The long-term objectives of this research are to elucidate molecular mechanisms of attachment for important oral bacteria to teeth, and to develop approaches for modulating attachment and colonization of prominent periodontopathogens in the oral cavity.
The Specific Aims of the proposed project: (1) identify, isolate and characterize the adhesion associated with Actinomyces type 1 fimbriae which interacts with proline-rich proteins thought to be receptors in the experimental salivary pellicle; (2) establish relevance of the adhesion on attachment to experimental tooth surfaces and natural teeth in vivo; (3) establish functional relevance (i.e., adherence inhibition activity (ALA), and modulation of infection in vivo) of genetically regulated variations in specificity of serum antibodies from inbred mice immunized with A.viscosus T14V, and investigate the possibility that AIA is genetically regulated in humans. Data obtained from this project will hopefully serve as a model for future studies investigating molecular mechanisms of attachment and colonization of other oral microorganisms associated with periodontal diseases in humans. In addition, if AIA has functional relevance regarding actinomyces colonization and is genetically regulated in humans, it is hoped that principles and methods developed to establish this association can be applied to prominent periodontopathogens as well. These studies should lead to development of better approaches for preventing colonization of periodontopathogens by immune modulation. To identify the adhesion, antibody-mediated adsorption inhibition will be studied in an in vitro hydroxyapatite-bacterial adsorption assay; and biochemical (Fast Protein Liquid Chromatography, differential and sucrose- gradient centrifugation, etc.) and immunologic (affinity-chromatography using polyclonal or monoclonal anti-adhesion antibodies) methods will be used for the purification of the adhesion. Molecular mechanisms established in vitro will be confirmed in humans where possible by studying the adsorption of antibiotic-resistant fimbrial-deficient and adherence- defective mutants of A.viscosus T14V to enamel slabs cut from extracted 3rd molars. The functional relevance (i.e., AIA) of genetically regulated variations in the specificity of serum antibodies from inbred mice will be evaluated by testing various spectrotypes and idiotypes of antibodies purified from sera of immunized mice using isoelec-tric focusing and idiotype-specific ELISAs, in AIA assays. Preliminary experiments will also investigate the possibility that AIA is genetically regulated in humans by segregation and linkage analysis of AIA using sera from a group of families who have been previously typed for HLA on chromosome 5 and immunoglobulin allotype on chromosome 11. To establish whether genetic regulation of AIA influences oral colonization, inbred mice which produce high or low levels of AIA will be immunized with purified type 1 fimbriae-associated adhesion and challenged with strain T14V to determine if AIA levels correlate with the level of T14V colonization on teeth.
