Novel, acid-labile, hydroxydiether lipid cores in methanogenic bacteria.

Polar ether lipids extracted from 15 methanogenic bacteria, representative of seven genera, were screened by nuclear magnetic resonance and thin layer chromatography for the presence of hydroxyl groups on the C20-phytanyl moieties. Major amounts of hydroxydiether core lipid were confirmed for Methanosaeta concilii and discovered in two Methanosarcina species, Methanococcus voltae, and tentatively in several Methanobacterium species. Signals at 1.24 and 1.8-1.9 ppm in 1H NMR spectra are characteristic of Methanosaeta concilii lipids hydroxylated on carbon-3 (sn-3 chain). Related signals, which were shifted slightly, appeared in spectra of the polar lipids extracted from both Methanosarcina species. Following mild hydrolysis to remove the polar head groups, only two chromatographically distinct core lipids were found in significant amounts in Methanosarcina barkeri (and Methanosarcina mazei) consisting of 43% 2,3-di-O-phytanyl-sn-glycerol (C20,20-diether) and 57% C20,20-hydroxydiether. This latter core lipid differed from the hydroxydiether from M. concilii by hydroxylation, on carbon-3, of the phytanyl chain in ether linkage to the sn-2 carbon of glycerol. The structural assignment was based on identification of the novel hydroxydiether core and its methylation products by 1H NMR, 13C NMR, and mass spectroscopy. The hydroxy core lipid degraded to various products during standard methanolic HCl and sulfuric acid procedures, including a methoxy derivative (methanolic HCl) and the 3-mono-O-phytanyl-sn-glycerol

Activation of Dendritic Cells by Liposomes Prepared from Phosphatidylinositol Mannosides from Mycobacterium bovis Bacillus Calmette-Guérin and Adjuvant Activity In Vivo

Liposome vesicles could be formed at 65°C from the chloroform-soluble, total polar lipids (TPL) extracted from Mycobacterium bovis bacillus Calmette-Guérin (BCG). Mice immunized with ovalbumin (OVA) entrapped in TPL liposomes produced both anti-OVA antibody and cytotoxic T lymphocyte responses. Murine bone marrow-derived dendritic cells were activated to secrete interleukin-6 (IL-6), IL-12, and tumor necrosis factor upon exposure to antigen-free TPL liposomes. Three phosphoglycolipids and three phospholipids comprising 96% of TPL were identified as phosphatidylinositol dimannoside, palmitoyl-phosphatidylinositol dimannoside, dipalmitoyl-phosphatidylinositol dimannoside, phosphatidylinositol, phosphatidylethanolamine, and cardiolipin. The activation of dendritic cells by liposomes prepared from each purified lipid component of TPL was evaluated in vitro. A basal activity of phosphatidylinositol liposomes to activate proinflammatory cytokine production appeared to be attributable to the tuberculosteric fatty acyl 19:0 chain characteristic of mycobacterial glycerolipids, as similar lipids lacking tuberculosteric chains showed little activity. Phosphatidylinositol dimannoside was identified as the primary lipid that activated dendritic cells to produce amounts of proinflammatory cytokines several times higher than the basal level, indicating the importance of mannose residues. Although the activity of phosphatidylinositol dimannoside was little influenced by palmitoylation of mannose at C-6, a further palmitoylation at inositol C-3 diminished the induction levels of IL-6 and IL-12. Further, OVA entrapped in palmitoyl-phosphatidylinositol dimannoside liposomes was delivered to dendritic cells for major histocompatibility complex class I presentation more effectively than TPL OVA-liposomes. BCG liposomes containing mannose lipids caused up-regulation of costimulatory molecules and CD40. Thus, the inclusion of pure phosphatidylinositol mannosides of BCG in lipid vesicle vaccines represents a simple and efficient option for targeting antigen delivery and providing immune stimulation

Isopranoid- and dipalmitoyl-aminophospholipid adjuvants impact differently on longevity of CTL immune responses

The success of lipid membranes as cytotoxic T-cell (CTL) adjuvants requires targeted uptake by antigenpresenting cells (APCs) and delivery of the antigen cargo to the cytosol for processing. To target the phosphatidylserine (PS) receptor of APCs, we prepared antigen-loaded liposomes containing dipalmitoylphosphatidylserine and archaeal lipid liposomes (archaeosomes), containing an equivalent amount of archaetidylserine, and compared their ability to promote short and long-term CTL activity in animals. CTL responses were enhanced by the incorporation of PS into phosphatidylcholine/cholesterol liposomes and, to a lesser extent, into phosphatidylglycerol/cholesterol liposomes, that correlated to the amount of surface amino groups reactive with trinitrobenzoyl sulfonate. Archaeosomes contrasted to the liposome adjuvants by exhibiting higher amounts of surface amino groups and inducing superior shorter and, especially, longer-term CTL responses. The incorporation of dipalmitoyl lipids into archaeosomes induced instability and prevented long-term, but not short-term, CTL responses in mice. The importance of glycero-lipid cores (isopranoid versus dipalmitoyl) to the longevity of the CTL response achieved was shown further by incorporating dipalmitoyl phosphatidylethanolamine (DPPE) or equivalent amounts of synthetic archaetidylethanolamine (AE) into archaeosome adjuvants. Both DPPE and AE at equivalent (5 mol%) concentrations enhanced the rapidity of CTL responses in mice, indicating the importance of the head group in the short term. In the longer term, 5% of DPPE (but not 5% of AE) was detrimental. In addition to head-group effects critical to the potency of short-term CTL responses, the longer term CTL adjuvant properties of archaeosomes may be ascribed to stability imparted by the archaeal isopranoid core lipids.Peer reviewed: YesNRC publication: Ye

Archaeosomes varying in lipid composition differ in receptor-mediated endocytosis and differentially adjuvant immune responses to entrapped antigen

Archaeosomes prepared from total polar lipids extracted from six archaeal species with divergent lipid compositions had the capacity to deliver antigen for presentation via both MHC class I and class II pathways. Lipid extracts from Halobacterium halobium and from Halococcus morrhuae strains 14039 and 16008 contained archaetidylglycerol methylphosphate and sulfated glycolipids rich in mannose residues, and lacked archaetidylserine, whereas the opposite was found in Methanobrevibacter smithii, Methanosarcina mazei and Methanococcus jannaschii. Annexin V labeling revealed a surface orientation of phosphoserine head groups in M. smithii, M. mazei and M. jannaschii archaeosomes. Uptake of rhodamine-labeled M. smithii or M. jannaschii archaeosomes by murine peritoneal macrophages was inhibited by unlabeled liposomes containing phosphatidylserine, by the sulfhydryl inhibitor N-ethylmaleimide, and by ATP depletion using azide plus fluoride, but not by H. halobium archaeosomes. In contrast, N-ethylmaleimide failed to inhibit uptake of the four other rhodamine-labeled archaeosome types, and azide plus fluoride did not inhibit uptake of H. halobium or H. morrhuae archaeosomes. These results suggest endocytosis ofarchaeosomes rich in surface-exposed phosphoserine head groups via a phosphatidylserine receptor, and energy-independent surface adsorption of certain other archaeosome composition classes. Lipid composition affected not only the endocytic mechanism, but also served to differentially modulate the activation of dendritic cells. The induction of IL-12 secretion from dendritic cells exposed to H. morrhuae 14039 archaeosomes was striking compared with cells exposed to archaeosomes from 16008. Thus, archaeosome types uniquely modulate antigen delivery and dendritic cell activation