SummaryEntry into intestinal epithelial cells is an essential feature in the pathogenicity of Salmonella typhi, which causes typhoid fever in humans. This process requires intact motility and secretion of the invasion-promoting Sip proteins, which are targets of the type III secretion machinery encoded by the inv, spa and prg loci. During our investigations into the entry of S. typhi into cultured epithelial cells, we observed that the secretion of Sip proteins and flagellin was impaired in Vi-expressing strains. We report here that the production of Sip proteins, flagellin and Vi antigen is differentially modulated by the RcsB-RcsC regulatory system and osmolarity. This regulation occurs at both transcriptional and post-translational levels. Under low-osmolarity conditions, the transcription of iagA, invF and sipB genes is negatively controlled by the RcsB regulator, which probably acts in association with the viaB locusencoded TviA protein. The cell surface-associated Vi polysaccharide, which was maximally produced under these growth conditions, prevented the secretion of Sip proteins and flagellin. As the NaCl concentration in the growth medium was increased, transcription of iagA, invF and sipB was found to be markedly increased, whereas transcription of genes involved in Vi antigen biosynthesis was greatly reduced. The expression of iagA, whose product is involved in invF and sipB transcription, occurred selectively during the exponential growth phase and was maximal in the presence of 300 mM NaCl. At this osmolarity, large amounts of Sips and flagellin were secreted in culture supernatants. As expected from these results, and given the essential role of Sip proteins and motility in entry, RcsB and osmolarity modulated the invasive capacity of S. typhi. Together, these findings might reflect the adaptive response of S. typhi to the environments encountered during the different stages of pathogenesis.

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The Vi antigen is a capsular polysaccharide expressed by Salmonella typhi, the agent of human typhoid fever. Expression of this antigen is controlled by the viaA and viaB chromosomal loci. The viaB locus is composed of 11 genes designated tviA-tviE (typhi Vi), vexA-vex€ (Vi antigen export) and ORFl1. We constructed 5. typhi Ty2 strains carrying non-polar mutations in ten genes located at the viaB locus and examined the individual contribution of each gene to Vi phenotype. Phenotypes of the mutants and complementation experiments suggested that synthesis of Vi antigen monomer was catalysed by the TviB and TviC polypeptides. Subsequent polymerization of the polysaccharide might be catalysed by the TviE protein, but required functional TviD product. Proteins encoded by vexA, vexB and wexC directed transport of the polymer to the bacterial cell surface. Anchoring of the Vi antigen at the bacterial cell surface was dependent of the VexE protein. The TviA protein was not essential for Vi polymer synthesis. However, disruption of the tviA gene on 5. @phi Ty2 chromosome strongly decreased expression of Vi antigen. This defect was fully complemented by providing tviA in trans on a recombinant plasmid. By using lacZ transcriptional fusions, it was shown that the TviA product positively regulated co-transcription of the tviA and tviB genes from a promoter located upstream of tviA. Moreover, we showed that a tviAB-/acZ fusion was not expressed in a viaA (i'csB) mutant of S. typhi. However, expression of the tviAB-/acZfusion was restored in this wiaA mutant either by the rcsB gene of Escherichia coli, or by the tviA gene of 5. typhi when present in high copy number. This suggested that the tviA and wiaA products could be involved in the same regulatory pathway modulating Vi antigen expression in 5. typhi. Together these results demonstrated that proteins encoded by the viaB locus are not only involved in Vi polymer synthesis and translocation of the polysaccharide to the bacterial cell surface, but also in regulation of Vi antigen expression in 5. typhi.

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