A variety of stategies are used to limit the growth of oral pathogens that can lead to the development dental caries and peridontal disease. One mechanism many microorganisms use to escape control by the host is the elaboration of highly specific proteolytic enzymes which inactivate secretory immunoglobulin Al (sIgAl). Bacteria, relevant to dental disease, which produce IgAl protease include members of the genera Streptococcus, Bacteroides and Capnocytophaga. To clarify the role of Igal proteases in the growth of oral pathogens, a program which led to the development of specific inhibitors of the enzymes was initiated two years ago. In addition, gross features of sIgAl recognized by the proteases have been identified, the enzyme secreted by S. sanguis purified and the IgAl protease genes from two genera cloned and expressed. In the continuation period proposed in this application, research will focus on (1) preparation of high affinity IgAl protease inhibitors which can be used as research tools, (2) elucidation of the molecular mechanisms by which the proteases function, (3) comparison of the 1gA1 proteases produced by bacteria involved in dental caries with those associated with periodontitis and (4) studies aimed at identifying how the proteases affect function of the secretory immune system. Available IgAl protease inhibitors have IC50 values in the MuM range. The transition state inhibitor approach will be employed to increase affinity of these compounds. Stability of the protease inhibitors in the oral environment will also be tested. Mechanisms by which the proteases cleave IgAl will be studied using both standard enzymological techniques and by sequencing the DNA of the cloned enzyme. The amino acid sequence of the enzyme derived from this data may allow construction of a three dimensional model of the IgAl protease active site. Studies on the cleavage of naturally occurring IgA's lacking various domains will continue to help identify features required for inactivation of the immunoglobulin. Specific DNA probes with which to screen various oral pathogens for the existence of iga genes will be constructed. Last, a limited study of the effect of the IgAl proteinases on both the humoral and cellular immune systems will be undertaken.
