Role of LpnA in Francisella tularensis Susceptibility to Resazurin

Abstract

Francisella tularensis is classified by the Centers for Disease Control and Prevention as a Category A bioterrorism agent due to its low infectious dose, ease of aerosolization, and high mortality rate. Given there is no licensed tularemia vaccine and the possibility of antibiotic-resistant F. tularensis strains, there is an urgent need to develop new treatments against this bacterium. Resazurin (Rz) has bactericidal activity against F. tularensis and other gram-negative bacteria including the human pathogens Neisseria gonorrhoeae and Helicobacter pylori. However, the mechanism of action for Rz has yet to be determined. To identify targets of Rz, we screened for Rz-resistant (Rzr) mutants of F. tularensis LVS. Whole genome sequencing was performed on 42 Rzr isolates and all contained mutations within the coding regions of FTL_0421 and FTL_1504. FTL_0421 encodes for LpnA, the major lipoprotein in F. tularensis. The FTL_0421 mutation in Rzr1 resulted in loss of LpnA expression which correlated with increased sensitivity to osmotic stress. To investigate the individual role of lpnA in Rz-resistance, a deletion mutant (ΔlpnA) was generated using standard molecular genetics techniques.  An agar dilution assay was then performed to determine the Rz susceptibility of the ΔlpnA strain. A minimum inhibitory concentration (MIC) of 2.75μg/mL was achieved for the ΔlpnA and the LVS wild-type strain. This data suggests mutation of lpnA alone is not sufficient to confer resistance to resazurin. In the future, we will investigate whether lpnA in combination with other genes like FTL_1504 play a role in resazurin susceptibility in F. tularensis.  (Supported by NIH Grant P20GM103434 to the West Virginia IDeA Network for Biomedical Research Excellence & the NASA Space Grant Consortium)