Objective: The purpose of this investigation was to isolate the serine lipids of P. gingivalis in very high purity and verify their capacity to engage human TLR2.
Method: Total lipids were extracted from lyophilized P. gingivalis (ATCC 33277) previously grown in broth culture. The lipids were fractionated by normal phase HPLC using a semi preparative column eluted with a neutral HPLC solvent (hexane:isopropanol:water, 6/8/0.75, v/v/v). The fractions were screened for serine lipids using liquid chromatography-mass spectrometry (LC-MS) and the fractions containing serine lipids were pooled and refractionated using the same HPLC column but eluted with the HPLC solvent supplemented with 0.1% acetic acid. Fractions were evaluated for serine lipids and other contaminating lipids by LC-Multiple Reaction Monitoring (MRM) MS and the appropriate fractions were pooled and tested for biological activity using HEK293 cells stably transfected to express TLR2 and an NFkB-dependent secretory alkaline phosphatase reporter.
Result: The acidic solvent fractionation provided a substantial improvement in P. gingivalis serine lipid purity as determined by LC-MRM analysis. Contaminating phosphorylated dihydroceramide lipids were at least three orders of magnitude less abundant than the serine lipids. The highly purified serine lipids were shown to engage TLR2 as measured by HEK293 cell activation.
Conclusion: These results show that highly purified preparations of serine lipids from P. gingivalis are strong agonists for human TLR2 and should be considered potential virulence factors in pathogenesis of chronic peridodontitis.