THE MOLECULAR BASIS FOR GAMMA PHAGE TARGETING

Abstract

Bacteriophage γ is a highly specific virus that infects Bacillus anthracis, leading to its adoption bythe Centers for Disease Control and Prevention (CDC) as a presumptive anthrax detection tool. This dissertation investigates the molecular basis of γ phage targeting, focusing on the interactions of its receptor-binding proteins. Using a combination of artificial intelligence protein prediction software (AlphaFold3), biochemical assays, and genetic analysis, we characterize key phage proteins responsible for host recognition and infection. Our study identifies the previously reported tail protein GP14 as an evolved distal tail (EvoDit) hexameric hub that connects the tape measure protein to the receptor-binding protein (RBP) of γ phage. Additionally, we demonstrate for the first time that GP15 is a trimeric RBP containing an intramolecular chaperone that undergoes selfcleavage to ensure proper protein maturation. Both GP14 and GP15 exhibit strong affinity exclusively for γ phage-sensitive strains. Furthermore, we confirm that the previously identified receptor for γ phage's endolysin PlyG, the secondary cell wall polysaccharide (SCWP), plays a crucial role in γ phage adsorption, likely serving as the primary receptor. Structural and functional analyses reveal that GP14 and GP15 form a receptor-binding complex, facilitating irreversible attachment to B. anthracis. Additionally, we uncover that PlyG naturally exists as a dimer, with monomeric mutants exhibiting significantly reduced activity, highlighting dimerization as a key factor in its enhanced bacteriolytic function and therapeutic potential. We further explore the translational potential of γ phage components in developing rapid bacterial diagnostics, which have traditionally relied on antibodies. The binding domain of PlyG (CBD) is integrated into a lateral flow assay for anthrax detection, offering significant advantages over the CDC’s γ phage infection assay, which requires specialized expertise and takes several days to produce results. These findings advance our understanding of phage-host interactions through tail protein characterization and highlight the diagnostic potential of γ phage proteins. Future research will refine the structural characterization of these proteins and enhance the design of phage-based rapid diagnostic tests. By leveraging γ phage biochemistry, this work lays the foundation for innovative countermeasures against B. anthracis and other bacterial pathogens.