Recent evidence shows that the polymorphonuclear leukocyte (PMNL) protects the periodontium against periodontal bacteria. Severe periodontal disease is observed in young patients with chronic granulomatous disease, a condition in which oxygen-dependent killing mechanisms of PMNL are impaired. This clinical observation suggests that oxidative bactericidal mechanisms of PMNL are of key importance, and that compensation serum cidal and non-oxidative killing mechanisms of PMNL is inadequate. Myeloperoxidase is believed to be one of the most important mediators of oxidative killing, greatly potentiating the cidal activity of hydrogen peroxide by adsorbing to the bacterial surface and forming destructive compounds from hydrogen peroxide and halide. We propose to examine the killing of oral bacteria by hydrogen peroxide and myeloperoxidase. Specifically, we hypothesize that the adsorption of myeloperoxidase to bacteria may vary for different bacteria, thus imbue myeloperoxidase-mediated killing with selectivity. To study the relationship between myeloperoxidase-mediated killing and myeloperoxidase adsorption to bacteria, the susceptibility of a variety of oral bacteria to killing by myeloperoxidase will be determined using either standard plating techniques or a killing assay based on the uptake of acridine orange. Bacterial catalase activity will be quantified with a Clark-type oxygen electrode. Bacterial species examined will include strains from the genera Bacteroides, Fusobacterium, Capnocytophaga, Selenomonas, Wolinella, Haemophilus, Eubacteria, Actinobacillus, Actinobacillus, Actinomyces and some oral streptococci. The adsorption of myeloperoxidase from the suspending media will be screened initially using crossed-immunoelectrophoresis or SDS-PAGE. We will examine the adsorption of purified myeloperoxidase to bacteria at equilibrium using a direct binding assay with 125I myeloperoxidase or a radioimmunoassay in which bound myeloperoxidase is detected with rabbit 125I anti-myeloperoxidase. Bound myeloperoxidase will be visualized by electron microscopy and peroxidase staining with tetramethylbenzidine. Binding will be quantified by the Scatchard analysis, and apparent Kd and binding capacity determined. We will attempt to correlate a low Kd or a high binding capacity with increased susceptibility to killing by myeloperoxidase. Finally, we will examine the nature of the binding reaction usin pH, ionic strength, polyelectrolytes, detergents, glycosidases, and lectins.
