Purification and characterisation of lipoglycan macroamphiphiles from Propionibacterium acnes

Lipidated macroamphiphiles such as the lipoteichoic acids and mycobacterial lipoarabinomannans are cell envelope components of Gram-positive bacteria that have been extensively associated with the pathogenesis of disease. In order to study such associations, purification of these macroamphiphiles is essential for resolving their structures and diverse biological effects. We describe herein a method for purification of lipoglycan components from Propionibacterium acnes. This method uses the existing phenol-water extraction, followed by hydrophobic interaction chromatography and an additional purification step that utilises preparative electrophoresis for the separation of two lipoglycan components. Analysis of these lipoglycans revealed evidence for a lipid anchor based on fatty acids whilst the polysaccharide moiety contained significant amounts of mannose, glucose and galactose, together with an amino sugar suspected of being a diaminohexuronic acid. These latter components have been previously identified as components of the P. acnes cell wall polysaccharide. Consequently, it is proposed that there may be a relationship between the structures of these distinctive cell envelope polymers.     

Lipid A mutants of Salmonella typhimurium. Purification and characterization of a lipid A precursor produced by a mutant in 3-deoxy-D-mannooctulosonate-8-phosphate synthetase.

We describe here the isolation, purification, and structural characterization of a lipid A precursor synthesized under nonpermissive conditions by a mutant of Salmonella typhimurium conditionally defective in the synthesis of the 3-deoxy-D-mannoctulosonate (2-keto-3-deoxyoctonate, KDO) region of the lipopolysaccharide. The precursor was isolated free from lipopolysaccharide, murein, and phospholipids by extraction of delipidated cells with 90% phenol/CHCL3/petroleum ether. The molecule was recovered from the phenol phase after precipitation of lipopolysaccharide with H2O and subsequently purified by DEAE-cellulose chromatography. Structural analyses showed that the lipid A precursor is a phosphorylated glucosamine disaccharide containing one ester and two amide-linked residues of beta-hydroxymyristate. In contrast to lipid A, the precursor disaccharide lacks ester-linked 12:0 and 14:0 fatty acids as well as KDO. The molecule contains 2 phosphate residues both of which were identified as phosphomonoesters by 31P NMR spectroscopy. One of the phosphomonoesters is located in position 1 of the reducing terminal glucosamine residue; the location of the other phosphomonoester was not determined. The structure of the precursor provides strong support for the conclusion that KDO incorporation occurs at an early stage in lipid A biosynthesis prior to the incorporation of ester-linked saturated fatty acids.     

Improved preparation of lipoteichoic acids

A procedure is described for measuring the extraction of lipoteichoic acids from gram-positive bacteria in absolute terms. Virtually complete extraction was achieved from various bacteria by hot phenol/water if the cells were disrupted. Extraction of whole and delipidated cells and of the membrane fraction gave considerably lower yields. Most of the nucleic acids co-extracted from disrupted cells was removed by treatment with nucleases. Nuclease-resistant nucleic acid, protein, polysaccharide, and teichoic acid were separated from lipoteichoic acid by anionexchange chromatography on DEAE-Sephacel or hydrophobic interaction chromatography on octyl-Sepharose. Purified preparations were essentially free of polymeric contaminants, retained their alanine ester substitution, and were in the sodium salt form. Hydrophobic interaction chromatography also made it possible to recognize contamination of lipoteichoic acid with its deacylated and lyso-form, and to discriminate molecular species containing two and three, or two and four acyl groups.     

Black lipid membranes of tetraether lipids from Thermoplasma acidophilum.

Black lipid membranes were formed of tetraether lipids from Thermoplasma acidophilum and compared to the bilayer forming lipids diphytanoylphosphatidylcholine and diphythanylglucosylglycerol. Bilayer-forming lipids varied in thickness of black lipid membranes due to the organic solvent used. Measurements of the specific membrane capacitance (Cm = 0.744 microF/cm2) showed that the membrane-spanning tetraether lipids from Thermoplasma acidophilum form a monolayer of a constant thickness of 2.5-3.0 nm no matter from which solvent. This finding corresponds to the results of Gliozzi et al. for the lipids of another archaebacterium, Sulfolobus solfataricus. Black lipid membranes were formed at room temperature with a torus from bilayer-forming lipids, however, the torus could also be formed by the tetraether-lipid itself at room temperature and at defined concentration. In these stable black lipid membranes, conductance was measured in the presence of valinomycin, nonactin, and gramicidin. At 10(-7) M concentration, valinomycin mediated higher conductance in membranes from tetraether lipids (200-1200 microS/cm2) than from bilayer-forming lipids (125-480 microS/cm2). Nonactin, at 10(-6) M concentration, mediated a 6-fold higher conductance in a tetraether lipid membrane than in a bilayer, whereas conductance, in the presence of 5 x 10(-11) M gramicidin could reach higher values in bilayers than in tetraether lipid monolayers of comparable thickness. Monensin did not increase the conductance of black lipid membranes from tetraether lipids under all conditions applied in our experiments. Poly(L-lysine) destroyed black lipid membranes. Lipopolysaccharides from Thermoplasma acidophilum were not able to form stable black lipid membranes by themselves. The lipopolysaccharide complexes from Thermoplasma acidophilum and from Escherichia coli decreased the valinomycin-mediated conductance of monolayer and bilayer membranes. This influence was stronger than that of the polysaccharide dextran.     

Bacterial cell wall macroamphiphiles: Pathogen-/microbe-associated molecular patterns detected by mammalian innate immune system

Innate immune system is the first line of host defense against invading microorganisms. It relies on a limited number of germline-encoded pattern recognition receptors that recognize conserved molecular structures of microbes, referred to as pathogen-/microbe-associated molecular patterns (PAMPs/MAMPs). Bacterial cell wall macroamphiphiles, namely Gram-negative bacteria lipopolysaccharide (LPS), Gram-positive bacteria lipoteichoic acid (LTA), lipoproteins and mycobacterial lipoglycans, are important molecules for the physiology of bacteria and evidently meet PAMP/MAMP criteria. They are well suited to innate immune recognition and constitute non-self signatures detected by the innate immune system to signal the presence of an infective agent. They are notably recognized via their lipid anchor by Toll-like receptors (TLRs) 4 or 2. Here, we review our current knowledge of the molecular bases of macroamphiphile recognition by TLRs, with a special emphasis on mycobacterial lipoglycan detection by TLR2.     

Identification of a lipoarabinomannan-like lipoglycan in the actinomycete Gordonia bronchialis

The cell envelopes of actinomycetes contain lipidated macroamphiphiles, of which the most extensively characterised are the lipoarabinomannans of mycobacteria and related bacteria. We have investigated the mycolic acid-containing actinomycete Gordonia bronchialis and identified the presence of a lipoarabinomannan-like lipoglycan. The extraction and purification procedures recovered a second amphiphilic fraction with properties suggesting a phosphatidylinositol mannoside, consistent with studies of other Gordonia species.Dedicated to the memory of our former colleague Dr. David Bendell.     

'Lipoteichoic acid' of Bifidobacterium bifidum subspecies pennsylvanicum DSM 20239. A lipoglycan with monoglycerophosphate side chains

The lipid macroamphiphile of Bifidobacterium bifidum subsp. pennsylvanicum DSM 20239 was extracted with phenol/water and purified by treatment with nucleases and hydrophobic interaction chromatography. From analytical data, the results of Smith degradation, hydrolysis with HF and methylation studies, the following structure is proposed: (formula; see text) where n and m are approximately 7-10 and 8-15, respectively. The monoglycerophosphate residues have the sn-glycero-1-phosphate configuration; 20-50% of them are substituted with L-alanine in ester linkage. The lipid anchor is most likely a galactosyldiacylglycerol, part of which carries a third fatty acid. This is the first example among gram-positive bacteria of a glycerophosphate-containing lipid macroamphiphile that carries the glycerophosphate residues as monomeric side chains on a lipoglycan. Further, it contains L-alanine in place of the D-alanine found in lipoteichoic acids.     

Proteomics Analysis of Thermoplasma Quinone Droplets

A novel type of lipid droplet/lipoprotein (LD/LP) particle from Thermoplasma acidophilum has been identified recently, and based on biochemical evidences, it was named Thermoplasma Quinone Droplet (TaQD). The major components of TaQDs are menaquinones, and to some extent polar lipids, and the 153 amino acid long Ta0547 vitellogenin‐N domain protein. In this paper, the aim is to identify TaQD proteome components with 1D‐SDS‐PAGE/LC–MS/MS and cross reference them with Edman degradation. TaQD samples isolated with three different purification methods—column chromatography, immunoprecipitation, and LD ultracentrifugation—are analyzed. Proteins Ta0093, Ta0182, Ta0337, Ta0437, Ta0438, Ta0547, and Ta1223a are identified as constituents of the TaQD proteome. The majority of these proteins is uncharacterized and has low molecular weight, and none of them is predicted to take part in lipid metabolism. Bioinformatics analyses does not predict any interaction between these proteins, however, there are indications of interactions with proteins taking part in lipid metabolism. Whether if TaQDs provide platform for lipid metabolism and the interactions between TaQD proteins and lipid metabolism proteins occur in the reality remain for further studies.     

Purification and fractionation of lipopolysaccharide from gram-negative bacteria by hydrophobic interaction chromatography

By hydrophobic interaction chromatography on octyl-Sepharose, lipopolysaccharide (LPS) of Escherichia coli Re mutant and of wild-type smooth-form (S-form) Salmonella typhimurium and Salmonella abortus equi is fractionated according to increasing amount of fatty acids. Thereby a fractionation of S-form LPS according to the length of the O-polysaccharide chain also occurs, because with increasing of fatty acids there is a decrease in the mean length of the O-polysaccharide chain from approximately 30 to 4 repeating units. Molecular species of Re-mutant LPS contain four 3-hydroxytetradecanoyl residues in addition to which dodecanoic, tetradecanoic and possibly hexadecanoic acid, appear in this sequence. Among the molecular species of S-form LPS, dodecanoic, tetradecanoic and hexadecanoic acids appear in the same order, but in contrast to Re-mutant LPS a significant fraction of S-form LPS contains less than four 3-hydroxytetradecanoyl residues. Hydrophobic interaction chromatography also proved an effective one-step purification procedure of LPS as was shown with a crude preparation from S-form S. typhimurium.     

Studies on the chemical composition of lipopolysaccharide from Neisseria meningitidis group B.

A lipopolysaccharide was isolated from Neisseria meningitidis group B by phenol/water extraction and purified by differential ultracentrifugation. This preparation exhibited endotoxic properties as shown by the limulus-lysate assay. Mild acid hydrolysis of the lipopolysaccharides yielded a lipid A fraction and a polysaccharide fraction. The lipid A fraction contained fatty acids, phosphorus and glucosamine. Analysis of the polysaccharide fraction revealed the presence of glucose, galactose, glucosamine, 2-keto-3-deoxyoctonic acid and phosphorus. There was no heptose.     

Structure of macroamphiphiles from several Bifidobacterium strains.

Lipoteichoic acid-like substances, macroamphiphiles, were isolated from cell homogenates of Bifidobacterium bifidum YIT 4007 and YIT 4013, Bifidobacterium breve YIT 4010 and YIT 4014, and Bifidobacterium longum YIT 4021 by phenol extraction followed by nuclease digestion, gel chromatography, ion-exchange chromatography, and hydrophobic interaction chromatography. The macroamphiphile preparations from these five strains contained D-glucose, D-galactose, glycerol, phosphorus, L-alanine, and fatty acids in molar ratios of 1.00, 1.57 to 1.95, 1.02 to 1.99, 0.97 to 1.72, 0.15 to 0.46, and 0.16 to 0.43. Data from structural analyses including methylation, 1H nuclear magnetic resonance measurement, alkaline hydrolysis, mild acid hydrolysis, and hydrogen fluoride treatment led to the most likely common structure for the macroamphiphiles of the examined strains, (formula; see text) where Gro-P is glycerophosphate, m is the number of repeating units of galactofuranan, and n is the number of repeating units of glucan. Whereas the polymers from the respective strains differed in the numbers of repeating units of the galactofuranan and glucan moieties and in the number of fatty acid residues, the proposed structure is essentially the same as that reported previously for the macroamphiphile of B. bifidum subsp. pennsylvanicum DSM 20239 by W. Fischer (Eur. J. Biochem. 165:639-646, 1987).     

Characterisation of a lipomannan lipoglycan from the mycolic acid containing actinomycete Dietzia maris

Lipoglycans such as the mycobacterial lipoarabinomannans (LAM) are important cell envelope components of actinomycetes. To further our understanding of the diversity of these enigmatic macromolecules the lipoglycan composition of Dietzia maris has been investigated. Phenol-water extraction and hydrophobic interaction chromatography were used to purify a lipoglycan which was unusually small and predominantly lipomannan in nature. The presence of minor levels of arabinose along with components consistent with the presence of a phosphatidylinositol anchor suggest that this lipoglycan is a novel representative of the lipomannan/LAM structural archetype. This was further supported by the observed cross-reaction of the D. maris lipoglycan with an antiserum raised against LAM from Mycobacterium tuberculosis. These findings reveal a previously unsuspected diversity in the lipoglycan composition of the mycolic acid containing actinomycetes and are further discussed in relation to the apparent absence of phosphatidylinositolmannoside glycolipids in D. maris.     

Structural analysis of the lipopolysaccharide from Chlamydophila psittaci strain 6BC.

The lipopolysaccaride of Chlamydophila psittaci 6BC was isolated from tissue culture-grown elementary bodies using a modified phenol/water procedure followed by extraction with phenol/chloroform/light petroleum. Compositional analyses indicated the presence of 3-deoxy-Dmanno-oct-2-ulosonic acid, GlcN, organic bound phosphate and fatty acids in a molar ratio of approximately 3. 3 : 2 : 1.8 : 4.6. Deacylated lipopolysaccharide was obtained after successive microscale treatment with hydrazine and potassium hydroxide, and was then separated by high performance anion-exchange chromatography into two major fractions, the structures of which were determined by 600 MHz NMR spectroscopy as alpha-Kdo-(2-->8)-alpha-Kdo-(2-->4)-alpha-Kdo-(2-->6)-beta-D-GlcpN -(1 -->6)-alpha-D-GlcpN 1,4'-bisphosphate and alpha-Kdo-(2-->4)-[alpha-Kdo-(2-->8)]-alpha-Kdo-(2-->4)-alpha-Kdo-(2- ->6)-beta-D-GlcpN-(1-->6)-alpha-D-GlcpN 1,4'-bisphosphate. The distribution of fatty acids in lipid A was determined by compositional analyses and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry experiments on lipid A and de-O-acylated lipid A. It was shown that the carbohydrate backbone of lipid A is replaced by a complex mixture of fatty acids, including long-chain and branched (R)-configured 3-hydroxy fatty acids, the latter being exclusively present in an amide linkage.     

Studies in lipid histochemistry

Summary Conditions for a selective extraction of nonpolar lipids from tissue sections with acetone were investigated using methods of lipid chromatography, histochemistry and quantitative determination of lipid phosphorus.Extraction of nonpolar lipids is selective when water (present either in acetone or in tissue sections) is completely excluded from the extraction procedure and the extraction is carried out at 0–4° C for 20–30 minutes. Under these conditions a negligible amount of polar lipids is extracted which cannot be demonstrated histochemically. A very small amount of nonpolar lipids remaining in sections cannot be demonstrated histochemically either.A method for the preparation of anhydrous acetone was recommended and an extraction procedure devised. This is to be applied in cases where nonpolar and polar lipids are to be distinguished and as an integral part of all types of phospholipid stains.When water is present during the extraction procedure (either in acetone or in tissue sections) significant extraction of all polar lipids occurs which is proportional to the content of water, to the length of extraction and to the temperature. Under these conditions the extraction of nonpolar lipids is somewhat slower.The significance of selective extraction with anhydrous acetone in histochemical analysis of lipids is discussed particularly with reference to lipids in atherosclerotic plaques.     

Lipids of Thermoplasma acidophilum

Cells of Thermoplasma acidophilum contain about 3% total lipid on a dry weight basis. Total lipid was found to contain 17.5% neutral lipid, 25.1% glycolipid, and 56.6% phospholipid by chromatography on silicic acid. The lipids contain almost no fatty acid ester groups but appear to have long-chain alkyl groups in ether linkages to glycerol. The phospholipid fraction includes a major component which represents about 80% of the lipid phosphorus and 46% of the total lipids. We believe this component to be a long-chain isopranol glycerol diether analogue of glycerolphosphoryl monoglycosyl diglyceride. The glycolipids appear to contain isopranol diether analogues. Several components of the complex, neutral lipid fraction have been identified as hydrocarbons, vitamin K(2)-7, and isopranol glycerol diether analogues. Sterols are present in the neutral lipids but do not appear to be synthesized by the organism.     

Immunochemical characteristics of a lipopolysaccharide from Pseudomonas aurantiaca

A lipopolysaccharide was isolated from Pseudomonas aurantiaca IMB 31 by extraction with aqueous phenol and purified by ultracentrifugation. The lipopolysaccharide was confined to the phenol phase. Fucosamine (2-amino-2,6-dideoxygalactose) (36%) and bacillosamine (2,4-diamino-3,4,6-trideoxyglucose) (23%) were identified as hypothetic components of the O-chain in the carbohydrate moiety of the macromolecule using the techniques of paper chromatography, gas-liquid chromatography and ion-exchange chromatography on an amino acid analyser. Rhamnose, glucose, galactose, glucosamine and galactosamine were detected as hypothetical components of the core in the lipopolysaccharide composition, as well as 2-keto-3-deoxyoctonic acid, heptose, alpha-alanine and phosphorus, usual components of the core in Pseudomonas. The following predominant fatty acids were identified in the composition of lipid A using the techniques of gas-liquid chromatography with standard compounds and gas-liquid mass spectrometry: 3-OH C10:0 (14.4%), C12:0 (30.5%), 2-OH C12:0 (14.9%), 3-OH C12:0 (17.4%), C16:0 (9.9%). The serological relationship between P. aurantiaca strains was studied, and their phylogenetic relationship with P. fluorescens is discussed.     

Identification of lipoglycan antigens from the Acholeplasma laidlawii cell membrane in crossed immunoelectrophoresis

Membranes from the wall-less prokaryote Acholeplasma laidlawii contain a component termed lipoglycan or lipopolysaccharide (LPS). The lipoglycan has extraction properties, which are similar to those of LPS of gram-negative bacteria, but it is chemically distinct from bacterial LPS. The membrane-bound lipoglycan of A. laidlawii did not seem to be particularly immunogenic and antibodies against it could not always be detected by rocket immunoelectrophoresis (RIE) or crossed immunoelectrophoresis (CIE) in hyperimmune sera raised against membranes. The immunoprecipitate corresponding to the lipoglycan, obtained by CIE of Tween 20-solubilized A. laidlawii membranes, has been identified and shown to be both a cathodically and anodically migrating component at pH 8.6. The shape of the immunoprecipitate in both RIE and CIE showed that the lipoglycan antigen is composed of at least two components, which are immunologically related.     

Characterization of the precursor of tetraether lipid biosynthesis in the thermoacidophilic archaeon<Emphasis Type="Italic"> Thermoplasma acidophilum</Emphasis>

Polar lipid biosynthesis in the thermoacidophilic archaeon Thermoplasma acidophilum was analyzed using terbinafine, an inhibitor of tetraether lipid biosynthesis. Cells of T. acidophilum were labeled with [(14)C]mevalonic acid, and their lipids were extracted and analyzed by two-dimensional thin-layer chromatography. Lipids labeled with [(14)C]mevalonic acid, [(14)C]glycerol, and [(32)P]orthophosphoric acid were extracted and hydrolyzed under different conditions to determine the structure of polar lipids. The polar lipids were estimated to be archaetidylglycerol, glycerophosphatidylcaldarchaetidylglycerol, caldarchaetidylglycerol, and beta- l-gulopyranosylcaldarchaetidylglycerol, the main polar lipid of T. acidophilum. Pulse and chase experiments with terbinafine revealed that one tetraether lipid molecule is synthesized by head-to-head condensation of two molecules of archaetidylglycerol and that a sugar group of tetraether phosphoglycolipid is expected to attach to the tetraether lipid core after head-to-head condensation in T. acidophilum. A precursor accumulated in the presence of terbinafine with a fast-atom-bombardment mass spectrometry peak m/z 806 was compatible with archaetidylglycerol. The relative height of the peak m/z 806 decreased after removal of the inhibitor. The results suggest that most of the precursor, archaetidylglycerol, is in fully saturated form.     

Detergent-solubilized proteoglycans in rat testicular Sertoli cells

Rat Sertoli cells were cultured for 48 h in the presence of [35S]sulfate and extracted with 4 M guanidine chloride. In this extract, a Sepharose CL-2B Kav 0.10 proteoheparan appeared lipid associated, since after addition of detergent it emerged at Kav = 0.65 on Sepharose CL-2B. Treatment of cells with 0.2% Triton X-100 released 35S-labeled material which was purified by ion-exchange chromatography and hydrophobic interaction chromatography on octyl-Sepharose. Proteoglycan with affinity for octyl-Sepharose (Kav = 0.30 and 0.12 on Sepharose CL-4B and CL-6B, respectively) mostly carried heparan sulfate chains with Kav = 0.38 and minor proportion of heparan chains with Kav = 0.77 on Sepharose CL-6B. An association with lipids was confirmed by intercalation into liposomes of this proteoheparan which might be anchored in the plasma membrane, via an hydrophobic segment and/or covalently linked to an inositol-containing phospholipid. Non-hydrophobic material consisted of: (i) proteoheparan slightly smaller in size than lipophilic proteoheparan and possibly deriving from this one and (ii) two heparan sulfate glycosaminoglycan populations (Kav = 0.38 and 0.86 on Sepharose CL-6B) corresponding to single glycosaminoglycan chains and their degradation products.     

Stomatococcus mucilaginosus produces a mannose-containing lipoglycan rather than lipoteichoic acid

Five strains of Stomatococcus mucilaginosus were investigated to determine whether the organism produces a lipoteichoic acid or a lipoglycan. Crude phenol extracts were purified by hydrophobic interaction chromatography and shown to contain lipoglycan. The major carbohydrate component present was mannose, indicating that the macroamphiphile is a lipomannan. The fatty acid composition of the lipoglycan was similar to that of stomatococcal whole cells. These data provide additional chemotaxonomic evidence supporting the suprageneric classification of the genus Stomatococcus within a group of actinomycete genera that also includes the genus Micrococcus.Abbreviations LTA Lipoteichoic acid - HIC Hydrophobic interaction chromatography - FAMEs Fatty acid methyl esters