Abstract
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.
Highlights
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
Given that our findings indicated that SE-1 interacted with the relatively conserved DNA-binding domains of these AraC family proteins, we tested whether it might inhibit VirF, an AraC family activator required for virulence in Shigella
As in our growth assays with E. coli, we found that 0.3% dimethyl sulfoxide (DMSO) did not detectably slow the growth of Shigella, and SE-1 resulted in no detectable decrease in the Shigella growth rate at concentrations as high as 40 M
Summary
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. Anti-infective strategies that target several individual AraC family bacterial virulence gene activator proteins have been reported and have the potential to be developed into alternatives to traditional antibiotics [24,25,26,27,28]. AraC family proteins activate virulence gene expression in many pathogenic bacteria, including Shigella flexneri (VirF), Vibrio cholerae (ToxT), enterotoxigenic Escherichia coli (ETEC) (Rns/CfaD), and Pseudomonas aeruginosa (ExsA) [29]. Inhibition of AraC family virulence activators is expected to exert less selective pressure on bacteria to develop resistance than currently available antibiotics [22, 32,33,34]. VirF expression has been shown to be temperature dependent, with 3- to 4-fold-lower expression at 30°C than at 37°C
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