Fluorescence High-Throughput Screening for Inhibitors of TonB Action

Gram-negative bacteria acquire ferric siderophores through TonB-dependent outer membrane transporters (TBDT). By fluorescence spectroscopic hgh-throughput screening (FLHTS), we identified inhibitors of TonB-dependent ferric enterobactin (FeEnt) uptake throughEscherichia coli FepA (EcoFepA). Among 165 inhibitors found in a primary screen of 17,441 compounds, we evaluated 20 in secondary tests: TonB-dependent ferric siderophore uptake and colicin killing and proton motive force-dependent lactose transport. Six of 20 primary hits inhibited TonB-dependent activity in all tests. Comparison of their effects on [59 Fe]Ent and [14 C]lactose accumulation suggested several as proton ionophores, but two chemicals, ebselen and ST0082990, are likely not proton ionophores and may inhibit TonB-ExbBD. The facility of FLHTS againstE. coli led us to adapt it toAcinetobacter baumannii . We identified its FepA ortholog (AbaFepA), deleted and cloned its structural gene, genetically engineered 8 Cys substitutions in its surface loops, labeled them with fluorescein, and made fluorescence spectroscopic observations of FeEnt uptake inA. baumannii . Several Cys substitutions in AbaFepA (S279C, T562C, and S665C) were readily fluoresceinated and then suitable as sensors of FeEnt transport. As inE. coli , the test monitored TonB-dependent FeEnt uptake by AbaFepA. In microtiter format withA. baumannii , FLHTS produced Z′ factors 0.6 to 0.8. These data validated the FLHTS strategy against even distantly related Gram-negative bacterial pathogens. Overall, it discovered agents that block TonB-dependent transport and showed the potential to find compounds that act against Gram-negative CRE (carbapenem-resistantEnterobacteriaceae) /ESKAPE (Enterococcus faecium ,Staphylococcus aureus ,Klebsiella pneumoniae ,Acinetobacter baumannii ,Pseudomonas aeruginosa , andEnterobacter species) pathogens. Our results suggest that hundreds of such chemicals may exist in larger compound libraries.IMPORTANCE Antibiotic resistance in Gram-negative bacteria has spurred efforts to find novel compounds against new targets. The CRE/ESKAPE pathogens are resistant bacteria that includeAcinetobacter baumannii , a common cause of ventilator-associated pneumonia and sepsis. We performed fluorescence high-throughput screening (FLHTS) againstEscherichia coli to find inhibitors of TonB-dependent iron transport, tested them againstA. baumannii , and then adapted the FLHTS technology to allow direct screening againstA. baumannii . This methodology is expandable to other drug-resistant Gram-negative pathogens. Compounds that block TonB action may interfere with iron acquisition from eukaryotic hosts and thereby constitute bacteriostatic antibiotics that prevent microbial colonization of human and animals. The FLHTS method may identify both species-specific and broad-spectrum agents against Gram-negative bacteria.