Possible association of segregated lipid domains of Mycoplasma gallisepticum membranes with cell resistance to osmotic lysis.

Freeze-fracturing of cholesterol-rich Mycoplasma gallisepticum membranes from cells grown in a medium containing horse serum revealed particle-free patches. The patches appeared in cells quenched from either 4 or 37 degrees C. Particle-free patches also occurred in membranes of cells grown in a serum-free medium supplemented with egg-phosphatidylcholine but not in membranes of cells grown with dioleoylphosphatidylcholine. The appearance of particle-free patches was attributed to the presence of disaturated phosphatidylcholine (PC) molecules in M. gallisepticum membranes, which were synthesized by the insertion of a saturated fatty acid at position 2 of lysophosphatidylcholine derived from exogenous PC present in the growth medium. Consequences of the synthesis of the disaturated PC also included a decrease in osmotic fragility and the ability of the cells to be permeated by K+. Electron paramagnetic resonance and fluorescence polarization measurements revealed that the fluidity of the lipid domain in the protein-rich M. gallisepticum membranes was almost identical to that of an aqueous dispersion of M. gallisepticum membrane lipids. Furthermore, the electron paramagnetic resonance spectra of the membranes were single-component spectra showing no indication of immobilized regions. The possibility that the osmotic resistance of M. gallisepticum cells is associated with the particle-free patches rather than with a restricted membrane fluidity caused by membrane proteins is discussed.     

Incorporation and modification of exogenous phosphatidylcholines by mycoplasmas.

The uptake and modification of exogenous phosphatidylcholine (PC) by several Mycoplasma and Spiroplasma species was investigated. While in most Mycoplasma species and in all Spiroplasma species tested the PC appears to be incorporated unchanged from the growth medium, the PC of M. gallisepticum, M. pulmonis, and M. pneumoniae was disaturated PC, apparently formed by modification of 1-saturated-2-unsaturated PC from the growth medium. The modification of the exogenous PC by M. gallisepticum was inhibited by chloramphenicol under conditions that did not affect de novo synthesis of phosphatidylglycerol. A low activity of an endogenous phospholipase A was detected in native M. gallisepticum membranes. The activity was markedly stimulated by treating the membranes with low concentrations of the nonionic detergents. The PC modification was affected by the fatty acid composition of the exogenous PC species. Diunsaturated, 1-saturated-2-unsaturated, and 1-unsaturated-2-saturated PCs were modified to various extents, whereas the disaturated dipalmitoyl PC (DPPC) was not. Both modified and unmodified PCs were incorporated by the cells, but the unmodified DPPC was incorporated at a lower rate and to a lesser extent. The possibility that the incorporation of DPPC into M. gallisepticum cells is associated with the formation of intracytoplasmic membranes is discussed.     

Ultrastructural visualization of anionic sites on mycoplasma membranes by polycationic ferritin.

Anionic sites on mycoplasma membranes were visualized in the electron microscope by a polycationized ferritin derivative. The technique of thin sectioning was used. Staining prior to fixation led to clustering of ferritin granules on the mycoplasma cell surface. On glutaraldehyde-fixed Mycoplasma mycoides subsp. capri, M. gallisepticum, M. pneumoniae, and Acholeplasma laidlawii, the anionic sites were uniformly distributed over the entire membrane surface. M. hominis did not bind the polycationic ferritin label. Chemical and enzymatic treatments of the mycoplasmas indicated that the anionic sites may be lipid phosphate groups. Isolated M. mycoides subsp. capri membranes were labeled exclusively on only one membrane surface, presumably the outer one. Liposomes prepared from diphosphatidylglycerol and phosphatidylcholine were also labeled by the polycationic ferritin.     

Studies with Lectins on the Surface Carbohydrate Structures of Mycoplasma Membranes

The surface carbohydrate structures on the cell membranes of various mycoplasma species have been investigated by using lectins, which are sugar-specific proteins. Carbohydrate structures presumably bound to glycolipids, with both galactose and glucose units, were found to be exposed on the surface of Mycoplasma pneumoniae and its temperature-sensitive mutants, M. mycoides var. mycoides and capri, M. pulmonis, M. gallinarum, and M. gallisepticum. Lipid-bound glucose was found on M. neurolyticum. The possible relationship of the lipid-bound surface carbohydrate groups to the known serological cross-reactions and lipid compositions of the various mycoplasma species is discussed. Intact Acholeplasma laidlawii and M. fermentans have no lectin-binding sites exposed on their surfaces; galactose groups were discovered only after Pronase digestion of the organisms, suggesting that their glycolipids are hidden under a protein layer. Neither intact nor Pronase-digested M. hominis reacted with the lectins; this is fully consistent with the lipid composition of this organism, which contains glycolipids. The lectins from Vicia cracca and Phaseolus vulgaris, which react with N-acetyl-galactosamine groups, agglutinated M. gallinarum, M. gallisepticum, M. mycoides var. capri, and M. pulmonis. The agglutinability was lost after Pronase treatment, indicating that the corresponding carbohydrates are presumably protein bound. They may be correlated with the extracellular structures observed by electron microscopy of both sectioned and negatively stained mycoplasma species.     

Lysophospholipase-catalyzed hydrolysis of lysophospholipids in Mycoplasma gallisepticum membranes.

Mycoplasma gallisepticum strains have a membrane-bound lysophospholipase which hydrolyzes lysophospholipid generated in these membranes by treatment with an external phospholipase. This paper studies the hydrolysis of the membranous lysophospholipids by an enzyme residing in the same membrane (intramembrane utilization) or in adjacent membranes (intermembrane utilization). To study intermembrane hydrolysis, the phospholipids of M. gallisepticum were labeled with [3H]oleic acid. Membranes were prepared, heated at 65 degrees C, and subsequently treated with pancreatic phospholipase A2. This resulted in membranes whose enzyme was heat inactivated, but which contained lysophospholipid. When these membranes were mixed with M. gallisepticum cells or membranes, the lysophospholipid was hydrolyzed by the membranous lysophospholipase. To study intramembrane hydrolysis, [3H]oleyl-labeled membranes of M. gallisepticum were treated with pancreatic phospholipase A2 at pH 5.0. At this pH, lysophospholipid was generated but not hydrolyzed. Adjustment of the pH to 7.4 resulted in hydrolysis of the lysophospholipid by the membranous lysophospholipase. These procedures permitted measuring the initial rates of intramembrane and intermembrane hydrolysis of the lysophospholipid, showing that the time course and dependence on endogenous substrate concentration were different in the intramembrane and intermembrane modes of utilization. They also permitted calculation of the molar concentration of the lysophospholipid in the membrane and its rate of hydrolysis, expressed as moles per minute per cell or per square centimeter of cell surface.     

Interaction of insecticides with lipid membranes.

The permeability of liposome membranes is increased by organophosphorus and organochlorinated insecticides at concentrations of 10(-5)--10(-4) M. The order of effectiveness is similar to the toxicity of the compounds to mammals, and is the following for permeation of non-electrolytes and for valinomycin-induced permeation of K+: parathion greater than 1,1,1-trichloro-2,2-bis(p-chlorophenyl) ethane (DDT) approximately aldrin greater than malathion greater than lindane. The degree of effectiveness for X-537A-induced permeation of Ca2+ was the following: aldrin greater than or equal to DDT greater than parathion greater than malathion greater than lindane. The organophosphorus compound, ethyl azinphos (10(-4) M), dramatically increases the permeability of liposome membranes to all the tested substances, probably as a consequence of surfactant effects. Some organochlorinated insecticides appear to react with cation ionophores and modulate their motion across lipid membranes. It is suggested that the insecticides may exert some of their toxic actions by modifying certain mechanisms in the cell membrane.     

Localization of an immunodominant 64kDa lipoprotein (LP 64) in the membrane of Mycoplasma gallisepticum and its role in cytadherence

A 64 kDa lipoprotein (LP 64) haemagglutinin (pI 4.9-5.0) was isolated from the membrane of Mycoplasma gallisepticum. Triton X-114 phase partitioning has demonstrated that the hydrophobic nature of this haemagglutinin is due to a lipid portion of the molecule. Autoradiography of [3H]-palmitate-labelled M. gallisepticum revealed the presence of several additional lipoproteins. Immunoelectron microscopy demonstrated the localization of LP 64 to the base of the terminal structure. Densitometric scans of stained polyacrylamide gels of M. gallisepticum showed that LP 64 constitutes 1.7% of the total protein. Scans of immunoblots of M. gallisepticum indicate that LP 64 is highly immunogenic in chickens, accounting for 7.4% of the total serum IgG response at four weeks post-infection. A quantitative value for the IgG response to LP 64, relative to the percentage of total protein (the Relative Immunogenicity Index) was 4.4. LP 64 is conserved among several strains of M. gallisepticum, but its presence could not be detected in Mycoplasma synoviae. Antiserum raised to electroeluted LP 64 reacted specifically with this lipoprotein when assessed on either one- or two-dimensional immunoblots of M. gallisepticum. This antiserum, as well as Fab fragments, inhibited haemagglutination of chicken erythrocytes and inhibited the attachment of 14C-labelled M. gallisepticum to chicken tracheal epithelium in vitro by 62%.     

Isolation of mycoplasma membranes by dicyclohexylcarbodiimide-induced lysis.

A simple procedure was devised to prepared membranes from Mycoplasma gallisepticum cells. The cells were lysed in an isosmotic NaCl solution by dicyclohexylcarbodiimide, which blocks ATPase activity and interferes with the regulation of cell volume. The procedure can be used to isolate membranes of other osmotically resistant mycoplasmas.     

Cholesterol distribution and movement in the Mycoplasma gallisepticum cell membrane.

The time course and extent of transfer of [14C]-cholesterol from resting Mycoplasma gallisepticum cells or membrane preparations to high-density lipoproteins were studied. More than 90% of the total cholesterol in isolated, unsealed membrane preparations was exchanged in a single kinetic process. In intact cells, however, cholesterol exists in two different environments. Cholesterol in one environment, representing approximately 50% of the total unesterified cholesterol, is readily exchanged with the cholesterol of high-density lipoproteins, with a half-time of about 4 h at 37 degrees C. The rate of exchange of [14C]cholesterol from the other environment was exceedingly slow, with a half-time of about 18 days. The fraction of the total cholesterol in the readily exchangeable cholesterol pool in intact cells increased somewhat upon aging of the culture. Electron spin resonance spectra of nitroxide-labeled stearic acids incorporated into membranes of M. gallisepticum cells indicated increased rigidity at the late exponential phase of growth. These results suggest that cholesterol is present in approximately equal concentrations on both surfaces of the M. gallisepticum membrane and that in resting cells the rate of movement of cholesterol molecules from the inner to outer halves of the lipid bilayer is exceedingly slow or nonexistent.     

Influence of the growth at high osmolality on the lipid composition, water permeability and osmotic response of Lactobacillus bulgaricus

Changes in water permeability and membrane packing were measured in cells of Lactobacillus bulgaricus and in vesicles prepared with lipids extracted from them. The osmotic response of whole cells and vesicles is compared with the one of bacteria grown in a high osmolal medium. Both bacteria and vesicles, behave as osmometers. This means that the volume decrease is promoted by the outflow of water, driven by the NaCl concentration difference, arguing that neither Na+ nor Cl- permeates the cell or the lipid membrane in these conditions. Therefore, the volume changes can be correlated with the rate of water permeation across the cell or the vesicle membranes. The permeation of water was analyzed as a function of the lipid species by measuring the volume changes and the saturation ratio of the lipids. To put into relevance the membrane processes, the permeation properties of lipid vesicles prepared with lipids extracted from bacteria grown in normal and high osmolality conditions were also analyzed. The permeation response was correlated with the physical properties of the membrane of whole cells and vesicles, by means of fluorescence anisotropy of diphenyl hexatriene (DPH). The modifications in membrane properties are related with the changes in the membrane composition triggered by the growth in a high osmolal medium. The changes appear related to an increase in the sugar content of the whole pool of lipids and in the saturated fatty acid residues.     

Cholesterol prevents interaction of the cell‐penetrating peptide transportan with model lipid membranes

Interaction of the cell‐penetrating peptide (CPP) cysteine‐transportan (Cys‐TP) with model lipid membranes was examined by spin‐label electron paramagnetic resonance (EPR). Membranes were labeled with lipophilic spin probes and the influence of Cys‐TP on membrane structure was studied. The influence of Cys‐TP on membrane permeability was monitored by the reduction of a liposome‐trapped water‐soluble spin probe. Cys‐TP caused lipid ordering in membranes prepared from pure dimyristoylphosphatidylcholine (DMPC) and in DMPC membranes with moderate cholesterol concentration. In addition, Cys‐TP caused a large increase in permeation of DMPC membranes. In contrast, with high cholesterol content, at which model lipid membranes are in the so‐called liquid‐ordered phase, no effect of Cys‐TP was observed, either on the membrane structure or on the membrane permeability. The interaction between Cys‐TP and the lipid membrane therefore depends on the lipid phase. This could be of great importance for understanding of the CPP–lipid interaction in laterally heterogeneous membranes, while it implies that the CPP–lipid interaction can be different at different points along the membrane. Copyright © 2008 European Peptide Society and John Wiley & Sons, Ltd.     

Kinetics of cholesterol and phospholipid exchange from membranes containing cross-linked proteins or cross-linked phosphatidylethanolamines

Mono- and dipalmitoylphosphatidylethanolamine derivatives have been synthesized and used to evaluate the role of cross-links between the amino groups of two phospholipid molecules in the rate of cholesterol movement between membranes. Incorporation of the cross-linked phospholipids into small unilamellar vesicles (the donor species) decreased the rate of spontaneous cholesterol exchange with acceptor membranes (small unilamellar vesicles or Mycoplasma gallisepticum cells). These results suggest that the cross-linking of aminophospholipids by reactive intermediates, which may be one of the degenerative transformations associated with peroxidation of unsaturated lipids and cellular aging, can inhibit cholesterol exchangeability in biological membranes. The rates of spontaneous [14C]cholesterol and protein-mediated 14C-labeled phospholipid exchange from diamide-treated mycoplasma and erythrocyte membranes have also been measured. The formation of extensive disulfide bonds in the membrane proteins of M. gallisepticum enhanced the 14C-labeled phospholipid exchange rate but did not affect the rate of [14C]cholesterol exchange. The rates of radiolabeled cholesterol and phospholipid exchange between erythrocyte ghosts and vesicles were both enhanced (but to different extents) when ghosts were treated with diamide. These observations suggest that diamide-induced oxidative cross-linking of sulfhydryl groups in membrane proteins does not lead to random defects in the lipid domain.     

Interaction of insecticides with lipid membranes

The permeability of liposome membranes is increased by organophosphorus and organochlorinated insecticides at concentrations of 10⁻⁵−10⁻⁴ M. The order of effectiveness is similar to the toxicity of the compounds to mammals, and is the following for permeation of non-electrolytes and for valinomycin-induced permeation of K⁺: parathion > 1,1,1-trichloro-2,2-bis(p-chlorophenyl) ethane (DDT) ≈ aldrin malathion > lindane. The degree of effectiveness for X-537A-induced permeation of Ca²⁺ was the following: aldrin DDT > parathion malathion > lindane. The organophosphorus compound, ethyl azinphos (10⁻⁴ M), dramatically increases the permeability of liposome membranes to all the tested substances, probably as a consequence of surfactant effects. Some organochlorinated insecticides appear to react with cation ionophores and modulate their motion across lipid membranes.It is suggested that the insecticides may exert some of their toxic actions by modifying certain mechanisms in the cell membrane.     

The Temperature Dependence of Lipid Membrane Permeability, its Quantized Nature, and the Influence of Anesthetics

We investigate the permeability of lipid membranes for fluorescence dyes and ions. We find that permeability reaches a maximum close to the chain melting transition of the membranes. Close to transitions, fluctuations in area and compressibility are high, leading to an increased likelihood of spontaneous lipid pore formation. Fluorescence correlation spectroscopy reveals the permeability for rhodamine dyes across 100-nm vesicles. Using fluorescence correlation spectroscopy, we find that the permeability of vesicle membranes for fluorescence dyes is within error proportional to the excess heat capacity. To estimate defect size we measure the conductance of solvent-free planar lipid bilayer. Microscopically, we show that permeation events appear as quantized current events very similar to those reported for channel proteins. Further, we demonstrate that anesthetics lead to a change in membrane permeability that can be predicted from their effect on heat capacity profiles. Depending on temperature, the permeability can be enhanced or reduced. We demonstrate that anesthetics decrease channel conductance and ultimately lead to blocking of the lipid pores in experiments performed at or above the chain melting transition. Our data suggest that the macroscopic increase in permeability close to transitions and microscopic lipid ion channel formation are the same physical process.     

Cultivation of Mycoplasmas on Cellulose Ester Substrates

The ability of mycoplasmas to grow on cellulose ester substrates was evaluated. Mycoplasma pneumoniae, M. hominis, M. arthritidis, M. gallisepticum, and Acholeplasma laidlawii grew on Millipore (mixed cellulose ester) filters and Sepraphore III (cellulose polyacetate) membranes.     

Recombinant DNA probes and polymerase chain reaction for detection of Mycoplasma gallisepticum strains

Abstract Two recombinant DNA clones, pMG286.2 and pMG301.1, were isolated from the partial genomic library of Mycoplasma gallisepticum strain S6. Recombinant M. gallisepticum specific fragments were used as probes in Southern hybridisation with 10 M. gallisepticum strains whose DNA was digested by Eco RI, Hin dIII, Bgl II, Rsa I and Bam HI. The 1.5 kb fragment pMG301.1 did not show polymorphism in hybridisation patterns with M. gallisepticum strains, while the 3.5 kb fragment pMG286.2 enabled differentiation of M. gallisepticum strains into clusters. The DNA sequence of pMG301.1 was used to design a pair of 27-mer oligonucleotides flanking a 1.3 kb genomic region. These two primers directed specific in vitro amplification of all M. gallisepticum strains assayed giving an expected 1.3 kb product. Digestion of polymerase chain reaction products by Dde I enabled simple differentiation between clusters of M. gallisepticum strains and may be useful for improved epizootiological studies of M. gallisepticum infections in poultry.     

Molecular species of phosphatidylcholine and phosphatidylethanolamine in subcellular membranes from rat liver.

Four subfractions of phosphatidycholine and phosphyatidylethanolamine according to the degree of unsaturation of their fatty acids have been separated from lipid extracts of microsomes, and inner and outer mitochondrial membranes. The predominant species found in the three membranes contained one saturated and one unsaturated fatty acid. In microsomes completely saturated species of both phosphatidylcholine and phosphatideylethanolamine were practically nonexistent. In outer mitochondrial membranes species with two unsaturated fatty acids were absent. In the inner mitochondrial membranes, however, disaturated species and those with two unsaturated fatty acids were found.     

Permeation of aromatic carboxylic acids across lipid bilayers: the pH-partition hypothesis revisited

According to the pH-partition hypothesis the charged species of organic compounds do not contribute to lipid bilayer permeation as they generally show negligible partitioning into n-octanol. With this assumption, membrane permeation is related to the molar fraction of the neutral species at a particular pH. A recently developed permeation assay permits us to directly determine pH-dependent permeation of aromatic carboxylic acids. Tb(3+)-loaded liposomes are incubated with aromatic carboxylic acids and upon excitation at the absorption wavelength of the acid, permeation kinetics can be measured as an increase in Tb(3+) luminescence. The anions of the tested acids permeated egg phosphatidylcholine membranes only 12 (2-hydroxynicotinic acid), 66 (salicylic acid), and 155 (dipicolinic acid) times slower than the net neutral species. The anions, therefore, controlled the total permeation already at 1-2 pH units above their pK(a). These results indicate that in contrast to the expectations of the pH-partition hypothesis, lipid bilayer permeation of an acidic compound can be completely controlled by the anion at physiological pH.