Possible role of bacterial siderophores in inflammation. Iron bound to the Pseudomonas siderophore pyochelin can function as a hydroxyl radical catalyst

Abstract

Tissue injury has been linked to neutrophil associated hydroxyl radical (.OH) generation, a process that requires an exogenous transition metal catalyst such as iron. In vivo most iron is bound in a noncatalytic form. To obtain iron required for growth, many bacteria secrete iron chelators (siderophores). Since Pseudomonas aeruginosa infections are associated with considerable tissue destruction, we examined whether iron bound to the Pseudomonas siderophores pyochelin (PCH) and pyoverdin (PVD) could act as .OH catalysts. Purified PCH and PVD were iron loaded (Fe-PCH, Fe-PVD) and added to a hypoxanthine/xanthine oxidase superoxide- (.O2-) and hydrogen peroxide (H2O2)-generating system. Evidence for .OH generation was then sought using two different spin-trapping agents (5.5 dimethyl-pyrroline-1-oxide or N-t-butyl-alpha-phenylnitrone), as well as the deoxyribose oxidation assay. Regardless of methodology, .OH generation was detected in the presence of Fe-PCH but not Fe-PVD. Inhibition of the process by catalase and/or SOD suggested .OH formation with Fe-PCH occurred via the Haber-Weiss reaction. Similar results were obtained when stimulated neutrophils were used as the source of .O2- and H2O2. Addition of Fe-PCH but not Fe-PVD to stimulated neutrophils yielded .OH as detected by the above assay systems. Since PCH and PVD bind ferric (Fe3+) but not ferrous (Fe2+) iron, .OH catalysis with Fe-PCH would likely involve .O2(-)-mediated reduction of Fe3+ to Fe2+ with subsequent release of "free" Fe2+. This was confirmed by measuring formation of the Fe2(+)-ferrozine complex after exposure of Fe-PCH, but not Fe-PVD, to enzymatically generated .O2-. These data show that Fe-PCH, but not Fe-PVD, is capable of catalyzing generation of .OH. Such a process could represent as yet another mechanism of tissue injury at sites of infection with P. aeruginosa.