Demonstration of Porphyromonas gingivalis Phosphorylated Dihydroceramides at Periodontitis Disease Sites

The structures of novel phosphorylated dihydroceramides of Porphyromonas gingivalis were recently defined and the demonstrated capacity of these lipids to stimulate prostaglandin secretory responses and changes in fibroblast morphology indicate their potential to act as host virulence factors. Objectives: The present investigation sought to determine whether these phosphoceramide lipids of P. gingivalis are present on teeth contaminated with subgingival calculus and/or whether these lipids are recovered from chronic periodontitis gingival tissue samples. Methods: Lipids were extracted from teeth contaminated with gross deposits of subgingival calculus or were extracted from pooled gingival tissue samples excised from chronic severe periodontitis sites. A sample of each lipid extract was treated to form volatile trimethylsilyl derivatives and subjected to selected ion monitoring using electron impact GC-mass spectrometry (MS) analysis. The total lipid extract of calculus-contaminated teeth was evaluated using negative ion electrospray-MS analysis. The periodontitis tissue lipid extract was treated with sodium methoxide followed by HPLC fractionation and electrospray-MS analysis. Results: GC-MS analysis revealed that teeth layered with gross subgingival calculus contain both phosphoethanolamine and phosphoglycerol dihydroceramides of P. gingivalis. Electrospray-MS analysis confirmed that phosphoglycerol dihydroceramides of P. gingivalis are prevalent in lipid extracts of diseased teeth and are detected in diseased gingival tissues. However, only the dominant phosphoglycerol dihydroceramide of P. gingivalis was detected in gingival tissue lipids by GC-MS analysis. By contrast, the phosphoethanolamine dihydroceramides that are prominent products of P. gingivalis were not observed in lipid extracts of diseased teeth using electrospray-MS analysis. Conclusions: This study confirms that P. gingivalis lipids are present on calculus-laden teeth and are present in diseased periodontal tissues. By demonstrating these bacterial lipid products on diseased teeth and in diseased gingival tissue samples, this study provides direct evidence implicating these substances as virulence factors in the pathogenesis of chronic periodontitis.