Lipidomic analysis of Porphyromonas gingivalis reveals novel glycerol bisphosphoceramide, phosphatidyl-, and phosphoglycerol dipeptide lipid families

Porphyromonas gingivalis, like other members of the phylum Bacteroidetes (synonym Bacteroidota), synthesizes several classes of dihydroceramides and peptidolipids. Using a similar strategy as that recently used to delimit the lipidome of its close relative Bacteroides fragilis, we applied linear ion trap multiple-stage mass spectrometry (linear ion trap MSn) with high-resolution mass spectrometry, to structurally characterize the complete lipidome of P. gingivalis and compare it to B. fragilis. This analysis discovered that the P. gingivalis lipidome consists of several previously unidentified lipid families, including dihydroceramide-1-phosphophate, acylated dihydroceramide-1-phosphophate, phosphoglycerol glycylserine lipid, and bis(phosphodihydroceramide) glycerol. Interestingly, we also found a novel sphingolipid family containing a polyunsaturated long–chain base, and a new lipoglycylserine phosphatic acid containing unsaturated acyl chains not reported for the lipid family. The comprehensive coverage of the lipidome of P. gingivalis conducted in this study has revealed more than 140 lipid species including several novel lipids in over 20 lipid families/subfamilies.

Porphyromonas gingivalis is a Gram-negative, oral anaerobe belonging to the phylum Bacteroidetes (1).This bacterium is strongly associated with development of destructive periodontal disease in adults (2)(3)(4), and infections with this bacterium are also correlated with a variety of systemic diseases, including atherosclerosisassociated cardiovascular diseases and Alzheimer's disease (AD) (5)(6)(7)(8)(9)(10)(11). P. gingivalis synthesizes several classes of dihydroceramides (DHCs), including phosphoglycerol DHC (PG DHC), phosphoethanolamine DHC (PE DHC), as well as lipopeptides, including glycine lipid (G-lipid), lipoglycylserine (GS-lipid), and lipoglycylserine phosphatidic acid (GS-PA).At least one of these lipid classes have been shown to promote proinflammatory secretory reactions in gingival fibroblasts as well as alter fibroblast morphology in culture (12)(13)(14).Purified Gand GS-lipid were shown to promote toll-like receptor 2-dependent TNF-α release from bone marrow macrophages, and activate human embryonic kidney cells through toll-like receptor 2 and TLR6 but not TLR1 (15), and purified PG DHC has been shown to induce apoptosis, hence DHCs are thought to be important virulence determinants of P. gingivalis (16)(17)(18).In addition, recent studies have shown that synthesis of sphingolipids (SLs) by P. gingivalis is central to its ability to evade the host inflammatory response via the production of SL-containing outer membrane vesicles (19)(20)(21), indicating that the ratio of membrane lipids is important for homeostasis.Lastly, cell surface virulence determinants from P. gingivalis and Bacteroides fragilis have been detected in human AD brains (22,23), and it has been hypothesized that infections with P. gingivalis or B. fragilis play a role in AD pathogenesis (23,24).DHC dihydrosphingosine (contains a unique methyl side chain (iso and anteiso) 17-, 18-, or 19-carbon sphinganine) base, to which a major iso-17:0 (3-OH) FA is linked to the 2-amino group.In PG DHC and PE DHC, an additional iso-15:0 FA can also be linked by an ester bond ("piggy back") to the hydroxyl group of the 3hydroxy 17:0-FA to form a 3-O-acyl-PG DHC (acylated-PG DHC) (25) and 3-O-acyl PE DHC (acylated-PE DHC), respectively (see Supporting material for identification).Structural characterization of lipid classes including PG DHC, PE DHC, serine dipeptide, and diacylated phosphoserine-glycine lipodipeptide specific to P. gingivalis was previously described by Nichols et al. (13,14,26).They applied chromatographic separation to isolate the lipid families, combined with chemical reactions, GC/MS, LC/MS, and NMR spectroscopic *For correspondence: Fong-Fu Hsu, fhsu@wustl.edu.

Mass spectrometry
Both high-resolution (R = 100,000 at m/z 400) and lowenergy collision-induced dissociation (CID) LIT MS n analyses were conducted on a Thermo Fisher Scientific (San Jose, CA) LTQ Orbitrap Velos MS with Xcalibur operating system.Lipid extracts were dissolved in 1% NH 4 OH in methanol and infused or injected (via a loop) onto the ESI source and analyzed in the negative-ion mode.The skimmer of the source was set at ground potential, the electrospray needle was set at 4 kV, and temperature of the heated capillary was 300 • C. The automatic gain control of the ion trap was set to 5 × 10 4 , with a maximum injection time of 50 ms.Helium was used as the buffer and collision gas at a pressure of 1 × 10 −3 mbar (0.75 mTorr).The MS n experiments were carried out with an optimized relative collision energy ranging from 25% to 45%, an activation q value of 0.25, and an activation time of 10 ms that leave minimal residual precursor ions with abundance around 20%.The mass selection window for the precursor ions was set at 1 Da wide to admit the monoisotopic ion to the ion trap (for CID) for unit resolution detection in the ion trap or HR accurate mass detection in the Orbitrap mass analyzer.Mass spectra were accumulated in the profile mode, typically for 2-10 min for MS n spectra (n = 2, 3, 4).

PtO 2 /H 2 hydrogenation
For further insight into the structure of the polyunsaturated LCB substituent in glycerol phosphoryl ceramide (GPC) lipids, GPC fraction (c.a. 10 ug in 500 uL methanol) isolated by aminopropyl Sepak column as described previously (27) was placed in a tube, and 15 mg PtO 2 was added, vortexed, and a stream of H 2 was bubbled into the slurry at room temperature for 30 min.After reaction, the tube was centrifuged, and the methanol layer was transferred to another vial, and injected into mass spectrometer for HR ESI/ MS analysis.
Acid hydrolysis, free (FA) extraction, preparation of FA-N-(4-aminomethylphenyl)pyridinium derivatives, and tandem mass spectrometric analysis of FA-AMPP derivative for characterization of the FA substituents of the molecules See Supplementary Material 2.

Characterization of acylated PE-DHC by LIT MS n
See Supplementary Material 2.

RESULTS
We profiled the lipids extracted from P. gingivalis cells by high resolution (R = 100,000 at m/z 400) ESI MS scan in the negative-ion mode via loop injection, similar to the methodology previously used for B. fragilis (27).Accurate mass measurements permit extraction of elemental composition of the molecules, and when combined with MS n that allows further insight into the fragmentation processes readily afford accurate assignments of the lipid structure and the entire lipid repertoire can be depicted (Table 1) (See supplemental Fig. S1A for HR full-scan ESI-MS).

Characterization of GS-lipid, G-lipid, serine lipid, and a new GS-PG lipid family
The serine-glycine dipeptide lipids produced by P. gingivalis were previously defined as lipid 654 (31), which was detected at m/z 653 and a homologous ions at m/z 611, 625, and 677, as the [M -H] -ions in the negativeion mode.GS-lipids also contain ions at m/z 415 and 429, in which the acyl chain attached to the β-OH FA substituent is absent.G-lipids termed lipid 567 and lipid 342 consisted of similar FA substituents and have also been reported (15).In this study, serine lipids (S-lipids) were identified, which were seen at m/z 596.4898 (calculated C 35 H 66 O 6 N:596.4896) and m/z 582.4740 (calculated C 34 H 64 O 6 N: 582.4739) in the negative-ion mode, identical to those found in B. fragilis group (27).Structural characterization of these peptidolipids applying LIT MS n is exemplified by HR MS 2 on the S-lipid ion of m/z 596 (Fig. 1A), which gave rise to a major ion of m/z 354, arising from elimination of β-hydroxy C15-acyl chains as FA (15:0-FA) to form a N-C17:1-acyl-S.The MS 3 spectrum of m/z 354 (596 →354; Fig. 1B) contained the ion at m/z 267 arising from loss of [Ser -H 2 O] (87 Da), consistent with the presence of m/z 104 representing a serine anion.The spectrum also contained a major ion at m/z 324 arising from loss of HCHO (Scheme 1), and an ion at m/z 280 from further loss of CO 2 .This fragmentation process was supported by the MS 4   1C) gave rise to ions at m/z 653 (loss of 154 Da) and 635 (653 -H 2 O) arising from loss of phosphoglycerol residue, indicating the attachment of a PG residue to the GS-lipid.The MS 3 spectrum of the ion at m/z 653 (807 →653; Fig .1D) is identical to that observed for 15:0-βh17:0-GS (27), consistent with the notion that the molecule contains a GS-core structure.The results led us to define a 15:0-βh17:0-GS PG structure (GS-PG) in which the PG tail most likely attached to the -OH group of the serine residue (Scheme 2).
Characterization of PE DHC (EPC), PG DHC (GPC), new PS DHC (SPC), and DHC-1-phosphate families Nichols and colleagues identified and isolated the dominant members of DHC phospholipid family in P. gingivalis, including PG DHC, PE DHC, and 3-O-acylated PG DHC (substituted PG DHC) in which the LCB is fully saturated (i.e., sphinganine LCB) (26).However, PS DHC and DHC-1-phosphate lipid families that were detected in this study were not previously reported.We also found 3-O-acylated PE DHC and 3-O-acylated DHC-1-P lipid (Table 1).Interestingly, a novel PG ceramide and PE ceramide species with polyunsaturated LCB (d20:4-LCB) were also present (supplemental Fig. S1).The structures of these ceramide phospholipids (Table 1) were characterized by LIT MS n approaches with high-resolution MS.For example, higher energy CID (HCD) on the [M -H] -ion at m/z 722 (Fig. 2A) gave rise to major ions at 153 and 171, consistent with the notion that the molecule consists of PG head group (32).By contrast, the LIT MS 2 spectrum of m/z 722 (Fig. 2B) contained ions at m/z 648 (loss of [glycerol -H 2 O]) and 630 (loss of glycerol), indicating the presence of glycerol head group (32), and the ions at m/z 496 arising from cleavage of the N-β-hydroxyheptadecanoyl substituent as an aldehyde (loss of C 14 H 29 CHO; 226 Da) and at m/z 454 from further loss of an acetylene (CH 2 =CO) (Scheme 3).This fragmentation process is supported by the MS 3 spectrum of the ion of m/z 496 (722 → 496; Fig. 2C), which contained ions of m/z 454, along with ions of m/z 422 (loss of [glycerol -H 2 O]; 74 Da) and 404 (loss of glycerol; 92 Da) and ions of m/z 171 and 153 that signify the presence of the PG head group (Scheme 3).The above structural information readily led to the assignment of a d18:0/ βh17:0-GPC structure.The spectrum (Fig. 2B) also contained the ion of m/z 510, arising from the analogous loss of N-β-hydroxy-hexadecanoyl substituent as an aldehyde (loss of C 13 H 27 CHO; 212 Da), indicating the presence of a d19:0/βh16:0-GPC minor isomer.Similarly, the HCD MS 2 spectrum of the [M -H] -ion at m/z 742 (Fig. 2D), and CID MS 2 spectrum of the ion at m/z 742 (Fig. 2E) contained ions of m/z 668 (742 -74) and 650 (742 -92) from loss of glycerol, and ions of m/z 171 and 153 (Fig. 2D) representing PG head group, together with m/z 516 (loss of C 14 H 29 CHO; 226 Da) indicating the presence of a βh17:0 substituent.The MS 3 spectrum of m/z 516 (742 → 516; Fig. 2F) contained ions at m/z 442/ 424 (loss of glycerol), and ions at m/z 171 and 153; and the spectrum profile is similar to that of Fig. 2C.Taken    together, the results indicated the presence of a d20:4/ βh17:0-GPC, a new GPC subfamily with a d20:4-LCB.ESI high-resolution mass measurement also showed the presence of the ion series of m/z 890.6856, …, 946.7481, 960.7637, and 974.7794 (supplemental Fig. S1D) (Table 1), which are 224 Da (C 13 H 27 CH=CO) heavier than the ceramide PG lipids seen at m/z 666.4716, 680.4873, .. , 736.5499, and 756.5187, indicating the presence of an acylated Cer-PG family in which a 15:0fatty acyl group is ester bonded to the 3-hydroxy fatty acyl chain.High-resolution CID MS 2 spectrum of m/z 946 (Fig. 3A) and HCD MS 2 spectrum of m/z 946 (Fig. 3B) indicate that the major fragment ion at m/z 704 arose from elimination of the 15:0-fatty acyl substituent (loss of 15:0-FA) to form an d18:0/Nα,β-unsaturated 17:1-GPC (d18:0/17:1-GPC) (Scheme 4).The MS 3 spectrum of the ion of m/z 704 (946 →704; Fig. 3C) is dominated by ions at m/z 630 (loss of [glycerol -H 2 O]) and 612 (loss of glycerol) arising from loss of the glycerol head group, consistent with the GPC structure.The spectrum also contained m/z 454 arising from further loss of the 17:1-fatty acyl ketene (loss of C 14 H 29 CH=C=C=O; 250 Da), along with ions at m/z 362/380 from further loss of the glycerol head (from m/z 454).This latter fragmentation process is supported by the MS 4 spectrum of m/z 454 (946 →704 →454; Fig. 3D).The above results readily identify a d18:0/15:0-β17:0-GPC structure.A similar acyl-GPC subfamily consisting of the same fatty acyl substituent but with d20:4-LCB were also observed at m/z 952.7012, 966.7167, 980.7325, and 994.7481.For example, MS 2 on the ion at m/z 966 (Fig. 3E) gave rise to a major ion at m/z 724 arising from loss of 15:0-FA.The MS 3 spectrum of m/z 724 (966 →724; Fig. 3F) contained the major ion at m/z 474 arising from similar loss of the 17:1-fatty acyl ketene, indicating that the molecule contained the same N-15:0-β17:0-fatty acyl chain attached to the d20:4-LCB.The results are in accord with the earlier notion of the presence of the GPC with polyunsaturated LCB moieties.
To provide further insight into the unsaturation status of the LCB of the molecules (e.g., d20:4-LCB), we applied HRMS analysis on the reaction product of the above lipid families after hydrogenation with PtO 2 /H 2 at room temperature.The high-resolution ESI mass spectrum showed that the ions at m/z 742 and 966, the speculated d20:4/βh17:0-GPC and d20:4/15:0-βh17:0-GPC, respectively, vanished; while new ions appeared at m/z 748 and 750 that are 6 and 8 hydrogens heavier (supported by high-resolution mass measurements), than m/z 742; and ions at m/z 972 and 974 that are also 6 and 8 hydrogens heavier than m/z 966, respectively, were observed.The hydrogenation of 3 and 4 alkene bonds is consistent with the earlier notion of the presence of GPC lipids with d20:4-LCB, a polyunsaturated alkenyl amine, rather than a LCB with attachment of a benzene ring [the ring and double bond equivalent (RDB) of a benzene ring is equal to 4].
The presence of GS-PA species with even more unsaturated bonds, for example, 11.5 RDB was seen by the [M -H] -ion at m/z 1315.94, which is 2 H lighter than m/z 1317.96.The MS 2 spectrum of m/z 1315.9, again is dominated by ion of m/z 679 (data not shown) arising from similar loss of 15:0-βh17:0-GS residue.The MS 3 spectrum of m/z 679 (1315 → 679; Fig. 7H) is equivalent The LIT MS n spectra that showed the GS-PA lipid species containing 1, (A and B), 2 (C and D), 3 (E), 4 (F and G), and 5 (H) unsaturated bond in sn-1 or sn-2 of the fatty acyl chains on PA.The MS 2 spectra of the [M -H] -ions at m/z 1295 (A) and 1293 (C) yielded a major ion equivalent to a deprotonated PA anion arising from loss of 15:0-βh17:0-GS residues.These [M -H] -ions of diacyl-PA underwent the fragmentation processes identical to PA and gave rise to (1) ions from loss of sn-1 FA as acid and ketene, respectively; (2) ions from loss of sn-2 FA as acid and ketene, respectively; (3) the carboxylate anions (RCO 2 -) representing the sn-1 and sn-2 FA chains, respectively.The ions from losses of the FA chain at sn-2 are more abundant than the corresponding losses of the FA chain at sn-1, and the R 1 CO 2 -is more abundant than the R 2 CO 2 -, leading to the assignment of the FA on the glycerol backbone.Therefore, MS 3  to that of 15:0/20:5-PA.Taken together, the results define a 15:0-βh17:0-GS-15:0/20:5-PA structure.

DISCUSSION
B. fragilis and P. gingivalis belong to the Cytophaga-Flavobacteria-Bacteroides phylum and share similar genomes (37).Using MS-based shotgun lipidomic approaches, we found that several lipid families including DHC-1-P and GS-PA are present in the membranes of both B. fragilis (27) and P. gingivalis (this study).Given their close relationship, it is not surprising that these two bacteria contain common lipids, yet key differences in the lipid structure and lipid families/subfamilies were found in their lipid repertoires.For example, the GS-PA lipid of m/z 1227.9109([M -H] -) observed for B. fragilis mainly represents a 15:0/βh16:0-GS-14:0/15:0-PA, while it represents a 15:0/βh17:0-GS-15:0/13:0-PA in P. gingivalis.The PA residues in GS-PA lipid in the B. fragilis group are all saturated and branched.On the other hand, P. gingivalis produces several previously unreported GS-PA lipid subfamilies whose PAs contain 1, 2, 3, 4, or 5 unsaturated bonds.In B. fragilis, both phosphatidylinositol (PI) and PI DHC (inositol phosphoryl ceramide (IPC)) lipids are the most prominent with GS-PA lipid family also being abundant, but PG DHC (GPC) and acylated GPC are absent.In contrast, P. gingivalis lacked PI and PI DHC (IPC) lipids, with PG DHC (GPC) and acylated GPC lipids being the most prominent.The observation of the new DHC-PGP-DHC lipid family and abundant GPC in P. gingivalis in the present study, and of the recent new DHC-PIP-DHC lipid family and the abundant IPC lipids exclusively found in B. vulgatus (27) is also interesting.Therefore, it may not be that far-fetched to speculate that G(PDHC) 2 and I(PDHC) 2 are formed by condensation of two molecules of GPC and GPI, respectively, similar to the pathway by which cardiolipins in bacteria are synthesized by condensation of two molecules of phosphatidylglycerols (38).
The observation of the new SL subfamily that consists of polyunsaturated LCB found in this study is also worth attention.Although further study to determine the unsaturation status, such as the location of double bonds is required, to our knowledge, ceramides with polyunsaturated bonds (i.e., ≥ 3) in LCB have not been reported.
Nichols and coworkers reported the involvement of purified DHC-PG and GS lipids in the proinflammatory secretory reactions in gingival fibroblasts (10)(11)(12).Whether the new lipid families/subfamilies found in this study are virulence determinants contributing to human diseases remains to be tested.Nevertheless, this study to envision the entire lipidome of P. gingivalis may provide a conceptual basis for further research to achieve a better understanding of the roles the new lipids in the oral pathogen may play in the development of periodontal and other human diseases.
spectrum of m/z 324 (596 →354 →324; data not shown), which is dominated by ion of m/z 280.Loss of CO 2 from m/z 354 also gave rise to m/z 310, which further dissociated to m/ z 280 (310 -HCHO) and 292 (310 -H 2 O) by further losses of HCHO and H 2 O, respectively.The above re-

TABLE 1 .
The lipid repertoire of Porphyromona gingivalis obtained by LIT MSn with high-resolution MS