The role of collagenases produced by Bacteroides gingivalis in the etiology of bacterial infections will be examined utilizing biochemical, immunological, and genetic techniques. The enzyme produced by a human oral strain of B. gingivalis will be initially purified and characterized. The properties of the enzyme will be examined relative to its possible role in the initiation of periodontal lesions. In addition, antibodies produced against the purified enzyme will be utilized to compare the enzyme with collagenases produced by other oral bacteria. The gene coding for collagenase activity will be isolated in a lambda phage cloning system. The gene will be subjected to sequence analysis in order to selectively mutagenize the gene. Protein domains essential for activity will be determined following deletion analysis and immunological methods. This information will be utilized to carry out site-directed mutagenesis of the collagenase gene. In this manner it should be possible to determine the mechanism of action of the enzyme at the sequence level. Gene probes will also be constructed from the cloned gene in order to examine evolutionary relatedness between organisms expressing collagenase activity as well as serving as a convenient means of detecting B. gingivalis in clinical samples.
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