Periodontal disease is present in almost all individuals with teeth and is the major cause of loss of teeth after forty. Bacteriodes gingivalis has been implicated as an etiologic agent of chronic periodontitis, which is the common form of periodontal disease in adults. As yet, it is not known what factor or factors render this organism virulent and how these factors contribute in the pathogenesis of disease. Understanding of virulence factors in periodontopathic organisms is important for the development of further methods for controlling the disease process. B. gingivalis produces a collagenase and it is thought that proteolytic enzymes play a role in the pathogenesis of a variety of disease processes. This proposal will investigate the role of B. gingivalis collagenase in the production of disease in the mouse abscess model. The following specific aims are proposed. 1. preparation of monoclonal antibody to B. gingivalis collagenase. Microbial collagenase will be partially purified by inhibitor affinity chromatography and FPLC prior to immunization of the mice. Immunodot techniques will be used to select positive clones. Transblot and zymogram techniques will identify the specificity of monoclonal antibodies. 2. purification and characterization of B. gingivalis collagenase. The enzyme will be purified using affinity chromatography with anti-collagenase monoclonal antibodies and other techniques. The enzyme will be characterized as to substrate specificity and sites of cleavage, response to inhibitors as well as other characteristics. 3. demonstration of collagenase in vivo. B. gingivalis will be used to induce abscesses in mice and the aspirated abscess will be examined for microbial collagenase production by immunofluorescent methods using monoclonal antibodies. Antibodies to collagenase will be identified in serum using tranbslot and zymogram methods. 4. studies of the effects of B. gingivalis collagenase on the disease process. Collagenase negative mutants will be produced and compared with collagenase positive B. gingivalis organisms in a mouse abscess model. The protection from abscess formation by active immunization with collagenase and passive immunization with monoclonal antibodies will be investigated.
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