Optimal oxygen-dependent microbicidal activity of human polymorphonuclear leukocytes (PMNs) depends on quick and deliberate generation of reactive species in the phagosome. To that end, assembly and activation of the NADPH-dependent oxidase and delivery of granule contents must be precisely orchestrated both spatially and temporally. Our studies demonstrate that phagocytosis by PMNs, from initial internalization of the particle to complete fusion with the lysosomal compartment, proceeds in an orderly fashion, with components of the cytoskeleton and NADPH oxidase associating and disassociating in a precise and regulated sequence. Moreover peak production of reactive oxygen species (ROS) coincides with assembly of the oxidase at the phagosome and termination of ROS generation parallels the dissociation and/or degradation of cytosolic oxidase components at the periphagosomal space. Taken together, the elements of the PMN cytoskeleton may participate in directing, maintaining, and terminating O2-dependent microbicidal events during phagocytosis. However, currently unknown are: (1) the identity and function of cytoskeletal elements participating in oxidase localization at and assembly on the phagosome, (2) the specific contribution of the cytoskeleton to the regulation of phagosome-lysosome maturation, and (3) the mechanism(s) underlying termination of phagosomal oxidase activity. We propose to define the contributions of the submembranous cytoskeleton to the O2-dependent microbicidal system. The proposed studies test the hypotheses: (a) that cytoskeletal elements, directly or indirectly, are essential for the phagosomal localization and assembly of the NADPH oxidase; (b) that cytoskeletal proteins regulate phagosomal maturation, and (c) that structural modification of membrane and/or cytosolic oxidase components mediates ice. disassembly and inactivation. To test these hypotheses we will pursue three Specific Aims: (1) to determine the functional significance of the submembranous cytoskeleton in O2-dependent microbicidal activity; (2) to identify the mechanism(s) for disassembly and termination of NADPH oxidase activity at the phagosomal membrane; and (3) to determine the structural basis for, and functional importance of, the interaction of p57 with proteins at the phagosome. Findings from these studies will provide novel insights into the regulation of the O2-dependent microbicidal activity, including PMN oxidase as well as phagosome-lysosome fusion, within the physiologic context of phagocytosis. Fundamental principles elucidated already have been applied to studies of specific host-pathogen interactions.
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