The role of pilD in Francisella tularensis susceptibility to resazomycins.

Abstract

The CDC classifies Francisella tularensis as a Category A bioterrorism agent. If used during a terror attack, the inhalation of a single F. tularensis bacterium can cause the fatal disease tularemia. Due to the potential release of antibiotic-resistant F. tularensis strains, new therapeutics against F. tularensis must be developed. Resazomycins are resazurin-based compounds that exhibit antimicrobial activity against F. tularensis as well as other Gram-negative bacteria including Neisseria gonorrhoeae, the causative agent of gonorrhea. The mode of action of these antibiotics is not understood. To identify potential targets of resazomycins, we selected for mutants of F. tularensis that were capable of growing in the presence of 20x the minimal inhibitory concentration of resazurin (Rz). Approximately 50% of these Rz-resistant F. tularensis isolates had a mutation in the genes FTL_0959 (pilD) and FTL_1306 (dipA). In F. tularensis, pilD encodes for the cytoplasmic membrane peptidase responsible for processing the prepilin subunits in type IV pilin assembly proteins. To investigate the role of pilD in Rz susceptibility, we generated a pilD disruption mutant in wild-type F. tularensis and tested its sensitivity to Rz. In preliminary experiments, growth of the pilD disruption mutant is inhibited by Rz, although to a lesser degree than wild-type LVS suggesting pilD may play a minor role in resazomycin susceptibility. We are currently working to generate a complementation construct to express wild-type pilD in select Rz-resistant Ft mutants to further investigate the role of this gene in Rz susceptibility.