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

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

Structural and Serological Studies of the O6-Related Antigen of Aeromonas veronii bv. sobria Strain K557 Isolated from Cyprinus carpio on a Polish Fish Farm, which Contains l-perosamine (4-amino-4,6-dideoxy-l-mannose), a Unique Sugar Characteristic for Aeromonas Serogroup O6

Amongst Aeromonas spp. strains that are pathogenic to fish in Polish aquacultures, serogroup O6 was one of the five most commonly identified immunotypes especially among carp isolates. Here, we report immunochemical studies of the lipopolysaccharide (LPS) including the O-specific polysaccharide (O-antigen) of A. veronii bv. sobria strain K557, serogroup O6, isolated from a common carp during an outbreak of motile aeromonad septicemia (MAS) on a Polish fish farm. The O-polysaccharide was obtained by mild acid degradation of the LPS and studied by chemical analyses, mass spectrometry, and 1H and 13C NMR spectroscopy. It was revealed that the O-antigen was composed of two O-polysaccharides, both containing a unique sugar 4-amino-4,6-dideoxy-l-mannose (N-acetyl-l-perosamine, l-Rhap4NAc). The following structures of the O-polysaccharides (O-PS 1 and O-PS 2) were established: O-PS 1: →2)-α-l-Rhap4NAc-(1→; O-PS 2: →2)-α-l-Rhap4NAc-(1→3)-α-l-Rhap4NAc-(1→3)-α-l-Rhap4NAc-(1→. Western blotting and an enzyme-linked immunosorbent assay (ELISA) showed that the cross-reactivity between the LPS of A. veronii bv. sobria K557 and the A. hydrophila JCM 3968 O6 antiserum, and vice versa, is caused by the occurrence of common α-l-Rhap4NAc-(1→2)-α-l-Rhap4NAc and α-l-Rhap4NAc-(1→3)-α-l-Rhap4NAc disaccharides, whereas an additional →4)-α-d-GalpNAc-associated epitope defines the specificity of the O6 reference antiserum. Investigations of the serological and structural similarities and differences in the O-antigens provide knowledge of the immunospecificity of Aeromonas bacteria and are relevant in epidemiological studies and for the elucidation of the routes of transmission and relationships with pathogenicity

Correction: Structural Studies of the Lipopolysaccharide from the Fish Pathogen Aeromonas veronii Strain Bs19, Serotype O16. Mar. Drugs 2014, 12, 1298–1316

We found one editorial mistake in our published paper [1]. In Line 2 of Table 4, the same composition of sugars is given for the C4 and C5 species (in the C5 species, one residue: 6dHexNAc has been missed). [...

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

Structural Studies of the Lipopolysaccharide of Aeromonas veronii bv. sobria Strain K133 Which Represents New Provisional Serogroup PGO1 Prevailing among Mesophilic Aeromonads on Polish Fish Farms

In the present work, we performed immunochemical studies of LPS, especially the O-specific polysaccharide (O-PS) of Aeromonas veronii bv. sobria strain K133, which was isolated from the kidney of carp (Cyprinus carpio L.) during an outbreak of motile aeromonad infection/motile aeromonad septicemia (MAI/MAS) on a Polish fish farm. The structural characterization of the O-PS, which was obtained by mild acid degradation of the LPS, was performed with chemical methods, MALDI-TOF mass spectrometry, and 1H and 13C NMR spectroscopy. It was revealed that the O-PS has a unique composition of a linear tetrasaccharide repeating unit and contains a rarely occurring sugar 2,4-diamino-2,4,6-trideoxy-D-glucose (bacillosamine), which may determine the specificity of the serogroup. Western blotting and ELISA confirmed that A. veronii bv. sobria strain K133 belongs to the new serogroup PGO1, which is one of the most commonly represented immunotypes among carp and trout isolates of Aeromonas sp. in Polish aquacultures. Considering the increase in the MAI/MAS incidences and their impact on freshwater species, also with economic importance, and in the absence of an effective immunoprophylaxis, studies of the Aeromonas O-antigens are relevant in the light of epidemiological data and monitoring emergent pathogens representing unknown antigenic variants and serotypes

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

A Unique Sugar l-Perosamine (4-Amino-4,6-dideoxy-l-mannose) Is a Compound Building Two O-Chain Polysaccharides in the Lipopolysaccharide of Aeromonas hydrophila Strain JCM 3968, Serogroup O6

Lipopolysaccharide (LPS) is the major glycolipid and virulence factor of Gram-negative bacteria, including Aeromonas spp. The O-specific polysaccharide (O-PS, O-chain, O-antigen), i.e., the surface-exposed part of LPS, which is a hetero- or homopolysaccharide, determines the serospecificity of bacterial strains. Here, chemical analyses, mass spectrometry, and 1H and 13C NMR spectroscopy techniques were employed to study the O-PS of Aeromonas hydrophila strain JCM 3968, serogroup O6. MALDI-TOF mass spectrometry revealed that the LPS of A. hydrophila JCM 3968 has a hexaacylated lipid A with conserved architecture of the backbone and a core oligosaccharide composed of Hep6Hex1HexN1HexNAc1Kdo1P1. To liberate the O-antigen, LPS was subjected to mild acid hydrolysis followed by gel-permeation-chromatography and revealed two O-polysaccharides that were found to contain a unique sugar 4-amino-4,6-dideoxy-l-mannose (N-acetyl-l-perosamine, l-Rhap4NAc), which may further determine the specificity of the serogroup. The first O-polysaccharide (O-PS1) was built up of trisaccharide repeating units composed of one α-d-GalpNAc and two α-l-Rhap4NAc residues, whereas the other one, O-PS2, is an α1→2 linked homopolymer of l-Rhap4NAc. The following structures of the O-polysaccharides were established: O-PS1 →3)-α-l-Rhap4NAc-(1→4)-α-d-GalpNAc-(1→3)-α-l-Rhap4NAc-(1→ O-PS2 →2)-α-l-Rhap4NAc-(1→ The present paper is the first work that reveals the occurrence of perosamine in the l-configuration as a component of bacterial O-chain polysaccharides