Investigation of potential dominant negative dipA mutants in susceptibility of Francisella tularensis to resazomycins.

Abstract

Multidrug-resistant bacteria pose a significant threat to global public health. According to the CDC, more than 2.8 million antibiotic-resistant infections occur in the United States each year, and more than 35,000 people die as a result. The development of new antibiotics is necessary to combat this public health crisis. Recently, we identified a novel family of compounds, resazomycins, which exhibit antimicrobial activity against select Gram-negative bacteria such as Francisella tularensis. The mode of action of resazomycins is not well understood, but through a screen for Rz-resistant (RZR) isolates of F. tularensis, one gene, dipA, was identified to be mutated in half of the strains that were sequenced. Complementation with wild-type dipA did not restore sensitivity to Rz in the RZR isolates, suggesting dipA does not play a role in Rz susceptibility or mutant dipA may be exhibiting a dominant negative effect. To explore the latter possibility, the dipA gene was amplified by PCR from spontaneous Rz mutants Rzr43 and Rzr46 and cloned into the F. tularensis shuttle vector pABST. These recombinant plasmids were then introduced into wild-type LVS. Upon confirmation of the successful introduction of each dipA allele into LVS, the resulting strains were then cultivated on chocolate agar containing Rz to determine their susceptibility to resazomycins. Preliminary results suggest that expression of mutant dipA in LVS did not confer resistance to Rz indicating dipA may not play a role in Rz susceptibility. Further investigation is needed to fully elucidate the contribution of dipA to the bactericidal action of resazomycins.