McGillivray, ShaunaKlingemann, Lauren2022-07-222022-07-222022https://repository.tcu.edu/handle/116099117/54206Bacillus anthracis is the causative agent of anthrax disease, a serious disease that presents itself in the form of skin ulcers and systemic infections with a high mortality rate. This bacterium is dangerous to humans due to its virulence factors that help it infect a host organism. Much research in the field has focused on the importance of the plasmid-encoded anthrax toxins and capsule; both are critical for virulence. However, the goal of this project is to identify new chromosomal genes that also contribute to virulence in B. anthracis. Previous screening of a transposon library identified 11 B. anthracis mutants attenuated in infecting Caenorhabditis elegans, an invertebrate worm model. To further validate virulence phenotypes and prioritize transposon mutants for follow-up, a Galleria mellonella invertebrate infection model was used to assess survival of the 11 transposon mutants originally identified in C. elegans. One mutant, TN2, showed virulence attenuation in both models. TN2 has a disruption in a promoter region that we hypothesize controls two genes: a putative BNR repeat domain protein (2A) and a glycosyl-like 2 transferase family protein (2B).  For my project, I attempted insertional mutagenesis to inactivate the 2A gene with the goal of confirming that this gene is linked to virulence, rather than unintended mutations elsewhere in the genome. Unfortunately, an insertional mutant disrupting 2A (D2A) was not able to be constructed. However, we still compared the ability of the wild-type and the original transposon mutant to survive exposure to hydrogen peroxide and determined that the transposon mutant is not susceptible to reactive oxygen species. This research identified a potential novel bacterial virulence factor and more information on its mechanism of action thus expanding our understanding of bacterial pathogenesis.Bacillus anthracisanthraxvirulencewaxwormtransposonGalleria mellonellaputative BNR repeat domain proteinbioterrorisminsertional mutagenesis