This proposal concerns the role and regulation of defined phospholipases during phagocytosis by PMN and during the antibacterial action of potent membrane-active proteins present in inflammatory fluids or purified in this laboratory from polymorphonuclear leukocytes (PMN).
Our specific aims are to examine: (1) The molecular determinants of the action of selective phospholipases A2 (PLA2) (e.g. PMN, inflammatory fluid) against phospholipase A-less mutants of Escherichia coli and the structural basis of the functional diversity of this conserved family of 14 kDa PLA2; (2) The regulation of bacterial (phospho)lipid degradation during phagocytosis and its role in intracellular bacterial killing and disassembly; and (3) The nature and possible functional consequences of the interaction of extracellular PLA2 with PMN or other """"""""inflammatory"""""""" host cells.
These aims have their origin in our earlier work and their pursuit will therefore rest heavily on well-tested methods used in this laboratory, including: collection of PMN and inflammatory exudates; functional assays of PMN (protein)-bacteria interactions such as phagocytosis, bacterial killing, bacterial phospholipid degradation and biosynthesis; purification of proteins, including PLA2; construction and expression of recombinant PLA2 variants; and construction and use of bacterial mutants. The long-term objectives of this proposal concern two fundamental questions: (l) What regulates the action of defined endogenous and exogenous phospholipases on the phospholipids of natural membranes? (2) What determines the extent of digestion and disassembly of bacteria killed by phagocytes? The proposed studies are likely to provide new insights related to the function of host defenses in infection and of membranes in general.
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