The goal is this research is to understand how Pseudomonas aeruginosa obtains extracellular iron to support growth during human infections. Our long-term goal is to use this understanding toward the construction of growth-inhibiting or bactericidal compounds. To be a successful pathogen, P. aeruginosa must mobilize iron from unavailable mammalian iron reserves, serum transferrin and secretory lactoferrin into forms which are available to bacterial metabolism. This bacterium depends upon three extracellular products to mobilize unavailable iron. Pyocyanin is a reducing agent which can catalyze the removal of Fe(II) from transferrin. Pyochelin and pyoverdin (siderophores) are iron binding compounds which supply iron in utilizable forms to high affinity transport systems. Pyoverdin appears to be the most effective siderophore. The first specific aim of this proposal will be to follow the fates of both iron and pyoverdin during transport using radiolabelling studies and to compare these transport mechanisms with those used for ferripyochelin. The concerted activities of all three extracellular products will be investigated during transport assays. High pressure liquid chromatography (HPLC) and electrophoresis will be used in conjunction with transport assays to determine the participation of the bacterial surface and of proteolytic enzymes in iron mobilization from transferrin. The biosynthesis of pyoverdin will be investigated by both amino acid analysis of pigments isolated by isoelectric focusing and HPLC, and by labelling the forms of pyoverdin with radioactive amino acids. Mutants which produce abbreviated segments of the peptide portion of the siderophore will be fed radiolabelled amino acids and their products will be analyzed to yield a sequential perspective of peptide formation. Completion of pigment synthesis will be studied by following the incorporation of (14C)shikimate into the quinoline derivatives and finally into the dihydroxyquinoline of pyoverdin. NMR and mass spectroscopy will be employed to analyze possible alteration in the dihydroxyquinoline moiety of pyoverdin. This information and these compounds will be used in our research for analogues of siderophores and precursors which inhibit bacterial iron acquisition.
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