The application deals with IgA proteases, extracellular bacterial proteolytic enzymes that cleave and inactivate human mucosal and salivary IgAl antibody proteins. IgA proteases of various human pathogenic bacteria are all post-proline cleaving and have very high, possibly unique specificity for human IgA heavy chain as substrate. Bacteria making this enzyme include oral streptococci e.g. S. sanguis that colonizes human dental enamel to form plaque, a complex, adherent microbial aggregate (continaing abundant protease activity) that leads to caries. We will test the general hypothesis that during their extracellular secretion the IgA proteases of dividing bacteria interact with mucosal or salivary IgA anti-protease antibodies in a way that fosters, and regulates, bacterial aggregation during the complex process of colonization of host mucosal tissues and the proteinaceous pellicle on dental enamel. We will test the biological role of these proteases by growing recombinant streptococcal strains in human saliva bathing hydroxyapatite similar to that on natural teeth. We have recently cloned and sequenced the entire S. sanguis iqa gene that encodes IgA protease, and it will be analyzed using gene fusions and related recombinant techniques. This is to establish the role of a long tandem repeat that has been found in the enzyme protein structure and to localize its human IgA-binding, zinc-binding and antigenic domains, and to understand the genetic and biochemical relationship of streptococcal to other IgA proteases of Gram negative pathogens. We also will design, synthesize, and characterize by NMR spectroscopy peptide boronic acid-type and other potent inhibitors of the streptococcal and oral Bacteroides enzymes similar to those we recently developed for the serine-type IgA proteases of Neisserea and Hemophilus pathogens. These will be used to probe the role of the enzymes in colonization of dental enamel, with a view to drugs that may limit this process. Such compounds will also serve as guides to synthesis of effective substrates for the metallo-enzyme-type IgA proteases like those of S. sanguis, a longstanding and unsolved problem that has blocked study of their role in pathogenesis.
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