Genetic Analysis of Activation of the Vibrio cholerae Cpx Pathway

Abstract

The Cpx two-component system is thought to mediate envelope stress responses in many gram-negative bacteria and has been implicated in the pathogenicity of several enteric pathogens. While cues that activate the Escherichia coli Cpx system have been identified, the nature of the molecular signals that stimulate this pathway is not well understood. Here, we investigated stimuli that trigger this system in Vibrio cholerae, a facultative pathogen that adapts to various niches during its life cycle. In contrast to E. coli, there was no basal activity of the V. cholerae Cpx pathway under standard laboratory conditions. Furthermore, several known stimuli of the E. coli pathway did not induce expression of this system in V. cholerae. There were no defects in intestinal growth in V. cholerae cpx mutants, arguing against the idea that this pathway promotes V. cholerae adaptation to conditions in the mammalian host. We discovered that chloride ions activate the V. cholerae Cpx pathway, raising the possibility that this signal transduction system provides a means for V. cholerae to sense and respond to alterations in salinity. We used a genetic approach to screen for mutants in which the Cpx pathway is activated. We found that mutations in genes whose products are required for periplasmic disulfide bond isomerization result in activation of the Cpx pathway, suggesting that periplasmic accumulation of proteins with aberrant disulfide bonds triggers the V. cholerae Cpx pathway.

The Vibrio cholerae Cpx Envelope Stress Response Senses and Mediates Adaptation to Low Iron

“…These included genes that encode components of the TonB1 energy transduction system (VCA0910 to VCA0912), genes involved in heme utilization, such as those encoding the HutBCD ABC transport system (VCA0913 to VCA0915), the hutWXZ operon (VCA0907 to VCA0909) (70), and the heme receptor protein HutA gene (VCA0576), genes for vibriobactin siderophore biosynthesis (VC0771) and transport (VCA0227, VCA0229, and VCA0230), and genes for ferrichrome transport (VC0200 and VC0201). Interestingly, a previous study showed that a transposon insertion in the hutA gene activates the Cpx pathway in V. cholerae (24). To confirm the observed changes in gene expression in the microarray for some of these genes, qRT-PCR was performed, and the relative quantification results were in accordance with the microarray data ( Fig.…”

“…The V. cholerae C6706 Cpx response is induced by iron limitation and inhibition of efflux pumps. Previously, Slamti and Waldor (24) reported that the activation of the Cpx pathway in V. cholerae is required to mediate cellular responses toward changes in salinity and oxidative stress (24). For example, they found an increase in the signal of a cpxP-lacZ reporter when different concentrations of sodium chloride and CuSO 4 were added to a basal medium of yeast extract and tryptone (24).…”

“…Previously, Slamti and Waldor (24) reported that the activation of the Cpx pathway in V. cholerae is required to mediate cellular responses toward changes in salinity and oxidative stress (24). For example, they found an increase in the signal of a cpxP-lacZ reporter when different concentrations of sodium chloride and CuSO 4 were added to a basal medium of yeast extract and tryptone (24). To test if the previously described inducing cues (i.e., chloride ions and CuSO 4 ) of the Cpx pathway in V. cholerae El Tor strain N16961 also activate the Cpx pathway in the C6706 strain background, we constructed a cpxP-lux transcriptional reporter by using the previously described reporter plasmid pJW15 (53).…”

“…Since the E. coli and V. cholerae CpxR proteins are very similar (60.3% identity) (24) and the DNA binding domain is almost 100% conserved, we used the previously described E. coli CpxR binding site [5=-G(T/C)AA(A/C)(N) 5 (G/ C)(T/A)(A/C/T)(A/C)(A/G/C)-3=] (57) to search for Cpx regulon members in V. cholerae El Tor N16961, using the Virtual Footprint tool (58; http://prodoric.tu-bs.de/vfp/). We scanned the V. cholerae El Tor N16961 genome because it is closely related to V. cholerae El Tor strain C6706, as found by phylogenetic analysis of the whole genome (59).…”

“…For example, in Haemophilus ducreyi, the Cpx response appears not to be as tightly linked to protein misfolding but rather was shown to control the expression of many genes, including several major virulence determinants, predominantly in a negative fashion (22,23). Similarly, for Vibrio cholerae El Tor N16961, recent evidence indicates that the Cpx pathway may be involved in sensing and mediating adaptation to high salinity in addition to responding to misfolded envelope proteins that contain aberrant disulfide bonds (24). A recent microarray study reported that in V. cholerae El Tor N16961, a major function of the Cpx response is to control efflux pump expression (25), as has been shown in E. coli (15) and Klebsiella pneumoniae (26).…”