Live-Cell Profiling of Penicillin-Binding Protein Inhibitors in Escherichia coli MG1655.
ACS Infect Dis
; 8(7): 1241-1252, 2022 07 08.
Article
in En
| MEDLINE
| ID: mdl-35763562
ABSTRACT
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 ß-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 ß-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.
Key words
Full text:
1
Collection:
01-internacional
Database:
MEDLINE
Main subject:
Beta-Lactams
/
Escherichia coli
Language:
En
Journal:
ACS Infect Dis
Year:
2022
Document type:
Article
Affiliation country: