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.

Agency
National Institute of Health (NIH)
Institute
National Institute of Dental & Craniofacial Research (NIDCR)
Type
Research Project (R01)
Project #
5R01DE013664-08
Application #
7774387
Study Section
Special Emphasis Panel (ZRG1-MOSS-K (02))
Program Officer
Lunsford, Dwayne
Project Start
2000-04-01
Project End
2012-02-29
Budget Start
2010-03-01
Budget End
2011-02-28
Support Year
8
Fiscal Year
2010
Total Cost
$381,082
Indirect Cost
Name
Loma Linda University
Department
Other Basic Sciences
Type
Schools of Medicine
DUNS #
009656273
City
Loma Linda
State
CA
Country
United States
Zip Code
92350
Dou, Y; Aruni, W; Muthiah, A et al. (2016) Studies of the extracytoplasmic function sigma factor PG0162 in Porphyromonas gingivalis. Mol Oral Microbiol 31:270-83
Boutrin, M-C; Yu, Y; Wang, C et al. (2016) A putative TetR regulator is involved in nitric oxide stress resistance in Porphyromonas gingivalis. Mol Oral Microbiol 31:340-53
McKenzie, R M E; Aruni, W; Johnson, N A et al. (2015) Metabolome variations in the Porphyromonas gingivalis vimA mutant during hydrogen peroxide-induced oxidative stress. Mol Oral Microbiol 30:111-27
Aruni, A Wilson; Dou, Yuetan; Mishra, Arunima et al. (2015) The Biofilm Community-Rebels with a Cause. Curr Oral Health Rep 2:48-56
Dou, Y; Robles, A; Roy, F et al. (2015) The roles of RgpB and Kgp in late onset gingipain activity in the vimA-defective mutant of Porphyromonas gingivalis W83. Mol Oral Microbiol 30:347-60
Aruni, A Wilson; Mishra, Arunima; Dou, Yuetan et al. (2015) Filifactor alocis--a new emerging periodontal pathogen. Microbes Infect 17:517-30
Aruni, W; Chioma, O; Fletcher, H M (2014) Filifactor alocis: The Newly Discovered Kid on the Block with Special Talents. J Dent Res 93:725-32
Dou, Yuetan; Aruni, Wilson; Luo, Tianlong et al. (2014) Involvement of PG2212 zinc finger protein in the regulation of oxidative stress resistance in Porphyromonas gingivalis W83. J Bacteriol 196:4057-70
Aruni, A Wilson; Zhang, Kangling; Dou, Yuetan et al. (2014) Proteome analysis of coinfection of epithelial cells with Filifactor alocis and Porphyromonas gingivalis shows modulation of pathogen and host regulatory pathways. Infect Immun 82:3261-74
Henry, Leroy G; Boutrin, Marie-Claire; Aruni, Wilson et al. (2014) Life in a Diverse Oral Community - Strategies for Oxidative Stress Survival. J Oral Biosci 56:63-71

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