Freeze-etch electron microscopy of erythrocytes,Acholeplasma laidlawii cells and liposomal membranes after the action of filipin and amphotericin B

Freeze-etch electron microscopy demonstrated that filipin induces the formation of aggregates 150–250Åin diameter, in the membranes of rat erythrocytes, in cholesterol-containing membranes ofAcholeplasma laidlawii cells and in egg lecithin-cholesterol liposomes. No change in fracture faces was observed when cholesterol was absent in the membranes ofA. laidlawii, and lecithin liposomes.Amphotericin B does not visibly affect the freeze-etch morphology of erythrocytes, cholesterol-containingA. laidlawii cells and lecithin-cholesterol liposomes.     

Lipid and protein membrane components associated with cholesterol uptake by mycoplasmas

Membranes of Mycoplasma species take up 2–4 times more exogenous cholesterol than membranes of Acholeplasma species. To test whether the lower cholesterol uptake capacity of Acholeplasma is due to the high glycolipid content of their membranes, the phospholipids of Acholeplasma laidlawii and Mycoplasma capricolum membranes were hydrolyzed by phospholipase A². Digestion removed about 30% of the polar lipids of A. laidlawii, leaving the glycolipids and phospholglycolipids intact, and about 70% of the polar lipids of M. capricolum, the residue consisting mostly of sphingomyelin. Cholesterol uptake by the treated membranes from phosphatidylcholine/cholesterol vesicles decreased in rough proportion to the amount of polar lipid removed, indicating that the glycolipids in A. laidlawii membranes can participate in cholesterol uptake.Trypsin digestion of growing cells and isolated membranes of M. capricolum decreased cholesterol uptake by about one-half. Similar treatment of A. laidlawii cells and membranes had no effect on cholesterol uptake. These findings suggest the existence of protease-sensitive receptors on the cell surface of M. capricolum responsible for tighter contact with the cholesterol/phosphatidylcholine vesicles. It is proposed that the ability of Mycoplasma species to take up large quantities of exogenous cholesterol and phospholipids depends on the presence of protein receptors for cholesterol donors, receptors which are absent in Acholeplasma species.     

Cholesterol-phosphatidylcholine dispersions as donors of cholesterol to Mycoplasma membranes

Growing cells of sterol-requiring Mycoplasma hominis and sterol non-requiring Acholeplasma laidlawii were used to test the ability of cholesterol-dipalmitoyl phosphatidylcholine dispersions to serve as cholesterol donors to these organisms. Dispersions with high cholesterol to phosphatidylcholine ratios were more effective than dispersions with low cholesterol to phosphatidylcholine ratios in donating cholesterol to the membranes of both mycoplasmas and in promoting growth of the sterol-requiring species. M. hominis took up almost three times as much cholesterol as did A. laidlawii. In addition, significant quantities of the phosphatidylcholine component of the dispersions were found to be associated with M. hominis membranes as against none in the A. laidlawii membrane preparations. In all cases, the percentage of cholesterol taken up by M. hominis from the dispersions exceeded that of phosphatidylcholine by a factor of 3–5. These results were interpreted to suggest that all the cholesterol taken up by A. laidlawii is transferred from the dispersion to the membranes by a process which involves only a transient contact between the organisms and the lipid dispersions, whereas a certain amount of the cholesterol taken up by M. hominis may also be derived from lipid dispersions adhering to or fusing with the cell membranes.     

A cytolytic protein from the edible mushroom, Pleurotus ostreatus

Aqueous extracts of the edible mushroom, Pleurotus ostreatus, contain a substance that is lytic in vitro for mammalian erthrocytes. The hemolytic agent, pleurotolysin, was purified to homogeneity and found to be a protein lacking seven of the amino acids commonly found in proteins. In the presence of sodium dodecyl sulfate it exists as monomers of molecular weight 12 050 whereas under non-dissociating conditions it appears to exist as dimers. It is isoelectric at about pH 6.4. The sensitivity of erythrocytes from different animals correlates with sphingomyelin content of the erythrocyte membranes. Sheep erythrocyte membranes inhibit pleurotolysin-induced hemolysis and the inhibition is time and temperature dependent. Ability of membranes to inhibit hemolysis is abolished by prior treatment of membranes with specific phospholipases. Pleurotolysin-induced hemolysis is inhibited by liposomes prepared from cholesterol, dicetyl phosphate adn sphingomyelin derived from sheep erythrocytes whereas a variety of other lipid preparations fail to inhibit. It is concluded that sphingomyelin plays a key role in the hemolytic reaction.     

Chronic exposure to inhaled anesthetics increases cholesterol content in Acholeplasma laidlawii

Acholeplasma laidlawii cells were grown in cholesterol-enriched medium and exposed continuously to either air (control), 4.0 vol.% halothane in air at 1 atm pressure (4% atm halothane), or 80% cyclopropane in oxygen for 24 h at 37°C. Cells grown in the presence of 4% atm halothane or 80% cyclopropane had approximately twice as much membrane cholesterol content/mg protein as the control cells. Cells grown in an anesthetic environment also tended to have a higher membrane cholesterol/phospholipid molar ratio compared to control cells. Membranes isolated from halothane-exposed cells grown in a cholesterol-enriched medium were more ordered at 37°C (measurements were made with no anesthetic present) than membranes from control cells grown in an identically enriched medium. This difference in membrane physical state between control and anesthetic-exposed cells decreased as the temperature decreased, and disappeared at approx. 23°C. Continuous exposure of A. laidlawii to 4% atm halothane or 80% cyclopropane for 24 h did not markedly affect membrane fatty acid composition, either in cells grown on an unsupplemented medium or in cells grown in a medium enriched in myristic, palmitic or stearic acids. These results further support the hypothesis that an increased membrane cholesterol content may play a role in the tolerance or dependence that develops after chronic exposure to anesthetic agents.     

Susceptibility of erythrocytes from several animal species to Vibrio vulnificus hemolysin

The hemolytic activity of Vibrio vulnificus hemolysin (VVH) against erythrocytes from several animal species (sheep, horse, cow, rabbit, chicken) was investigated. VVH was active against erythrocytes from all species, but the amount of VVH causing 50% hemolysis under identical conditions (hemolytic susceptibility to VVH) differed. The degree of 125I-labeled VVH (125I-VVH) binding to each erythrocyte species correlated with the susceptibility of the cells to hemolysis. However, marked differences in the binding ability of 125I-VVH were not observed against liposomes constructed with lipids from each erythrocyte membrane. On the other hand, release of hemoglobin (Hb) differed for each of the erythrocyte species despite administration of approximately the same hemolytic VVH concentration to each species. Furthermore, under hypotonic conditions, the stability of each erythrocyte species varied markedly; the more susceptible the erythrocyte to VVH, the more unstable it was under such conditions. These results, therefore, suggest that the susceptibility of erythrocytes to VVH may be closely associated with the binding ability of VVH and erythrocyte membrane stability.     

Detection of Cholesterol in Cell Membranes by Use of Bacterial Toxins

A method is described for the detection of cholesterol in membranes from erythrocytes, mycoplasmas, and bacterial cells by a ferritin-labeling technique. Membranes treated with cereolysin, a bacterial hemolysin which specifically binds to cholesterol, and then treated with ferritin-antitetanolysin, were specifically ferritin-labeled for cholesterol. A similar antigen-antibody system, streptolysin O-ferritin-antistreptolysin, was also used successfully with erythrocyte membranes. There was an uneven distribution of ferritin in erythrocyte membranes suggesting that the distribution of cholesterol may not be entirely random. Mycoplasma gallisepticum was intensely labeled, but Acholeplasma laidlawii with or without cholesterol in the membranes was not labeled, suggesting an unusual location for cholesterol in A. laidlawii membranes. As controls, two of three species of bacterial membranes lacking cholesterol were not ferritin-labeled.     

The influence of retinal on complement-dependent immune damage to liposomes

Retinal was incorporated into liposomes containing dipalmitoyllecithin, cholesterol, dicetyl phosphate and galactocerebroside; the latter substance served as antigen. They were compared to control liposomes, lacking retinal, with regard to glucose release due to complement-dependent immune damage in the presence of anticerebroside serum. The liposomes were indistinguishable from each other in the amount of total glucose trapped, light scattering characteristics and phosphate content. The rate and extent of glucose release in 30 min was inhibited by the incorporation of retinal. In addition, inhibition was directyl related to retinal concentration and was also observed in the presence of a wide range of concentrations of antigen and complement. Damage to liposomes in the presence of either guinea pig or human complement was inhibited by retinal; this was in contrast to the erythrocyte system in which the hemolytic activity of guinea pig complement was inhibited while that of human complement was enhanced by retinal. Addition of retinal to performed liposomes did not influence complement-dependent damage. Inhibition occurred only when retinal was present during the initial formation of the model membranes. Inhibition persisted even after washing the liposomes free of any unincorporated retinal. The data indicate that liposomes may be an excellent model for studying the influences of retinal on complement mechanism in membranes.     

Cloning and expression in Escherichia coli and Bacillus subtilis of the hemolysin (cereolysin) determinant from Bacillus cereus.

From a cosmid gene bank of Bacillus cereus GP4 in Escherichia coli we isolated clones which, after several days of incubation, formed hemolysis zones on erythrocyte agar plates. These clones contained recombinant cosmids with B. cereus DNA insertions of varying lengths which shared some common restriction fragments. The smallest insertion was recloned as a PstI fragment into pJKK3-1, a shuttle vector which replicates in Bacillus subtilis and E. coli. When this recombinant plasmid (pJKK3-1 hly-1) was transformed into E. coli, it caused hemolysis on erythrocyte agar plates, but in liquid assays no external or internal hemolytic activity could be detected with the E. coli transformants. B. subtilis carrying the same plasmid exhibited hemolytic activity at levels comparable to those of the B. cereus donor strain. The hemolysin produced in B. subtilis seemed to be indistinguishable from cereolysin in its sensitivity to cholesterol, activation by dithiothreitol, and inactivation by antibodies raised against cereolysin. When the recombinant DNA carrying the cereolysin gene was used as a probe in hybridization experiments with chromosomal DNA from a streptolysin O-producing strain of Streptococcus pyogenes or from listeriolysin-producing strains of Listeria monocytogenes, no positive hybridization signals were obtained. These data suggest that the genes for these three SH-activated cytolysins do not have extended sequence homology.     

Effect of Cholesterol Content on Gramicidin S-Induced Hemolysis of Erythrocytes

Gramicidin S (GS) is a cyclo-decapeptide antibiotic with wide Gram+ and Gram− antimicrobial spectrum. However, its therapeutic application is very limited due to hemolytic activity of GS. The presence of cholesterol defines one of the most significant differences between eukaryotic plasma membranes and bacterial inner membranes. To find out the cholesterol effect on the GS hemolytic efficiency we compared GS-induced hemolysis of erythrocytes extracted from the blood of healthy donors against donors with atherosclerosis, “naturally” enriched with cholesterol. Our results show that increased cholesterol levels significantly attenuates yet does not abolishes the GS hemolytic activity. High levels of cholesterol content in erythrocyte membranes results in a decrease in the membrane fluidity and deformability leading to a decrease in the rate of GS interaction with membranes. The results obtained confirm that hydrophobic as well as electrostatic interactions must be involved in the binding of GS to cell membranes. Lipid peroxidation occurring within atherosclerotic erythrocytes leads to considerable decrease in the degree of GS-induced erythrocyte hemolysis in vitro. These results can be applied to the rational design of GS analogs with increased antibacterial efficiency but reduced hemolytic activity.     

Phospholipid and cholesterol uptake by mycoplasma cells and membranes

The ability of growing mycoplasma cells and their isolated membranes to take up exogenous phospholipids was correlated with their ability to take up cholesterol. Horse serum or vesicles made of phosphatidylcholine and cholesterol served as lipid donors. Growing cells of five Mycoplasma species took up significant quantities of phosphatidylcholine and sphingomyelin as well as free and esterified cholesterol. In contrast, growing cells of three Acholeplasma species failed to take up any of the exogenous phospholipids, and only incorporated low amounts of free cholesterol and no esterified cholesterol. Hence, the ability of mycoplasmas to take up large quantities of cholesterol appears to be correlated with an ability to take up exogenous phospholipids. Isolated membranes of Mycoplasma capricolum and Acholeplasma laidlawii took up lower amounts of cholesterol than did membranes of growing cells and did not take up phospholipids. Inhibition of M. capricolum growth decreased the ability of the cells to take up exogenous phospholipids and cholesterol. The possibility that the contact between the lipid donors and the membrane involves specific receptors best exposed in actively growing cells is discussed.     

Role of phospholipids in the DDT-induced efflux of potassium in human erythrocytes

Incubation of human erythrocytes for 1–2 h at 37°C in a suspension of dipalmitoylphosphatidylcholine (DPPC) liposomes results in a phospholipid enrichment of erythrocyte membranes by 45–55% and a depletion of cholesterol by 19–24%. The enrichment by DPPC was time and concentration dependent. By contrast, dioleoylphosphatidylcholine (DOPC) liposomes were less effective in enriching the membranes with phospholipid and in depleting the membranes of cholesterol. Concomitantly, the DDT-induced efflux of K⁺ was reduced in the case of DPPC-enriched erythrocytes but enhanced in DOPC-enriched erythrocytes. These results suggest that DDT partitions more readily into the unsaturated than the saturated phospholipids of the erythrocyte membrane. It is concluded that the extent to which DDT affects the flux of K⁺ across the membrane is dependent on the fluidity of the lipid phase. We also report here a rapid method for cholesterol depletion of red blood cells in comparison to previously reported methods.     

Hemolytic Activity of Membrane-Active Peptides Correlates with the Thermodynamics of Binding to 1-Palmitoyl-2-Oleoyl-sn-Glycero-3-Phosphocholine Bilayers

Understanding the mechanisms of antimicrobial, cytolytic and cell-penetrating peptides is important for the design of new peptides to be used as cargo-delivery systems or antimicrobials. But these peptides should not be hemolytic. Recently, we designed a series of such membrane-active peptides and tested several hypotheses about their mechanisms on model membranes. To that end, the Gibbs free energy of binding to 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) vesicles was determined experimentally. Because the main lipid components of the outermost monolayer of erythrocyte membranes are zwitterionic, like POPC, we hypothesized that the Gibbs free energy of binding of these peptides to POPC would also be a good indicator of their hemolytic activity. Now, the hemolytic activity of those synthetic peptides was examined by measuring the lysis of sheep erythrocyte suspensions after peptide addition. Indeed, the Gibbs free energy of binding was in good correlation with the hemolytic activity, which was represented by the concentration of peptide in solution that produced 50 % hemolysis. Furthermore, with two exceptions, those peptides that caused graded dye release from POPC vesicles were also hemolytic, while most of those that caused all-or-none release were not.     

Characterization of the mycoplasma membrane proteins: V. Release and localization of membrane-bound enzymes in Acholeplasma laidlawii

The peripheral membrane protein fraction released by washing Acholeplasma laidlawii membranes with low-ionic strength buffers contained about 50 % of the total membrane-bound ribonuclease and deoxyribonuclease activities. The ATPase, NADH oxidase and p-nitrophenylphosphatase activities remained bound to the membrane even when EDTA was added to the wash fluids, and thus appear to belong to the integral membrane protein group.Serving as a marker for peripheral membrane proteins, the membrane-bound ribonuclease activity was solubilized by bile salts much more effectively than the integral membrane-bound enzymes. On the other hand, the solubilized ribonuclease showed a much lower capacity to reaggregate with other solubilized membrane components to membranous structures. Yet, most of the ribonuclease molecules which were bound to the reaggregated membranes could not be released by low-ionic strength buffer. The reaggregated membranes differed from the native membranes in the absence of particles on their fracture faces obtained by freeze cleaving, and by their much higher labeling by the [¹²⁵I]lactoperoxidase iodination system. These results suggest that most of the proteins are exposed on the reaggregated membrane surfaces, with very little, if any, protein embedded in its lipid bilayer core.Enzyme disposition in the A. laidlawii membrane was studied by comparing the activity of isolated membranes with that of membranes of intact cells after treatment with pronase or with an antiserum to membranes. The data indicate the asymmetrical disposition of these activities, the ATPase and NADH oxidase being localized on the inner membrane surface, while the nucleases are exposed on the external membrane surface.     

Effects of cholesterol on physical properties of human erythrocyte membranes: impact on susceptibility to hydrolysis by secretory phospholipase A2

The ability of secretory phospholipase A₂ (sPLA₂) to hydrolyze cell membranes is highly dependent on the physical properties of the membrane. The effects of cholesterol on these properties have been characterized in artificial bilayers and found to alter sPLA₂ activity significantly. It is hypothesized that the natural difference in cholesterol content between erythrocytes and leukocytes is in part responsible for their differing susceptibility to hydrolysis by sPLA₂. To test this hypothesis, defined amounts of cholesterol were removed from erythrocyte membranes using methyl-β-cyclodextrin. Treatment of cells with methyl-β-cyclodextrin increased the hydrolysis rate and total substrate hydrolyzed by sPLA₂. In general, this effect of cholesterol removal was more pronounced at higher temperatures. Comparison of the level of membrane order (assessed with the fluorescent probe laurdan) with hydrolysis rate revealed that sPLA₂ activity was greatly enhanced upon significant reductions in lipid order. Additional treatment of the cells with calcium ionophore further enhanced the hydrolysis rate and altered the relationship with membrane order. These data demonstrated that interactions with sPLA₂ observed in artificial bilayers apply to biological membranes. It is also proposed that the high level of cholesterol in erythrocyte membranes is a protective mechanism to guard against hydrolytic enzymes.     

Binding of cytochrome b5 to cholesterol-containing phosphatidylcholine vesicles

Cytochrome b₅ was found to bind readily to sonicated vesicles containing as much as 0.8 mol cholesterol per mol egg phosphatidylcholine. This observation conflicts with the suggestion of Enomoto and Sato ((1977) Biochim. Biophys. Acta 466, 136–147) that cholesterol prevents binding of this protein to erythrocyte membranes.     

Survival of Frozen Mycoplasmas

Cooling to -70 C killed a higher percentage of Acholeplasma laidlawii and Mycoplasma mycoides var. capri cells than cooling to -20 C. However, to preserve cell viability for prolonged periods storage at -70 C was much more preferable. The percentage of cells surviving freezing could be increased by increasing the initial cell concentration or by the addition of dimethyl sulfoxide or glycerol as cryoprotective agents. In the presence of 1.5 M of any one of these agents survival rates of up to 100% could be obtained. The optimal cooling rates for maximal survival of A. laidlawii under the experimental conditions tested were 11 C/min for cooling to -20 C and about 15 C/min for cooling to -70 C. Increasing the warming rate during thawing from 0.6 to 67 C/min increased survival by 3 log. Oleic acid enrichment of A. laidlawii membrane lipids, or reduction in the cholesterol content of M. mycoides var. capri membranes, increased the percentage of organisms surviving freezing. Hence, the composition of membrane lipids appears to have a marked influence on the susceptibility of mycoplasmas to freezing injury.     

The temperature dependence of molecular order and the influence of cholesterol in Acholeplasma laidlawii membranes

²H nuclear magnetic resonance (NMR) of Acholesplasma laidlawii membranes grown on a medium supplemented with perdeuterated palmitic acid shows that at 42°C or above, the membrane lipids are entirely in a fluid state, exhibiting the characteristic ‘plateau’ in the variation of deuterium quadrupolar splitting with chain position. Between 42 and 34°C there is a well-defined gel-to-fluid phase transition encompassing the growth temperature of 37°C, and at lower temperatures the membranes are in a highly ordered gel state. The ²H-NMR spectra of the gel phase membranes are similar to those of multilamellar dispersions of chain perdeuterated dipalmitoyl phosphatidylcholine (Davis, J.H. (1979) Biophys. J. 27, 339) as are the temperature dependences of the spectra and their moments. The incorporation of large amounts of cholesterol into the membrane removes the gel to fluid phase transition. Between 20 and 42°C, the position dependence of the orientational order of the hydrocarbon chains of the membranes is similar to that of the fluid phase of the membranes without cholesterol, i.e., they exhibit the plateau in the deuterium quadrupolar splittings. However, the cholesterol-containing membranes have a higher average order, with the increases in order being greater for positions near the carbonyl group of the acyl chains. Below 20°C the ²H spectra of the membranes containing cholesterol change dramatically in a fashion suggestive of complex motional and/or phase behaviour.