Pseudomonas aeruginosa causes serious infections in patients whose innate defense mechanisms are compromised. These patients are typically the burned, traumatized, immunosuppressed, and cancer patients. The first steps in bacterial infection are colonization and growth. Bacterial growth in tissue and plasma occurs in the presence of an effective innate defense mechanism, nutritional immunity. Nutritional immunity depends upon transferrin to withhold essential iron from bacteria and impede growth. Most strains of P. aeruginosa are sensitive to this growth inhibition. However, there are many strains from human infections which resist nutritional immunity and grow rapidly in human plasma. A clinical survey in progress is being used to investigate whether growth in human plasma can be related to the virulence of P. aeruginosa strains. The two extracellular processes in bacterial aqcuisition of iron which would be most sensitive to inhibition are siderophore chelation of iron and ferrisiderophore binding to proteins on the bacterial surface during iron transport. The research in this proposal will elucidate the mechanisms of iron acquisition in P. aeruginosa, particularly in those strains which are resistant to nutritional immunity and grow rapidly in human plasma. Two aspects of iron metabolism will be examined during this comparison: siderophore activity and binding during transport. P. aeruginosa produces two siderophores, pyochelin and pyoverdin. High pressure liquid (HPLC) and thinlayer (TLC) chromatographic techniques will be used to analyze siderophore synthesis in addition to ferrisiderophore formation in plasma and media. Secondly, electrophoresis (SDS-PAGE) and chromatography on high performance protein columns will be used to analyze ferrisiderophore and ferritransferrin binding activities on bacterial surfaces. These techniques are being used to attain the long-term goal of supporting nutritional immunity during infections of patients by inhibiting bacterial growth. Currently we are searching for siderophore analogues which inhibit bacterial growth in plasma and in mice.
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