Small-molecule inhibitor of the Shigella flexneri master virulence regulator VirF.

VirF is an AraC family transcriptional activator that is required for the expression of virulence genes associated with invasion and cell-to-cell spread by Shigella flexneri, including multiple components of the type three secretion system (T3SS) machinery and effectors. We tested a small-molecule compound, SE-1 (formerly designated OSSL_051168), which we had identified as an effective inhibitor of the AraC family proteins RhaS and RhaR, for its ability to inhibit VirF. Cell-based reporter gene assays with Escherichia coli and Shigella, as well as in vitro DNA binding assays with purified VirF, demonstrated that SE-1 inhibited DNA binding and transcription activation (likely by blocking DNA binding) by VirF. Analysis of mRNA levels using real-time quantitative reverse transcription-PCR (qRT-PCR) further demonstrated that SE-1 reduced the expression of the VirF-dependent virulence genes icsA, virB, icsB, and ipaB in Shigella. We also performed eukaryotic cell invasion assays and found that SE-1 reduced invasion by Shigella. The effect of SE-1 on invasion required preincubation of Shigella with SE-1, in agreement with the hypothesis that SE-1 inhibited the expression of VirF-activated genes required for the formation of the T3SS apparatus and invasion. We found that the same concentrations of SE-1 had no detectable effects on the growth or metabolism of the bacterial cells or the eukaryotic host cells, respectively, indicating that the inhibition of invasion was not due to general toxicity. Overall, SE-1 appears to inhibit transcription activation by VirF, exhibits selectivity toward AraC family proteins, and has the potential to be developed into a novel antibacterial agent.

Identification of a small-molecule inhibitor of bacterial AraC family activators.

Protein members of the AraC family of bacterial transcriptional activators have great promise as targets for the development of novel antibacterial agents. Here, we describe an in vivo high-throughput screen to identify inhibitors of the AraC family activator protein RhaS. The screen used two Escherichia coli reporter fusions: one to identify potential RhaS inhibitors and a second to eliminate nonspecific inhibitors from consideration. One compound with excellent selectivity, OSSL_051168, was chosen for further study. OSSL_051168 inhibited in vivo transcription activation by the RhaS DNA-binding domain to the same extent as the full-length protein, indicating that this domain was the target of its inhibition. Growth curves showed that OSSL_051168 did not affect bacterial cell growth at the concentrations used in this study. In vitro DNA-binding assays with purified protein suggest that OSSL_051168 inhibits DNA binding by RhaS. In addition, we found that it inhibits DNA binding by a second AraC family protein, RhaR, which shares 30% amino acid identity with RhaS. OSSL_051168 did not have a significant impact on DNA binding by the non-AraC family proteins CRP and LacI, suggesting that the inhibition is likely specific for RhaS, RhaR, and possibly additional AraC family activator proteins.

The AraC/XylS family activator RhaS negatively autoregulates rhaSR expression by preventing cyclic AMP receptor protein activation.

The Escherichia coli RhaR protein activates expression of the rhaSR operon in the presence of its effector, L-rhamnose. The resulting RhaS protein (plus L-rhamnose) activates expression of the L-rhamnose catabolic and transport operons, rhaBAD and rhaT, respectively. Here, we further investigated our previous finding that rhaS deletion resulted in a threefold increase in rhaSR promoter activity, suggesting RhaS negative autoregulation of rhaSR. We found that RhaS autoregulation required the cyclic AMP receptor protein (CRP) binding site at rhaSR and that RhaS was able to bind to the RhaR binding site at rhaSR. In contrast to the expected repression, we found that in the absence of both RhaR and the CRP binding site at the rhaSR promoter, RhaS activated expression to a level comparable with RhaR activation of the same promoter. However, when the promoter included the RhaR and CRP binding sites, the level of activation by RhaS and CRP was much lower than that by RhaR and CRP, suggesting that CRP could not fully coactivate with RhaS. Taken together, our results indicate that RhaS negative autoregulation involves RhaS competition with RhaR for binding to the RhaR binding site at rhaSR. Although RhaS and RhaR activate rhaSR transcription to similar levels, CRP cannot effectively coactivate with RhaS. Therefore, once RhaS reaches a relatively high protein concentration, presumably sufficient to saturate the RhaS-activated promoters, there will be a decrease in rhaSR transcription. We propose a model in which differential DNA bending by RhaS and RhaR may be the basis for the difference in CRP coactivation.

Differences in the mechanism of the allosteric l-rhamnose responses of the AraC/XylS family transcription activators RhaS and RhaR.

Proteins in the largest subset of AraC/XylS family transcription activators, including RhaS and RhaR, have C-terminal domains (CTDs) that mediate DNA-binding and transcription activation, and N-terminal domains (NTDs) that mediate dimerization and effector binding. The mechanism of the allosteric effector response in this family has been identified only for AraC. Here, we investigated the mechanism by which RhaS and RhaR respond to their effector, l-rhamnose. Unlike AraC, N-terminal truncations suggested that RhaS and RhaR do not use an N-terminal arm to inhibit activity in the absence of effector. We used random mutagenesis to isolate RhaS and RhaR variants with enhanced activation in the absence of l-rhamnose. NTD substitutions largely clustered around the predicted l-rhamnose-binding pockets, suggesting that they mimic the structural outcome of effector binding to the wild-type proteins. RhaS-CTD substitutions clustered in the first HTH motif, and suggested that l-rhamnose induces improved DNA binding. In contrast, RhaR-CTD substitutions clustered at a single residue in the second HTH motif, at a position consistent with improved RNAP contacts. We propose separate allosteric mechanisms for the two proteins: Without l-rhamnose, RhaS does not effectively bind DNA while RhaR does not effectively contact RNAP. Upon l-rhamnose binding, both proteins undergo structural changes that enable transcription activation.

Transcription activation by the DNA-binding domain of the AraC family protein RhaS in the absence of its effector-binding domain.

The Escherichia coli L-rhamnose-responsive transcription activators RhaS and RhaR both consist of two domains, a C-terminal DNA-binding domain and an N-terminal dimerization domain. Both function as dimers and only activate transcription in the presence of L-rhamnose. Here, we examined the ability of the DNA-binding domains of RhaS (RhaS-CTD) and RhaR (RhaR-CTD) to bind to DNA and activate transcription. RhaS-CTD and RhaR-CTD were both shown by DNase I footprinting to be capable of binding specifically to the appropriate DNA sites. In vivo as well as in vitro transcription assays showed that RhaS-CTD could activate transcription to high levels, whereas RhaR-CTD was capable of only very low levels of transcription activation. As expected, RhaS-CTD did not require the presence of L-rhamnose to activate transcription. The upstream half-site at rhaBAD and the downstream half-site at rhaT were found to be the strongest of the known RhaS half-sites, and a new putative RhaS half-site with comparable strength to known sites was identified. Given that cyclic AMP receptor protein (CRP), the second activator required for full rhaBAD expression, cannot activate rhaBAD expression in a DeltarhaS strain, it was of interest to test whether CRP could activate transcription in combination with RhaS-CTD. We found that RhaS-CTD allowed significant activation by CRP, both in vivo and in vitro, although full-length RhaS allowed somewhat greater CRP activation. We conclude that RhaS-CTD contains all of the determinants necessary for transcription activation by RhaS.