Novel ceramides recovered from Porphyromonas gingivalis: relationship to adult periodontitis

Abstract

The primary purpose of this study was to characterize the major structural features of ceramides recovered from Porphyromonas gingivalis , a suspected periodontal pathogen. Complex lipids extracted from P. gingivalis were treated with N,O-bis(trimethylsilyl)-trifluoroacetamide and analyzed using gas chromatography-mass spectrometry. Mass spectra of lipid derivatives revealed cleavage products consistent with structures of four major ceramides. Two of the major ceramides are proposed to contain long chain bases of either 2-amino-1,3-octadecanediol or 2-amino-1,3-nonadecanediol in amide linkage to 3-hydroxy iso branched C 17:0 . The remaining major ceramides are proposed to contain either 2-amino-1,3-octadecanediol or 2-amino-1,3-nonadecanediol in amide linkage to C 17:1 . Alkaline hydrolysis of P. gingivalis lipids and subsequent formation of suitable derivatives revealed 3-hydroxy iso branched C 17:0 , C 17:1 , 2-amino-1,3-octadecanediol, and 2-amino-1,3-nonadecanediol as hydrolysis products. Therefore, the constitutive fatty acids and long chain bases recovered in alkaline hydrolysis products of P. gingivalis lipids are consistent with the proposed ceramide structures. The next goal of this study was to investigate whether these bacterial ceramides exist in lipid extracts of human teeth and gingival tissue at sites of severe adult periodontitis. Using selected ion monitoring of characteristic ions and retention times for each ceramide described above, lipids from teeth and gingival tissue were shown to contain primarily the ceramides containing C 17:1 . It is concluded that P. gingivalis synthesizes at least four major ceramides and two of these ceramides are selectively adsorbed to diseased tooth surfaces and may penetrate into diseased gingival tissue.-Nichols, F. C. Novel ceramides recovered from Porphyromonas gingivalis : relationship to adult periodontitis.

Structural confirmation of the dihydrosphinganine and fatty acid constituents of the dental pathogen Porphyromonas gingivalis

“…The aqueous layer was extracted with CH 2 Cl 2 and the combined organic layers were washed with saturated NaHCO 3 , dried over MgSO 4 , filtered, and evaporated to yield a white solid, 8 15 (3.80 g, 15.30 mmol, 81% over 2 steps), which was used without further purification. 1 [2-Hydroxy-1-(methoxymethylcarbamoyl)-ethyl]-carbamic acid tert-butyl ester (8, 0.82 g, 3.3 mmol) was dissolved in dry THF (7 mL) under N 2 . 15 The resulting solution was cooled to −15 • C and sec-BuMgBr [prepared from 2-bromobutane (0.73 mL, 6.6 mmol) and Mg (0.19 g, 7.92 mmol) in 8 mL dry THF] was added dropwise at −15 • C. After 10 minutes, 12-methyltridecylmagnesium bromide [prepared from 5 (1.83 g, 6.6 mmol) and Mg (0.19 g, 7.92 mmol) in 8 mL dry THF] was added at −15 • C. The resulting solution was allowed to warm to room temperature overnight.…”

“…3, 63.2, 61.6, 60.4, 39.9, 39.0, 29.9, 29.7, 29.6, 29.5, 29.4, 29.1, 28.3, 28.0, 27.4, 23.4, 22.6, 21.0, and 14. 0.62 mmol, 89%). 1 H NMR (400 MHz, CDCl 3 with methanol-d 4 ): d 3.88-3.84 (dd, J = 3.9, 11.8 Hz, 1H), 3.67-3.61 (d, J = 7.8 Hz, 2H), 3.43 (bs, 1H), 1.50-1.38 (m, 12H), 1.30-1.06 (m, 20H), and 0.87-0.84 ppm (d, J = 6.7 Hz, 6H); 13 C NMR (100 MHz, CDCl 3 with methanol-d 4 ): d 79. 7, 76.7, 73.6, 62.1, 39.0, 34.2, 29.9, 29.6, 28.3, 27.9, 27.4, 25.9, and 22.6…”

“…The solid was filtered and washed with water, giving 10b as a tan solid (76.7 mg, 0.27 mmol, 57%). 1 H NMR (400 MHz, pyridine-d 5 ) 22 d 5.92 (bs, 1H), 4.89 (bs, 3H), 4.28-4.24 (dd, J = 4.1, 10.2 Hz, 1H), 4.10-4.06 (dd, J = 7.5, 10.4 Hz, 1H), 4.00 (m, 1H), 3.30-3.28 (m, 1H), 1.83-1.12 (m, 23H), and 0.86-0.84 ppm (t, J = 6.7 Hz, 6H); 13 C NMR (100 MHz, pyridine-d 5 ): d 73.6, 64. 4, 58.3, 39.0, 34.1, 30.0, 29.8, 29.7, 28.0, 27.5, 26.4, and 22.5…”

“…Purification by column flash chromatography on silica gel (CH 2 Cl 2 -EtOAc, 100 : 0 to 50 : 50) gave a white solid, 12b (0.33 g, 0.81 mmol, 94%). 1 H NMR (400 MHz, pyridine-d 5 ): 23 d 4.96-4.89 (bs, 3H), 4.47-4.45 (m, 1H), 4.33-4.30 (m, 3H), 1.94-1.16 (m, 36H), and 0.89-0.88 ppm (d, J = 6.5 Hz, 6H); 13 C (100 MHz; 150 MHz, pyridined 5 ): d 72. 7,62.8,58.3,39.7,35.4,30.6,30.4,29.0,28.6,28.1,and 23.2 ppm;HRMS (ESI+): m/z (M + H) + , calcd for C 24 H 49 NO 2 : 416.3740.…”

“…Previous work used GC-mass 1 and electrospraymass 2 spectroscopic analysis to show that the lipid extracts of P. gingivalis are similar to lipid profiles in extracts of diseased tooth roots and diseased gingival tissues. 1 Lipid extracts of P. gingivalis are known to potentiate prostaglandin secretion from gingival fibroblasts when co-treated with interleukin-1b, and more recent work demonstrated that phosphorylated dihydroceramides are the primary biologically active lipid constituents. 3 Beyond the GC-MS data, little is known about the specific lipids responsible for this biological activity.…”