TcpH influences virulence gene expression in Vibrio cholerae by inhibiting degradation of the transcription activator TcpP

Abstract

Expression of toxT, the transcription activator of cholera toxin and pilus production in Vibrio cholerae, is the consequence of a complex cascade of regulatory events that culminates in activation of the toxT promoter by TcpP and ToxR, two membrane-localized transcription factors. Both are encoded in operons with genes whose products, TcpH and ToxS, which are also membrane localized, are hypothesized to control their activity. In this study we analyzed the role of TcpH in controlling TcpP function. We show that a mutant of V. cholerae lacking TcpH expressed virtually undetectable levels of TcpP, although tcpP mRNA levels remain unaffected. A time course experiment showed that levels of TcpP, expressed from a plasmid, are dramatically reduced over time without co-overexpression of TcpH. By contrast, deletion of toxS did not affect ToxR protein levels. A fusion protein in which the TcpP periplasmic domain is replaced with that of ToxR remains stable, suggesting that the periplasmic domain of TcpP is the target for degradation of the protein. Placement of the periplasmic domain of TcpP on ToxR, an otherwise stable protein, results in instability, providing further evidence for the hypothesis that the periplasmic domain of TcpP is a target for degradation. Consistent with this interpretation is our finding that derivatives of TcpP lacking a periplasmic domain are more stable in V. cholerae than are derivatives in which the periplasmic domain has been truncated. This work identifies at least one role for the periplasmic domain of TcpP, i.e., to act as a target for a protein degradation pathway that regulates TcpP levels. It also provides a rationale for why the V. cholerae tcpH mutant strain is avirulent. We hypothesize that regulator degradation may be an important mechanism for regulating virulence gene expression in V. cholerae.

FIG. 1.

Effect of membrane localization on stability and activity of TcpP. (A) Schematic diagram of the TcpP truncates constructed. The truncates were expressed from the inducible plasmid pBAD18. (B) Western blot analysis of the ΔtcpP strain transformed with the truncates illustrated above. (C) β-Galactosidase assay of ΔtcpP strains containing a chromosomal toxT-lacZ reporter fusion transformed with the truncates described above.

FIG. 2.

Effect of tcpH and toxS deletion on TcpP and ToxR, respectively. (A) Western blot analysis of TcpP levels in wild-type (WT), ΔtcpH and ΔtcpH + tcpH(pACYC184) cultures. (B) Western blot analysis of ToxR levels in the wild type (WT), ΔtoxS, and ΔtoxR. For both analyses, cells were grown to mid-log phase in LB medium at pH 6.5 at 30° C.

FIG. 3.

Effect of tcpH deletion on the level of tcpP transcript. RT-PCR analysis of RNA transcript from wild-type (WT), ΔtcpP, ΔtcpH, and ΔtcpH + tcpH (pACYC184) cultures RNA was harvested from cultures grown to mid-log phase in LB medium at pH 6.5 and 30°C. (A) The TcpP-specific product is shown. (B) The RNA polymerase α-subunit (rpoA)-specific product is shown to demonstrate that levels of RNA have been normalized. +RT, with reverse transcriptase; −RT, without reverse transcriptase.

FIG. 4.

Co-overexpression of TcpH increases the stability of TcpP. Western blot analysis of the ΔtcpP tcpH⁺ strain transformed with tcpP alone (ΔtcpP + P) expressed from the inducible plasmid tcpP (pBAD18) or cotransformed with tcpP(pBAD18) and tcpH (ΔtcpP + P + H) expressed from pACYC184. Rifampin was added to stop transcription of mid-log cultures. Cultures remained at 30°C after the addition of rifampin.

FIG. 5.

Effect of the periplasmic domain on TcpP stability and activity. (A) The ΔtcpP mutant strain was transformed with either tcpP_wt (pBAD18) containing the full-length wild-type version of tcpP or tcpP_R (pBAD18) containing a chimeric version of tcpP in which the periplasmic domain has been replaced with that of ToxR. Western blot analysis of mid-log cultures was performed after the addition of rifampin to stop transcription. Cultures remained at 30°C after the addition of rifampin through the end of the time course. (B) The same experiment as described for panel A was performed with the ΔtoxR mutant strain transformed with either toxR_wt-pBAD18 or toxR_P (pBAD18).