Decoding the Penicillin-Binding Proteins with Activity-Based Probes

The bacterial cell wall is a complex structure that is primarily composed of peptidoglycan (PG), which provides protection from the environment and structural rigidity for the cell. As such, PG plays an important role in bacterial survival, which has made its biosynthesis a crucial target for antibiotic development for many decades. Despite long-standing efforts to inhibit PG construction, much remains unknown about the enzymes required for PG biosynthesis or how PG composition and architecture are altered to enable adaptation to environmental stressors. This knowledge will be crucial in the identification of new ways to interfere with PG construction that could overcome widespread resistance to cell wall-targeting antibacterial agents.All bacterial species possess a suite of penicillin-binding proteins (PBPs), which are critical actors in PG construction and remodeling, as well as the main targets of β-lactam antibiotics. While the importance of the PBPs is well-known, the field lacks a complete understanding of PBP activity regulation, localization, and critical protein-protein interactions during the growth and division process. Bacteria possess between 4 and 16 PBP homologues with only one or several being genetically essential in each cell. A key outstanding question about these proteins is why bacteria expend the energy required to maintain this relatively large number of related proteins when so few are required to maintain life. The Carlson lab focuses on the development of chemical tools to address this fundamental question. In particular, we have generated a suite of chemical probes to selectively target one or a small number of PBP homologues in their catalytically active state. These activity-based probes (ABPs) have and will continue to enable a deeper understanding of the traits that differentiate the PBPs over the bacterial lifespan.In this account, we discuss the development of selective chemical tools to study the PBPs. Key to our success has been assessment of the PBP inhibition profiles of an expansive set of commercially available β-lactams in both Gram-positive and Gram-negative bacteria. This work has directly identified molecules that can be used in chemical genetic studies and provided scaffolds for the generation of PBP-selective ABPs. We also discovered a novel β-lactone scaffold that is exquisitely selective for PBPs over other protein classes and targets a different subclass of these proteins than related β-lactams. Using these probes, we have explored PG biosynthesis in Streptococcus pneumoniae, Escherichia coli and Bacillus subtilis yielding new insights about their cell wall construction and remodeling processes, as well as specialized activities under stress.