UCLA

Many species of pathogenic Gram-positive bacteria have become resistant to commonly used antibiotics, creating a pressing need for the development of novel antimicrobial therapies. These pathogenic bacteria are surrounded by a complex peptidoglycan cell wall that is decorated with a range of macromolecules such as surface proteins, protein-based oligomeric pili, and surface glycopolymers. These structures enable the microbe to effectively interact with their environment, and in pathogenetic bacteria are frequently virulence factors that are involved in: immune system modulation, bacterial adhesion, nutrient acquisition, and spore formation. Two distinct enzyme superfamilies are primarily responsible for covalently attaching macromolecules to the Gram-positive cell wall: sortase enzymes that attach proteins and assemble pili, and LytR-Psr-CpsA (LCP) enzymes that attach glycopolymers. This dissertation presents my efforts to discover a sortase inhibitor, structural and mechanistic investigations of both sortase and LCP enzymes, and the development of a new sortase mediated ligation technology. I describe our efforts to find inhibitors against the S. aureus sortase transpeptidase. We utilized both experimental and computational methods to discover potent inhibitor scaffolds. This led to structure-activity studies that not only improved inhibitor potency, but also yielded molecules that can inhibit bacterial protein display in vivo, and appear to be non-toxic to human cells. Sortase enzymes have also proven to be valuable biotechnological conjugation tools. I describe the development of a novel sortase-mediated ligation tool. We demonstrate a 15-fold rate enhancement for existing sortase mediated ligation strategies. Additionally, I discuss studies on sortase pilus assembly and display. To gain insight into the molecular mechanism of sortase pilus assembly, we reconstituted an archetypal Gram-positive pilus assembly process in vitro. This has allowed us to generate molecular models of various intermediates of pilus assembly and define the molecular determinants of this assembly process. We have identified novel features on both the sortase and pilin precursor proteins that are essential for pilus covalent isopeptide assembly. In the concluding chapter of this dissertation, structural work characterizing the unique LCP enzyme that mediates surface protein glycosylation is presented. Work on this project reveals that LCP enzymes use a conserved phosphotransferase mechanism to attach glycopolymers to surface displayed proteins, and this may be a mechanism for actinobacteria wall teichoic acid display, a key macromolecule in the bacterial cell envelope. Combined, this work serves to further define the mechanism of Gram-positive bacterial macromolecular surface display, improved sortase-mediated bioconjugation strategies, and steps towards novel anti-infective sortase inhibitors.