The neutral serine proteases (NSP) of neutrophils (PMN) are among the most potent host defense molecules active against periodontal pathogens in vitro. We already know (i) that NSP kill periodontal bacterial by two distinct mechanisms, (ii) that periodontal bacteria are differentially sensitive to distinct domains of NSP, and (iii) that certain NSP, such as elastase and azurocidin, interact to exert microbicidal effect against periodontal bacteria. We now hypothesize that NSP interact to expose internal domains and activate latent proantibiotic molecules and the defects in this dynamic, phagocyte-based protein processing system may contribute to the pathogen-specific, sluggish killing which has been observed in localized juvenile periodontitis (LJP). Our specific research aims are:
Aim 1 --to identify the direct and interactive bactericidal activities of the NSP against a panel of significant periodontal bacteria including strains of Porphyromonas gingivalis, Fusobacterium nucleoatum, Prevotella intermedia, Actinobacillus actinomycetemcomitans, Capnocytophaga sputigena and Eikenella corredens:
Aim 2 --to examine postphagocytic protein processing in normal PMN which have ingested periodontal pathogens;
Aim 3 --to delineate the antibiotic domains of two NSP (cathepsin G and elastase) which are active against periodontal pathogens;
and Aim 4 --to compare the activities of neutrophils from healthy, normal individuals with those from individuals with early onset forms of periodontitis (including LJP) and adult periodontitis with respect to their ability to activate latent proantibiotic domains of NSP. To accomplish Aim 1, we will assess the sensitivity of a panel of periodontal bacteria to PMN granule fractions and purified NSP, determine the role of enzyme-dependent mechanisms, and characterize the interactions among NSP which result in the killing of each panel microbe. To accomplish Aim 2, we will monitor postphagocytic protein/NSP processing by NSP in normal human PMN that have ingested panel bacterial under aerobic and anaerobic conditions, using HPLC, electrophoresis, and determinations of antimicrobial activities using the radial diffusion and overlay agar methods. To accomplish Aim 3, we will use synthetic peptides to identify the antibiotic domains of two representative NSP (elastase, a serprocidin; and cathepsin B, a granzyme B-related protein) active against pathogens. To accomplish Aim 4, we will compare postphagocytic protein processing in neutrophils from healthy individuals and individuals with forms of periodontitis. These studies will help define how normal host leukocytes control periodontal pathogens and will point to novel approaches for predicting and preventing periodontal disease. In addition, they could lead to the development of novel antibiotic peptides, based upon naturally-occurring host defense molecules, for selective control periodontal organisms in the future.
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