UCLA

Pathogenic Gram-positive bacteria have acquired resistance to many commonly used antibiotics. One potential strategy to combat this problem is to develop small molecule therapeutics that target bacterial virulence mechanisms which enable pathogens to cause disease. The bacterial cell surface contains many virulence factors that have key roles in host-pathogen interactions, but the mechanisms by which they are synthesized and displayed remains incompletely understood. The work described in this dissertation is concentrated on understanding how bacteria synthesize wall teichoic acid (WTA) glycopolymers. WTAs are peptidoglycan-anchored alditol-phosphate polymers that have important functions in host immune system evasion, biofilm formation, and antibiotic resistance, among other roles. This research is specifically focused on the TagA glycosyltransferase, which catalyzes the first committed enzymatic step in WTA biosynthesis. Small molecule TagA inhibitors might function as novel antivirulence therapeutics because cells that lack the enzyme in methicillin-resistant Staphylococcus aureus (MRSA) maintain viability but become re-sensitized to β-lactam antibiotics. The research described in this dissertation advances our understanding of the enzymatic mechanism of TagA and its role in controlling the architecture of the bacterial cell wall. Chapter 1 surveys the biosynthetic pathways that are used to produce peptidoglycan and WTA polymers in Bacillus subtilis and S. aureus. Chapter 2 details the determination of the first crystal structure of the soluble portion of TagA from Thermoanaerobacter italicus, which is shown to adopt a novel glycosyltransferase structural fold. This study identified two conserved residues in TagA that are important for catalysis and demonstrated that C-terminal tail residues in the enzyme are essential for enzymatic activity in vitro and membrane association in cells. Chapter 3 describes the construction of a solubility-enhanced TagA variant and crystal structures of this enzyme bound to its native substrate, UDP-ManNAc, and an epimer of the substrate, UDP-GlcNAc. These structures provide insight into stereospecific protein-ligand contacts that confer substrate specificity. Molecular dynamics simulations of full-length TagA models with and without its bound substrates are also presented which demonstrate that UDP-ManNAc stabilizes construction of the enzyme’s active site through interactions with key catalytic residues in the C-terminal tail. Collectively, these data suggest a model of enzyme function in which membrane association via the C-terminal tail triggers a conformational change in TagA that is further stabilized by stereospecific contacts with its UDP-ManNAc substrate. Lastly, Chapter 4 details ongoing progress in capturing the active, monomeric form of the protein for structural investigations and studying the influence of TagA activity on cell morphology in B. subtilis using transmission electron microscopy. This work contributes to our knowledge of WTA biogenesis in bacteria and lays a foundation for the structure-guided development of TagA-specific inhibitors that could function as antivirulence agents.