Defensins are small, cysteine-rich, antimicrobial peptides (Mr 3,500- 4,000). They are among the principal constituents of mammalian neutrophils (PMN) and certain mammalian macrophages and also appear to be ancestral members of the immune system. Recently, Ouellette and his associates cloned an abundant mRNA species from murine small intestine, and found that it coded for a peptide(""""""""cryptdin"""""""") whose amino aid sequence identified it as a typical defensin. Although cryptdin mRNA was virtually absent at birth, it was heavily expressed by epithelial cells at the base of intestinal crypts (Paneth cells) in 20d old mice. In older mice, mononuclear cells in the small intestine's lamina propria also contained mRNA that hybridized with cryptdin cDNA. It is known that certain defensins obtained from myeloid cells can distinguish between mouse- virulent and mouse avirulent strains of S. typhimurium. Although the functions of Paneth cells in man and animals have long been an enigma, the observation that defensin mRNA is abundantly expressed by these long-lived, secretory and phagocytic cells of the small intestine suggests that they may regulate the small intestine's microbial flora and defend against invasion by microbes. We will isolate cryptdin and other small-intestinal defensins from rodents, determine their amino acid sequences, and examine their antimicrobial activities against phoP-positive (virulent) and phoP- negative (avirulent) isogenic strains of S. typhimurium and selected other microorganisms. Defensins expressed within rodent PMN. will serve as controls in these studies. """"""""Cryptdin"""""""" and selected natural or designed congeners will be produced by solid-phase chemical synthesis and used to study defensin congeners will be produced by solid-phase chemical synthesis and used to study defensin structure, function and mechanism(s). Enteric and myeloid defensins will be cloned to enable us to examine their tissue- specific expression, and to identify novel defensins for potential solid- phase synthesis. Benefits that may accrue from this work include new insights into: 1) the functions of Paneth cells, 2) the antimicrobial defenses of the gastrointestinal tract, 3) the mechanisms responsible for neonatal susceptibility to enteric infections 4) The distribution and tissue-specific expression of defensins and 5) the mechanisms of defensin- mediated antimicrobial activity. If Paneth cells prove to be the small- intestines's principal guardians against microorganisms, this work may also lead to increased understanding of gastrointestinal inflammation and infection in humans, and to the development of therapeutic antibiotics specifically designed to work in the small intestine.
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