The ability of neutrophils kill ingested microorganisms is essential for host defense against microbial infection. Our long term goal is to identify the endogenous antibiotic peptides of human neutrophils. The present application has six specific aims. l) To purify """"""""granulins"""""""" from human neutrophils, test their antimicrobial, growth-regulatory and cytotoxic properties, and determine their subcellular localization and their mode of release. 2) To determine if human neutrophils contain antimicrobial members of the """"""""four-disulphide-core family"""""""" of basic proteins. 3) To establish the prevalence of neutrophil defensin deficiency in inbred strains of mice and to purify and identify the non- defensin antimicrobial proteins of murine neutrophils. 4) To examine selected properties of human defensin HNP-4 relevant to its antimicrobial, cytotoxic and corticostatic functions. 5) To express human defensins in otherwise defensin-deficient murine phagocytes and to determine the consequences for antimicrobial activity against H. capsulatum and C. albicans. 6) To characterize the binding of human defensins to bacteria and human mononuclear cells, and to study the uptake of exogenous defensins by human monocytes and macrophages and determine its consequences for antimicrobial activity against H. capsulatum and C. albicans. The methods used in Specific Aims 1-4 involve cellular and subcellular fractionations, analytical and preparative protein chemistry and various microbiological assays.
Specific Aim 5 combines molecular biology and microbiology and will use recently constructed murine cell lines that express and process the human defensin HNP-1.
Specific Aim 6 will combine immunoelectron microscopy with measurements of binding and uptake of defensins and quantitative measurements of macrophage-mediated antimicrobial activity. Overall, these studies will define the role of defensins and other cysteine-rich antimicrobial peptides (granulins and four-disulphide-core congeners) in the antimicrobial activity of human neutrophils and assess the potential of defensins to control infections caused by organisms that replicate within macrophages. The work with murine neutrophils, whose content of endogenous antimicrobial polypeptides differs greatly from those of humans, will provide essential information relevant to the interpretation of murine models of human infection.
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