37 Porphyromonas Infection Upregulates Antioxidant Response in Primary Gingival Epithelial Cells

Wednesday, March 21, 2012: 2:30 p.m. - 4 p.m.
Presentation Type: Oral Session
R. SPOONER, C. CHOI, J. DEGUZMAN, and O. YILMAZ, Periodontology, University of Florida, Gainesville, FL
Porphyromonas gingivalis infection has the ability to modulate and inhibit “danger signal” extracellular ATP (eATP)-induced Reactive-Oxygen-Species (ROS) production and the associated oxidative stress in Gingival Epithelial Cells. The major antioxidant system utilized by eukaryotic cells to combat oxidative stress is production of Glutathione (GSH), which can   directly reduce ROS. Objectives: To study GSH antioxidant response and its mechanisms in primary GECs treated w±/o  eATP during P. gingivalis infection. Methods: Fluorescent ThiolTracker Violet was used to quantify intracellular reduced GSH levels. Total cellular GSH and oxidized glutathione (GSSG) levels were measured via chemiluminescent detection. Glutamate-cysteine ligase (GCL), Glutathione synthase (GS), and Glutathione reductase (GR) mRNA expression levels were determined via real time Q-PCR. Results: ThiolTracker staining shows that 24h infected cells were able to overcome eATP-mediated loss of reduced GSH which was statistically significant at P < 0.05, t-test compared to eATP-treated uninfected cells. eATP-treated cells exhibited a reduced  GSH/GSSG ratio over the time course, while P. gingivalis-infected cells w±/o  eATP pre-treatment showed elevated GSH/GSSG ratios greater than untreated-control levels at 6h, 24h p.i. The GS mRNA levels exhibited slight elevation over the time course for eATP-treated cells, while infected cells w±/o  eATP had a more dramatic increase in mRNA at 3h p.i. returning  to untreated-control levels at later time points. The GR mRNA levels were elevated in infected cells w±/o  eATP as well as in eATP-treated cells relative to untreated-control at 6h. The GCLc and GCLm mRNA were elevated in infected cells w±/o  eATP at 6h p.i. Conclusion:   Results suggest that the GSH antioxidant pathway is likely an important mechanism for successful intracellular life of  P. gingivalis  in GECs and may contribute to observed infection-mediated inhibition of ROS and protection of host cells from eATP-mediated oxidative stress.
This abstract is based on research that was funded entirely or partially by an outside source: This research was supported by UFCD Student Summer Research Program, NIDCR-R01DE016593, R01DE019444

Keywords: Epithelium/epithelial, Host-microbial interactions, Infection and Periodontal organisms
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