Virstatin inhibits dimerization of the transcriptional activator ToxT - PubMed (original) (raw)

Virstatin inhibits dimerization of the transcriptional activator ToxT

Elizabeth A Shakhnovich et al. Proc Natl Acad Sci U S A. 2007.

Abstract

The development of antimicrobials is critical in this time of increasing antibiotic resistance of most clinically relevant bacteria. To date, all current antibiotics focus on inhibiting crucial enzymatic activities of their protein targets (i.e., trimethoprim for dihydrofolate reductase), thus disrupting in vitro essential gene functions. In contrast, we have previously reported the identification of virstatin, a small molecule that inhibits virulence regulation in Vibrio cholerae, thereby preventing intestinal colonization in an infant mouse model for cholera. Virstatin prevents expression of the two major V. cholerae virulence factors, cholera toxin (CT) and the toxin coregulated pilus, by inhibiting the virulence transcriptional activator ToxT. It has previously been described that the N-terminal domain of ToxT has the ability to form homodimers. We now demonstrate that virstatin inhibits ToxT dimerization, thus demonstrating that it further falls into a unique class of inhibitors that works by disrupting protein-protein interactions, particularly homodimerization. Using virstatin, truncation mutants of ToxT, and a virstatin-resistant mutant, we show that dimerization is required for ToxT activation of the ctx promoter. In contrast, ToxT dimerization does not appear to be required at all of the other ToxT-regulated promoters, suggesting multiple mechanisms may exist for its transcriptional activity.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Fig. 1.

The N-terminal domain of ToxT dimerizes. (A) (Upper) Activity of ToxT at the ctx promoter when it is expressed from a plasmid in O395Δ_toxT_ as measured by CT ELISA. ToxTWT6 (aa6–276) is active, whereas ToxTWT10 (aa10–267) does not complement the toxT deletion. Full-length ToxT is 276 aa. (Lower) Western blot with α-His demonstrates that all portions of ToxT were expressed to equal levels. (B) Portions of, but not full-length, ToxT drive a protein–protein interaction in a bacterial two-hybrid system in E. coli when induced with IPTG as measured by β-galactosidase assay. Truncation of the first five amino acids allows lacZ transcription, whereas truncation of the first nine abolishes activity. Full-length and N-terminal portion of ToxTL113P point mutant (2) are able to dimerize in this system. Vector control is a transformant containing empty pACTrAp-Zif and pBRGpω plasmids. White, no IPTG; black, 10 μM IPTG. (C) (Upper) Only N-terminal portions of ToxT that can induce a protein–protein interaction can exert a dominant negative effect on CT production. ToxT truncations were expressed from a plasmid in wild-type O395. (Lower) Western blot with α-His demonstrates that all N-terminal portions were expressed to equal levels.

Fig. 2.

Fig. 2.

(A) Virstatin inhibits ToxT dimerization in bacterial two-hybrid system in E. coli. The addition of increasing concentrations of virstatin inhibited the protein–protein interaction of the ToxT N terminus with itself as measured by β-galactosidase assay. Point mutant ToxTL113P was more resistant to virstatin. Activity is presented as a percentage of reporter activity above background in the presence of virstatin compared with no virstatin. Solid line, ToxT; dashed line, ToxTL113P. (B) Chemical structure of virstatin base with the R group labeled in bold. (C) Inhibition of ToxT dimerization in the bacterial two-hybrid system and CT production in V. cholerae by analogs of virstatin is correlated. Each compound is designated by its R group and inhibition is graphed as the percentage of control activity. Virstatin is the first compound listed. White, β-galactosidase production; black, CT.

Fig. 3.

Fig. 3.

Virstatin favors ToxT monomers. (A) MBP–ToxT–His6 fusion construct. (B) The MBP–ToxT fusions complement a toxT deletion in O395 as well as wild-type ToxT. MBP–ToxTWT is inhibited by virstatin, whereas MBP–ToxTL113P is resistant to virstatin. No virstatin, black; virstatin, white. (C) (Upper) ToxTWT and ToxTL113P amounts in the absence and presence of virstatin in FPLC fractions are quantified by ELISA using Ni+2-coated plates and an anti-MBP antibody (molecular mass standards are indicated with arrows above the graph). (Lower) Western blot analysis of each of the fractions was performed by using an α-MBP antibody, demonstrating that monomeric ToxTWT can be isolated only in the presence of virstatin (bands shown are all 74 kDa).

Fig. 4.

Fig. 4.

ToxT function varies at different promoters. (A) Virstatin inhibited activity of ToxT at the ctxAB, tcpA, acfD, and tagA promoters to a greater extent than at the acfA, aldA, and tcpI promoters. Activity at each of the promoters was assayed by measuring β-galactosidase activity by using a lacZ transcriptional reporter in O395Δ_lacZ_. Data are presented as the percentage of activity in the presence of 100 μM virstatin compared with control activity. (B) Map of ToxT-binding sites at acfA and acfD promoters.

Fig. 5.

Fig. 5.

Model of ToxT activation of the ctx promoter and inhibition with virstatin. (A) The C-terminal domain of ToxT blocks the homodimerization site on the N-terminal domain in the absence of a hypothetical small molecule activator. (B) In V. cholerae, this putative activator (cube) binds to either the N- or C- terminal domains, resulting in a conformational change in ToxT, which exposes the dimerization site. (C) Dimerization of ToxT occurs, allowing binding and transcriptional activation of the ctx promoter. (D) Virstatin (red star) prevents dimerization of the N-terminal domains and transcription at the ctx promoter.

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