Molecular Analysis of ToxR-TcpP Functional Interaction and Activation of the Virulence Cascade in Vibrio cholerae.

Abstract

Vibrio cholerae, the causative agent of cholera, regulates expression of virulence factors such as the cholera toxin and the Toxin Co-regulated Pilus through the master virulence regulator ToxT. Transcriptional activation of toxT is in turn co-activated by the transmembrane winged helix-turn-helix (w-HTH) transcription factors ToxR and TcpP. ToxR is a fully functional transcriptional activator and is able to directly activate transcription of the outer membrane porin encoded by ompU. However, ToxR does not activate transcription of toxT directly, but instead assists TcpP-mediated activation of the toxT promoter. We have found, through mutational studies that ToxR binds the toxT promoter 3-helical turns upstream of the promoter proximal TcpP binding site. This is in contrast to the ToxR binding site on the ompU promoter, which is just upstream of the -35 element and in the opposite orientation. Additionally, different faces of the ToxR protein are preferentially required for co-activation of toxT as compared to direct activation of ompU. This highlights the different functions of ToxR as a co-activator and a direct activator. Although, w-HTH proteins often interact through the wing, the wing of ToxR is not required for ToxR interaction with TcpP. However, the wing of ToxR is required for binding to, and transcriptional activation of, both toxT and ompU promoters. Finally, we investigated the regulation of TcpP stability in transcriptional activation of toxT. Controlled proteolytic degradation of TcpP has been previously shown to downregulate activation of toxT under non-inducing conditions. We have found that the periplasmic cysteines in TcpP form an intramolecular disulfide bond, and that this bond protects TcpP from proteolytic degradation, allowing TcpP to activate transcription of toxT. Similar periplasmic cysteine bonds are present in ToxR, however they are not critical for stability or transcriptional activation. ToxR may co-activate the toxT promoter by binding to, and altering the structure of, the toxT promoter thereby enhancing TcpP binding. Additionally, ToxR-TcpP interactions may help recruit TcpP to the promoter. Co-activation of toxT by ToxR and TcpP allows for integration of many environmental signals, which insures that virulence gene expression will only be activated under appropriate conditions.