The goal of the proposed studies is to understand mechanisms of mucosal defense by characterizing antibacterial peptide components of mouse Paneth cells and their effects on pathogenic microorganisms. Cryptdin is one of several Paneth cell defensins, and cryptdin and other enteric defensins are hypothesized to constitute a family of antimicrobial peptides produced by these epithelial granulocytes to protect against invasion and colonization of the small bowel by bacteria. Experiments are proposed to isolate cryptdin and intestinal defensins from small bowel and determine their antibacterial activities in vitro, to characterize enteric defensin genes in Paneth cells, and to define genetic loci in Salmonella typhimurium that confer resistance to cryptdin and enteric defensins. Specifically, one series of objectives is to purify cellular and secreted forms of intestinal defensins from mouse small bowel and determine their peptide sequences, to characterize natural and synthetic intestinal defensins with regard to antimicrobial activity against virulent and avirulent strains of enteric pathogens, and to determine their cellular, sub cellular, and extracellular distribution using light and electron microscopic immunohistochemical techniques. Secondly, cDNAs corresponding to enteric defensins will be isolated and characterized, the intestinal cells that express such sequence will be identified by in situ hybridization, and the structure and organization of the murine cryptdin gene family will be defined. Thirdly, the relationship between resistance to enteric defensins and Salmonella virulence will be investigated by analyzing genetic loci of S. typhimurium that confer resistance to cryptdins, by identifying S. typhimurium phoP mutants with suppressor mutations of the cryptdin sensitive phenotype and determining the genetic basis of the reversion, by characterizing bacterial genes and proteins that confer resistance to cryptdins, and by constructing S. typhimurium strains with mutations in genes essential to cryptdin resistance. By delineating the role of enteric defensins in the mucosal barrier, the factors that regulate their expression, and specific mechanisms of resistance to cryptdins, a component of mucosal host defense will have been characterized at the level of the peptide effectors, the genes encoding the peptides, and the molecular targets of the peptides in the microorganism. Completion of these aims should provide insight into molecular mechanisms of intestinal defense against enteric bacterial pathogens.
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