Identifying Novel Mutants with Increased Susceptibility to Hydrogen Peroxide and Reduced Virulence in Bacillus anthracis Sterne

Abstract

Bacillus anthracis is a gram-positive bacterial pathogen that causes the deadly infectious disease anthrax. B. anthracis contains over 5,000 chromosomal genes, and we believe there are unidentified chromosomal genes important for virulence. To identify novel virulence genes, our lab constructed a transposon mutant library with random disruptions in the B. anthracis Sterne genome and screened for virulence-associated phenotypes. Previous screens successfully identified two novel virulence genes, clpX and yceGH, using this library. In this screen, we used H2O2, a reactive oxygen species involved in innate immune defense, and screened around 1000 mutants. We obtained three mutants that were susceptible to hydrogen peroxide in vitro: 11F11, LV1, and LV2. To determine whether they also had phenotypes in vivo, we infected Galleria mellonella to study their virulence in an invertebrate animal infection model. LV2 showed reduced virulence in the in vivo survival assay, and all three mutants showed reduced virulence in the in vivo competition assay. I have determined the site of the transposon insertion in 11F11 and LV1, and the transposon has inserted in the genes for catalase and a collagenase-like protein, respectively. I attempted to create an independent insertional mutation in LV1 to confirm that the observed phenotypes are linked to the disruption of the collagenase-like protein; however homologous recombination has not been successful. Future directions include creating a complementation plasmid for LV1 and determining the insertion site of LV2. The findings of this research could be used as potential therapeutic drug targets and will offer insight into the mechanisms that B. anthracis uses for its pathogenesis.