Contribution of Francisella tularensis FTL_1306 (dipA) in resazomycin susceptibility

Abstract

The CDC classifies Francisella tularensis as a Category A bioterrorism agent. Due to the growing global threat of antibiotic resistant bacteria, novel therapeutics against F. tularensis must be developed.  Resazomycins are resazurin (Rz)-based compounds that exhibit antimicrobial activity against F. tularensis and other gram-negative bacteria. The action of resazomycins is not well understood, but potential targets of the antibiotic were identified in a high throughput screen for Rz-resistant isolates. The dipA (FTL_1306) gene was identified as mutated in half of the 48 Rz-resistant (RZR) strains sequenced. To further investigate the role of dipA in Rz susceptibility, we introduced a wild-type copy of dipA into select RZR isolates (RZR1, 5, 43, and 46) that contain dipA mutations. The dipA gene was amplified by PCR from wild-type F. tularensis and cloned into the F. tularensis shuttle vector pABST to generate a construct (pABST-dipA) in which dipA will be constitutively expressed under control of the groEL promoter. The pABST-dipA plasmid was mobilized into each of the selected RZR isolates by electroporation. Western blotting indicated that expression of wild-type dipA was restored in RZR strains with the introduction of the pABST-dipA construct. The MIC of resazurin for the resulting RZR dipA-complemented strains was equivalent to that wild-type F. tularensis and significantly different from resistant mutants. Further investigation is needed to fully elucidate the contribution of dipA to the bactericidal action of resazomycins.