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Abstract

Gram (-) bacteria acquire ferric siderophores through TonB-dependent transporters (TBDT) in their outer membrane. By fluorescence spectroscopic high-throughput screening (FLHTS) we identified inhibitors of TonB-dependent ferric enterobactin (FeEnt) uptake through E. coli FepA (EcoFepA). Among 165 inhibitors found in a primary screen of 17,441 compounds, we evaluated 20 candidates in secondary tests of TonB activity, including ferric siderophore uptake and colicin killing. 6 of the 20 primary hits inhibited TonB action in all the tests. Further analysis of the inhibitors in [59Fe] Ent and [14C]- lactose accumulation experiments suggested several as proton ionophores, but two chemicals, ebselen and ST0082990, did not behave like proton ionophores and may inhibit TonB-ExbBD. The success of FLHTS against E. coli led us to adapt it to the ESKAPE pathogen Acinetobacter baumannii. We identified its FepA ortholog (AbaFepA), confirmed its involvement in FeEnt uptake by deleting the structural gene, cloned AbafepA, genetically engineered 8 Cys substitutions in its surface loops, modified them with fluorescein and made fluorescence spectroscopic observations of FeEnt uptake in A. baumannii. Among the Cys substitutions in AbaFepA, several (S279C, T562C, S665C) were well labeled by fluorescein and suitable for measurements of FeEnt transport. As in E. coli, the test monitored TonB-dependent FeEnt uptake by AbaFepA. In micro titer format FLHTS with A. baumannii produced Z� factors from 0.6-0.8. Overall these experiments both identified agents that block TonB action, and revealed the potential of FLHTS for larger screens of bigger libraries to find novel antimicrobial compounds against Gram (-) bacteria, including the CRE/ESKAPE pathogens.

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