Modulation of Expression and Antibacterial Targeting of Shigella flexneri VirF.

Abstract

Shigella flexneri is a human enteropathogen that invades the intestinal mucosa and results in severe bacillary dysentery. With the emergence of multiantibiotic-resistant strains, the development of novel therapeutics against critical S. flexneri target proteins promises a more effective treatment regimen for shigellosis. VirF is the positive regulator of transcription in the Shigella spp. virulence cascade, and ∆virF mutant strains of S. flexneri are avirulent. Although many cellular factors are associated with expression of the VirF protein (i.e., temperature, pH), previous studies demonstrated that the concentration of VirF protein is decreased by 60% in ∆vacC 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 Shigella flexneri, with a focus on both modulation of VirF expression by TGT and expression and activity of the VirF protein itself. 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 codon usage (NAU versus NAC codons) in the 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 (∆vacC) mutant results from the absence of queuine-modified tRNA. In addition, we report that the virF mRNA is itself a substrate for the eubacterial TGT in vitro. Modification of RNA sequences (termed riboswitches) with small molecule metabolites has 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-β-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 – β-galactosidase assay. Further studies are pending to identify selective VirF inhibitors.