Peptidoglycan (PG) is a mesh-like heteropolymer made up of glycan chains cross-linked by short peptides and is the major scaffold of eubacterial cell walls, determining cell shape, size, and chaining. This structure, which is required for growth and survival, is located outside of the cytoplasmic membrane of bacterial cells, making it highly accessible to antibiotics. Penicillin-binding proteins (PBPs) are essential for construction of PG and perform transglycosylase activities to generate the glycan strands and transpeptidation to cross-link the appended peptides. The β-lactam antibiotics, which are among the most clinically effctive antibiotics for the treatment of bacterial infections, inhibit PBP transpeptidation, ultimately leading to cell lysis. Despite this importance, the discrete functions of individual PBP homologues have been difficult to determine. These major gaps in understanding of PBP activation and macromolecular interactions largely result from a lack of tools to assess the functional state of specific PBPs in bacterial cells. We have identified β-lactones as a privileged scaffold for the generation of PBP-selective probes and utilized these compounds for imaging of the essential proteins, PBP2x and PBP2b, in Streptococcus pneumoniae. We demonstrated that while PBP2b activity is restricted to a ring surrounding the division sites, PBP2x activity is present both at the septal center and at the surrounding ring. These spatially separate regions of PBP2x activity could not be detected by previous activity-based approaches, which highlights a critical strength of our PBP-selective imaging strategy.

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ABPP methods have been utilized for the last two decades as a means to investigate complex proteomes in all three domains of life. Extensive use in eukaryotes has provided a more fundamental understanding of the biological processes involved in numerous diseases and has driven drug discovery and treatment campaigns. However, the use of ABPP in prokaryotes has been less common, although it has gained more attention over the last decade. The urgent need for understanding bacteriophysiology and bacterial pathogenicity at a foundational level has never been more apparent, as the rise in antibiotic resistance has resulted in the inadequate and ineffective treatment of infections. This is not only a result of resistance to clinically used antibiotics, but also a lack of new drugs and equally as important, new drug targets. ABPP provides a means for which new, clinically relevant drug targets may be identified through gaining insight into biological processes. In this chapter, we place particular focus on the discussion of ABPP strategies that have been applied to study different classes of bacterial enzymes.

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