Bicarbonate Increases Binding Affinity of Vibrio cholerae ToxT to Virulence Gene Promoters

Abstract

The major Vibrio cholerae virulence gene transcription activator, ToxT, is responsible for the production of the diarrhea-inducing cholera toxin (CT) and the major colonization factor, toxin coregulated pilus (TCP). In addition to the two primary virulence factors mentioned, ToxT is responsible for the activation of accessory virulence genes, such as aldA, tagA, acfA, acfD, tcpI, and tarAB. ToxT activity is negatively modulated by bile and unsaturated fatty acids found in the upper small intestine. Conversely, previous work identified another intestinal signal, bicarbonate, which enhances the ability of ToxT to activate production of CT and TCP. The work presented here further elucidates the mechanism for the enhancement of ToxT activity by bicarbonate. Bicarbonate was found to increase the activation of ToxT-dependent accessory virulence promoters in addition to those that produce CT and TCP. Bicarbonate is taken up into the V. cholerae cell, where it positively affects ToxT activity by increasing DNA binding affinity for the virulence gene promoters that ToxT activates regardless of toxbox configuration. The increase in ToxT binding affinity in the presence of bicarbonate explains the elevated level of virulence gene transcription.

Mechanism for Inhibition of Vibrio cholerae ToxT Activity by the Unsaturated Fatty Acid Components of Bile

“…Autoradiographs were analyzed using ImageJ software (NIH) as previously described, with nonspecific binding omitted from further analysis (32). Briefly, to determine the K D (equilibrium dissociation constant) for samples containing either linoleic acid or virstatin and compared to protein bound to DNA without inhibitor, the percentage of protein bound with labeled DNA was determined for each lane.…”

“…Strains were maintained in Luria broth (LB) containing 20% glycerol and stored at Ϫ70°C. All promoter::lacZ fusions used for ␤-galactosidase assays were made in plasmid pTL61T as described for previous studies (26,32). Overnight cultures were grown for ϳ16 h at 37°C in LB and then diluted 1:40 into LB (pH 6.5) at 30°C for virulence-inducing conditions in the presence or absence of freshly prepared 0.05% bile (sodium choleate), 160 M linoleic acid, or 100 M virstatin.…”

“…Maltose binding protein-ToxT fusion (MBPToxT) and MBP-cyclic AMP (cAMP) receptor protein (MBP-CRP) purification was performed as previously described using Escherichia coli strain BL21(DE3) with plasmid pMAL-c2E carrying either MBP-ToxT or MBP-CRP (32,35). Briefly, after MBP-ToxT or MBP-CRP induction by isopropyl-␤-D-thiogalactopyranoside (IPTG), cells were lysed by French press and run over an amylose column, and fractions containing MBPToxT or MBP-CRP were saved and dialyzed into 50 mM Na 2 HPO 4 (pH 8.0) and 100 mM NaCl and then dialyzed into the same solution containing 20% glycerol to save as a freezer stock at Ϫ80°C.…”

A Small Unstructured Region in Vibrio cholerae ToxT Mediates the Response to Positive and Negative Effectors and ToxT Proteolysis

“…Previous work has suggested that palmitoleic acid decreases the ability of ToxT to bind to P tcpA (19). Additionally, we have shown that bicarbonate increases the binding affinity of ToxT to virulence gene promoters regardless of toxbox orientation (37). To assess the ability of linoleic acid and bicarbonate to alter the binding affinity of ToxT N106F to P tcpA , we used electrophoretic mobility shift assays (EMSAs) to determine the equilibrium binding affinity of WT MBP-ToxT and the N106F derivative for the major virulence gene promoter, P tcpA , in the absence and presence of effector.…”

“…Autoradiographs were analyzed using ImageJ software (NIH) as previously described (37). Briefly, the percentage of labeled DNA bound by MBP-ToxT WT or N106F was determined for each lane, and GraphPad Prism 5 software was used for curve fitting to the…”