A fadD mutant of Vibrio cholerae Is Impaired in the Production of Virulence Factors and Membrane Localization of the Virulence Regulatory Protein TcpP

Abstract

In the enteric pathogen Vibrio cholerae, expression of the major virulence factors is controlled by the hierarchical expression of several regulatory proteins comprising the ToxR regulon. In this study, we demonstrate that disruption of the fadD gene encoding a long-chain fatty acyl coenzyme A ligase has marked effects on expression of the ToxR virulence regulon, motility, and in vivo lethality of V. cholerae. In the V. cholerae fadD mutant, expression of the major virulence genes ctxAB and tcpA, encoding cholera toxin (CT), and the major subunit of the toxin-coregulated pilus (TCP) was drastically repressed and a growth-phase-dependent reduction in the expression of toxT, encoding the transcriptional activator of ctxAB and tcpA, was observed. Expression of toxT from an inducible promoter completely restored CT to wild-type levels in the V. cholerae fadD mutant, suggesting that FadD probably acts upstream of toxT expression. Expression of toxT is activated by the synergistic effect of two transcriptional regulators, TcpP and ToxR. Reverse transcription-PCR and Western blot analysis indicated that although gene expression and production of both TcpP and ToxR are unaffected in the fadD mutant strain, membrane localization of TcpP, but not ToxR, is severely impaired in the fadD mutant strain from the mid-logarithmic phase of growth. Since the decrease in toxT expression occurred concomitantly with the reduction in membrane localization of TcpP, a direct correlation between the defect in membrane localization of TcpP and reduced toxT expression in the fadD mutant strain is suggested.

The Fatty Acid Regulator FadR Influences the Expression of the Virulence Cascade in the El Tor Biotype of Vibrio cholerae by Modulating the Levels of ToxT via Two Different Mechanisms

“…Although prior studies in the classical biotype of V. cholerae did not reveal a role for FadR in the expression of the virulence cascade (36), the results here show that in the El Tor biotype of V. cholerae, FadR is also required for expression of TCP and CT. Examination of a V. cholerae strain C6706 ΔfadR mutant revealed a modest reduction in the transcription of ToxT.…”

“…Although no role for FadR in the regulation of virulence was observed in the classical biotype of V. cholerae (36), we show here that FadR is required for the expression of the cascade in the El Tor biotype. The influence of FadR on the expression of both the tcpA and ctx genes in this biotype suggested a possible effect on ToxT, the regulator that directly activates both of these genes.…”

“…It has previously been shown that loss of FadR does not influence the expression of the virulence cascade in the classical biotype of V. cholerae (36). Since the conditions that induce the expression of the virulence cascade in the El Tor biotype (static growth in a peptone-based medium, AKI [37], at 37°C in the presence of bicarbonate) are different from those that induce its expression in the classical biotype, we assessed the loss of FadR in both biotypes on the expression of the virulence cascade under AKI conditions.…”

“…1) influences the virulence cascade in V. cholerae by promoting the localization of TcpP into the membrane (36). In the absence of FadD, induction of the E -dependent extracytoplasmic stress response results in proteolysis of membrane-localized TcpP by the integral membrane protease RseP and reduces expression from the toxT promoter (39).…”

Host Cell Contact Induces Expression of Virulence Factors and VieA, a Cyclic di-GMP Phosphodiesterase, in Vibrio cholerae

“…3). Complementation of strains JJM43 and O395 ⌬tcpP with plasmids pVM7 (6) and ptcpP (21), carrying the cloned toxR and tcpP genes, respectively, restored toxT expression in both control and adhered bacteria (Fig. 3).…”