Modulation of expression and antibacterial targeting of Shigella flexneri VirF

Shigella flexneri is a human enteropathogen that invades the intestinal mucosa and results in severe bacillary dysentery. VirF is the positive regulator of transcription in the Shigella spp. virulence cascade, and DeltavirF mutant strains of S. flexneri are avirulent. Although many cellular factors are associated with expression of the VirF protein, previous studies demonstrated that the concentration of VirF is decreased by 60% in DeltavacC mutant strains of S. flexneri. VacC is tRNA-guanine transglycosylase (TGT), an RNA-modification enzyme that catalyzes the incorporation of the modified nucleoside queuosine (Q) in substrate tRNA. We have studied the VirF protein as a novel antibacterial target in S. flexneri to provide proof of principle of targeting virulence factor expression and to combat the emergence of multi-drug resistant strains, with a focus on both modulation of VirF expression by TGT and expression and activity of the VirF protein itself.;Based on previous studies of differential redundant codon recognition by Q- versus G-tRNA and that TGT will recognize RNA species other than tRNA, we hypothesized that TGT modulates VirF protein expression through queuine modification at both the level of tRNA and the virF mRNA. We report that the modification state of tRNA with queuine and the Q-cognate redundant codon usage (NAU versus NAC codons) in a target mRNA alters the rate of protein expression. VirF mRNA contains an overall bias of 80% for the NAU Q-cognate codons, and it is conceivable that the subtle decrease in VirF protein expression in the S. flexneri (Delta vacC) mutant results from the absence of queuine-modified tRNA. In addition, we report that the virF mRNA is itself a site-specific substrate for the eubacterial TGT in vitro. Altered conformations of RNA sequences (termed riboswitches) upon binding small molecule metabolites have been shown to modulate either transcription or translation of the target RNA.;To measure VirF activity in the presence of small molecules, we report the development of a high-throughput cell-based reporter assay using a virB operator-beta-galactosidase fusion. We have screened approximately 42,000 small molecules and report the identification of 7 compounds with low to mid-micromolar IC50 values in the VirF-beta-galactosidase assay.