R Maurer

Intestinal short‐chain fatty acids alter Salmonella typhimurium invasion gene expression and virulence through BarA/SirA

Salmonella 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 acetyl‐phosphate. 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 Δ(ackA–pta) mutation alone had no effect on virulence but, in combination with Δ(barA), it increased the oral LD50 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.

Characterization of two novel regulatory genes affecting Salmonella invasion gene expression.

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.

Cloning and characterization of dnaE, encoding the catalytic subunit of replicative DNA polymerase 111, from Vibrio cholerae strain C6706

We report that Vibrio cholerae (Vc) contains a gene homologous to Escherichia coli dnaE, the structural gene for the alpha (catalytic) subunit of replicative DNA polymerase III (PolIII). Despite 24% amino acid (aa) differences in the encoded proteins, the Vc gene strongly complements an E. coli dnaE temperature sensitive (ts) mutant, indicating that all functional features essential for replication are conserved.