The long range goal of this project is to systematically study the structural and molecular biology of """"""""defensins and bactenecins"""""""", the antimicrobial cationic polypeptides present in the granules of neutrophils. These studies will help identify, design, develop and evaluate a new class of nontoxic antibiotics to be used to defend against periodontal pathogens including Actinobacillus actinomycetemcomitans and Porphyromonas gingivalis as well as opportunistic pathogens such as Candida albicans. Defensins and bactenecins are delivered to vacuoles containing the ingested microorganisms during phagocytosis and are released into the extracellular fluids when neutrophils are stimulated by secretagogues. They interact with the membranes of microbes, alter membrane permeability and ionic gradient, and further cause impairment of the function of the respiratory chain and other energy dependent activities in the inner membranes. They play a major role in host defense, inflammation and restoration of the altered homeostatic condition by their ability to kill invading microbes. This suggests a rationale for selection of these molecules for our present studies and the importance of these molecules in controlling oral infections by these organisms including periodontal disease. The highly conserved disulfides, glycines and the charged residues in defensins, and the repeating Pro-Arg-Pro triplets spaced by a single hydrophobic residue in bactenecins, provide rigid structures for these molecules to overcome the adaptive microbial resistance to organic antibiotics. These unique conserved structural elements also suggest that their microbicidal functions are indeed dependent on a specific structural feature and a particular charge distribution. The structural diversity observed in the primary sequence of these polypeptides is reflected in wide variations in their biological activity. This functional diversity is clearly a consequence of subtle variations in the size, sequence, fine structural motifs and side-chain topography of these molecules. This project involves synthesis of defensins, bactenecins and peptide analogs by both chemical and genetic engineering techniques, structure determination by spectroscopic methods (NMR, FTIR, CD), computer modeling and crystal structure analysis, and assessment of in vitro cidal activity of these molecules. The information to be obtained from structure-function analyses will permit the design and synthesis of stereochemically constrained peptide analogs of defensins and bactenecins which could elicit enhanced and prolonged activity. These new oral antibiotics may be useful for the control and treatment of periodontal disease and other oral infections. In addition, the molecular biology studies would delineate the cDNA and the recombinant DNA encoding the sequences of the active analogs and lead to gene therapy approaches for similar clinical applications.
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