Small-Molecule Inhibitor of the Shigella flexneri Master Virulence Regulator VirF

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.

Antibiotic resistance in Enterobacteriaceae: mechanisms and clinical implications

“…202 Newer antibiotics New antibiotic classes for which resistance is expected to be slow or difficult to develop are welcome arrivals. 203 Agents that suppress virulence characteristics, [204][205][206] rather than kill the microbe, should also select less strongly for resistance and are attractive candidates for co-administration with current antimicrobials, [207][208][209][210][211] although none is immediately available.…”

Marching towards Making Evolution-Proof Antibiotics

“…A group of researchers lead by Dr. Susan Egan selectively inhibited an important AraC family Shigella virulence regulatory protein VirF that regulates a type III secretion system (T3SS) required for bacterial invasion. 3 A highly potent molecule "SE-1" ( 1-butyl-4-nitromethyl-3-quinolin-2-yl-4H-quinoline) is found to not impact the growth of the bacteria but prevent bacteria's ability to invade and infect cultured human intestinal cells. The efficacy and solubility of the molecule SE-1 is high enough to be developed as novel-antibacterial agent.…”

Burkholderia pseudomallei Type III Secretion System Cluster 3 ATPase BsaS, a Chemotherapeutic Target for Small-Molecule ATPase Inhibitors

“…Over the last decade many natural product and synthetic compound inhibitors targeting bacterial TTSS have been identified by high-throughput screening or rational design methods (39)(40)(41). However, in the majority of cases the TTSS component targeted by the inhibitor remains unknown, and thus very few have been fully investigated for their mechanism of action (42). In the present study, we used compound 939, which has been identified as an efficient inhibitor of Y. pestis YscN and B. mallei BsaS TTSS ATPase in vitro (31).…”