Porphyromonas gingivalis, a black-pigmented, gram-negative anaerobe, is an important etiological agent of periodontal disease and is also linked to cardiovascular disease and other systemic diseases. The gingipains are considered the major factor that contributes to its pathogenicity. How these gingipains are regulated/activated however is poorly understood. The long term objective of our research program is to elucidate the molecular mechanism(s) for virulence regulation in P. gingivalis as a prerequisite to the development of novel therapeutic interventions to aid in the control and prevention of periodontal disease and other P. gingivalis-associated diseases (e.g. cardiovascular disease). The specific hypothesis to be addressed in this application is that the novel vim (virulence modulating) genes are involved in specific glycosylation modifications that are essential for gingipain maturation/activation. That hypothesis is based on the observations made in the previous funding period where we have identified three novel genes, vimA, vimE and vimF, which when inactivated inhibit the maturation/activation of the gingipains and other virulence factors of P. gingivalis. Those studies were among the first evidence of posttranslational regulation of protease activity in P. gingivalis. Further, the presence of the inactive proenzyme gingipain species which also displayed altered carbohydrate modification in the wim-defective isogenic mutants link glycosylation with gingipain maturation/activation. In this project, we wish to extend these findings and to more fully clarify the vim-dependent mechanism(s) of gingipain biogenesis.
The specific aims are designed to use our unique model system for a comprehensive assessment of gingipain biogenesis in P. gingivalis.
The specific aims are: 1. To characterize the glycosylation of the gingipains in the vim-defective isogenic mutants of P. gingivalis and correlate the effects on structure/function. We will evaluate the specific carbohydrate defects (including N-linked and O-linked sugars) in the proenzyme gingipain species of the vim-defective isogenic mutants. We will identify the specific sites for the attachment of the sugars and correlate their effects on the physical properties of the gingipain. 2. To confirm the glycosyltransferase function of VimF and to evaluate its specific role in glycosylation. Because VimF is a putative glycosyl transferase we will correlate its function with the specific carbohydrate defect observed in gingipains from the vwnF-defective mutant. This will allow us to test the hypothesis that regions of the gingipain require specific glycosylation to facilitate activation/maturation. 3. To characterize the interaction of the gingipains and the VimA protein in P. gingivalis. Our data suggest that vimA may be part of a complex that is involved in gingipain glycoslylation/activation. To test this hypothesis, we will assess the ability of VimA to interact with the gingpains by defining the kinetics of its interaction and the identification of its specific binding domain. Site-specific mutations will be performed to confirm its effect on gingipain glycosylation, level of gingipain activity and its impact on the virulence potential of the organism. ? ? ?
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