Discipline: Chemistry and Chemical Sciences
Subcategory: Cell and Molecular Biology
Room: Exhibit Hall A
Caleb McIver - Oakwood University
Co-Author(s): Dr. Elaine Vanterpool, Oakwood University, Huntsville, AL
Porphyromonas gingivalis W83, a periodontal pathogen, is linked to systemic inflammatory disorders and cardiovascular events. P. gingivalis produces powerful proteases that can damage host tissues and promote apoptosis. Previous studies show that inactivation of the virulence modulation gene (vimF), a putative glycosyltransferase gene, resulted in decreased virulence factor activation. The impact of the vimF-defective mutant (FLL95) on human cells has not been evaluated. The purpose of this study is to further evaluate the mechanisms of P. gingivalis-induced apoptosis and how vimF may impact these mechanisms. It is hypothesized that that vimF is essential for P. gingivalis to induce apoptosis in host cells. To test this hypothesis, wild-type P gingivalis W83 and isogenic-defective mutant FLL95 (vimF-defective mutant) strains were grown in BHI broth. Membrane proteins were extracted using french press methods and extracellular proteins were acetone precipitated. P. gingivalis proteins were then incubated with Human Umbilical Vein Endothelial Cells (HUVEC) for up to 24 hours (as well as the untreated control). HUVEC morphology was evaluated using the FLoid cell imager and cell signaling mechanisms using Toll-like receptor 2 (TLR-2, immunomodulatory receptor protein), ryanodine receptor 1 (RyR1, calcium research channel on the ER membrane) and c-fos (a proto-oncogene that can regulate cell proliferation, differentiation and transformation and may change expression in response to cell damage) expression were quantified using ELISA. Results show that the vimF-defective mutant does not induce visible apoptotic morphology in HUVEC cells comparison to HUVEC incubated with W83. All data were normalized to internal control GAPDH. Changes in protein expression are observed for c-fos, RyR1 and TLR2. HUVEC cells incubated with W83 show no detectable change in RyR1 expression however more than a 20% downregulation is seen in the presence of FLL95 after 6 hours of incubation and 50% decrease after 24 hours. P. gingivalis W83 proteins incubated with HUVEC causes an upregulation (188%) in c-fos expression at 24 hours where FLL95 causes a downregulation by 68%. TLR2 expression is upregulated (171%) by W83 and downregulated by FLL95 (76%) after 24 hours. Collectively, the data of this study supports our hypothesis that vimF is essential for P. gingivalis to induced apoptosis and cell damage. We will continue to further investigate the mechanisms of vimE in virulence modulation of P. gingivalis. Data from these findings can be used to prevent or neutralize the damaging effects of P. gingivalis-induced infections. Vanterpool E, Roy F, Fletcher HM. Inactivation of vimF, a putative glycosyltransferase gene downstream of vimE, alters glycosylation and activation of the gingipains in Porphyromonas gingivalis W83.?Infect Immun. 2005 Jul;73(7):3971-82. doi: 10.1128/IAI.73.7.3971-3982.
Funder Acknowledgement(s): This study was supported by a grant from NSF/HBCU-UP and UNCF McBay awarded to Elaine Vanterpool PhD, chairperson for the Biology Department, Oakwood University, Huntsville, AL.
Faculty Advisor: Dr. Elaine Vanterpool, firstname.lastname@example.org
Role: Experimentation and analysis.