Most bacteria are surrounded by a cell wall composed of a complex polymeric structure called peptidoglycan that is essential for cell survival. The biosynthetic pathway for production of peptidoglycan and the proteins required for its assembly have been the targets for many antibacterial agents. For example, penicillin-binding proteins (PBPs), which polymerize and crosslink strands of peptidoglycan also have affinity for the beta-lactam antibiotic penicillin. PBPs are classified into three groups based upon their molecular weight, conserved amino acid motifs and function. These proteins all contain a serine in their peptidase domain that is required for catalysis. Penicillin hinders PBP function by forming a stable acyl-enzyme intermediate with this residue, which in turn inhibits crosslinking of peptidoglycan.
Despite the effectiveness of beta-lactam antibiotics, bacterial resistance has arisen very rapidly. A more detailed understanding of the mechanism of peptidoglycan synthesis may be the key for design of new and more effective antibiotics. We have developed fluorescently-labeled small molecule probes to visualize the activity of PBPs directly in live cells. Small molecule-conjugated fluorophores can provide superior temporal resolution than more traditional strategies, such as protein fusion, and their activity can be modulated by dose. These compounds are also generally easy to use and enable visualization in a broad range of organisms to explore the construction and remodeling of peptidoglycan.
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