Development of Activity-Based Probes and Biochemical Methods for the Study of Penicillin-Binding Proteins in Live Bacteria

Abstract

Bacterial cells are surrounded by a polymeric, mesh-like structure known as the peptidoglycan, and is an essential component of all eubacteria. Multi-protein machinery complexes function to carry out highly orchestrated synthesis and remodeling of the peptidoglycan throughout cell growth and division. One of the key components of these machinery complexes is the class of essential bacterial enzymes known as the penicillin-binding proteins (PBPs). PBPs are a highly conserved class of membrane-associate enzymes that function to carry out the final steps of peptidoglycan biosynthesis. All PBPs have a highly homologous transpeptidation domain, which contains a conserved, catalytic serine, to enable cross-linking of adjacent stem-peptide chains within the peptidoglycan. The catalytic serine residue has been exploited by the β-lactam class of antibiotics for ~ 100 years. Despite the success of the β-lactams as the most clinically used class of antibiotics, significant gaps in knowledge regarding the PBPs remain. The specific roles and regulations of individual PBP homologs is poorly understood and this can be attributed to a lack of appropriate tools to enable these studies. Our group has undertaken a chemical biology approach to addressing this gap, through the development of activity-based probes and biochemical methods that enable the visualization of PBP activities in native environments. The work presented in this thesis is focused on the expansion of available tools and methods that we have at our disposable to study the PBPs within live cells of both Gram-negative and Gram-positive bacteria. Efforts focused on the expansion of a β-lactone library of activity-based probes demonstrated that bioorthogonal probes retained the same activity as previously synthesized fluorophore-conjugated molecules but have increased utility in protein pull-down experiments to investigate the protein-protein interactions of specific PBP homologs. Next, the development of a live-cell kinetics assay in Streptococcus pneumoniae has provided a novel means to determine the potency values of inhibitors against the entire complement of an organism’s PBPs in one assay. The data that will be generated from future work will enable quantitative structure-activity-relationship studies to be performed, which in turn will inform us on the rational design of future PBP-selective molecules. Finally, the development of a live-cell method to study inhibitors of the PBPs in non-hypersusceptible Gram-negative species provides a means to identify molecules that are selective for the PBPs in these species and enable the development of activity-based probes for PBP homologs in understudied bacteria. In sum, we present new tools and methods that when combined with existing strategies will provide a more complete understanding of how individual PBPs function within live cells, ultimately enabling us to identify targets for the next generations of antibiotics.