TcpH Influences Virulence Gene Expression inVibrio choleraeby 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.

Degradation of the membrane-localized virulence activator TcpP by the YaeL protease in Vibrio cholerae

“…When TcpP levels were monitored in the presence and absence of TcpH, TcpP was significantly more stable over time in cells expressing TcpH compared with those not expressing TcpH, indicating that TcpH protects TcpP from turnover in V. cholerae (13). Inhibition of proteolysis does not appear to be a general mechanism of action for effector proteins such as TcpH and ToxS because deletion of toxS did not result in ToxR instability.…”

“…TcpP interacts with TcpH, which is also a membrane protein but lacks an extensive cytoplasmic domain (9,10). The association of TcpP and TcpH in V. cholerae stabilizes TcpP from degradation, thereby enabling formation of a proposed transcription activation complex of TcpP͞TcpH and ToxR͞ToxS (11)(12)(13). Although ToxR stability is enhanced by ToxS when the proteins are overexpressed in Escherichia coli, ToxS is not required for ToxR stability in V. cholerae (13,14).…”

“…The association of TcpP and TcpH in V. cholerae stabilizes TcpP from degradation, thereby enabling formation of a proposed transcription activation complex of TcpP͞TcpH and ToxR͞ToxS (11)(12)(13). Although ToxR stability is enhanced by ToxS when the proteins are overexpressed in Escherichia coli, ToxS is not required for ToxR stability in V. cholerae (13,14). The role of ToxS may be to stimulate dimer formation by ToxR (14,15).…”

“…Further analysis of the molecular mechanism by which TcpH controls gene expression led to the observation that the periplasmic domain of TcpP is a determinant of instability that is protected from degradation by TcpH (13). When TcpP levels were monitored in the presence and absence of TcpH, TcpP was significantly more stable over time in cells expressing TcpH compared with those not expressing TcpH, indicating that TcpH protects TcpP from turnover in V. cholerae (13).…”

“…Inhibition of proteolysis does not appear to be a general mechanism of action for effector proteins such as TcpH and ToxS because deletion of toxS did not result in ToxR instability. From the observation that a chimeric protein in which the TcpP periplasmic domain was replaced by that of ToxR was stable irrespective of TcpH expression, it was determined that the periplasmic domain contributes to TcpP instability in the absence of TcpH (13). These results led us to investigate the mechanism of TcpP proteolysis and attempt to identify the protease responsible for TcpP degradation.…”