Proteolysis of ToxR is controlled by cysteine‐thiol redox state and bile salts in Vibrio cholerae

Abstract

Abbreviated Summary: Virulence gene regulation in Vibrio cholerae is highly complex, comprising several transcriptional activators and decision check points. The ToxRS complex is a key regulator that is subjected to regulated intramembrane proteolysis (RIP). The participating interaction partners were characterized, addressing DegS, DegP, ToxS and factors influencing ToxR-RIP, such as bile acids and the cysteine redox state of ToxR. In Vibrio cholerae, virulence gene expression is regulated by a transmembrane-localized transcription factor complex designated as ToxRS. ToxR harbours two cysteines in the periplasmic domain that can form inter- and intramolecular disulfide bonds. In this study, we investigated the σE−dependent inner membrane proteolysis of ToxR, which occurs via the periplasmic-localized proteases DegS and DegP. Both proteases respond to the redox state of the two cysteine thiol groups of ToxR. Interestingly, in the presence of sodium deoxycholate, ToxR proteolysis is blocked independently of ToxS, whereas ToxR activation by bile salts requires ToxS function. From these data, we identified at least two levels of control for ToxR activation by sodium-deoxycholate. First, bile inhibits ToxR degradation under starvation and alkaline pH or under conditions in which DegPS responds to the reduced disulfide bonds of ToxR. The second level links bile to ToxRS complex formation and further activation of its transcription factor activity. Overall, our data suggest a comprehensive bile sensory function for the ToxRS complex during host colonization.

A disulfide constrains the ToxR periplasmic domain structure, altering its interactions with ToxS and bile-salts

“…This, based on structural homology with VtrAC, stabilizes the binding interface between ToxR and ToxS. Such an increase in stability is consistent with observations regarding the double cysteine mutant degradation in Lembke et al (Lembke et al, 2018). Interestingly it has been observed that ToxS enhances ToxR intra-chain disulfide bond formation (Ottemann and Mekalanos, 1996).…”

“…ToxS, an integral membrane protein with a periplasmic domain, augments ToxR activity (Miller et al, 1989) and protects ToxR from degradation in conditions of stationary growth and alkaline pH (Almagro-Moreno et al, 2015;Lembke et al, 2018).…”

“…Investigations into the contribution of cysteines C236 and C293 on V. cholerae ToxR function showed single cysteine mutants were nonfunctional (Fan et al, 2014), whereas a double mutant had decreased function in porin regulation, but no defects in virulence gene expression (Fengler et al, 2012). Moreover, the double mutant was shown to be more susceptible to degradation than wild-type ToxR, even in the stabilizing presence of ToxS (Lembke et al, 2018). In wild type ToxR, these cysteines are known to form either an intra-chain disulfide bond or a disulfide linked homodimer, which is present when toxS is deleted in V. cholerae (Ottemann and Mekalanos, 1995;Fengler et al, 2012).…”

Host stimuli and operator binding sites controlling protein interactions between virulence master regulator ToxR and ToxS in Vibrio cholerae

“…Finally, the cysteine residues in ToxR are associated with its transcriptional activity through intramolecular disulfide bond formation. Moreover, they also provide a signal for proteolysis once they appear in their reduced form (Ottemann and Mekalanos, 1996;Fengler et al, 2012;Lembke et al, 2018).…”

Virulence Regulation and Innate Host Response in the Pathogenicity of Vibrio cholerae

“…Red circles in ompU and ompT indicate the ToxR binding sites and the transcriptional start sites are marked with arrows for each promoter. (Mey et al, 2015;Lembke et al, 2018). ToxR activation by bile salts depends on the function of ToxS.…”