Chemical Proteomic Methods To Study Bacterial Cell Wall Biosynthesis

Abstract

Bacterial cells are surrounded by an external layer called the cell wall that defines cellular shape and protects them from osmolysis. A major component of this protective coating, which is unique to prokaryotes, is the peptidoglycan (PG), a heteropolymeric structure composed of saccharide backbones that are cross-linked by peptide subunits. This exclusive presence in bacteria makes the cell wall an ideal antimicrobial target due to the elimination of side effects in humans and other eukaryotes. Indeed, the largest class of clinically-utilized antibiotics, β-lactams such as the penicillins, target the final stages of PG biosynthesis. Penicillin-binding proteins (PBPs) are membrane-associated proteins that are involved in the final stages of bacterial cell wall synthesis and assembly. Discovered as the target of β-lactam antibiotics, PBPs have received much attention for many decades as crucial antibacterial targets and for their role in antibiotic resistance. However, specific roles of individual family members in each bacterial strain, as well as their protein-protein interactions, are yet to be understood. Besides their therapeutic applications, β-lactams have long been used as chemical probes to study the PBPs. Bocillin-FL, for example is a commercially available fluorescent analog of penicillin V with global affinity for all PBPs. Our group is interested in developing PBP-selective activity-based probes to explore these proteins. This dissertation expands on my efforts in developing such probes to specifically target individual PBPs, based on selected β-lactam antibiotics, as well as a β-lactone scaffold that we designed and showed that specifically targets PBPs. Using our probe toolbox, we visualized the activity of specific PBPs in Bacillus subtilis and S. pneumoniae. With the established potency of these probes, we intend to study PBP-associated proteins via pulldown assays. Ultimately, these studies will shed light on bacterial cell wall construction and division, which would potentially lead to discovery of novel key players involved in these processes that could be harnessed as novel antibacterial targets.