Date of Completion

6-18-2014

Embargo Period

6-13-2014

Advisors

Dr. Qiang Zhu, Dr. Kamran Safavi

Field of Study

Dental Science

Degree

Master of Science

Open Access

Open Access

Abstract

Porphymonas endodontalis has been shown to populate necrotic root canal systems and may contribute to apical bone loss. Recent work has shown that P. endodontalis synthesizes a novel serine lipid class (Lipid 654) similar to that produced by P. gingivalis. Objective: The purpose of this study was to isolate P. endodontalis Lipid 654 in very high purity and characterize its structure and biological activity
 in RAW 264.7 cells and HEK293 cells. Methods: P. endodontalis (ATCC 35406, type strain) was grown under anaerobic conditions and centrifuged. P. endodontalis total lipids were recovered using the Bligh and Dyer phospholipid extraction procedure and were fractionated by semipreparative normal phase HPLC using hexane-isopropanol-water (6:8:0.75, vol/vol/vol). Fractions containing Lipid 654 were identified by mass spectrometric (MS) analysis and were pooled, refractionated using an acidic HPLC solvent, and tested for biological activity. Results: MS analysis revealed that repurified Lipid 654 contained P. endodontalis and P. gingivalis Lipid 654 preparations and was significantly attenuated by anti-mouse TLR2 neutralizing antibody. Treatment of HEK293 cells, stably transfected to express TLR2 and an NFκB-dependent secretory alkaline phosphatase reporter, revealed that both P. endodontalis and P. gingivalis Lipid 654 preparations engage human TLR2 with equal potency. Conclusions: Our results show that P. endodontalis Lipid 654 engages TLR2 with stimulation of TNF-α secretion from macrophages and activation of the transcription factor NF-κB, and these responses may contribute to apical bone destruction associated with necrotic pulps.

Major Advisor

Dr. Frank Nichols

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