages at 500 × g. The values represent the average values obtained from three independent experiments, including the standard deviation between these measurements. For each sample a minimum of 500 cells were examined. w.t., wild type.

FIG. 4

Effect of mutations in fliA and flgM on the transcription of components of the SPI-1 type III secretion system. The levels of transcription of the different reporter gene fusions in different S. enterica serovar Typhimurium and (A) and Typhi (B) backgrounds were measured by assaying C2,3O activity in bacterial cell lysates as indicated in Materials and Methods. The values represent the means ± standard deviations from one representative experiment performed with triplicate samples. Equivalent results were obtained in several repetitions of this experiment. w.t., wild type.

FIG. 5

Western blot analysis of SipC production and secretion. Proteins from culture supernatants and whole-cell lysates of wild-type S. enterica serovars Typhimurium (SL1344) (A) and Typhi (ISP1820) (B) and isogenic strains carrying transposon insertions in the three regulatory classes of flagellar genes were resolved by SDS–10% PAGE, transferred to nitrocellulose, and probed with monoclonal antibodies directed to SipC as indicated in Materials and Methods.

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The flagellar sigma factor FliA (sigma(28)) regulates the expression of Salmonella genes associated with the centisome 63 type III secretion system - PubMed (original) (raw)