This proposal concerns the role and regulation of the action of mammalian Group IIA phospholipase A2 (PLA2) during inflammation and the determinants of the degradation of Gram-negative bacterial lipopolysaccharides (LPS) during host antibacterial action.
The specific aims are to examine: (1) the molecular determinants of the antibacterial actions of mammalian Group IIA PLA2; (2) the regulation and role of PLA2 antibacterial action at inflammatory sites; (3) the cellular and molecular determinants of LPS degradation during intraphagocytic and extracellular antibacterial action; (4) the molecular determinants and possible physiologic role of Group IIA PLA2 action against host cell-derived membrane phospholipids (PL).
These aims have their origin in the applicant's earlier work and their pursuit will therefore rest heavily on well-tested methods used in his laboratory including: collection of phagocytes and inflammatory exudates from experimental animals; in vitro and ex vivo functional assays of phagocyte (or cell-free protein)-bacteria interactions such as phagocytosis, bacterial killing, and (bacterial) PL and LPS degradation; expression and purification of recombinant proteins including PLA2 variants; and construction and use of bacterial mutants. Planned studies will also include assessment of PLA2 function in vivo to further establish the contribution of this enzyme to innate host antibacterial defenses. The long-term objectives of this proposal concern two fundamental questions: (1) What determines the extent of bacterial digestion and disassembly during host antibacterial action? (2) What regulates the action of defined phospholipases on the PL of biological membranes? The proposed studies are likely to provide new insights related to the mechanisms of host defenses in infection and of the function of membranes in general.
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