Calcium hydroxide inactivates lipoteichoic acid from Enterococcus faecalis through deacylation of the lipid moiety.
BACKGROUND
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.


CONCLUSIONS
We suggest that calcium hydroxide attenuates the inflammatory activity of E. faecalis LTA through deacylation of the LTA.
Department of Oral Microbiology & Immunology, Seoul National University, Seoul, Republic of Korea.
Jung Eun
JE
Baik
Kyoung-Soon
KS
Jang
Seok-Seong
SS
Kang
Cheol-Heui
CH
Yun
Kangseok
K
Lee
Byung-Gee
BG
Kim
Kee-Yeon
KY
Kum
Seung Hyun
SH
Han
D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism; D002126:Calcium Hydroxide / Q000494:pharmacology*; D002460:Cell Line; D054405:Chemokine CCL3 / Q000378:metabolism; D054357:Chemokine CXCL10 / Q000378:metabolism; D013293:Enterococcus faecalis / Q000187:drug effects* / Q000378:metabolism; D006017:Glycolipids / Q000737:chemistry / Q000378:metabolism; D008070:Lipopolysaccharides / Q000737:chemistry / Q000378:metabolism*; D008262:Macrophage Activation / Q000187:drug effects; D008264:Macrophages / Q000187:drug effects*; D008562:Membrane Glycoproteins / Q000737:chemistry / Q000187:drug effects* / Q000378:metabolism; D051379:Mice; D009569:Nitric Oxide / Q000378:metabolism; D012388:Root Canal Irrigants / Q000494:pharmacology*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013682:Teichoic Acids / Q000737:chemistry / Q000378:metabolism*
D001426:Bacterial Proteins; D054405:Chemokine CCL3; D054357:Chemokine CXCL10; D006017:Glycolipids; D008070:Lipopolysaccharides; D008562:Membrane Glycoproteins; D012388:Root Canal Irrigants; D013682:Teichoic Acids; D009569:Nitric Oxide; C009900:lipoteichoic acid; D002126:Calcium Hydroxide