Penicillin-binding proteins (PBPs) make up an essential class of bacterial enzymes that carry out the final steps of peptidoglycan synthesis and regulate the recycling of this polymeric structure. PBPs are an excellent drug target and have been the most clinically relevant antibacterial target since the 1940s with the introduction of β-lactams. Despite this, a large gap in knowledge remains regarding the individual function and regulation of each PBP homologue in most bacteria. This can be attributed to a lack of chemical tools and methods that enable the study of individual PBPs in an activity-dependent manner and in their native environment. The development of such methods in Gram-negative bacteria has been particularly challenging due to the presence of an outer membrane and numerous resistance mechanisms. To address this, we have developed an optimized live-cell assay for screening inhibitors of the PBPs in Escherichia coli MG1655. We utilized EDTA to permeabilize Gram-negative cells, enabling increased penetration of our readout probe, Bocillin-FL, and subsequent analysis of PBP-inhibition profiles. To identify scaffolds for future development of PBP-selective activity-based probes, we screened ten β-lactams, one diazabicyclooctane, and one monobactam for their PBP-selectivity profiles in E. coli MG1655. These results demonstrate the utility of our assay for the screening of inhibitors in live, non-hypersusceptible Gram-negative organisms.
Bibliographical noteFunding Information:
This work was supported by the National Institutes of Health (R01 GM128439 A1, E.E.C.) and the University of Minnesota, Department of Chemistry. J.D.S. was supported by the National Institutes of Health’s National Center for Advancing Translational Sciences, grants TL1R002493 and UL1TR002494. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health’s National Center for Advancing Translation Sciences. Graphical figures created with GraphPad Prism and BioRender.
© 2022 American Chemical Society.
- Escherichia coli
- Gram-negative bacteria
- penicillin-binding proteins