Molecular Mechanisms and Inhibition of Transcription Activation by Bacterial AraC Family Activator Proteins

AraC family proteins are transcriptional regulators that are defined by the presence of a conserved DNA binding domain (DBD). My research focused on three AraC family activators: Rns (activator of virulence genes in diarrhea-causing ETEC), VirF (activator of virulence genes in diarrhea-causing Shigella) and RhaR (activator of L-rhamnose catabolic operons in Escherichia coli). With the ultimate goal of discovery of novel antibacterial agents that inhibit the AraC family proteins, here I have investigated the molecular mechanism of transcription activation by Rns and RhaR. Site-directed mutagenesis of residues in the ETEC Rns N-terminal domain (NTD) identified three residues (N15, N16 and I17) that are required for the transcription activation function of Rns. Site-directed mutagenesis of residues in the Rns DBD (predicted to be contacted by the NTD residues) identified three residues (K216, Y251 and G252) that are required for transcription activation, and one residue (H250) that is required for both DNA binding and transcription activation. We propose that transcription activation by Rns involves contacts between RS2 and AS2 region residues and these contacts may impart the structure or dynamics required by Rns to activate transcription. In RhaR, I investigated the role of the RhaR Arm in transmission of the signal that effector (L-rhamnose) is bound from the NTD to the DBD, converting RhaR to its activating state. Site-directed mutagenesis results suggested that the RhaR Arm is involved in maintaining RhaR in its non-activating state. Our results suggest that residue L35 in the Arm makes inter-domain interactions with the RhaR DBD to reduce transcription activation by RhaR in the absence of L-rhamnose. To identify novel agents that target AraC family proteins, I tested the small molecule SE-1, which our lab identified as an effective inhibitor of the AraC family proteins RhaS and RhaR. Despite limited sequence identity, SE-1 was also shown to inhibit VirF and Rns activity in cell-based assays in E. coli. I showed that SE-1 blocked in vitro DNA binding by VirF and Rns, and expression of VirF-dependent virulence genes in Shigella. A collaborator showed that SE-1 inhibited invasion of Shigella into eukaryotic host cells. SE-1 did not detectably inhibit the growth or metabolism of the bacterial or eukaryotic host cells, respectively, indicating that the inhibition of invasion was not due to general toxicity. Overall, SE-1 appears to exhibit selectivity toward AraC family proteins, and has 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 E. coli reporter fusions; one to identify potential RhaS inhibitors, and a second to eliminate non-specific 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 impact 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

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

This is the publisher's version, also available electronically from http://iai.asm.org/content/81/11/4220VirF 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

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

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

Identification of a Small Molecule Inhibitor of Bacterial AraC Family Activators.pdf

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

Role of in Biofuel Production

Increased energy consumption coupled with depleting petroleum reserves and increased greenhouse gas emissions have renewed our interest in generating fuels from renewable energy sources via microbial fermentation. Central to this problem is the choice of microorganism that catalyzes the production of fuels at high volumetric productivity and yield from cheap and abundantly available renewable energy sources. Microorganisms that are metabolically engineered to redirect renewable carbon sources into desired fuel products are contemplated as best choices to obtain high volumetric productivity and yield. Considering the availability of vast knowledge in genomic and metabolic fronts, Escherichia coli is regarded as a primary choice for the production of biofuels. Here, we reviewed the microbial production of liquid biofuels that have the potential to be used either alone or in combination with the present-day fuels. We specifically highlighted the metabolic engineering and synthetic biology approaches used to improve the production of biofuels from E. coli over the past few years. We also discussed the challenges that still exist for the biofuel production from E. coli and their possible solutions