Fluorescence Sensors for Detection, Discrimination and Quantification of Siderophores

Abstract

To proliferate in the host environment during colonization or infection, bacteria acquire iron from eukaryotic tissues, fluids, cells and proteins. To do so, they secrete siderophores that chelate iron, and they express membrane transporters that recognize and internalize ferric siderophores. The production of siderophores and the acquisition of ferric siderophores are determinants of microbial pathogenesis. For example, the E. coli outer membrane protein FepA actively transports ferric enterobactin, a prototypic catecholate iron complex of the Enterobacteriaceae, whose utilization promotes colonization of the mouse gut. We genetically engineered several fluorescent sensors to detect, discriminate and quantify ferric siderophores, in purified form or in complex mixtures of metabolites and other biochemicals. By substituting single Cys residues in TonB-dependent outer membrane transporters, and modifying them with maleimide fluorophores in living cells, we created sensors that recognized different metal complexes: native, glucosylated and degraded ferric enterobactin; the hydroxamates ferric aerobactin, ferrichrome and ferrioxamine B: the porphyrins hemin and vitamin B12. In spectroscopic assays these constructs sensitively detected and quantified the different metal chelates in solution. These sensors facilitate assays of biochemical specificity, affinity, and membrane transport. They may also detect the presence and activities of bacterial pathogens, that often show a particular profile of siderophore production or ferric siderophore utilization. The sensors are useful for both basic research and drug discovery, because they rapidly detect and identify siderophores in both clinical and food samples