SummarySalmonella typhimurium causes enteric and systemic disease by invading the intestinal epithelium of the distal ileum, a process requiring the invasion genes of Salmonella pathogenicity island 1 (SPI-1). BarA, a sensor kinase postulated to interact with the response regulator SirA, is required for the expression of SPI-1 invasion genes. We found, however, that a barA null mutation had little effect on virulence using the mouse model for septicaemia. This confounding result led us to seek environmental signals present in the distal ileum that might supplant the need for BarA. We found that acetate restored the expression of invasion genes in the barA mutant, but had no effect on a sirA mutant. Acetate had its effect only at a pH that allowed its accumulation within the bacterial cytoplasm and not with the deletion of ackA and pta , the two genes required to produce acetylphosphate. These results suggest that the rising concentration of acetate in the distal ileum provides a signal for invasion gene expression by the production of acetyl-phosphate in the bacterial cytoplasm, a pathway that bypasses barA . We also found that a D D D D ( ackA-pta ) mutation alone had no effect on virulence but, in combination with D D D D ( barA ), it increased the oral LD 50 24-fold. Thus, the combined loss of the BarA-and acetate-dependent pathways is required to reduce virulence. Two other short-chain fatty acids (SCFA), propionate and butyrate, present in high concentrations in the caecum and colon, had effects opposite to those of acetate: neither restored invasion gene expression in the barA mutant, and both, in fact, reduced expression in the wild-type strain. Further, a combination of SCFAs found in the distal ileum restored invasion gene expression in the barA mutant, whereas colonic conditions failed to do so and also reduced expression in the wild-type strain. These results suggest that the concentration and composition of SCFAs in the distal ileum provide a signal for productive infection by Salmonella , whereas those of the large intestine inhibit invasion.

SummaryCsrA is a regulator of invasion genes in Salmonella enterica serovar Typhimurium. To investigate the wider role of CsrA in gene regulation, we compared the expression of Salmonella genes in a csrA mutant with those in the wild type using a DNA microarray. As expected, we found that expression of Salmonella pathogenicity island 1 (SPI-1) invasion genes was greatly reduced in the csrA mutant, as were genes outside the island that encode proteins translocated into eukaryotic cells by the SPI-1 type III secretion apparatus. The flagellar synthesis operons, flg and fli , were also poorly expressed, and the csrA mutant was aflagellate and non-motile. The genes of two metabolic pathways likely to be used by Salmonella in the intestinal milieu also showed reduced expression: the pdu operon for utilization of 1,2-propanediol and the eut operon for ethanolamine catabolism. Reduced expression of reporter fusions in these two operons confirmed the microarray data. Moreover, csrA was found to regulate co-ordinately the cob operon for synthesis of vitamin B 12 , required for the metabolism of either 1,2-propanediol or ethanolamine. Additionally, the csrA mutant poorly expressed the genes of the mal operon, required for transport and use of maltose and maltodextrins, and had reduced amounts of maltoporin, normally a dominant protein of the outer membrane. These results show that csrA controls a number of gene classes in addition to those required for invasion, some of them unique to Salmonella , and suggests a co-ordinated bacterial response to conditions that exist at the site of bacterial invasion, the intestinal tract of a host animal.

Penetration of intestinal epithelial cells by Salmonella enterica serovar Typhimurium requires the expression of invasion genes, found in Salmonella pathogenicity island 1 (SPI1), that encode components of a type III secretion apparatus. These genes are controlled in a complex manner by regulators within SPI1, including HilA and InvF, and those outside SPI1, such as the two-component regulators PhoP/PhoQ and BarA/SirA. We report here that epithelial cell invasion requires the serovar Typhimurium homologue of Escherichia coli csrA, which encodes a regulator that alters the stability of specific mRNA targets. A deletion mutant of csrA was unable to efficiently invade cultured epithelial cells and showed reduced expression of four tested SPI1 genes, hilA, invF, sipC, and prgH. Overexpression of csrA from an induced araBAD promoter also negatively affected the expression of these genes, indicating that CsrA can act as both a positive and a negative regulator of SPI1 genes and suggesting that the bacterium must tightly control the level or activity of CsrA to achieve maximal invasion. We found that CsrA affected hilA, a regulator of the other three genes we tested, probably by controlling one or more genetic elements that regulate hilA. We also found that both the loss and the overexpression of csrA reduced the expression of two regulators of hilA, hilC and hilD, suggesting that csrA exerts its control of hilA through one or both of these regulators. We further found, however, that CsrA could affect the expression of both invF and sipC independent of its effects on hilA. One additional striking phenotype of the csrA mutant, not observed in a comparable E. coli mutant, was its slow growth. Phenotypic revertants that had normal growth rates, while maintaining the csrA mutation, were common. These suppressed strains, however, did not recover the ability to invade cultured cells, indicating that the csrA-mediated loss of invasion cannot be attributed simply to poor growth and that the growth and invasion deficits of the csrA mutant arise from effects of CsrA on different targets.

A Salmonella typhimurium chromosomal deletion removing ≈19 kb of DNA at centisome 65 reduces invasion of cultured epithelial cells as well as the expression of lacZY operon fusions to several genes required for the invasive phenotype. As the deleted region contains no genes previously known to affect Salmonella invasion, we investigated the roles of individual genes in the deleted region using a combination of cloning, complementation and directed mutation. We find that the deletion includes two unrelated regulatory genes. One is the Salmonella homologue of Escherichia coli barA (airS ), which encodes a member of the multistep phosphorelay subgroup of two‐component sensor kinases. The action of BarA is coupled to that of SirA, a member of the phosphorylated response regulator family of proteins, and includes both HilA‐dependent and HilA‐independent components. The other regulatory gene removed by the deletion is the Salmonella homologue of E. coli csrB, which specifies a regulatory RNA implicated in controlling specific message turnover in E. coli. These results identify a protein that is likely to play a key role in the environmental control of Salmonella invasion gene expression, and they also suggest that transcriptional control of invasion genes could be subject to refinement at the level of message turnover.

The csr regulatory system of Salmonella regulates the expression of the genes of Salmonella pathogenicity island 1 (SPI1) required for the invasion of epithelial cells. This system consists of the posttranscriptional regulator CsrA and an untranslated regulatory RNA, CsrB, that opposes the action of CsrA. Here we identify and characterize the role of a second regulatory RNA, CsrC, whose ortholog was discovered previously in Escherichia coli. We show that a mutant of csrC has only mild defects in invasion and the expression of SPI1 genes, as does a mutant of csrB, but that a double csrB csrC mutant is markedly deficient in these properties, suggesting that the two regulatory RNAs play redundant roles in the control of invasion. We further show that CsrC, like CsrB, is controlled by the BarA/SirA two-component regulator but that a csrB csrC mutant exhibits a loss of invasion equivalent to that of a barA or sirA mutant, indicating that much of the effect of BarA/SirA on invasion functions through its control of CsrB and CsrC. In addition to their control by BarA/SirA, each regulatory RNA is also controlled by other components of the csr system. The loss of csrB was found to increase the level of CsrC by sevenfold, while the loss of csrC increased CsrB by nearly twofold. Similarly, the overexpression of csrA increased CsrC by nearly 11-fold and CsrB by 3-fold and also significantly increased the stability of both RNAs.