The molecular analysis of neutrophil function has brought to the field of leukocyte biology important insights into the normal mechanisms of host defense, as well as an understanding of certain dysfunctional syndromes. The goal of this proposal is to clucidate the molecular mechanisms of a component of the """"""""oxygen-independent"""""""" antimicrobial system of mammalian neutrophils, namely the microbicidal peptides of these cells. Recent studies have demonstrated that one family of neutrophil antimicrobial peptides, the defensins, possesses broad spectrum antimicrobial activity in vitro. The discovery that neutrophils also contain very potent non-defensin microbicidal peptides suggests that peptide-mediated host defense may be more prevalent than previously thought. This proposal focuses on several aspects of the structures and functions of these remarkable antimicrobial peptides. The research plan is designed to be interactive, and is comprised of four parts, each of which is defined by a specific aim: The goal os Specific Aim 1 is to dissect the structure-function relationships of members of the defensin peptide family using three complementary approaches: analysis of the natural diversity of defensin structure as it correlates with antimicrobial potency, analysis of the effects of site-specific chemical modifications, and the design and testing of congeners synthesized by solid-phase technology.
Specific Aim 2 addresses the structural and functional features of eight microbicidal peptides isolated from bovine neutrophil granules. Two of the peptides, recently characterized, are the first non-defensin antimicrobial peptides isolated from mammalian neutrophils. The remaining six peptides, which appear to be defensin-like will be characterized and sequenced. The antimicrobial functions of all eight peptides will be determined and compared with the well characterized defensin activities.
Specific Aim 3 addresses the recent observation that defensin peptides permeabilize phospholipid bilayers, supporting the hypothesis that they act at the target cell membrane. The interactions of defensin and non-defensin antimicrobial peptides with model membranes will be analyzed in experiments using planar lipid bilayers and a liposome lysis assay. The goal of Specific Aim 4 is to isolate from human neutrophils analogs of the recently discovered non-defensin peptides of bovine granulocytes. The strategy for isolating such peptides make use of some novel properties of the bovine peptides. Determination of the mechanisms of peptide-mediated host defense may provide important insight into the dynamic interactions between the host and pathogenic microbes, and may contribute to the analysis of previously uncharacterized phagocyte disorders.
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