LOOP DELETION MUTAGENESIS OF FEPA

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DESCRIPTION (Adapted from applicant's abstract): The investigators will study the transport mechanism of ferric enterobactin, a bacterial iron chelator (siderophore, through its outer membrane (GM) receptor protein, FepA. The research program will demarcate and characterize FepA surface loops by assessing the effects of their deletion on two biochemical stages of FepA physiology: binding of the siderophore on the external surface of the GM, and internalization through the GM to the periplasm. They will assay the following aspects of FepA function: 1. Specific permeability: ferric enterobactin binding and transport. 2. Non- specific permeability: accessibility of the FepA channel to antibiotics and oligosaccharides. 3. Conformation: binding of antibodies and colicins to surface loops, and maintenance of trimeric structure. 4. Surface loop exchange among the FepA proteins of Escherichia, Salmonella, and Pseudomonas. These experiments will use site-directed mutagenesis, siderophore binding and transport studies, flow cytometric measurement of surface epitopes, solid phase immunoassays, microbiological tests, and electron spin resonance spectroscopy. Iron acquisition is essential to the growth of bacteria in mammalian hosts. The investigators will study the functions of the ferric enterobactin receptor proteins from Escherichia, Salmonella, and Pseudomonas. Siderophore transport systems play an important role in the infectivity of bacteria because they concentrate iron without compromising the barrier properties of the GM, thereby enabling the survival of bacterial pathogens in mammalian hosts. The proposed research will clarify basic principles of iron transport through the gram-negative GM, and thereby advance the understanding of infectious bacterial disease. Ferric enterobactin transport through FepA is TonB-dependent. Their experiments will identify surface loops of FepA that recognize siderophores, and/or facilitate their passage through the GM. This goal has not been accomplished for any TonB-dependent receptor protein in any organism. Since gram-negative bacterial pathogens obtain iron with transport systems that are functionally similar at least in their dependence on TonB, the understanding of the FepA transport mechanism is generally relevant to efforts against bacterial pathogenesis. Besides the knowledge gained about ferric enterobactin passage through FepA, this study will generate methods for comparable investigations of other bacterial GM proteins and other organisms.