Phosphorylated Dihydroceramides from Common Human Bacteria Are Recovered in Human Tissues

Abstract

Novel phosphorylated dihydroceramide (PDHC) lipids produced by the periodontal pathogen Porphyromonas gingivalis include phosphoethanolamine (PE DHC) and phosphoglycerol dihydroceramides (PG DHC) lipids. These PDHC lipids mediate cellular effects through Toll-like receptor 2 (TLR2) including promotion of IL-6 secretion from dendritic cells and inhibition of osteoblast differentiation and function in vitro and in vivo. The PE DHC lipids also enhance (TLR2)-dependent murine experimental autoimmune encephalomyelitis (EAE), a model for multiple sclerosis. The unique non-mammalian structures of these lipids allows for their specific quantification in bacteria and human tissues using multiple reaction monitoring (MRM)-mass spectrometry (MS). Synthesis of these lipids by other common human bacteria and the presence of these lipids in human tissues have not yet been determined. We now report that synthesis of these lipids can be attributed to a small number of intestinal and oral organisms within the Bacteroides, Parabacteroides, Prevotella, Tannerella and Porphyromonas genera. Additionally, the PDHCs are not only present in gingival tissues, but are also present in human blood, vasculature tissues and brain. Finally, the distribution of these TLR2-activating lipids in human tissues varies with both the tissue site and disease status of the tissue suggesting a role for PDHCs in human disease.

Macrophage Depletion Abates Porphyromonas gingivalis–Induced Alveolar Bone Resorption in Mice

“…In addition to P. gingivalis proteins and LPS penetrating gingival tissue, P. gingivalis phosphorylated dihydoceramide lipids have been isolated from gingival tissue and subgingival plaque of chronic periodontitis patients (52)(53)(54). Whole P. gingivalis cells and certain components, including the RgpA-Kgp complexes and lipids, induce secretion of a number of proinflammatory cytokines from epithelial and fibroblast cells in vitro, such as IL-1, IL-6, IL-8, PGE 2 , macrophage chemoattractant protein (MCP)-1a, and ICAM-1 (50,54,55).…”

Free Lipid A Isolated from Porphyromonas gingivalis Lipopolysaccharide Is Contaminated with Phosphorylated Dihydroceramide Lipids: Recovery in Diseased Dental Samples

“…Instead, we reported that a unique fatty acid constituent of both lipid A and LPS of P. gingivalis, called 3-hydroxy isobranched C 17:0 (3-OH iso C 17: 0), exists in lipid products recovered predominantly from organic solvent extracts of subgingival plaque, periodontally diseased tissues, and diseased teeth, with little recovered in aqueous extracts of these samples (26,29). Since LPS partitions essentially only into the aqueous phase with this extraction technique (39), we concluded that LPS of P. gingivalis and other related Bacteroidetes bacteria (33), including Prevotella intermedia and Tannerella forsythia, is not present to a significant extent in diseased periodontal tissues. By analogy, it is presumed that LPS from nonBacteroidetes subgingival organisms also does not contaminate diseased periodontal tissues.…”

“…The possibility exists that the 3-OH iso C 17:0 recovered from organic extracts of subgingival plaque samples and periodontally diseased tissues could include free lipid A species devoid of the O-polysaccharide component of LPS. The first aim of this investigation was to isolate lipid A from P. gingivalis, and after characterization of the molecular structures using matrix-assisted laser desorption ionization tandem mass spectrometry (MALDI-MS/MS) and electrospray-MS/MS, we determined whether the dominant phosphorylated lipid A species are present in lipid extracts of periodontally diseased teeth and subgingival plaque samples.The phosphorylated dihydroceramide lipid classes of P. gingivalis that promote proinflammatory reactions and morphological changes in fibroblasts (31) are also readily detected on periodontally diseased teeth (32) and are recovered from gingival tissue samples from periodontitis sites (26,32,33). This observation is important because the phosphorylated dihydroceramide lipids of P. gingivalis are reported to engage Toll-like receptor 2 (TLR2) when promoting dendritic cell secretion of interleukin-6 (IL-6) (29) and inhibiting osteoblast function and mineral deposition in vivo and in vitro (43).…”

“…The phosphorylated dihydroceramide lipid classes of P. gingivalis that promote proinflammatory reactions and morphological changes in fibroblasts (31) are also readily detected on periodontally diseased teeth (32) and are recovered from gingival tissue samples from periodontitis sites (26,32,33). This observation is important because the phosphorylated dihydroceramide lipids of P. gingivalis are reported to engage Toll-like receptor 2 (TLR2) when promoting dendritic cell secretion of interleukin-6 (IL-6) (29) and inhibiting osteoblast function and mineral deposition in vivo and in vitro (43).…”

“…By analogy, it is presumed that LPS from nonBacteroidetes subgingival organisms also does not contaminate diseased periodontal tissues. We have since shown that organic solvent extracts of diseased periodontal tissues contain phosphorylated dihydroceramide (PDHC) lipids of P. gingivalis (32,33). These phosphorylated dihydroceramide lipids contain 3-OH iso C 17:0 .…”