Occurrence of New Polyenoic Very Long Chain Acyl Residues in Lipids from Acanthamoeba castellanii

The cellular fatty acid composition of Acanthamoeba castellanii, a unicellular bacteriovorous organism, was reinvestigated. Lipids from amoebae grown axenically in proteose peptone-yeast extract-glucose medium were extracted with chloroform–methanol and separated by silicic acid column chromatography into non-polar and polar fractions. The fatty acid composition of the lipids and the double-bond position of the unsaturated acids have been determined by capillary gas chromatography-mass spectrometry (GC-MS) of their corresponding methyl esters, 2-alkenyl-4,4-dimethyloxazoline (DMOX) derivatives and dimethyldisulfide (DMDS) adducts. Evidence is given that lipids from A. castellanii in addition to the three already identified saturated straight chain fatty acids: tetradecanoic (C14:0), hexadecanoic (C16:0), octadecanoic (C18:0), and six preponderant unsaturated fatty acids: hexadecenoic (C16:1 Δ7), octadecenoic (C18:1 Δ9), octadecadienoic (C18:2 Δ9,12), eicosadienoic (C20:2 Δ11,14), eicosatrienoic (C20:3 Δ8,11,14), and eicosatetraenoic (C20:4 Δ5,8,11,14), contain additionally four very long chain unsaturated fatty acids: octacosenoic (C28:1 Δ21), octacosadienoic (C28:2 Δ5,21), triacontadienoic (30:2 Δ21,24), and triacontatrienoic (C30:3 Δ5,21,24) previously unreported in lipids of A. castellanii. These new long chain fatty acids account for approximately 25% of total fatty acids. To our knowledge, this is the first report of very long chain polyenoic fatty acids present in lipids extracted from A. castellanii cells

Localization of the attachment site of oligoglucans to Mesorhizobium

The location and nature of the linkage between peptidoglycan and oligoglucans in the cell wall of Mesorhizobium loti HAMBI 1148 have been defined by the analysis of nitrous acid deamination of peptidoglycan glucosaminyl residues. The MurN

Alteration of O-specific polysaccharide structure of symbiotically defective Mesorhizobium loti mutant 2213.1 derived from strain NZP2213

Mesorhizobium loti mutant 2213.1 derived from the wild-type strain NZP2213 by Tn5 mutagenesis showed impaired effectiveness of symbiosis with the host plant Lotus corniculatus (Turska-Szewczuk et al., 2007 Microbiol Res, in press). The inability of lipopolysaccharide (LPS) isolated from the mutant 2213.1 strain or de-O-acetylated LPS of the parental cells to inactivate phage A1 particles implicated alterations in the LPS structure. The O-specific polysaccharide of the mutant was studied by chemical analyses along with 1H and 13C NMR spectroscopy, which clearly confirmed alterations in the O-chain structure. 2D NMR data showed that the mutant O-polysaccharide consists of a tetrasaccharide repeating unit containing non-substituted as well as O-acetylated or O-methylated 6-deoxytalopyranose residues. Additionally, an immunogold assay revealed a reduced number of gold particles on the mutant bacteroid cell surface, which could result from both a diminished amount of an O-antigenic determinant in mutant LPS and modifications of structural epitopes caused by alterations in O-acetylation or O-methylation of sugar residues. Western immunoblot assay of alkaline de-O-acetylated lipophilic M. loti NZP2213 LPS showed no reactivity with homologous serum indicating a role of O-acetyl groups in its O-specificity

Occurrence of New Polyenoic Very Long Chain Acyl Residues in Lipids from Acanthamoeba castellanii

The cellular fatty acid composition of Acanthamoeba castellanii, a unicellular bacteriovorous organism, was reinvestigated. Lipids from amoebae grown axenically in proteose peptone-yeast extract-glucose medium were extracted with chloroform–methanol and separated by silicic acid column chromatography into non-polar and polar fractions. The fatty acid composition of the lipids and the double-bond position of the unsaturated acids have been determined by capillary gas chromatography-mass spectrometry (GC-MS) of their corresponding methyl esters, 2-alkenyl-4,4-dimethyloxazoline (DMOX) derivatives and dimethyldisulfide (DMDS) adducts. Evidence is given that lipids from A. castellanii in addition to the three already identified saturated straight chain fatty acids: tetradecanoic (C14:0), hexadecanoic (C16:0), octadecanoic (C18:0), and six preponderant unsaturated fatty acids: hexadecenoic (C16:1 Δ7), octadecenoic (C18:1 Δ9), octadecadienoic (C18:2 Δ9,12), eicosadienoic (C20:2 Δ11,14), eicosatrienoic (C20:3 Δ8,11,14), and eicosatetraenoic (C20:4 Δ5,8,11,14), contain additionally four very long chain unsaturated fatty acids: octacosenoic (C28:1 Δ21), octacosadienoic (C28:2 Δ5,21), triacontadienoic (30:2 Δ21,24), and triacontatrienoic (C30:3 Δ5,21,24) previously unreported in lipids of A. castellanii. These new long chain fatty acids account for approximately 25% of total fatty acids. To our knowledge, this is the first report of very long chain polyenoic fatty acids present in lipids extracted from A. castellanii cells

Identification of unusual phospholipid fatty acyl compositions of Acanthamoeba castellanii.

Acanthamoeba are opportunistic protozoan pathogens that may lead to sight-threatening keratitis and fatal granulomatous encephalitis. The successful prognosis requires early diagnosis and differentiation of pathogenic Acanthamoeba followed by aggressive treatment regimen. The plasma membrane of Acanthamoeba consists of 25% phospholipids (PL). The presence of C20 and, recently reported, 28- and 30-carbon fatty acyl residues is characteristic of amoeba PL. A detailed knowledge about this unusual PL composition could help to differentiate Acanthamoeba from other parasites, e.g. bacteria and develop more efficient treatment strategies. Therefore, the detailed PL composition of Acanthamoeba castellanii was investigated by 31P nuclear magnetic resonance spectroscopy, thin-layer chromatography, gas chromatography, high performance liquid chromatography and liquid chromatography-mass spectrometry. Normal and reversed phase liquid chromatography coupled with mass spectrometric detection was used for detailed characterization of the fatty acyl composition of each detected PL. The most abundant fatty acyl residues in each PL class were octadecanoyl (18∶0), octadecenoyl (18∶1 Δ9) and hexadecanoyl (16∶0). However, some selected PLs contained also very long fatty acyl chains: the presence of 28- and 30-carbon fatty acyl residues was confirmed in phosphatidylethanolamine (PE), phosphatidylserine, phosphatidic acid and cardiolipin. The majority of these fatty acyl residues were also identified in PE that resulted in the following composition: 28∶1/20∶2, 30∶2/18∶1, 28∶0/20∶2, 30∶2/20∶4 and 30∶3/20∶3. The PL of amoebae are significantly different in comparison to other cells: we describe here for the first time unusual, very long chain fatty acids with Δ5-unsaturation (30∶35,21,24) and 30∶221,24 localized exclusively in specific phospholipid classes of A. castellanii protozoa that could serve as specific biomarkers for the presence of these microorganisms