The CpxAR (Cpx) two-component regulator controls the expression of genes in response to a variety of environmental cues. The Cpx regulator has been implicated in the virulence of several gram-negative pathogens, although a role for Cpx in vivo has not been demonstrated directly. Here we investigate whether positive or negative control of gene expression by Cpx is important for the pathogenesis of Salmonella enterica serotype Typhimurium. The Cpx signal pathway in serotype Typhimurium was disrupted by insertional inactivation of the cpxA and cpxR genes. We also constitutively activated the Cpx pathway by making an internal in-frame deletion in cpxA (a cpxA* mutation). Activation of the Cpx pathway inhibited induction of the envelope stress response pathway controlled by the alternative sigma factor E (encoded by rpoE). Conversely, the Cpx pathway was highly up-regulated (>40-fold) in a serotype Typhimurium rpoE mutant. The cpxA* mutation, but not the cpxA or the cpxR mutation, significantly reduced the capacity of serotype Typhimurium to adhere to and invade eucaryotic cells, although intracellular replication was not affected. The cpxA and cpxA* mutations significantly impaired the ability of serotype Typhimurium to grow in vivo in mice. To our knowledge, this is the first demonstration that the Cpx system is important for a bacterial pathogen in vivo.

In Escherichia coli, extracytoplasmic stress is partially controlled by the alternative sigma factor, RpoE (ςE). In response to environmental stress or alteration in the protein content of the cell envelope, ςEupregulates the expression of a number of genes, includinghtrA. It has been shown that htrA is required for intramacrophage survival and virulence in Salmonella typhimurium. To investigate whether ςE-regulated genes other than htrA are involved in salmonella virulence, we inactivated the rpoE gene of S. typhimurium SL1344 by allelic exchange and compared the phenotype of the mutant (GVB311) in vitro and in vivo with its parent and an isogenic htrA mutant (BRD915). UnlikeE. coli, ςE is not required for the growth and survival of S. typhimurium at high temperatures. However, GVB311 did display a defect in its ability to utilize carbon sources other than glucose. GVB311 was more sensitive to hydrogen peroxide, superoxide, and antimicrobial peptides than SL1344 and BRD915. Although able to invade both macrophage and epithelial cell lines normally, the rpoE mutant was defective in its ability to survive and proliferate in both cell lines. The effect of the rpoE mutation on the intracellular behavior of S. typhimurium was greater than that of the htrA mutation. Both GVB311 and BRD915 were highly attenuated in mice. Neither strain was able to kill mice via the oral route, and the 50% lethal dose (LD50) for both strains via the intravenous (i.v.) route was very high. The i.v. LD50s for SL1344, BRD915, and GVB311 were <10, 5.5 × 105, and 1.24 × 107 CFU, respectively. Growth in murine tissues after oral and i.v. inoculation was impaired for both thehtrA and rpoE mutant, with the latter mutant being more severely affected. Neither mutant was able to translocate successfully from the Peyer’s patches to other organs after oral infection or to proliferate in the liver and spleen after i.v. inoculation. However, the htrA mutant efficiently colonized the livers and spleens of mice infected i.v., but the rpoE mutant did not. Previous studies have shown that salmonella htrA mutants are excellent live vaccines. In contrast, oral immunization of mice with GVB311 was unable to protect any of the mice from oral challenge with SL1344. Furthermore, i.v. immunization with a large dose (∼106 CFU) of GVB311 protected less than half of the orally challenged mice. Thus, our results indicate that genes in the ςE regulon other than htrA play a critical role in the virulence and immunogenicity of S. typhimurium.

Starvation ofFurthermore, evidence suggests that the σ E and σ S regulons function through separate mechanisms in the SSR. In contrast, C-starvation does not appear to generate signals required for Cpx regulon induction which support the findings that it is not required for LT-CSS or cross-resistance to H 2 O 2 , pH 31 or PmB challenges. However, it was required to achieve maximal cross-resistance to 55 SC. Therefore, σ E is a key regulatory component of the SSR and represents an additional σ factor required for the SSR of Salmonella.

The rpoE gene of Salmonella enterica serovar Typhimurium (S. Typhimurium), which encodes the extracytoplasmic stress response sigma factor c E , is critically important for the virulence of S. Typhimurium. We analysed expression of rpoE by wild-type and mutant bacteria grown in different conditions by S1-nuclease mapping using RNA, and using in vivo reporter gene fusions. Three promoters, rpoEp1, rpoEp2 and rpoEp3, were located upstream of the S. Typhimurium rpoE gene. The promoters were differentially expressed during growth and under several stress conditions including cold shock. Expression from the rpoEp3 promoter was absent in an S. Typhimurium rpoE mutant, demonstrating its dependence upon c E . The level of mRNA corresponding to rpoEp3 was also higher in a cpxR mutant, indicating a negative regulation of the promoter by the Cpx system. Using this rpoE-dependent promoter, we optimised a two-plasmid system for identification of promoters recognised by S. Typhimurium c E . The rpoEp3 promoter was active in the Escherichia coli twoplasmid system and has an identical transcription start point as in S. Typhimurium but only after induction of S. Typhimurium rpoE expression.

To survive and multiply in different environments, Vibrio cholerae has to coordinately regulate the expression of genes involved in adaptive responses. In many pathogens, adaptive responses, including pathogenic responses, are regulated by two-component regulator (TCR) systems. It is likely that members of a TCR family play a role in the regulation of processes involved in intestinal colonization, and therefore pathogenesis, in V. cholerae. We have identified and characterized a TCR system of V. cholerae : this system is a homologue of Escherichia coli PhoBR. The presence of a putative Pho box suggests that the V. cholerae phoBR operon is regulated by inorganic phosphate levels. The phoR and phoB genes are organized the same way as in E. coli. Mutation of the V. cholerae phoB gene affected the expression of the putative Pho regulon, including PhoA, but did not affect the production of cholera toxin. V. cholerae phoB mutants are less able to colonize rabbit intestine than wild-type V. cholerae. The addition of inorganic phosphate at a high concentration to the inoculum only partially restored the ability of the mutants to colonize the intestine, suggesting that the V. cholerae Pho regulon in vivo may not be regulated by inorganic phosphate levels alone.

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FkpA is a peptidylprolyl isomerase whose expression is regulated by the alternative sigma factor, sigma factor E ( E ). In contrast to the results of a previous report, inactivation of fkpA was found to have only a minor effect on the ability of Salmonella enterica serovar Typhimurium to invade and survive within epithelial and macrophage cell lines and cause infection in mice. However, an effect of the fkpA mutation on serovar Typhimurium virulence was seen if the mutation was combined with mutations in surA or htrA, two other E -regulated genes, which encode proteins involved in protein folding and/or degradation in the periplasm.

Carbon-energy source (C)-starved cells of Salmonella enterica serovar Typhimurium (S. Typhimurium) are remarkably more resistant to stress than actively growing ones. Carbon-starved S. Typhimurium is capable of withstanding extended periods of starvation and assault from a number of different stresses that rapidly kill growing cells. These unique properties of the C-starved cell are the direct result of a series of genetic and physiological adaptations referred to as the starvation-stress response (SSR). Previous work established that the SSR of S. Typhimurium is partially regulated by the extracytoplasmic function sigma factor σE. As part of an effort to identify σE-regulated SSR genes, we investigated surA and fkpA, encoding two different classes of peptidyl-prolyl isomerase that function in folding cell envelope proteins. Both surA and fkpA are members of the heat-shock-inducible σE regulon of Escherichia coli. Although both genes are expressed in C-starved Salmonella cells, evidence indicates that surA and fkpA are not C-starvation-inducible. Furthermore, their expression during C-starvation does not appear to be σE-dependent. Nonetheless, surA and fkpA proved to be important, to differing degrees, for long-term C-starvation survival and for the cross-resistance of C-starved cells to high temperature, acidic pH, and the antimicrobial peptide polymyxin B, but neither were required for cross-resistance to oxidative stress. These results point to fundamental differences between heat-shock-inducible and C-starvation-inducible genes regulated by σE and suggest that genes other than surA and fkpA are involved in the σE-regulated branch of the SSR in Salmonella.