Extraction and purification of lipoteichoic acids from gram-positive bacteria

Hot and cold, 80% aqueous phenol extraction procedures together with an aqueous extraction technique have been evaluated for the isolation of lipoteichoic acids from the cytoplasmic membrane of Gram-positive bacteria. Lipoteichoic acids of Staphylococcus aureusH, Micrococcus 2102, Bacillus subtilis 168, and Bacillus subtilis W-23 were examined as each of them emphasises a different problem of contamination. The purity of the lipoteichoic acids with respect to cell-wall material, nucleic acid, and protein is discussed together with the criteria of purity which enables critical structural analysis of lipoteichoic acids to be carried out.     

Comparative Studies on the Isolation of Membrane Lipoteichoic Acid from Lactobacillus fermenti

Preparations of membrane lipoteichoic acid containing different amounts of protein were isolated from intact organisms of Lactobacillus fermenti NCTC 6991 by various procedures chosen for their ability to disrupt the hydrophobic interaction of lipoteichoic acid with other membrane components. The highest yield of lipoteichoic acid was obtained with hot aqueous phenol, and this preparation contained very little protein. Partial removal of cell lipids with chloroform-methanol followed by extraction with water at 100 C gave a lipoteichoic acid-protein complex that was a very effective immunogen; immunogenicity was shown to relate to protein content, though the specificity of the antibodies was directed against the teichoic acid component.     

Lipoteichoic Acid Localization in Mesosomal Vesicles of Staphylococcus aureus

Mesosomal vesicles and plasma membranes of Staphylococcus aureus ATCC 6538P have been prepared and examined for the presence of lipoteichoic acid. Lipids were first removed by treatment with pyridine-acetic acid-butanol (22:31:100, vol/vol/vol) and chloroform-methanol (2:1, vol/vol). Subsequently, lipoteichoic acid was removed with 40% phenol in water. The lipoteichoic acid from mesosomal vesicles was characterized by (i) equimolar glycerol and phosphate, (ii) alanine upon hydrolysis (2 N NH₄OH, 18 h, 22 C), and (iii) fatty acids, diglycerol triphosphate, glycerol monophosphate, and glycerol diphosphate upon alkaline hydrolysis (1 N NaOH, 3h, 100 C). The plasma membranes contained no lipoteichoic acid. The presence in mesosomal vesicles of 18% of the dry weight as lipoteichoic acid and its absence from plasma membranes provide the first major chemical differences between these organelles. A study of the lipoteichoic acid content in various fractions of the cell showed that the mesosomal vesicles were the major and probably the sole site for the localization of the lipoteichoic acid in these organisms. A new method for the preparation of mesosomes in increased yields is reported. A theory for the control of cell division involving lipoteichoic acid and the mesosome is proposed.     

Immunochemical studies on the lipoteichoic acids of Bifidobacterium bifidum subsp. pennsylvanicum

Antisera to lipoteichoic acid of Bifidobacterium bifidum subsp. pennsylvanicum were obtained by injecting lipoteichoic acid/methylated BSA complexes into rabbits. Precipitin tests showed that the glycerol phosphate backbone is primarily responsible for serological specificity while the polysaccharide part of the molecule plays a minor role. Whole cells of B. bifidum subsp. pennsylvanicum were capable of absorbing antibodies, indicating the presence of lipoteichoic acid (14% of the total content) at or near the bacterial surface. Cross-reactivity with strains of the genera Bifidobacterium and Lactobacillus was tested using absorption of antiserum by whole bacteria and reactivity of phenol extracts. The results indicated that lipoteichoic acid is a common antigen within the genus Bifidobacterium. The cross-reactivity with the lactobacilli tested was very low.     

Studies on glucosyltransferase and endogenous glucosyl acceptor in Bacillus cereus AHU 1030 membranes

A glucosyltransferase, extracted from the membranes of Bacillus cereus AHU 1030 with Tris-HCl buffer containing 0.1% Triton X-100 at pH 9.5, was separated from an endogenous glucosyl acceptor by chromatography on DEAE-Sepharose CL-6B subsequent to chromatography on Sepharose 6B. Structural analysis data showed that the glucosyl acceptor was a glycerol phosphate polymer linked to beta-gentiobiosyl diglyceride. The enzyme catalyzed the transfer of glucosyl residues from UDP-glucose to C-2 of the glycerol residues of repeating units of the acceptor. On the other hand, a lipoteichoic acid which contained 0.3 D-alanine residue per phosphorus was isolated from the cells by phenol treatment at pH 4.6. Except for the presence of D-alanine, this lipoteichoic acid had the same structure as the glucosyl acceptor. The rate of glucosylation observed with the D-alanine-containing lipoteichoic acid as the substrate was less than 40% of that observed with the D-alanine-free lipoteichoic acid, indicating that the ester-linked D-alanine in the lipoteichoic acid interferes with the action of the glucosyltransferase. The enzyme also catalyzed glucosylation of poly(glycerol phosphate) which was synthesized in the reaction of a separate enzyme fraction with CDP-glycerol. Thus, it is likely that the glucosyltransferase functions in the synthesis of cell wall teichoic acid.     

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.     

The wall associated lipoteichoic acid ofStreptococcus sanguis

A competitive ELISA is described for the measurement of lipoteichoic acid. The assay was used to determine the wall associated lipoteichoic acid ofStreptococcus sanguis which was found to represent only 2–4% of the phenol extractable content. Extracellular lipoteichoic acid was detected even after exhaustive cell washing. This material was not the result ofde novo synthesis because membrane de-polarization had no effect on the amount detected. Since extracellular lipoteichoic acid interfered with the measurement of cell surface antigen, cells were fixed with glutaraldehyde prior to assay. Lipoteichoic acid was demonstrated on the surface of fixed cells which did not leak antigen. The relevance of fixation used in antigen location studies by electron microscopy of immune-labelled cells is discussed.     

Structure of the lipoteichoic acids from Bifidobacterium bifidum spp. pennsylvanicum

The lipoteichoic acids from Bifidobacterium bifidum spp. pennsylvanicum were extracted from cytoplasmic membranes or from disintegrated bacteria with aqueous phenol and purified by gel chromatography. The lipoteichoic acid preparations contained phosphate, glycerol, galactose, glucose and fatty acids in a molar ratio of 1.0:1.0:1.3:1.2:0.3. Chemical analysis and NMR studies of the native preparations and of products from various acid and alkaline hydrolysis procedures gave evidence for the structure of two lipoteichoic acids. The lipid anchor appeared to be 3-O-(6'-(sn-glycero-1-phosphoryl)diacyl-beta-D-galactofuranosyl)-sn-1, 2-diacylglycerol. The polar part showed two structural features not previously described for lipoteichoic acids. A 1,2-(instead of the usual 1,3-) phosphodiester-linked sn-glycerol phosphate chain is only used substituted at the terminal glycerol unit with a linear polysaccharide, containing either beta(1----5)-linked D-galactofuranosyl groups or beta(1----6)-linked D-glucopyranosyl groups.     

Glycerol teichoic acid as an antigenic determinant in a Gram-negative bacterium Butyrivibrio fibrisolvens.

An antigenic determinant isolated from a strain of the Gram-negative bacterium Butyrivibrio fibrisolvens reacted with specific antisera to the polyglycerophosphate backbone of membrane teichoic acids of lactobacilli. It gave a reaction of identity with membrane glycerol lipoteichoic acid and glycerol teichoic acid preparations from lactobacilli, and with phenol extracts of other Gram-positive bacteria. The antigen-antibody reactions was strongly inhibited by glycerol-phosphoryl-glycerol-phosphoryl-glycerol and the chemical composition was consistent with glycerol teichoic acid. It was concluded that this Gram-negative bacterium contained a glycerol teichoic acid whose polyglycerophospate backbone was acting as antigenic determinant. Extracts of 33 out of 52 other strains of butyrivibrios examined gave similar reactions.     

Effect of carrier molecules on production and properties of extracellular hemolysin produced byStreptococcus agalactiae

Streptococcus agalactiae type la strain 090 produced a cell-associated hemolysin during exponential growth in medium lacking proteins. Growth of the organism in medium containing proteins or medium supplemented with Tween 40 resulted in the appearance of extracellular hemolytic activity that was filterable. Maximum extracellular hemolytic activity was obtained in the late exponential phase of growth corresponding to the maximum number of cells. Extracellular hemolysin released in medium containing proteins could be precipitated by ammonium sulfate. Cell-associated hemolysin could be extracted in the cold by purified lipoteichoic acid from the organism. Purification and characterization of the extracellular hemolysin by column chromatography showed that the hemolysin was associated with molecules eliciting its release. Hemolysin associated with lipoteichoic acid or Tween 40 had an apparent molecular weight of 1,800,000 or 60,000 daltons, respectively.     

Apolipophorin-III and the interactions of lipoteichoic acids with the immediate immune responses of Galleria mellonella

We investigated the effects of lipoteichoic acids, surface components of Gram-positive bacteria, on the hemocytes and phenoloxidase activity in last instar Galleria mellonella larvae, as well as the binding of apolipophorin-III, an insect lipid-binding protein, to lipoteichoic acids. Binding of apolipophorin-III to lipoteichoic acid was studied using an assay based on 1,9-dimethylmethylene blue. Apolipophorin-III bound the lipoteichoic acids from Bacillus subtilis, Enterococcus hirae, and Streptococcus pyogenes and to intact cells of E. hirae. E. hirae lipoteichoic acid promoted the binding of apolipophorin-III to the cells of this species. All lipoteichoic acids tested caused a dose- and time-dependent drop in the total counts of hemocytes and, depending on the species of lipoteichoic acid, partial or complete depletion of plasmatocytes. Granulocyte counts were not affected. Apolipophorin-III prevented partially the loss of plasmatocytes due to B. subtilis lipoteichoic acid. All three lipoteichoic acids studied activated phenoloxidase in vitro; injections of B. subtilis lipoteichoic acid into the larvae elevated the phenoloxidase activity, whereas injections of E. hirae or S. pyogenes lipoteichoic acid, or apolipophorin-III alone, suppressed it. Apolipophorin-III decreased the activation of phenoloxidase by B. subtilis lipoteichoic acid.     

Increased carbohydrate substitution of lipoteichoic acid during inhibition of protein synthesis.

Decreases in electrophoretic mobilities of intracellular lipoteichoic acid, intracellular deacylated lipoteichoic acid, and extracellular deacylated lipoteichoic acid were observed during inhibition of protein synthesis in Streptococcus faecium after exposure to chloramphenicol or valine deprivation. Increased carbohydrate content, and thus an increased mass-to-charge ratio, rather than changes in ester alanine content or novel fatty acid substitutions, appeared to account for the decreased electrophoretic mobilities. The increase in carbohydrate content, as judged from mobility measurements, was progressive over time and appeared to occur on biosynthetically new lipoteichoic acid as well as on lipoteichoic acid made before inhibition of protein synthesis.     

Analysis of the membrane lipocarbohydrate antigen of Clostridium difficile by polyacrylamide gel electrophoresis and immunoblotting

Abstract The membrane lipocarbohydrate antigen (lipoteichoic acid analogue) of Clostridium difficile has been purified by aqueous phenol extraction and Sepharose 6B chromatography. After analysis by polyacrylamide gel electrophoresis (PAGE) and immunoblotting it has been shown to consist of a series of components of differing M _r. It appears as a regularly spaced ladder pattern similar to those shown for the lipopolysaccharide (LPS) of many Gram-negative bacteria.     

The function of beta-N-acetyl-D-glucosaminyl monophosphorylundecaprenol in biosynthesis of lipoteichoic acids in a group of Bacillus strains

Membrane preparations, obtained from Bacillus strains which have N-acetylglucosamine-linked lipoteichoic acids in their membranes, were shown to catalyze the transfer of N-[14C]acetylglucosamine (GlcNAc) from beta-[14C]GlcNAc-P-undecaprenol to endogenous polymer. In this reaction, alpha-GlcNAc-P-undecaprenol or alpha-GlcNAc-PP-undecaprenol could not substitute for beta-GlcNAc-P-undecaprenol as the N-acetylglucosamine donor. This enzyme was most active at pH 6.0 and in the presence of 40 mM MgCl2. The apparent Km for beta-GlcNAc-P-undecaprenol was 2 microM. The radioactive polymer products, solubilized by hot phenol treatment, coincided with lipoteichoic acids in chromatographic behavior. Hydrogen fluoride treatment of the polymer products gave a major fragment identical with GlcNAc(alpha 1----2)glycerol, which corresponded to the dephosphorylated repeating units of the lipoteichoic acids in the examined strains. Thus it is concluded that beta-GlcNAc-P-undecaprenol serves as the donor of N-acetylglucosamine in the biosynthesis of lipoteichoic acids in a group of Bacillus strains.     

Occurrence of ribitol-containing lipoteichoic acid in Staphylococcus aureus H and its glycosylation

A ribitol-containing lipoteichoic acid was obtained from the 20,000 x g supernatant fraction of Staphylococcus aureus H by extraction with Triton X-100 followed by fractionation on Sepharose 6B and DEAE-cellulose columns. The purified lipoteichoic acid was composed of phosphate, glycerol, glucose, glucosamine, ribitol, and fatty acids in a molar ratio of 1 : 0.9 : 0.06 : 0.03 : 0.09 : 0.07. Based on the structural analysis of fragments from alkali and HF hydrolysis, the lipoteichoic acid appears to consist of three moieties, namely a ribitol phosphate oligomer, poly(glycerol phosphate) which has about 30 glycerol phosphate units, and beta-glucosyl-beta-glucosyl(1 leads to 1)diacylglycerol. N-Acetylglucosamine was linked to the ribitol residues. The lipoteichoic acid serves as an acceptor of glycosyl moieties from UDP-glucose and UDP-N-acetylglucosamine in the enzyme reaction catalyzed by the membrane preparation. The rate of enzymatic glycosylation was increased by prior treatment of the lipoteichoic acid with N-acetyl-beta-D-glucosaminidase. The glycosylation seems to occur at the ribitol residues of the lipoteichoic acid.     

Structural studies on lipoteichoic acids from four Listeria strains.

The lipoteichoic acids were isolated from phenol extracts of four Listeria strains representing serotypes 4a, 4b, 6a, and 6 to compare the differences in structure of amphiphilic polysaccharides from various serotypes of Listeria spp. The lipoteichoic acids from the four strains examined had the same structure in both hydrophilic chains and lipid portions. On the basis of the results of nuclear magnetic resonance spectroscopy and Smith degradation, the hydrophilic chains were shown to be 1,3-linked poly(glycerol phosphate) in which some of the glycerol residues had alpha-galactosyl substituents. The lipid portions were released by treatment with 46% hydrogen fluoride or 98% acetic acid. They were determined to be 3(1)-(2'-O-alpha-D-galactopyranosyl-alpha-D-glucopyranosyl)-1(3), 2-diacylglycerol and 3(1)-[6'-phosphatidyl-2'-O-(alpha-D-galactopyranosyl)-alpha- D-glucopyranosyl]-1(3),2-diacylglycerol. The degrees of glycosyl substitution and proportions of the two lipids varied to some extent among these four strains.     

Membrane lipoteichoic acid of Streptococcus pyogenes and its stabilized L-form and the effect of two antibiotics upon its cellular content.

Membrane lipoteichoic acid continues to be synthesized by an osmotically fragile, stabilized L-form of Streptococcus pyogenes. Chromatographic and electrophoretic comparisons indicate that the lipid componenent of lipoteichoic acid in this L-form and its parental streptococcus is glycerophosphoryldiglucosyl diglyceride and not phosphatidylkojibiosyl diglyceride. Based upon dry weight determinations, the yield of lipoteichoic acid from the L-form is 0.19%, as compared with 0.97% from the streptococcus. When grown with bacitracin the L-form contains the same amount of teichoic acid as when grown without this antibiotic; however, its lipoteichoic acid content is reduced by 85%. Similarly, the L-form grown with novobiocin for 10 h contains only 17% of the teichoic acid found in control cells.     

Lipoteichoic acid in Streptomyces hygroscopicus: structural model and immunomodulatory activities

Gram positive bacteria produce cell envelope macroamphiphile glycopolymers, i.e. lipoteichoic acids or lipoglycans, whose functions and biosynthesis are not yet fully understood. We report for the first time a detailed structure of lipoteichoic acid isolated from a Streptomyces species, i.e. Streptomyces hygroscopicus subsp. hygroscopicus NRRL 2387T. Chemical, MS and NMR analyses revealed a polyglycerolphosphate backbone substituted with α-glucosaminyl and α-N-acetyl-glucosaminyl residues but devoid of any amino-acid substituent. This structure is very close, if not identical, to that of the wall teichoic acid of this organism. These data not only contribute to the growing recognition that lipoteichoic acid is a cell envelope component of gram positive Actinobacteria but also strongly support the recently proposed hypothesis of an overlap between the pathways of lipoteichoic acid and wall teichoic acid synthesis in these bacteria. S. hygroscopicus lipoteichoic acid induced signalling by human innate immune receptor TLR2, confirming its role as a microbe-associated molecular pattern. Its activity was partially dependant on TLR1, TLR6 and CD14. Moreover, it stimulated TNF-α and IL-6 production by a human macrophage cell line to an extent similar to that of Staphylococcus aureus lipoteichoic acid. These results provide new clues on lipoteichoic acid structure/function relationships, most particularly on the role of the polyglycerolphosphate backbone substituents.     

Binding of Streptococcal Lipoteichoic Acid to Fatty Acid-Binding Sites on Human Plasma Fibronectin

The ability of Streptococcus pyogenes lipoteichoic acid and palmitic acid to bind to purified human plasma fibronectin was investigated. Initial studies indicated that intact fibronectin formed soluble complexes with lipoteichoic acid, resulting in a change in the mobility of fibronectin in an electrical field. Fibronectin covalently linked to agarose beads bound radiolabeled lipoteichoic acid in the acylated form but not in the deacylated form. An 18-M excess of fibronectin inhibited binding of lipoteichoic acid to the immobilized protein by 92%. Fibronectin-bound [(3)H]lipoteichoic acid could be specifically eluted with unlabeled lipoteichoic acid, as well as by fatty acid-free serum albumin. Serum albumin, which is known to contain fatty acid-binding sites capable of binding to the lipid moieties of lipoteichoic acid, inhibited the binding of lipoteichoic acid to fibronectin in a competitive fashion. The fibronectin-bound lipoteichoic acid could be eluted by 50% ethanol and various detergents but not by 1.0 M NaCl, various amino acids, or sugars. Similarly, radiolabeled palmitic acid adsorbed to fibronectin could be eluted with 50% ethanol but not with 1.0 M NaCl. Fibronectin adsorbed to a column of palmityl-Sepharose was eluted with 50% ethanol in 0.5% sodium dodecyl sulfate but not with 1.0 M NaCl or 1% sodium dodecyl sulfate alone. The binding of lipoteichoic acid to fibronectin followed first-order kinetics and was saturable. A Scatchard plot analysis of the binding data indicated a heterogeneity of lipoteichoic acid-binding sites similar to that previously found for serum albumin. Nevertheless, fibronectin contains at least one population of high-affinity binding sites for lipoteichoic acid. The binding affinity (nKa approximately 250 muM(-1)) is 2 orders of magnitude greater than the binding affinity of serum albumin. These data suggest that human plasma fibronectin contains specific binding sites for fatty acids and that lipoteichoic acid binds to these sites by way of its glycolipid moiety.     

31P-NMR studies of the oral pathogen Streptococcus mutans: Observation of lipoteichoic acid

We have used ³¹P-nuclear magnetic resonance spectroscopy to identify phosphorus-containing compounds in whole cells of two serotype c strains of the oral pathogen Streptococcus mutans. The major resonance, centered at 0 ppm in whole cells, was attributed to lipoteichoic acid on the basis of its chemical shift, insensitivity to pH changes, cellular localization and a comparison with spectra obtained with purified lipoteichoic acid from S. mutans. The linewidths of resonances observed for intact cells and purified lipoteichoic acid were moderately narrowed by increasing the ionic strength, and substantially broadened in the presence of the lectin concanavalin A. Experiments with purified lipoteichoic acid suggest that this compound in whole cells is complexed with divalent cations such as Mg²⁺. Intracellular pools of other phosphorus-containing metabolites were found to be low when compared to the lipoteichoic acid concentration in both starved and glycolyzing cells.