A lipoproten from the membranes of Streptococcucs pyogenes and its stabilized L-form

The lack of cell wall formation by a stabilized L-form from Streptococcus pyogenes may be related to, or reflected in, changes of a particular type of membrane lipid. Therefore, this study details the first comparative investigation of isolated membranes from this Streptococcus and its stabilized L-form of isoprenoid-containing components. A lipoprotein present in minute amounts in the membranes from both this Streptococcus and its derived L-form was detected, isolated, purified and partially characterized. Lipoprotein from both membrane sources appeared to be identical, contained phosphorus and was electrophoretically homogeneous. A ratio of streptococcal to L-form membrane lipoprotein of at least 10 was observed. Chemical, physical and chromatographic studies of isolated and nonsaponifiable lipid of lipoprotein protein indicated the absence of quinones but the presence of isoprenoid units and hydroxyl group(s). Also, the spectral characteristics of lipid of lipoprotein and its chromatographic behavior, before and after acetylation, were similar to those of an isoprenoid alcohol isolated from lactobacilli and Staphylococcus aureus by others and known to be involved in bacterial cell wall peptidolgycan biosynthesis. Protein of lipoprotein, seemingly covalently linked to lipid, was unique because of its high ornithine content: with all of the ornithine of the coccal and L-form membrane apparently concentrated within this membrane component. Approximately one-half of this lipoprotein was composed of protein. The possibility of lipoprotein being related to an inability of this L-form to synthesize a rigid cell wall is indicated.     

Identification of a novel insulin-sensitive glycophospholipid from H35 hepatoma cells

This study identifies and partially characterizes an insulin-sensitive glycophospholipid in H35 hepatoma cells. The incorporation of [3H]glucosamine into cell lipids was investigated. A major labeled lipid was purified by sequential thin layer chromatography using first an acid followed by a basic solvent system. After hydrochloric acid hydrolysis and sugar analysis by thin layer chromatography, 80% of the radioactivity in the purified lipid was found to comigrate with glucosamine. H35 cells were prelabeled with [3H]glucosamine for either 4 or 24 h and treated with insulin causing a dose-dependent stimulation of turnover of the glycophospholipid which was detected within 1 min. The purified glycolipid was cleaved by nitrous acid deamination indicating that the glucosamine C-1 was linked to the lipid moiety through a glycosidic bond. [14C]Ethanolamine, [3H]inositol, and [3H]sorbitol were not incorporated into the purified glycolipid. The incorporation of various fatty acids into this glycolipid was also studied. [3H]Palmitate was found to be preferentially incorporated while myristic acid, stearic acid, oleic acid, linoleic acid, linolenic acid, and arachidonic acid were either not incorporated or incorporated less than 10% of palmitate. The purified glycolipid labeled with [3H]palmitate was cleaved by treatment with phospholipase A2 but was resistant to mild alkali hydrolysis suggesting the presence of a 1-hexadecyl,2-palmitoyl-glyceryl moiety in the purified lipid. Treatment of labeled glycophospholipid with phosphatidylinositol-specific phospholipase C from Staphylococcus aureus generated a compound migrating as 1-alkyl,2-acyl-glycerol and a polar head group with a size in the range from 800 to 3500. These findings coupled with the nitrous acid deamination demonstrate that glucosamine was covalently linked through a phosphodiester bond to the glyceryl moiety of the purified glycolipid. These findings suggest that insulin acts on this glycophospholipid by stimulating an insulin-sensitive phospholipase C. This unique glycophospholipid may play an important role in insulin action by serving as precursor of insulin-generated mediators.     

Membrane Studies of Streptococcus pyogenes and its L-form growing in hypertonic and physiologically isotonic media. An electron spin resonance spectroscopy approach

Electron spin resonance spectroscopy (ESR) was used to compare the lipid organization, thermal stability and the physical state of the membrane of a human pathogen, Streptococcus pyogenes and its osmotically fragile L-form with this same L-form now adapted to grow under physiologically isotonic conditions (physiological L-form). Comparison of the hyperfine splittings of a derivative of 5-ketostearic acid spin label, I(1 2, 3), after incorporation into the membrane, revealed that the lipid chain rigidity of these membranes is in the order physiological L-form > osmotically fragile L-form > streptococcus. The signal intensity (of the center magnetic field line) versus temperature analysis showed two transitions for these membranes. The first with melting points of 45, 26 and 36 °C and second transition at 70, 63 and 60 °C for the physiological L-form, osmotically fragile L-form and streptococcal membranes, respectively. This same order of membrane lipid chain rigidity was seen from the cooperativities obtained for each of these systems from analysis based on the expression for an $\text{n-}\text{order}$ reaction. The I(12,3) and other probes with the paramagnetic group close to the methyl end of the molecule suggested that this difference in lipid chain rigidity between these organisms resides in the environment closer to the lipid head group region rather than in the hydrophobic lipid core. Another major finding was the binding of I(12, 3) at two or more different sites in each of the membranes examined. This change in lipid chain rigidity now provides an explanation to account for the survival of a previously osmotically fragile L-form in physiologically isotonic media by focusing on changes in the physical nature of its membrane. In so doing, it adds to and reinforces the speculation of the potential survival in vivo and involvement in pathogenesis of osmotically fragile aberrant forms of bacteria.     

Membrane studies of Streptococcus pyogenes and its L-form growing in hypertonic and physiologically isotonic media. An electron spin resonance spectroscopy approach.

Electron spin resonance spectroscopy (ESR) was used to compare the lipid organization, thermal stability and the physical state of the membrane of a human pathogen, Streptococcus pyogenes and its osmotically fragile L-form with this same L-form now adapted to grow under physiologically isotonic conditions (physiological L-form). Comparison of the hyperfine splittings of a derivative of 5-ketostearic acid spin label, I(12, 3), after incorporation into the membrane, revealed that the lipid chain rigidity of these membranes is in the order physiological L-form greater than osmotically fragile L-form greater than streptococcus. The signal intensity (of the center magnetic field line) versus temperature analysis showed two transitions for these membranes. The first with melting points of 45, 26 and 36 degrees C and second transition at 70, 63 and 60 degrees C for the physiological L-form, osmotically fragile L-form and streptococcal membranes, respectively. This same order of membrane lipid chain rigidity was seen from the cooperativities obtained for each of these systems from analysis based on the expression for an n-order reaction. The I(12, 3) and other probes with the paramagnetic group close to the methyl end of the molecule suggested that this difference in lipid chain rigidity between these organisms resides in the environment closer to the lipid head group region rather than in the hydrophobic lipid core. Another major finding was the binding of I(12, 3) at two or more different sites in each of the membranes examined. This change in lipid chain rigidity now provides an explanation to account for the survival of a previously osmotically fragile L-form in physiologically isotonic media by focusing on changes in the physical nature of its membrane. In so doing, it adds to and reinforces the speculation of the potential survival in vivo and involvement in pathogenesis of osmotically fragile aberrant forms of bacteria.     

A comparative study of the bacterial cell wall,protoplast membrane and L-form envelope ofStreptococcus pyogenes

The chemical composition of the cell wall, protoplast membrane and L-form envelope of Group A streptococci has been studied. The L-form envelope could not be identified with either the cell wall or the protoplast membrane. Although both the induced structures were mainly lipo-protein in nature, the protein/lipid ratio was much higher in the protoplast membrane (4 : 1) than in the L-form envelope (1.7 : 1). The L-form envelope differed from the bacterial cell wall in that it had a very small amount of mucopeptide which carried relatively fewer peptide chains, and also very little reducing sugar. Electrokinetic measurements in the presence of sodium dodecyl sulphate revealed that none of the lipid material was present on the surface of the L-form or protoplast. pH-mobility curves of all three structures indicated the presence of surface protein, and the absence of surface phosphate groups associated with the phospholipids found in the L-form envelope and the protoplast membrane.     

Defective synthesis of lipid intermediates for peptidoglycan formation in a stabilized L-form of Streptococcus pyogenes.

Membrane preparations obtained from a stabilized L-form of Streptococcus pyogenes are incapable of synthesizing peptidoglycan from uridine-5'-diphospho-N-acetyl-D-muramyl-L-Ala-D-iso-Glu-L-Lys-D-Ala-D-Ala and uridine-5'-diphospho-N-acetyl-D-glucosamine, in contrast with similar preparations from the parental streptococcus. Furthermore, 50-fold higher levels of lipid intermediates which serve as membrane-bound substrates for peptidoglycan synthesis are synthesized in reaction mixtures containing streptococcal membranes than with similar preparations from the L-form. These observations suggest that the inability of this stabilized L-form to form a cell wall in vivo lies, at least in part, in its failure to synthesize significant quantities of the lipid substrates for peptidoglycan synthesis.     

Teichoic acid of a stabilized L-form of Streptococcus pyogenes

A stabilized L-form of Streptococcus pyogenes continues to synthesize glycerol teichoic acid. This polymer was obtained from S. pyogenes and its L-form, treated in identical fashion, and compared. Highly purified glycerol teichoic acid from only the L-form was found to be devoid of d-alanine and to have a shorter chain length. Otherwise, the glycerol teichoic acid from these two organisms was found to be a 1,3-phosphodiester-linked glycerophosphate polymer substituted with d-glucose. Evidence is presented that most, if not all, of the glycerol teichoic acid in this streptococcus lies between the wall and membrane. A possible need for the continued synthesis of a minute amount of glycerol teichoic acid by this L-form for survival is discussed in terms of the known function of teichoic acids in bacteria.     

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.     

Morphology, growth and reversion in a stable L-form of Escherichia coli K12

An L-form isolated from Escherichia coli K12 by sequential treatment with N-methyl-N'-nitro-N-nitrosoguanidine and lysozyme was adapted to grow in hyperosmolar liquid cultures. It was stable in the absence of antibiotic when cultured in brain heart infusion (BHI) broth containing NaCl and CaCl2, the optimal concentrations being 0.34 M and 1 mM, respectively. No growth of the L-form was observed when CaCl2 was not added to BHI medium containing 0.34 M-NaCl. On the other hand, when KCl replaced NaCl as the osmotic stabilizer, growth of the L-form was repressed in the presence of CaCl2. Electron microscopy of the L-form confirmed the absence of a cell wall. A revertant strain derived from the L-form grew as a stable bacillary form in BHI medium without osmotic stabilizer. The growth characteristics of the revertant strain resembled those of the parent strain. The revertant strain produced L-forms in the presence of NaCl.     

A novel inositol glycophospholipid (IGPL) and the serum dependence of its metabolism in bovine adrenocortical cells

Adrenocortical cells in primary culture actively incorporated [3H]-inositol into phosphatidylinositol (PI) and its mono (PIP) and bisphosphate (PIP2) derivatives. In addition to these well known phosphoinositides, a inositol-containing component was detected in the cell lipid extract when analyzed by proper chromatographic systems. This component was also labeled when the cells were provided with 32P or radioactive fatty acids and a distinctive character was its ability to incorporate [3H]-glucosamine. This novel phospholipid was thus characterized as an inositol glycophospholipid (IGPL). Study of IGPL metabolism in adrenocortical cells disclosed that the presence of serum in the culture medium strikingly increased glucosamine as well as inositol incorporation by a factor of about 10 and 5, respectively, within 36 hours. These observations suggest that IGPL turnover rate, especially at the level of its inositol-glycan moiety may be regulated by extracellular signals. A possible role of IGPL in membrane signalling systems and cell regulation remains to be clarified.     

Lipid components of the hydrocarbon assimilating yeast Candida lipolytica (strain 10).

The utilization of n-hexadecane by Candida lipolytica (stain 10) was studied with respect to the lipid content, phospholipid and fatty acid profiles resulting at various growth times. Thin layer chromatography of the lipid extracts showed quantitative changes in the different lipid classes. The phospholipid fraction obtained at each growth time was separated into 8 classes: lysophosphatidylcholine, sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, glycophospholipid, phosphatidylglycerol, cardiolopin, and phosphatidic acid. Differences in the percentage fatty acid composition of the lipid extracts were observed at various stages of growth. The cellular fatty acids included palmitic, palmitoleic (35-52%), stearic, oleic, linoleic (26-39%), and pentadecanoic (2-12%) as major components. This indicates that fatty acid(s) of the same length as that of the substrate was the most abundant component, thus showing intact incorporation mechaism. Fatty acids having longer chain lengths were also formed in substantial amounts indicating C2 addition and beta-oxidation of the fatty acids formed in the yeast.     

An L-Form of Staphylococcus aureus Adapted to a Brain Heart Infusion Medium without Osmotic Stabilizers

An L-form derived from halotolerant Staphylococcus aureus Tasaki was adapted to growth in a brain heart infusion medium without any supplemental osmotically protective solutes (360 mOsm/kg). This L-form had no chemically detectable peptidoglycan residues on its surface. Electron microscopic observations confirmed morphologically the absence of the structures and also of other osmotically protective polymers within or exterior to the cytoplasmic membrane. The osmotic stability and susceptibility to bacitracin, d-cycloserine, and vancomycin of the L-form adapted to growth in 360 mOsm osmotically unprotective medium was higher than that of the L-form grown in 1,950 mOsm supplemented with 4.5% NaCl. The adapted L-form tended to be more sensitive to almost all of the antibiotics examined, other than the inhibitors for cell wall-synthesis, than the original L-form strain requiring osmotic protection for growth. Chemical analysis of the membrane of the adapted L-form indicated 16.3% total lipids and 20.6% proteins by dry weight of the membrane, and it contained larger amounts of lipid phosphorus (20.0 μ/mg).     

Initial characterization of a polyclonal antibody to an insulin-sensitive glycophospholipid

This paper describes the production of a rabbit polyclonal antibody against an insulin-sensitive glycophospholipid from rat liver membranes. The immunogen was a highly purified glycophospholipid-tetanus toxoid conjugate. Immunoglobulin purified from immune serum reacted with a glycophospholipid-ovalbumin conjugate, indicating specificity for the glycophospholipid hapten and not the protein carrier. By radioimmunoassay the antibody recognized the purified glycophospholipid antigen but not other phospholipids including phosphatidylethanolamine, phosphatidylcholine, phosphatidylserine, and phosphatidylinositol. The antigenic site appears to be the carbohydrate portion of the glycophospholipid. The antibody also reacted with glycophospholipid purified from two rat hepatoma cell lines. Analysis of partially purified liver glycophospholipid by thin-layer chromatography revealed over 20 orcinol- or fluorescamine-positive bands, but immunostaining identified only 1 band. The latter had an Rf identical to those of the original glycophospholipid isolated from rat liver and metabolically labeled material isolated from hepatoma cells. The antibody should prove useful in determining the role of the glycophospholipid and its metabolites in insulin action.     

Identification and structural analysis of a glycophospholipid component from the venom of ant Paraponera clavata

The venom of South American ant Paraponera clavata and its low-molecular-mass fraction were shown to possess insectotoxic and pore-forming activities. A number of glycophospholipid components were isolated from this ant venom by means of gel filtration and reversed-phase chromatography. Some of the compounds cause conductivity fluctuations in lipid bilayer membranes within the ranges 3-25 pS and 200-400 pS at concentrations of 10(-6) to 10(-7) M. N-Acetylglucosamine, a fatty acid, and phosphoric acid residues were found in their structures. A full structure, 3-myristoyl-2-acetamido-2-deoxy-alpha-D-glucopyranosyl phosphate, was elucidated for one of the compounds by the use of 1H, 13C, and 31P NMR spectroscopy and mass spectrometry.     

Induction of immunity against experimental tuberculosis with mycobacterial mannophosphoinositides encapsulated in liposomes containing lipid A

Abstract Mycobacterial mannophosphoinositides (PIMs) encapsulated in liposomes made of egg phosphatidylcholine (EPC) and cholesterol (CH) (2:1.5 molar ratio) were able to induce humoral and delayed type hypersensitivity (DTH, foot-pad swelling reaction) responses in mice without the help of any carrier protein. Animals immunized with this glycophospholipid antigen demonstrated enhanced percent survival on intravenous challenge with virulent M. tuberculosis . On fractionation of PIMs, pentamannophosphoinositide (PIM₅) was found to induce higher antibody and DTH reaction and proved to be more immunoprotective than other fractions. Inclusion of lipid A as immunomodulator in liposomes containing PIMs or PIM₅ resulted in a significantly increased immune response. Further, mice immunized with PIMs or PIM₅ in lipid A-containing liposomes exhibited decreased mortality on challenge with M. tuberculosis H₃₇Rv, which was comparable to BCG vaccinated animals.     

Determinants for glycophospholipid anchoring of the Saccharomyces cerevisiae GAS1 protein to the plasma membrane.

A 125-kDa glycoprotein exposed on the surface of Saccharomyces cerevisiae cells belongs to a class of eucaryotic membrane proteins anchored to the lipid bilayer by covalent linkage to an inositol-containing glycophospholipid. We have cloned the gene (GAS1) encoding the 125-kDa protein (Gas1p) and found that the function of Gas1p is not essential for cell viability. The nucleotide sequence of GAS1 predicts a 60-kDa polypeptide with a cleavable N-terminal signal sequence, potential sites for N- and O-linked glycosylation, and a C-terminal hydrophobic domain. Determination of the anchor attachment site revealed that the C-terminal hydrophobic domain of Gas1p is removed during anchor addition. However, this domain is essential for addition of the glycophospholipid anchor, since a truncated form of the protein failed to become attached to the membrane. Anchor addition was also abolished by a point mutation affecting the hydrophobic character of the C-terminal sequence. We conclude that glycophospholipid anchoring of Gas1p depends on the integrity of the C-terminal hydrophobic domain that is removed during anchor attachment.     

An ELISA for the detection of Bacillus subtilis L-form bacteria confirms their symbiosis in strawberry

AIMS: To develop an ELISA for the detection of antigens derived from stable Bacillus subtilis L-form bacteria and to detect these in plants injected with L-form bacteria. METHODS AND RESULTS: A sandwich ELISA was developed and its specificity was investigated using L-forms and cell-walled forms of B. subtilis, different Bacillus species and a range of bacteria isolated from glasshouse-grown strawberry plants. The detection limits of the ELISA were approximately 10(3) viable cells ml(-1) for L-forms compared with 10(7) viable cells ml(-1) for cell-walled forms. Results showed that L-forms survived and moved within strawberry tissues injected with L-form bacteria. CONCLUSION: An ELISA that selectively detects B. subtilis L-form bacteria was developed and shown to confirm the presence of L-forms in plants. SIGNIFICANCE AND IMPACT OF THE STUDY: This will be a valuable rapid method to further studies on L-form plant interactions.     

Cell-free synthesis of glycosyl-phosphatidylinositol precursors for the glycolipid membrane anchor of Trypanosoma brucei variant surface glycoproteins. Structural characterization of putative biosynthetic intermediates.

Trypanosome variant surface glycoproteins exemplify a class of eukaryotic cell surface glycoproteins that rely on a carboxyl-terminal covalently-attached inositol-containing glycophospholipid for membrane attachment. The glycolipid anchor is acquired soon after translation of the polypeptide, apparently by replacement of a short carboxyl-terminal peptide sequence with a prefabricated glycolipid. A candidate glycolipid precursor (referred to as P2), and a related glycolipid (P3) have been identified recently in polar lipid extracts from trypanosomes. In this paper we describe the synthesis of P2 and P3 by trypanosome membranes. Analyses of organic solvent extracts from membranes incubated with radioactive sugar nucleotides (GDP-[3H]mannose or UDP-[3H]GlcNAc) showed a spectrum of labelled lipids, ranging from partially glycosylated species to the final products, P2 and P3. Structural analyses of these putative biosynthetic intermediates suggest that glycolipid assembly occurs via the sequential glycosylation of phosphatidylinositol.     

Interaction of synthetic glycophospholipids with phospholipid bilayer membranes.

A series of glycophospholipids synthesized by coupling mono-, di-, or tri-saccharides to dioleoylphosphatidylethanolamine (DOPE) by reductive amination was used to investigate the interaction of glycophospholipids with phospholipid bilayer membranes. These synthetic glycophospholipids functioned as a stabilizer for the formation of DOPE bilayer vesicles. The minimal mol% of glycophospholipid needed to stabilize the DOPE vesicles were as follows: 8% N-neuraminlactosyl-DOPE (NANL-DOPE), 20% N-maltotriosyl-DOPE (MAT-DOPE), 30% N-lactosyl-DOPE (Lac-DOPE), and 42% N-galactosyl-DOPE (Gal-DOPE). The estimated hydration number of glycophospholipid in reverse micelles was 87, 73, 46, and 14 for NANL-DOPE, MAT-DOPE, Lac-DOPE, and Gal-DOPE, respectively. Thus, the hydration intensity of the glycophospholipid was directly related to the ability to stabilize the DOPE bilayer phase for vesicle formation. Glycophospholipids also reduced the transition temperature from gel to liquid-crystalline phase (Tm) of dipalmitoylphosphatidylcholine (DPPC) bilayers. Interestingly, incorporation of NANL-DOPE induced a decrease of membrane fluidity of DPPC bilayers in the gel phase while other glycophospholipids had no effect. Also, low level of NANL-DOPE but not other glycophospholipids increased the transition temperature (TH) from liquid-crystalline to hexagonal phase of dielaidoylphosphatidylethanolamine bilayers. These results showed that NANL-DOPE with a highly hydratable headgroup which provides a strong stabilization activity for the L alpha phase of phospholipid membranes, may also be involved in specific interactions with neighboring phospholipids via its saccharide moiety.     

Presence and synthesis of cholesterol in stable staphylococcal L-forms.

The sterol which was present in two strains of a stable staphylococcal L-form was analyzed by gas-liquid chromatography and combined gas-liquid chromatography-mass spectrometry. The retention time of the sterol on gas-liquid chromatography was the same as that of authentic cholesterol. Analysis of the sterol by mass spectrometry showed a molecular ion at an m/e of 386 and the same patterns of major ions above an m/e of 145 as those of authentic cholesterol. As a result, the sterol in staphylococcal L-form was identified as cholesterol. A parent strain and its L-forms were cultured in medium containing [14C]acetate, and the synthesis of cholesterol was examined. In the L-forms, 0.52% of the total lipid radioactivity was found in cholesterol fraction, whereas no significant radioactivity was detected in the cholesterol fraction of the parent strain, indicating that staphylococcal L-forms have acquired the capacity to synthesize cholesterol.