Regulated intramembrane proteolysis of the virulence activator TcpP in Vibrio cholerae is initiated by the tail-specific protease (Tsp)

Abstract

Summary: Vibrio cholerae uses a multiprotein transcriptional regulatory cascade to control expression of virulence factors cholera toxin and toxin-co-regulated pilus. Two proteins in this cascade are ToxR and TcpP - unusual membrane-localized transcription factors with relatively undefined periplasmic domains and transcription activator cytoplasmic domains. TcpP and ToxR function with each other and two other membrane-localized proteins, TcpH and ToxS, to activate transcription of toxT, encoding the direct activator of toxin and pilus genes. Under some conditions, TcpP is degraded in a two-step proteolytic pathway known as regulated intramembrane proteolysis (RIP), thereby inactivating the cascade. The second step in this proteolytic pathway involves the zinc metalloprotease YaeL; V.cholerae cells lacking YaeL accumulate a truncated yet active form of TcpP termed TcpP*. We hypothesized that a protease acting prior to YaeL degrades TcpP to TcpP*, which is the substrate of YaeL. In this study, we demonstrate that a C-terminal protease called Tsp degrades TcpP to form TcpP*, which is then acted upon by YaeL. We present evidence that TcpH and Tsp serve to protect full-length TcpP from spurious proteolysis by YaeL. Cleavage by Tsp occurs in the periplasmic domain of TcpP and requires residues TcpPA172 and TcpPI174 for wild-type activity. In Vibrio cholerae, the membrane-localized transcription factor TcpP undergoes regulated intramembrane proteolysis (RIP) in response to environmental conditions that are unfavorable for virulence gene expression. Here, we uncover the protease (Tsp) that generates the TcpP intermediate substrate for the previously discovered site-2 protease YaeL. We also present evidence that YaeL is able to cleave full-length TcpP independently of Tsp under some conditions.