Abstract
Sensitive assays of biochemical specificity, affinity, and capacity are valuable both for basic research and drug discovery. We created fluorescent sensors that monitor high-affinity binding reactions and used them to study iron acquisition by ESKAPE bacteria, which are frequently responsible for antibiotic-resistant infections. By introducing site-directed Cys residues in bacterial iron transporters and modifying them with maleimide fluorophores, we generated living cells or purified proteins that bind but do not transport target compounds. These constructs sensitively detected ligand concentrations in solution, enabling accurate, real-time spectroscopic analysis of membrane transport by other cells. We assessed the efficacy of these "fluorescent decoy" (FD) sensors by characterizing active iron transport in the ESKAPE bacteria. The FD sensors monitored uptake of both ferric siderophores and hemin by the pathogens. An FD sensor for a particular ligand was universally effective in observing the uptake of that compound by all organisms we tested. We adapted the FD sensors to microtiter format, where they allow high-throughput screens for chemicals that block iron uptake, without genetic manipulations of the virulent target organisms. Hence, screening assays with FD sensors facilitate studies of mechanistic biochemistry, as well as discovery of chemicals that inhibit prokaryotic membrane transport. With appropriate design, FD sensors are potentially applicable to any pro- or eukaryotic high-affinity ligand transport process.
Highlights
Sensitive assays of biochemical specificity, affinity, and capacity are valuable both for basic research and drug discovery
We originally devised species- or transporter-specific fluorescence assays of ferric enterobactin (FeEnt) uptake for FepA of E. coli [23] and A. baumannii [25]; the same approach was effective for FepA of K. pneumoniae (Fig. 2)
The rate of fluorescence recovery in the spectroscopic system correlates to the rate of ligand depletion by bacterial transport [23], and comparisons of test strains in universal fluorescent decoy” (FD) uptake assays reiterated this point
Summary
Sensitive assays of biochemical specificity, affinity, and capacity are valuable both for basic research and drug discovery. Microbial siderophores (10 –12) capture the metal from host proteins [13], and ferric siderophores enter bacterial cells through high-affinity acquisition systems [14] The balance of these competing processes influences the outcome of infection, so bacterial iron uptake systems are potential targets for antibiotics. Such genetically engineered cells or proteins comprise “fluorescent decoy” (FD) sensors, which report ligand concentrations in solution and monitor transport of the same ligand by other cells We illustrate these applications by observations of iron transport activity through Gram-negative bacterial TBDTs and Grampositive bacterial NEAT domain– dependent hemin (Hn) uptake systems. Both high-affinity pathways function at nanomolar concentrations, and both contribute to bacterial colonization of eukaryotic hosts [10, 12]. We adapted these fluorescence tests to a microtiter high-throughput screening format to enable discovery of inhibitors that retard iron acquisition and thereby prevent bacterial growth and pathogenesis
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