Formation of an Intramolecular Periplasmic Disulfide Bond in TcpP Protects TcpP and TcpH from Degradation in Vibrio cholerae

Abstract

TcpP and ToxR coordinately regulate transcription of toxT, the master regulator of numerous virulence factors in Vibrio cholerae. TcpP and ToxR are membrane-localized transcription factors, each with a periplasmic domain containing two cysteines. In ToxR, these cysteines form an intramolecular disulfide bond and a cysteine-to-serine substitution affects activity. We determined that the two periplasmic cysteines of TcpP also form an intramolecular disulfide bond. Disruption of this intramolecular disulfide bond by mutation of either cysteine resulted in formation of intermolecular disulfide bonds. Furthermore, disruption of the intramolecular disulfide bond in TcpP decreased the stability of TcpP. While the decreased stability of TcpP-C207S resulted in a nearly complete loss of toxT activation and cholera toxin (CT) production, the second cysteine mutant, TcpP-C218S, was partially resistant to proteolytic degradation and maintained ϳ50% toxT activation capacity. TcpP-C218S was also TcpH independent, since deletion of tcpH did not affect the stability of TcpP-C218S, whereas wild-type TcpP was degraded in the absence of TcpH. Finally, TcpH was also unstable when intramolecular disulfides could not be formed in TcpP, suggesting that the single periplasmic cysteine in TcpH may assist with disulfide bond formation in TcpP by interacting with the periplasmic cysteines of TcpP. Consistent with this finding, a TcpH-C114S mutant was unable to stabilize TcpP and was itself unstable. Our findings demonstrate a periplasmic disulfide bond in TcpP is critical for TcpP stability and virulence gene expression. IMPORTANCEThe Vibrio cholerae transcription factor TcpP, in conjunction with ToxR, regulates transcription of toxT, the master regulator of numerous virulence factors in Vibrio cholerae. TcpP is a membrane-localized transcription factor with a periplasmic domain containing two cysteines. We determined that the two periplasmic cysteines of TcpP form an intramolecular disulfide bond and disruption of the intramolecular disulfide bond in TcpP decreased the stability of TcpP and reduced virulence gene expression. Normally TcpH, another membrane-localized periplasmic protein, protects TcpP from degradation. However, we found that TcpH was also unstable when intramolecular disulfides could not be formed in TcpP, indicating that the periplasmic cysteines of TcpP are required for functional interaction with TcpH and that this interaction is required for both TcpP and TcpH stability. C holera is caused by ingestion of the aquatic Gram-negative bacterium Vibrio cholerae. The profuse watery diarrhea characteristic of cholera is induced by cholera toxin (CT), an ADPribosylating toxin that induces cyclic AMP production in intestinal epithelial cells, resulting in massive secretion of water and electrolytes. Microcolony formation and colonization of the intestine require expression of the toxin-coregulated pilus (TCP) (1). Transcription of the genes encoding CT and TCP, as well as several other virulence factors, is regulated by...

Bile salts and alkaline pHreciprocally modulate the interaction between the periplasmic domains ofVibrio choleraeToxRandToxS

“…Interestingly, the bile salts deoxycholate, chenodeoxycholate, as well as decanoate (Rosenberg et al ., ) 2,2‐ and 4,4‐dipyridle (Rosner et al ., ) activate the E. coli transcription factor Rob by binding to the regulatory domain causing an unspecified conformational change (Rosenberg et al ., ). In V. cholerae TcpP forms homodimeric disulfide bonds, increasing the transcription of ToxT, when exposed to disulfide stress (Morgan et al ., ). The activation of ToxR by bile salts appears to be not simply a transcription factor undergoing a conformational change to become active, but rather an evolutionary adaptation in which the detrimental effects of its signal (i.e., destabilization by detergent like molecules) has been compensated for by the chaperone like function of ToxS, which facilitates ToxR activation.…”

Transmembrane redox control and proteolysis of PdeC, a novel type of c‐di‐ GMP phosphodiesterase

“…Yet, this degradation involves different sets of proteases (Almagro‐Moreno et al , ; Teoh et al , ). Notably, ToxR and TcpP can escape degradation with the help of the accessory periplasmic proteins ToxS and TcpH, respectively (Almagro‐Moreno et al , ; Morgan et al , ). Taken together, PdeC, ToxS, and TcpH demonstrate the striking versatility of transmembrane signaling that combines DSB/free thiol switching with proteolytic processing.…”

“…In addition, ToxR and TcpP, which are involved in virulence and porin regulation of V. cholerae, are activated by DSB formation in their C-terminal periplasmic domains (Fengler et al, 2012;Fan et al, 2014). For TcpP, it has been shown that covalent dimerization via an intermolecular DSB results in activation (Yang et al, 2013 Morgan et al, 2015). Taken together, PdeC, ToxS, and TcpH demonstrate the striking versatility of transmembrane signaling that combines DSB/free thiol switching with proteolytic processing.…”

Genetic analysis of Vibrio parahaemolyticus intestinal colonization

“…In V. cholerae, ToxR forms a disulfide-linked complex with a related protein, TcpP, that governs transcription of the virulence regulator toxT. ToxR's regulatory role can be bypassed by overexpression of either TcpP or ToxT (35). The V. parahaemolyticus regulator VtrA lacks periplasmic cysteine residues, precluding disulfide bond formation like that required for TcpP-ToxR interaction in V. cholerae; however, as with TcpP, exogenous expression of VtrA overcomes the requirement for toxR, restoring vtrB transcription and T3SS2 protein expression.…”