The Discovery, Characterization, and Engineering of Autolytic Enzymes as Antimicrobials

In an age of rising levels of antibiotic resistance, we are quickly running out of tools to address some of the most virulent and widespread infectious bacteria. One group of enzymes that show significant potential for use as next-generation antibiotics are antimicrobial peptidoglycan hydrolases, often referred to as lysins. These enzymes are responsible for the breakdown of peptidoglycan within the bacterial cell wall, and, when exogenously applied, can result in dramatic destabilization of the cell wall leading to rapid lysis of the bacteria. While phage endolysins have been widely investigated as potential antimicrobials, the therapeutic potential of endogenous autolysins from pathogenic bacteria is relatively unexplored. This thesis focuses on three projects, the first two are aimed at the identification, characterization, and engineering of genomically derived autolysins with antimicrobial activity against Clostridioides difficile and Staphylococcus aureus, two pathogens with concerning antibiotic resistance, while the final project pushes the development of techniques to address a common issue for bacterial lysins, immunogenicity in the form of antidrug antibodies. The first project details the bioinformatics-driven discovery of six new anti-C. difficile lysins belonging to the amidase-3 family of enzymes, and we describe experimental comparison of these new variants against the leading candidates from the literature. Our quantitative analyses include metrics for expression level, inherent antibacterial activity, breadth of strain selectivity, killing of germinating spores, and structural and functional measures of thermal stability. In the second project we combine a bioinformatics pipeline to identify putative Staphylococcus aureus autolysin domains, with a combinatorial approach to generate a library of chimeric autolysin which were subsequently screened in a high-throughput manner resulting in the discovery and characterization of the novel antimicrobial chimeric autolysin Ssa2-301. In the third project we focus on utilizing a technique to eliminate B cell epitopes from the surface of lytic enzymes using the previously characterized bacteriocin lysostaphin as a proof of principle. These projects illustrate the value of expanding efforts in the exploration of autolysins as potential antimicrobials, and they illustrate one example of the often-necessary engineering to improve their translational potential