High‐throughput screening for inhibitors of iron transport in E. coli (LB238)

Abstract

Iron acquisition is a component of the pathogenesis of Gram‐negative bacteria, therefore as a form of 'nutritional immunity' host organisms sequester iron. To obtain iron bacteria secrete siderophores that scavenge iron. The E. coli outer membrane protein FepA actively transports the siderophore ferric enterobactin (FeEnt) into the periplasm. We observe this uptake reaction by fluorescently labeling FepA in live bacteria, monitoring quenching that occurs upon binding of FeEnt, and then fluorescence recovery during transport. We developed methods to screen for iron transport inhibitors using a cell‐based high‐throughput screening platform (HTS). This novel assay measures the fluorescence of bacterial cells before and after addition of FeEnt, and monitors its transport. Using this microtiter assay we differentiate between three possible events: no binding of FeEnt to FepA, binding without transport, and binding with transport into the periplasm. After optimizing the assay parameters (cell density, FeEnt concentration, and measurement timing) we found a Z' factor of ~0.7. This test allows for evaluation of libraries of compounds in 96‐well or 384‐well formats using live bacteria, in less than 1 hour.Grant Funding Source: Supported by: NIH GM53836