Interaction of Vibrio cholerae ToxR and its domains with virstatin

The diarrheal disease cholera caused by Gram negative bacteria Vibrio cholerae is still a potential threat in many developing countries. Though the most common approach to combat cholera is use of oral rehydrating solution, it has many limitations. Search for small molecules as anti-cholera drug by high throughput screening were continued, which identified virstatin as a potential drug. ToxR protein (Swiss-Prot entry P15795) of V. cholerae is a transcriptional activator of two major virulence factors for cholera pathogenesis, cholera toxin (CT) and toxin coregulated pilus (TCP). ToxR is a 32.5 kDa integral membrane protein comprising of a DNA binding cytoplasmic domain (1-180 amino acid; ToxRc), a periplasmic domain (197-294 amino acid; ToxRp) linked by a 16 amino acid transmembrane domain. Transcriptional activation by ToxR initiated by binding the dimeric form of the protein to the heptamer motif 5’-TTATGAT-3’, the tandemly repeat sequence present in ctxAB gene. Previous report indicated that virstatin prevents dimerization and activity of ToxT; another transcriptional activator [1]. Along this line, we wanted to explore the interaction and mechanism of binding of ToxR to virstatin. ToxR and its individual domains were cloned, expressed and purified [2]. In vitro transcription showed that virstatin inhibits ToxR dependent transcription of ctxAB gene in a dose dependent manner. However, ToxRc lacks this activity. Biophysical and biochemical studies revealed destabilization of ToxR in presence of virstatin. Isothermal titration calorimetry used to quantify the thermodynamics of binding of ToxR with both virstatin and DNA. It showed an exothermic binding isotherm. Interestingly, ToxRc was unable to bind DNA implying the role of periplasmic domain in the function of ToxR. Analytical gel filtration revealed that the oligomeric status of ToxR remains intact even in the presence of virstatin, indicating a different mode of binding unlike ToxT. The structure/function modulation of ToxR by virstatin (and other small molecules) may provide an alternative strategy to combat cholera.