Determination of Accumulation of Molecules inEscherichia coli

Abstract

AbstractThere is an urgent need for efficacious agents against Gram-negative bacterial pathogens because the presence of an outer membrane inherently reduces susceptibility to antibiotics. This asymmetric bilayer effectively diminishes the permeation of small molecules into bacterial cells. A significant complication to drug discovery and development is that few methods exist for measuring the permeation of molecules past the outer membrane. Our laboratory has bridged this technical gap by developing a robust assay to semi-quantitatively measure the accumulation of small molecules into Gram-negative bacteria. Our assay is based on anchoring bioorthogonal strained alkyne epitopes within HaloTag-expressing bacteria. Permeation is then gauged with azide-bearing test molecules in live cells followingin cytostrain-promoted azide-alkyne cycloaddition (SPAAC). Overall, the Bacterial Azide Penetration Assay (BAPA) operates by semi-quantitatively measuring small molecule accumulation in a facile manner using reagents that are readily attainable, requiring basic instrumentation, and are compatible with high-throughput analysis. To this point, we have demonstrated that we can analyze the permeation of 1000+ molecules using BAPA.