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.

FIG. 1.

The V. cholerae cpx locus. (A) Schematic of the E. coli Cpx pathway. OM, outer membrane; IM, inner membrane. (B) Genetic organization of the V. cholerae cpx locus. DNA Strider was used to align the predicted sequences of the V. cholerae and E. coli Cpx proteins. The domains of E. coli CpxA are as described in reference . The V. cholerae CpxA domains were inferred using the SMART bioinformatics resource (37, 54). SP, signal peptide; PL, periplasmic loop; CD, cytoplasmic domain.

FIG. 2.

Expression of a P_cpxP-lacZ transcriptional fusion in wild-type V. cholerae and cpx mutant strains. (A, C) The indicated strains (symbols), all of which harbor pP'Z, were grown in LB medium at 37°C and harvested every hour. The dotted gray lines represent growth curves. The black lines represent the β-galactosidase activities. ▪, N16961; ▴, LSΔP; ▾, LSΔR; ♦, LSΔA; •, LSA*. (B) LSP'Z cells harboring pCpxR were grown in LB medium at 37°C to an OD₆₀₀ of approximately 1.5. The culture was split in half, and arabinose (•) or glucose (▪) was added to a final concentration of 0.1%. The x axis shows the amount of time after the sugars were added to the cultures. Each graph is representative of the results of two independent experiments. Note that the y axis for OD₆₀₀ is a logarithmic scale. (D) Appearance of the indicated strains, all derivatives of N16961 harboring a chromosomal P_cpxP-lacZ fusion (LSP'Z), on LB plates containing X-Gal (240 μg/ml).

FIG. 3.

Comparison of the growth of wild-type N16961 with the growth of LSΔP, LSΔR, LSΔA, and LSA* in suckling mice. Strains were mixed 1:1 and inoculated into suckling mice. The ratios of the wild type to the mutant strains in intestinal homogenates were divided by the ratios of these strains in the inocula to yield the competition indices.

FIG. 4.

Stimuli triggering the V. cholerae Cpx pathway. (A) CuSO₄ stimulates the P_cpxP-lacZ fusion. LSP'Z and LSΔRP'Z were streaked on LB plates supplemented with CuSO₄ (125 μM) and/or l-cysteine (500 μM). (B) Cl⁻ activates the V. cholerae Cpx pathway. LSP'Z and LSΔRP'Z were streaked on yeast extract-plus-tryptone plates supplemented with X-Gal (240 μg/ml) and the indicated salts. The plates shown were incubated overnight at 37°C and are representative of at least four independent experiments.

FIG. 5.

Transposon insertions that activate the Cpx pathway. (A) Overnight cultures were spotted onto a plate supplemented with X-Gal (120 μg/ml) and then grown for an additional 20 h at 37°C. This plate is representative of two independent experiments. Mutants B06 and E04 likely represent sibs. (B) β-Galactosidase activities (Miller units) of the transposon insertion mutants grown on plates as described for panel A. The data represent the means of the results of two experiments. (C) Western blots of CpxR in the transposon mutants, as well as in LSP'Z and LSA*P'Z. CpxR was not detected in the wild-type strain LSP'Z with the exposure shown in the figure, but was detectable with longer exposure. The samples used for the immunoblots were grown on plates as described above. Ribosomal protein L1 was used as a loading control. α, anti. (D) Schematic representation of the predicted localization of the proteins encoded by the genes interrupted by the transposon. Localization was predicted by using Psort, except for AmiB; we deduced the localization of this protein from its function. Put. MDR, putative multidrug resistance protein; HP, hypothetical protein; CHP, conserved hypothetical protein; HK, histidine kinase; MCP, methyl-accepting chemotaxis proteins.

FIG. 6.

Complementation analysis of activation of the Cpx pathway in transposon mutants. Overnight cultures were spotted onto a plate supplemented with X-Gal (240 μg/ml) and arabinose (0.1%) and then grown overnight at 37°C. The + and − signs under Plasmid indicate the presence or absence, respectively, of the appropriate complementation vector.

FIG. 7.

Intersection of the disulfide bond formation/isomerization pathway and the Cpx pathway. Overnight cultures of the strains indicated in panels A and B were streaked onto LB plates supplemented with X-Gal (240 μg/ml) and incubated overnight at 37°C.