Calcium hydroxide inactivates lipoteichoic acid from enterococcus faecalis through deacylation of the lipid moiety

Lipoteichoic acid (LTA) is a major virulence factor of Enterococcus faecalis that is closely associated with refractory apical periodontitis. Recently, we have shown that calcium hydroxide, a commonly used intracanal medicament, abrogated the ability of LTA to stimulate the production of tumor necrosis factor α in a murine macrophage line, RAW 264.7. Because calcium hydroxide could potentially modify the glycolipid moiety of LTA, we examined if calcium hydroxide inactivates LTA through deacylation of the LTA. Methods: LTA was prepared from E. faecalis by organic solvent extraction followed by chromatography with the hydrophobic-interaction column and the ion-exchange column. RAW 264.7 cells were stimulated with intact LTA or calcium hydroxide-treated LTA for 24 hours, and the productions of nitric oxide (NO) and chemokines interferon-gamma-induced protein (IP-10) and macrophage inflammatory protein-1α (MIP-1α) were determined. The glycolipid structure of LTA was analyzed using matrix-assisted laser desorption ionization-time of flight mass spectrometry and thin layer chromatography (TLC). Results: The production of NO, IP-10, and MIP-1α was augmented in LTA-stimulated cells, whereas no such effect was observed upon stimulation with calcium hydroxide-pretreated LTA. Mass spectrometry showed that intact glycolipids of LTA yielded distinct mass peaks at 930 to 1,070 mass over charge (m/z) units, corresponding to dihexosyl-diacylglycerol consisting of two acyl chains with chain lengths of C ₁₆ to C ₂₂ and with one or two unsaturated double bonds. However, those peaks were not observed in the mass spectra of the calcium hydroxide-treated LTA. Furthermore, free fatty acids released from the calcium hydroxide-treated LTA were detected using TLC. Conclusion: We suggest that calcium hydroxide attenuates the inflammatory activity of E. faecalis LTA through deacylation of the LTA.