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Journal of Lipid Research, Vol. 45, 2317-2330, December 2004 Structures and biological activity of phosphorylated dihydroceramides of Porphyromonas gingivalis
* Department of Periodontology, University of Connecticut School of Dental Medicine, 263 Farmington Avenue, Farmington, CT Published, JLR Papers in Press, October 1, 2004. DOI 10.1194/jlr.M400278-JLR200
1 To whom correspondence should be addressed. e-mail: nichols{at}nso.uchc.edu
Porphyromonas gingivalis, a recognized periodontal pathogen, synthesizes free ceramides as well as other phosphorylated ceramide lipids. The purpose of this study was to separate complex lipids of P. gingivalis by high-performance liquid chromatography (HPLC) and determine the structures and biological activities of the major ceramide classes. Using gas chromatography-mass spectrometry, electrospray tandem mass spectrometry (ESI-MS/MS) and NMR analyses, three major classes of dihydroceramides were identified in specific HPLC fractions, with all classes containing the same dihydroceramide base structures (3-OH isoC17:0 in amide linkage to saturated long-chain bases of 17, 18, or 19 carbons). The free dihydroceramide class recovered in HPLC fractions 78 revealed little biological activity. HPLC fraction 20 dihydroceramides, substituted with 1-O-phosphoglycerol and isoC15:0 linked to the hydroxyl of 3-OH isoC17:0, significantly potentiated interleukin-1ß (IL-1ß)-mediated prostaglandin secretion and produced marked alterations in fibroblast morphology. HPLC fraction 28 dihydroceramides, substituted with 1-O-phosphoethanolamine, demonstrated little capacity to potentiate IL-1ß-mediated prostaglandin secretion. The novel phosphorylated dihydroceramides synthesized by P. gingivalis demonstrate varying biological activities based on the phosphorylated head group substitution and/or the addition of esterified fatty acid. These results also demonstrate the strong virulence capacity of phosphoglycerol dihydroceramides of P. gingivalis to promote inflammatory factor secretion from IL-1ß-treated fibroblasts and to produce marked alterations in cell morphology in culture.
Supplementary key words phosphoethanolamine ceramide phosphoglycerol dihydroceramide interleukin-1ß prostaglandin E2 gingival fibroblast long-chain base gas chromatography-mass spectrometry electrospray MS/MS
Inflammatory periodontal disease in adults is initiated with the accumulation of specific bacteria in the sulcus around the teeth followed by a chronic inflammatory reaction to these organisms by the host. The anaerobic, Gram-negative organism, Porphyromonas gingivalis, is thought to be a major periodontal pathogen (1) associated with inflammatory periodontal disease in adults. Dihydroceramide lipids of P. gingivalis and other phylogenetically related organisms have been identified in lipid extracts of diseased tooth roots and diseased gingival tissues (2). Lipid extract of P. gingivalis is known to potentiate prostaglandin secretion from gingival fibroblasts when cotreated with interleukin-1ß (IL-1ß) (3). However, little is known about the specific lipids responsible for this biological activity. Therefore, the primary goal of this study was to determine whether the dominant dihydroceramide lipids of P. gingivalis contribute to the observed biological activity. To clarify this question, the dihydroceramide lipids of P. gingivalis were first separated into constituent fractions before structural analysis and biological testing. The biological activities of P. gingivalis dihydroceramides are important to assess, given the recent evidence that other microbes synthesize ceramide lipids that induce apoptosis (4).
As originally described for Bacteroides fragilis (5), P. gingivalis synthesizes free dihydroceramides containing 3-hydroxy isobranched (iso) C17:0 linked to either an 18- or 19-carbon saturated long-chain base (2). On the basis of gas liquid chromatography-mass spectrometry (GC-MS) analysis of P. gingivalis lipid extracts, two additional free dihydroceramides of P. gingivalis were proposed to contain C17:1 (
P. gingivalis (ATCC 33277, type strain) was inoculated into cooked meat medium supplemented with trypticase, yeast extract, menadione, and hemin (BBL, #295982, Becton Dickinson, Franklin Lakes, NJ). Once growth was established, the initial culture was inoculated into enriched thioglycollate medium (BBL) and prereduced blood agar plates. Batch suspension cultures were established after verification of anaerobic growth of Gram-negative rods in thioglycollate medium and demonstration of black-pigmented bacterial colonies on blood agar plates under anaerobic culture conditions. Batch suspension cultures (1 l flasks) were grown in basal [peptone, trypticase, and yeast extract, (BBL)] medium supplemented with hemin and menadione (Sigma; St. Louis, MO) and brain heart infusion. The suspension cultures were incubated in an anaerobic chamber flushed with N2 (80%), CO2 (10%), and H2 (10%) at 37°C for five days, and the bacteria were harvested by centrifugation (3,000 g for 20 min). Following lyophilization, 4 g of P. gingivalis pellet was extracted overnight using a modification of the phospholipid extraction procedures of Bligh and Dyer (6) and Garbus et al. (7). Specifically, 4 ml of H20 + 16 ml of MeOH-CHCl3 (2:1; v/v) was added to the bacterial sample and vortexed. After 12 h, 3 ml of 2 N KCl + 0.5 M K2HPO4 and 3 ml CHCl3 were added and the sample vortexed. The lower organic phase was carefully removed and CHCl3 (3 ml) was added to each sample and vortexed. The CHCl3 phase was removed and combined with the previous organic solvent extract. The lipid extract from P. gingivalis was dried under nitrogen and stored frozen.
Fractionation of bacterial lipids by high-performance liquid chromatography (HPLC) was accomplished using a semipreparative HPLC column (1 x 25 cm silica gel, 5 µm; Supelco, Inc., Bellefonte, PA) and eluted isocratically with hexane-isopropanol-water (6:8:0.75; v/v/v: solvent A) (8). Lipid samples were dissolved in solvent A to achieve a concentration of
Lipid film preparation Primary cultures of gingival fibroblasts were obtained using a single gingival tissue explant harvested at the time of periodontal surgery and processed as previously described (9). Gingival tissue samples were obtained according to a protocol approved by the University of Connecticut Health Center Institutional Review Board, and participants provided written informed consent. Primary cultures of gingival fibroblasts were grown in Minimal Essential Medium (MEM) supplemented with 10% fetal calf serum, penicillin G (100 U/ml), streptomycin (100 µg/ml), and amphotyricin B (0.25 µg/ml) (Sigma), and cells were passaged fewer than 10 times for these experiments. The cells were harvested using trypsin (0.1%, w/v) in Ca2+-, Mg2+-free phosphate-buffered saline and resuspended in MEM medium to achieve a 1:3 dilution of cells for passaging. Cells were cultured in an H2O-saturated, 5% CO2 atmosphere at 37°C. When fibroblasts were treated with bacterial lipids, cells were inoculated into culture wells to achieve a cell number/surface area equivalent to confluent cultures. After 2 h of exposure to bacterial lipids, the culture medium was supplemented with either control medium or medium containing recombinant human IL-1ß (final concentration,10 ng/ml) (Immunex; Seattle, WA). The cultures were then incubated for an additional 24 h, after which the medium samples were harvested and frozen.
Quantification of prostaglandins in culture medium samples All derivatizing agents were obtained from Pierce Chemical Corp. (Rockford, IL). Prostaglandin samples were derivatized using the method of Waddell, Blair, and Wellby (11). Prostaglandin samples were first treated with 2% methoxylamine hydrochloride in pyridine (30 µl). After standing overnight at room temperature, the samples were dried under nitrogen, dissolved in acetonitrile (30 µl), and treated with pentafluorobenzyl bromide (35% v/v in acetonitrile, 10 µl) and diisopropylethylamine (10 µl). The samples were vortexed, incubated for 20 min at 40°C, and evaporated under nitrogen. The residue was then treated with bistrimethylsilyl-trifluoroacetamide (BSTFA, 50 µl) and allowed to stand at room temperature for 45 days.
Synthesis of isobranched long-chain bases
Analysis of bacterial fatty acid, long-chain base, and dihydroceramide lipid components in HPLC fractions
GC-MS analysis
TMS derivatives of bacterial fatty acids and long-chain bases were analyzed using gas chromatography combined with electron impact mass spectrometry. The sample was applied to an SPB-1 (12 m x 0.2 mm, 0.33 µm film thickness; Hewlett Packard) column held at 100°C. The fatty acid and long-chain base samples were injected using the splitless mode (30 s), and the column was heated at 10°C/min to 270°C. The injector block was held at 260°C, and the transfer tube was maintained at 280°C. The mass spectrometer was used with the ion source temperature at 150°C, the electron energy at 70 eV, and an emission current of
For dihydroceramide analyses, selected HPLC fractions were treated to form TMS derivatives and were applied to an SPB-1 column (15 m x 0.25 mm, 0.1 µm film thickness; Supelco, Inc., Bellefonte, PA). Complex lipid samples were injected with the inlet block at 310°C using the splitless mode with a temperature program of 10°C/min from 200°C to 300°C followed by 5°C/min to 310°C and 4 min at 310°C. The mass spectrometer was used in the electron impact ionization mode with the ion source temperature at 150°C, an electron energy of 70 eV, and an emission current of
Electrospray tandem mass spectrometry analysis of dihydroceramide lipids
NMR analyses
Data analysis
Lipids were extracted from P. gingivalis and separated by HPLC as shown in Fig. 1 . GC-MS analysis revealed three well-separated dihydroceramide lipid classes. The free dihydroceramides recovered in HPLC fractions 78 corresponded to the free dihydroceramides of P. gingivalis containing 3-OH C17:0 in amide linkage to saturated dihydroxy long-chain bases of either 17, 18, or 19 carbons in length (2). Treatment of gingival fibroblasts with fraction 78 lipids did not promote IL-1ß-mediated prostaglandin secretion, and this ceramide fraction was not further purified. GC-MS analysis of fractions 1520 and 3134 suggested dihydroceramide lipid structures termed phosphoglycerol (PG) ceramides and phosphoethanolamine (PEA) ceramides, respectively. Fractions 1520 were pooled and repurified by HPLC, and peak dihydroceramide lipid recovery was noted in HPLC fraction 20. Fractions 3134 were repurified by HPLC, and the peak dihydroceramide recovery was noted in HPLC fraction 28.
Positive- and negative-ion ESI-MS analysis of fraction 20 lipids produced the mass spectra shown in Fig. 2 . The negative-ion spectra (Fig. 2B) revealed three major ions of 960, 946, and 932 m/z. Therefore, the masses of the intact lipids are inferred to be greater by 1 amu. Analysis of NMR spectra provided proton and carbon assignments as shown in Table 1. The proton and carbon spectra are assigned by number to the dihydroceramide structures proposed (Fig. 2B) (see below). The positive-ion spectra (Fig. 2A) revealed high-mass ions (984, 970, and 956 m/z), consistent with sodium adducts of the negative ions shown in the lower frame. The positive-ion mass spectra also demonstrated two additional groups of positive ions (790/791, 776/777, and 762/763 m/z, and 548, 534, and 520 m/z). Positive-ion MS/MS analysis revealed that the 962, 948, and 934 m/z parent ions produce daughter ions of 548, 934, and 920 m/z, respectively (Fig. 3) . The 791, 777, and 763 parent ions also produced daughter ions of 548, 534, and 520 m/z, respectively (Fig. 4) . In all cases, the parent or daughter ions within each group differed in mass by 14 amu increments, suggesting methyl addition to a specific aliphatic chain within the parent lipids. The negative-ion MS/MS spectra revealed 171 and 158 m/z daughter ions from the 962, 948, and 934 parent ions (Fig. 5) . Therefore, the 791, 777, and 763 m/z ions noted in Fig. 2 represent the loss of a 171 m/z ion (phosphoglycerol moiety) from the parent lipid molecules.
NMR analysis of HPLC fraction 20 lipids revealed a proton resonance at 5.22 ppm with HMBC correlations to carbonyl carbons at 170.5 and 173.9 ppm (see Table 1). Combined with other NMR data, these resonances allowed the assignment of the fatty acid substitution on the ß hydroxyl of 3-OH-C17:0, with methyl branches at the terminal aliphatic chain regions of the fatty acids. TOCSY (long-range 1H-1H COSY) and COSY data related protons of the long-chain dihydroceramide base, and HMQC data were used to assign the 13C data to this backbone. Finally, 1H-31P (gHMBC) and TOCSY data were used to assign the phosphoglycerol attachment to the carbons at 64.2 and 66.4 ppm, as relayed by the protons at 4.20, 3.88, and 3.85 ppm. Electrospray MS analysis of HPLC fraction 20 lipids dissolved in uniformly labeled deuterated methanol increased positive parent ion masses by 6 amu and increased negative parent ion masses by 4 amu (data not shown). Negative-ion MS/MS with deuterated methanol increased the mass of the 171 daughter ions by 3 amu (data not shown), consistent with a phospoglycerol moiety within these HPLC fraction 20 lipids. GC-MS analysis further clarified the structures of the fraction 20 lipids. TMS derivatives of fraction 20 lipids revealed the mass spectra shown in Fig. 6 . The high-mass ions of the three dihydroceramide lipids are recovered as proton adducts of the M-15 ions (nitrogen forms a proton adduct, thus increasing the mass of each dihydroceramide lipid by 1 amu). Loss of the phosphoglycerol moiety due to thermal decomposition in the GC injection block is associated with desaturation of the long-chain base and formation of an imine (see Results, HPLC fraction 28). The HPLC fraction 20 lipids shown in Fig. 6 represent TMS derivatives minus a methyl group (605, 591, and 577 m/z) of the daughter ions shown in Figs. 3 and 4 (548, 534, and 520 m/z). Therefore, the lipid derivatives observed by GC-MS (Fig. 6) are consistent with the addition of a single TMS moiety to the smallest daughter ions (548, 534, and 520 m/z ions). Hydrolysis of fraction 20 lipids with sodium methoxide recovered only isoC15:0 methyl ester, as verified with a synthetic fatty acid standard (data not shown). Treatment of fraction 20 lipids with 4N KOH revealed saturated long-chain bases of 17, 18, and 19 carbons in length and 3-OH isoC17:0 (data not shown). Based on the retention times of synthetic straight-chain (18 and 20 carbons) and isobranched long-chain base lipid standards (17 and 19 carbons), the 17- and 19-carbon long-chain bases of P. gingivalis are isobranched saturated long-chain bases, and the 18-carbon long-chain base is a straight long-chain base (data not shown). 3-OH isoC17:0 was released only with strong alkaline hydrolysis, indicating that this fatty acid is held in amide linkage. The dihydroceramide lipids of fraction 20 therefore consist of 3-OH isoC17:0 in amide linkage to 17-, 18-, or 19-carbon saturated long-chain bases substituted with a phosphoglycerol moiety linked to the long-chain bases. Reconciliation of the smallest daughter ions of fraction 20 lipids (548, 534, and 520 m/z ions) and the position of the esterified isoC15:0 will be provided in the Discussion section. The structures of HPLC fraction 20 phosphoglycerol dihydroceramides were further clarified by analysis of HPLC fraction 28 lipids. GC-MS analysis of TMS derivatives of HPLC fraction 28 lipids revealed three major lipid products with associated mass spectra (Fig. 7) . These lipid derivatives show mass spectra identical to those shown for the previously reported unsaturated ceramides (2). Of note, treatment of fraction 20 lipids with sodium methoxide and GC-MS analysis of the resultant TMS dihydroceramide lipid derivatives demonstrated spectra identical to those depicted in Fig. 7 (data not shown). Therefore, the core lipid structures for dihydroceramides of fraction 20 are identical to the core structures of fraction 28 dihydroceramide lipids. As with fraction 20 lipids, GC-MS of TMS derivatives of fraction 28 lipids at the high injection temperature is associated with thermal decomposition and loss of the phosphorylated head group and desaturation of the proximal end of the long-chain base, resulting in formation of an imine. This was verified by subjecting sphingomyelin and phosphatidylcholine standards to GC-MS under identical conditions. For both lipid standards, the observed mass spectra were consistent with loss of the phosphorylated head group, together with desaturation of either the long-chain base or the glycerol moiety, respectively. Finally, alkaline hydrolysis of fraction 28 lipids released essentially only 3-OH isoC17:0 (data not shown), indicating that unsaturation does not exist in the fatty acid component of these lipids. It is concluded that the phosphoceramides of HPLC fractions 28 and 20 undergo thermal decomposition in the injection block held at 310°C, and this results in loss of the phosphorylated head group together with formation of a monounsaturated ceramide. ESI-MS analysis of fraction 28 lipids resulted in positive- and negative-ion spectra, as shown in Fig. 8 . Figure 8B depicts three negative-ion products differing in mass by 14 amu (705, 691, and 677 m/z), whereas Fig. 8A demonstrates the corresponding positive ions (707, 693, and 679 m/z), as well as three ions reduced in mass by 141 amu (566, 552, and 538 m/z, respectively). Positive-ion MS/MS revealed 566, 552, and 538 m/z daughter ions of the 707, 693, and 679 m/z parent ions, respectively, indicating loss of a phosphethanolamine head group (data not shown). Negative-ion MS/MS revealed 140 m/z daughter ions for the 705, 691, and 677 parent ions (Fig. 9) . ESI-MS/MS analysis of HPLC fraction 28 lipids dissolved in uniformly labeled deuterated methanol increased positive parent ion masses by 7 amu and negative parent ion masses by 5 amu (data not shown). Negative-ion ESI-MS/MS using deuterated methanol solvent increased the 140 m/z daughter ion masses by 3 amu (data not shown). Deuterium substitution findings are consistent with the number of exchangeable protons expected for the intact dihydroceramides and phosphoethanolamine head group of HPLC fraction 28 phosphoceramides. The phosphoethanolamine location in fraction 28 lipids is reconciled by identifying the position of the unsaturated moiety using GC-MS (Fig. 7). The location of the phosphoglycerol moiety in HPLC fraction 20 lipids is confirmed by applying the same reasoning (see Fig. 6). Therefore, fraction 28 lipids represent three distinct 1-O-phosphoethanolamine dihydroceramides containing long-chain bases of 17, 18, or 19 carbons in amide linkage with 3-OH isoC17:0.
Treatment of gingival fibroblasts with the bacterial lipid fractions demonstrated significant potentiation of IL-1ß-mediated PGE2 secretion from primary cultures of gingival fibroblasts (Fig. 10) . However, HPLC fraction 20 lipids significantly stimulated PGE2 release over controls, IL-1ß-treated cultures, and cultures treated with HPLC fraction 28 lipids (P < 0.0001 by Fisher PLSD and Scheffe F-tests). PGF2 secretion was unaffected by bacterial lipids, and 6-keto PGF1 secretion was modestly increased by the bacterial lipids, but the differences between the treatment groups were not statistically significant. Therefore, the primary effect of bacterial lipids on prostaglandin synthesis was to potentiate PGE2 secretion over other major prostaglandin species released from IL-1ß-stimulated fibroblasts. Of note, bacterial lipids alone did not stimulate prostaglandin secretion, when compared with vehicle controls (data not shown). The total lipid extract of P. gingivalis also potentiated prostaglandin secretory responses but to a lesser extent than HPLC fraction 20 lipids. Fibroblast morphology in culture was dramatically affected by fraction 20 lipids and, to a lesser extent, the total lipid extract of P. gingivalis (Fig. 11)
. The morphological changes in gingival fibroblasts were noted with or without IL-1ß treatment (data not shown).
The present study demonstrates that P. gingivalis synthesizes phosphoceramides with a core lipid structure consisting of either a 17-, 18-, or 19-carbon long-chain base in amide linkage to 3-OH isoC17:0. Phosphorylated head groups consist of either phosphoglycerol or phosphoethanolamine, and both head groups are linked to long-chain bases at carbon 1. Of particular note, only the phosphoglycerol dihydroceramide contained isoC15:0 in ester linkage to the hydroxyl group of 3-OH isoC17:0. Additional experiments will evaluate whether P. gingivalis synthesizes phosphoethanolamine dihydroceramides that contain isoC15:0 or synthesizes phosphoglycerol dihydroceramides that do not contain isoC15:0. Although P. gingivalis synthesizes a wide variety of fatty acid products, including 3-OH isoC15:0 and C14:0 to C18:0 (data not shown), only the fatty acids mentioned above are constituents of the phosphoceramides of P. gingivalis. The free dihydroceramides of P. gingivalis detected in HPLC fractions 78 (data not shown) and described in a previous report (2) likely serve as the substrate molecules for the synthesis of the phosphoceramides described here. Although P. gingivalis appears to synthesize phosphoceramides from free dihydroceramide substrates, the details of the synthetic pathways and the specificity of the ceramide substitutions remain for future study.
Both GC-MS and ESI-MS/MS of HPLC fraction 20 lipids produced expected ceramide ions consistent with loss of phosphoglycerol moiety (Figs. 3, 4, 6). However, both analytical methods produce lower-mass ions, consistent with McLafferty rearrangements (14), resulting in loss of esterified isoC15:0 from the ceramides. McLafferty rearrangements explain the generation of 548, 534, and 520 ions under positive-ion ESI-MS/MS analysis, as well as the formation of the 605, 591, and 577 ions observed with GC-MS analysis of fraction 20 lipids. McLafferty rearrangements produce a monounsaturation of 3-OH isoC17:0, with loss of the ß-position oxygen. An earlier report concluded that the unsaturated ceramides are synthesized by P. gingivalis because The phosphoceramide lipids presented in this report are consistent with phosphoglycerol and phosphoethanolamine ceramides previously described by White and Tucker for the species Bacteroides melaninogenicus (15). Like B. melaninogenicus, P. gingivalis phosphoceramides also contain isobranched 17- and 19-carbon long-chain bases and a saturated straight-chain 18-carbon long-chain base (16). However, we did not recover fatty acids other than amide-linked 3-OH isoC17:0 and ester-linked isoC15:0 in the biologically active phosphoceramides of P. gingivalis, in contrast to B. melaninogenicus phosphoceramides that contain a variety of saturated and monounsaturated fatty acids (15). Therefore, the phosphoceramides of P. gingivalis appear to have specific fatty acid substitutions when compared with B. melaninogenicus phosphoceramides. Other bacterial species are known to synthesize ceramide-like lipids that share structural characteristics with P. gingivalis ceramides. Sphingobacterium species are reported to synthesize phosphoethanolamine ceramides containing a 15-methyl hexadecanoic long-chain base in amide linkage to either isoC15:0 or 2-OH isoC15:0 (4, 17). Neither phosphoglycerol ceramides nor ester-linked fatty acid were reported for Sphingobacterium phosphoceramides (4, 17). These bacterial sphingolipids were recently shown to promote apoptosis in several cell types (4). Because of the structural similarities between P. gingivalis phosphoceramides and Sphingobacterium sp. phosphoceramides, it is expected that P. gingivalis phosphoceramides will induce apoptosis as well. More importantly, the phosphoglycerol ceramides of P. gingivalis demonstrate considerably greater biological activity than do the phosphoethanolamine ceramides. Other bacteria produce lipids that are more distantly related to the phosphoceramides of P. gingivalis. These include Flavobacterium meningosepticum, which synthesizes a flavolipin consisting of an amide-linked isobranched fatty acid linked to a serine-glycine isobranched aliphatic amino alcohol (18). Finally, Capnocytophaga species synthesize sulfonolipids (capnines) that contain sulfonate isobranched long-chain bases (19, 20). Despite this evidence, the phosphoceramides of P. gingivalis characterized in this report suggest novel lipids that are distinct from those produced by other organisms. The free dihydroceramides of P. gingivalis (2) recovered in fractions 78 are without the capacity to potentiate prostaglandin secretion from gingival fibroblasts, a surprising result, given the reported capacity of free ceramides to promote prostaglandin secretion from fibroblasts (21, 22) and other cells (23). However, those HPLC fractions shown to contain the phosphoglycerol dihydroceramides markedly promoted PGE2 secretion from IL-1ß-treated fibroblasts, indicating that these phosphoceramides account for at least a major portion of the biological activity observed in the total lipid extract of P. gingivalis. The biological relevance of the phosphoglycerol dihydroceramides relates to the capacity of these lipids to promote inflammatory processes associated with periodontal diseases and perhaps other systemic inflammatory diseases associated with bacterial lipid accumulation. It is suspected that the phosphoglycerol dihydroceramides of P. gingivalis produce their biological effects through a recognized pattern recognition system (Toll receptor) or interact with an as-yet uncharacterized pattern recognition system in human cells. Whether the highly lipophilic phosphoglycerol dihydroceramides affect cell activity through interaction with a cell surface receptor or an intramembranous protein constituent remains to be established. Penetration of bacterial lipid into inflamed periodontal tissues without coincident bacterial invasion (24) would be expected to intensify the inflammatory reaction such that bone and connective tissue destruction could be favored through locally increased synthesis of PGE2 (25, 26). GC-MS has shown that lipid extracts from periodontally diseased teeth and diseased periodontal soft tissues are contaminated ceramide structures, consistent with the phosphoglycerol ceramide products shown in Fig. 6 (data not shown). Therefore, the results of this study demonstrate that a major periodontal pathogen, P. gingivalis, synthesizes a group of unusual, complex lipids, specifically phosphoglycerol ceramides, that account in part for the ability of P. gingivalis lipid extract to potentiate IL-1ß-mediated prostaglandin secretion from fibroblasts. The marked potentiation of prostaglandin synthesis is not observed with the structurally similar phosphoethanolamine dihydroceramide class described herein, indicating that the specific structural characteristics of the phosphoglycerol dihydroceramides account for the marked potentiation of IL-1ß-mediated prostaglandin secretion. In addition to potentiation of prostaglandin secretion from gingival fibroblasts, the phosphoglycerol dihydroceramides of P. gingivalis dramatically alter fibroblast morphology in culture (Fig. 11) and alter gene expression for a substantial number of proinflammatory and signal transduction genes, as measured by Affymetrix focused gene array (data not shown). Furthermore, at least one additional lipid class of P. gingivalis promotes similar cell activation events. Space limitations prevent inclusion of these new findings. Understanding the basis for cell activation following exposure to phosphoglycerol dihydroceramide lipids or synthetic chemical analogs will be of considerable importance in understanding diseases associated with accumulation of these agents in tissues.
The authors would like to thank Ms. Jody Barry for her assistance in the microbiological preparation of P. gingivalis. The authors would also like to thank Mr. Marvin Thompson for his assistance in the electrospray mass spectral analysis of the lipid compounds. The authors also thank Dr. Clarence Trummel for providing funds to cover publication costs. This work was supported in part by a grant from the Patterson Trust Foundation. Manuscript received July 20, 2004 and in revised form September 21, 2004.
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