Free Lipid A Isolated from Porphyromonas gingivalis Lipopolysaccharide Is Contaminated with Phosphorylated Dihydroceramide Lipids: Recovery in Diseased Dental Samples

Abstract

ABSTRACTRecent reports indicate thatPorphyromonas gingivalismediates alveolar bone loss or osteoclast modulation through engagement of Toll-like receptor 2 (TLR2), though the factors responsible for TLR2 engagement have yet to be determined. Lipopolysaccharide (LPS) and lipid A, lipoprotein, fimbriae, and phosphorylated dihydroceramides ofP. gingivalishave been reported to activate host cell responses through engagement of TLR2. LPS and lipid A are the most controversial in this regard because conflicting evidence has been reported concerning the capacity ofP. gingivalisLPS or lipid A to engage TLR2 versus TLR4. In the present study, we first preparedP. gingivalisLPS by the Tri-Reagent method and evaluated this isolate for contamination with phosphorylated dihydroceramide lipids. Next, the lipid A prepared from this LPS was evaluated for the presence of phosphorylated dihydroceramide lipids. Finally, we characterized the lipid A by the matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) and electrospray-MS methods in order to quantify recovery of lipid A in lipid extracts from diseased teeth or subgingival plaque samples. Our results demonstrate that both the LPS and lipid A derived fromP. gingivalisare contaminated with phosphorylated dihydroceramide lipids. Furthermore, the lipid extracts derived from diseased teeth or subgingival plaque do not contain free lipid A constituents ofP. gingivalisbut contain substantial amounts of phosphorylated dihydroceramide lipids. Therefore, the free lipid A ofP. gingivalisis not present in measurable levels at periodontal disease sites. Our results also suggest that the TLR2 activation of host tissues attributed to LPS and lipid A ofP. gingivaliscould actually be mediated by phosphorylated dihydroceramides.