A Commensal Strain of Staphylococcus epidermidis Overexpresses Membrane Proteins Associated with Pathogenesis When Grown in Biofilms.

Staphylococcus epidermidis has emerged as one of the major nosocomial pathogens associated with infections of implanted medical devices. The most important factor in the pathogenesis of these infections is the formation of bacterial biofilms. Bacteria grown in biofilms are more resistant to antibiotics and to the immune defence system than planktonic bacteria. In these infections, the antimicrobial therapy usually fails and the removal of the biofilm-coated implanted device is the only effective solution. In this study, three proteomic approaches were performed to investigate membrane proteins associated to biofilm formation: (i) sample fractionation by gel electrophoresis, followed by isotopic labelling and LC-MS/MS analysis, (ii) in-solution sample preparation, followed by isotopic labelling and LC-MS/MS analysis and (iii) in-solution sample preparation and label-free LC-MS/MS analysis. We found that the commensal strain S. epidermidis CECT 231 grown in biofilms expressed higher levels of five membrane and membrane-associated proteins involved in pathogenesis: accumulation-associated protein, staphylococcal secretory antigen, signal transduction protein TRAP, ribonuclease Y and phenol soluble modulin beta 1 when compared with bacteria grown under planktonic conditions. These results indicate that a commensal strain can acquire a pathogenic phenotype depending on the mode of growth.

(1-Deoxy)ceramides in bilayers containing sphingomyelin and cholesterol.

The discovery of a novel sphingolipid subclass, the (1-deoxy)sphingolipids, which lack the 1-hydroxy group, attracted considerable attention in the last decade, mainly due to their involvement in disease. They differed in their physico-chemical properties from the canonical (or 1-hydroxy) sphingolipids and they were more toxic when accumulated in cells, inducing neurodegeneration and other dysfunctions. (1-Deoxy)ceramides, (1-deoxy)dihydroceramides, and (1- deoxymethyl)dihydroceramides, the latter two containing a saturated sphingoid chain, have been studied in this work using differential scanning calorimetry, confocal fluorescence and atomic force microscopy, to evaluate their behavior in bilayers composed of mixtures of three or four lipids. When compared to canonical ceramides (Cer), a C16:0 (1-deoxy)Cer shows a lower miscibility in mixtures of the kind C16:0 sphingomyelin/cholesterol/XCer, where XCer is any (1-deoxy)ceramide, giving rise to the coexistence of a liquid-ordered phase and a gel phase. The latter resembles, in terms of thermotropic behavior and nanomechanical resistance, the gel phase of the C16:0 sphingomyelin/cholesterol/C16:0 Cer mixture [Busto et al., Biophys. J. 2014, 106, 621-630]. Differences are seen between the various C16:0 XCer under study in terms of nanomechanical resistance, bilayer thickness and bilayer topography. When examined in a more fluid environment (bilayers based on C24:1 SM), segregated gel phases are still present. Probably related to such lateral separation, XCer preserve the capacity for membrane permeation, but their effects are significantly lower than those of canonical ceramides. Moreover, C24:1 XCer show significantly lower membrane permeation capacity than their C16:0 counterparts. The above data may be relevant in the pathogenesis of certain sphingolipid-related diseases, including certain neuropathies, diabetes, and glycogen storage diseases.

Structure and functional properties of diacylglycerols in membranes.

1. 1,2-Diacyl-sn-glycerols (DAG) are minor components of cell membranes (about 1 mole% of the lipids) and yet they are potent regulators of both the physical properties of the lipid bilayer and the catalytic behaviour of several membrane-related enzymes. 2. In the pure state DAG's present a considerable polymorphism, with several crystalline phases in addition to the neat fluid phase. The most stable crystalline phase is the so-called beta' phase, a monoclinic crystalline form with orthorhombic perpendicular subcell chain packing, in which both acyl chains lie parallel to each other in a hairpinlike configuration about the sn-1 and sn-2 glycerol carbon atoms. The molecules are organized in a bilayer, with the glycerol backbone roughly parallel to the plane of the bilayer, and the acyl chains tilted at approximately 60 degrees with respect to that plane. Acyl chain unsaturation, and particularly a single cis unsaturation, impairs chain packing in mixed-chain DAG's, and this results in an increased number of metastable crystalline phases. 3. DAG's mix with phospholipids in fluid bilayers when their melting temperature is below or close enough to the melting temperature of the bilayer system. When incorporated in phospholipid bilayers, the conformation of DAG is such that the glycerol backbone is nearly perpendicular to the bilayer, with the sn-1 chain extending from the glycerol Cl carbon into the hydrophobic matrix of the bilayer and the sn-2 chain first extending parallel to the bilayer surface, then making a 90 degrees bend at the position of the sn-1 carbonyl to become parallel to the sn-1 chain. DAG's are located in phospholipid bilayers about two CH2 units deeper than the adjacent phospholipids. DAG's mix nonideally with phospholipids, giving rise to in-plane separations of DAG-rich and -poor domains, even in the fluid state. DAG molecules also increase the separation between phospholipid headgroups, and decrease the hydration of the bilayer surface. Also, because the transversal section of the DAG headgroup is small when compared to that of the acyl chains, DAG favours the (negative) curvature of the lipid monolayers, and DAG-phospholipid mixtures tend to convert into inverted nonlamellar hexagonal or cubic phases. 4. A number of membrane enzyme activities are modulated (activated) by DAG, most notably protein kinase C, phospholipases and other enzymes of lipid metabolism. Protein kinase C activation (and perhaps that of other enzymes as well) occurs as the combined result of a number of DAG-induced modifications of lipid bilayers that include: changes in lipid headgroup conformation, interspacing and hydration, changes in the bilayer propensity to form inverted nonlamellar phases, and lateral phase separations of DAG-rich and -poor domains. Among the DAG-activated enzymes, phospholipases C show the peculiarity of yielding the activator DAG as their reaction product, and this allows the self-induced transition from a low- to a high-activity status. 5. DAG's induce or enhance membrane fusion in a number of ways, mainly through partial dehydration of the bilayer surface, increase in lipid monolayer curvature and perhaps lateral phase separation. DAG-increased fusion rates have been demonstrated in several instances of cation-induced fusion of model membranes, as well as in Ca(2+)-induced fusion of chromaffin granules with plasma membrane vesicles. Also phospholipase C has been shown to induce vesicle aggregation and fusion through the catalytic generation of DAG in the bilayers. A rather general property of DAG is that it promotes vesicular or interparticle aggregation. 6. In the living cell, DAG is often generated through phospholipid degradation in response to an extracellular agonist binding a specific receptor in the cell surface. DAG is said to act as an intracellular second messenger. (ABSTRACT TRUNCATED)

Spectroscopic techniques in the study of membrane solubilization, reconstitution and permeabilization by detergents.

This review focuses on the use of spectroscopic techniques for the study of membrane solubilization, reconstitution, and permeabilization by detergents. Turbidity and light scattering, visible and infrared spectroscopic methods, fluorescence, nuclear magnetic resonance, electron spin resonance and X-ray diffraction are examined from the point of view of their applicability to the above detergent-mediated phenomena. A short introduction is provided about each of the techniques, and references are given for further study.

Surfactant-induced release of liposomal contents. A survey of methods and results.

A systematic approach to the phenomenon of surfactant-dependent release of liposomal contents has been attempted. A variety of methods have been comparatively studied. The influence of the size of the entrapped molecule, nature of the surfactant, composition of bilayers and sonication of liposomes have been considered separately. In order to compare different results, a parameter has been defined, R50, as the phospholipid/surfactant mole ratio producing 50% release of the entrapped solute. This parameter appears to be, to a large extent, independent of time and liposome concentration. Surfactant-induced release of liposomal contents does not occur as a result of breakdown of phospholipid bilayers, but is rather a different phenomenon, occurring at detergent concentrations substantially lower (2-5 times) than solubilization. The required amount of surfactant appears to increase with the size of the entrapped solute. R50 depends clearly on the nature of the soluble amphiphile, but there is no obvious relationship with its critical micellar concentration. Liberation of vesicle content also depends on bilayer composition: phospholipids have various effects on the stability of the membrane, while the hydrophobic peptide, gramicidin A, appears to have little influence. Cholesterol is interesting, since at equimolar proportions with phosphatidylcholine, it decreases the stability of bilayer towards Triton X-100, while increasing it in the presence of cholate. Sonication also exerts an influence on the surfactant-dependent release of vesicle contents; it appears to decrease the bilayer stability, so that lower detergent concentrations are required to liberate the entrapped solutes. Finally, it should be noted that, although the decrease in self-quenching of 6-carboxyfluorescein is a convenient method for the study of solute liberation, glucose release, as detected by enzymatic methods, may be more reliable for accurate measurements.

Detergent solubilisation of phospholipid bilayers in the gel state: the role of polar and hydrophobic forces.

Testing the solubilisation of phosphatidylcholine (PC) bilayers by Triton X-100 reveals that in the gel state, but not in the fluid state, the amount of detergent required to solubilise the phospholipid is highly dependent on the chain length. Saturated C16 and C18 PC are virtually insoluble at 4 degreesC. However, addition of water-soluble reagents that perturb hydrogen bonding, e.g. urea, or of small proportions of non-bilayer lipids, make the bilayers amenable to detergent solubilisation, even at low temperatures. These results are relevant in the explanation of the origin of detergent-resistant membrane fragments as found, e.g. in caveolae or 'rafts'.

Laser light-scattering characterization of mitochondrial complex III-Triton X-100-phospholipid mixed micelles.

Bovine-heart mitochondrial complex III was purified in the presence of Triton X-100, and the size and shape of the resulting protein-surfactant-phospholipid mixed micelles were investigated by laser light-scattering. The protein appears to be present in the form of a dimer, irrespective of temperature (between 25 and 40 degrees C) and protein concentration (between 0.5 and 5 mg/ml). The molecular weight of the micelle increases with temperature from 600 000 (25 degrees C) to 692 000 (40 degrees C). The variation of the solvent second virial coefficient in this temperature range suggests that, with increasing temperature, some of the free surfactant molecules become integrated in the mixed micelles. The average quadratic radius of gyration of these is of 42 +/- 5 nm, corresponding in our case to an ellipsoidal shape.

Phospholipid oxidation catalyzed by cytochrome c in liposomes.

Oxidation of liposome phospholipids has been studied in the presence of cytochrome c. Sonicated vesicles of soya bean or egg-yolk lipids, or purified phospholipid preparations, were treated with oxidized cytochrome c at a 10:4 lipid/protein ratio (w/w). Lipid peroxidation was examined by oxygen polarography, gas-liquid chromatography (GLC) and the thiobarbituric acid test. Oxidized, but not reduced, cytochrome effectively catalyzes lipid oxidation under these conditions. Oxygen consumption and disappearance of unsaturated fatty acids follow closely similar patterns, the O2 consumption rate showing a maximum (1.53 mol O2/min per mol heme) shortly before fatty acid loss reaches its peak. GLC and O2 consumption data suggest that monohydroperoxides are the most abundant oxidized species in the system. The thiobarbituric acid reaction, however, appears only to be of qualitative value in peroxidation studies. In order to test the mechanism through which oxidation occurs in our system, the effect of liposome composition and the presence of antioxidants was tested, both on cytochrome c binding to bilayers and on O2 consumption. Oxidized and reduced cytochrome c bind the lipid bilayers with similar affinity, but only the oxidized form is active in autoxidation. Antioxidants do not modify either cytochrome c binding to sonicated liposomes. Lipid composition does influence considerably cytochrome binding, and O2 consumption is correspondingly altered. Studies with various antioxidants and inhibitors suggest that both free radicals and singlet oxygen may be involved in the process under study.

Infrared spectroscopy of phosphatidylcholines in aqueous suspension. A study of the phosphate group vibrations.

The 1000-1300 cm-1 region of the infrared spectrum of dipalmitoylphosphatidylcholine (DPPC) and other phosphate-containing molecules has been studied by the Fourier-transform technique. Three absorption bands have been assigned to various vibrational modes of the DPPC phosphate group, with maximum wavenumbers at 1060, 1086 and 1222 cm-1. These values are the same above and below Tc of the phospholipid. Dehydration produces band-shifts toward higher wavenumbers .

An infrared spectroscopic study of specifically deuterated fatty-acyl methyl groups in phosphatidylcholine liposomes.

The region of the infrared spectrum corresponding to C-2H stretching vibrations (2050-2250 cm-1) has been examined for liposomes composed of dimyristoylphosphatidylcholine deuterated specifically at the methyl ends of either one (sn-2) or both the fatty acyl chains. This label is intended to provide information on lipid dynamics in the contact region between monolayers. The two most prominent bands observed correspond, respectively, to antisymmetric (2212 cm-1) and symmetric (2075 cm-1) C-2H stretching vibration. The antisymmetric band consists of two overlapping peaks, whose positions vary with the gel or liquid-crystalline state of the lipid. The separation between the peaks making up the antisymmetric band increases with temperature, and is maximum above the Tc transition temperature; this rules out the previously proposed assignment of these two peaks to different rotational modes of the methyl group relative to the adjacent methylene. The position and width of the symmetric band at 2075 cm-1 are also sensitive to the physical state of the lipid. The presence of cholesterol at an equimolar ratio with the phospholipid abolishes all the phase-dependent changes observed. The intrinsic polypeptide gramicidin A, at a 5:1 lipid/peptide mol ratio, is seen to enlarge the lipid thermotropic transition, with small effects above Tc. Cytochrome c, an extrinsic protein, at a 10:1 mole ratio, does not modify the phase-dependent behaviour of the terminal methyl groups, but consistently shifts all the observed bands to lower-frequency positions, which suggests a long-range effect of the protein along the phospholipid fatty acyl chains.

Electrokinetic charge of the anesthetic-induced bR480 and bR380 spectral forms of bacteriorhodopsin.

The translational and rotational electrokinetics of the anesthetic-induced spectral transitions bR568-->bR480-->bR380 of bacteriorhodopsin have been investigated. Formation of the bR480 form is associated with an increase of the purple membrane negative electrokinetic charge, while the transformation of bR480 into bR380 is accompanied by a decrease of the membrane negative charge as compared to that of the 480 nm-absorbing form. Removal of anesthetics leads to the back transitions bR480-->bR568 and (in part) bR380-->bR568; however, the electrokinetic charge of the native membranes is not restored. A strong decrease in the electric polarizability and the appearance of a slow polarizability component are also observed in anesthetic-treated membranes. Comparison with the electrokinetic behaviour of partially delipidated membranes and with that of liposomes composed of purple membrane total lipids suggests that: (i) anesthetic molecules partition mainly at the protein/lipid interface inducing irreversible rearrangement of the boundary lipid layer, and (ii) different mode(s) or site(s) of interaction are responsible for the spectral and surface charge effects. The data are compatible with the hypothesis of anesthetics acting through partial dehydration of the membrane surface.

An assessment of the biochemical applications of the non-ionic surfactant Hecameg.

A number of properties and effects of the novel non-ionic detergent Hecameg (6-O-(N-heptylcarbamoyl)-methyl-alpha-D-glucopyranoside) have been examined in view of its possible biochemical applications. In particular, its critical micellar concentration has been measured, and its effects on pure lipid membranes, soluble and membrane-bound enzymes have been recorded. Hecameg has some advantageous and some less advantageous properties; its relatively high critical micellar concentration (16.5 mM), almost insensitive to pH or ionic strength changes, makes it suitable for reconstitution procedures in which detergent must be removed by dialysis. It is also an effective lipid-solubilizing agent, producing leakage of vesicle contents at detergent concentrations well below the solubilizing range. Among the drawbacks, the presence of an amide group in the molecule may interfere with the protein amide group in spectroscopic measurements. It also appears to be less gentle than other nonionic surfactants towards certain enzyme activities.

Calcium-dependent conformation of E. coli alpha-haemolysin. Implications for the mechanism of membrane insertion and lysis.

Previous studies from this laboratory had shown that calcium ions were essential for the membrane lytic activity of E. coli alpha-haemolysin (HlyA), while zinc ions did not sustain such a lytic activity. The present data indicate that calcium-binding does not lead to major changes in the secondary structure, judging from circular dichroism spectra. However binding to Ca2+ exposes new hydrophobic residues at the protein surface, as indicated by the increased binding of the fluorescent probe aniline naphtholsulphonate (ANS), and by the increased tendency of the Ca2+-bound protein to self-aggregate. In addition zinc ions are seen to decrease the thermal stability of HlyA which, according to intrinsic fluorescence and differential scanning calorimetry data, is stable below 95 degrees C when bound to calcium, while it undergoes irreversible denaturation above 60 degrees C in the zinc-bound form. Binding to phosphatidylcholine bilayers is quantitatively similar in the presence of both cations, but about one-third of the zinc-bound HlyA is released in the presence of 2 M NaCl. Differential scanning calorimetry of dimyristoylglycerophosphocholine large unilamellar vesicles reveals that Zn2+-HlyA interaction with the lipid bilayer has a strong polar component, while Ca2+-HlyA appears to interact mainly through hydrophobic forces. Experiments in which HIyA transfer is measured from phospholipid vesicles to red blood cells demonstrate that Ca2+ ions promote the irreversible binding of the toxin to bilayers. All these data can be interpreted in terms of a specific Ca2+ effect that increases the surface hydrophobicity of the protein, thus facilitating its irreversible bilayer insertion in the fashion of intrinsic membrane proteins.

The modulation of membrane fluidity by hydrogenation processes. II. Homogeneous catalysis and model biomembranes.

A homogeneous catalyst, chlorotris (triphenylphosphine) rhodium (I) has been incorporated into model biomembrane structures in the form of lipid bilayer dispersions in water. This enables the hydrogenation of the double bonds of the unsaturated lipids within the bilayers to be accomplished. To decide the optimum conditions for efficient hydrogenation the reaction conditions have been varied. The effect of catalyst concentration, hydrogen gas pressure and lipid composition (with and without cholesterol) have all been studied. The partition of the catalyst into the lipid medium was checked by rhodium analysis. The results show that an increase of catalyst concentration or an increase of hydrogen gas pressure leads to increasing rates of hydrogenation. Successful hydrogenation was accomplished with different types of lipid dispersions (mitochondrial, microsomal and erythrocyte lipids). A selectivity of the homogeneous hydrogenation process is indicated. The polyunsaturated fatty acyl residues are hydrogenated at an earlier stage and at a faster rate than the monoenoic acids. Furthermore, an increase in the proportion of cholesterol to lipid within the bilayer structures causes a progressive decrease in the rate of hydrogenation. The fluidity of the lipid bilayers can be altered to such an extent by the hydrogenation process that new sharp endotherms corresponding to the order-disorder transition of saturated lipids occur at temperatures as high as 319 K. Some potential uses of hydrogenation for the modulation of cell membrane fluidity are discussed as well as the design of new types of catalyst molecules.

Early and delayed stages in the solubilization of purple membrane by a polyoxyethylenic surfactant.

The purpose of this paper is to explore the reasons by which purple membrane solubilization by detergents takes hours, or even days, to reach equilibrium, while most biomembranes are solubilized in a matter of seconds, or minutes. With that aim, changes in the purple membrane absorption spectrum produced by hydrogenated Triton X-100 under equilibrium conditions (24 h) have been compared to those caused by the same surfactant in the minute, second and sub-second time scale. It is found that the various processes that accompany, or lead to, solubilization are already detected, and even reach an apparent equilibrium, in the 10 s that follow detergent addition. No new phenomena are detected in the following minutes, or hours, that are relevant to the process under study. This leads to the conclusion that the long solubilization process consists of the repeated operation of simple phenomena that are relatively fast in themselves. A hypothesis is proposed according to which the tight crystalline organization of the purple membrane prevents the insertion of detergent monomers in the lipid bilayer; instead, the surfactant would bind the periphery of the patches, i.e., the hydrocarbon-water contact region, and solubilization would take place gradually, from the periphery towards the core of the membrane patches, at a progressively lower rate as the amounts of free detergent and detergent-binding sites are decreased by the previous solubilization steps.

The influence of membrane composition on the solubilizing effects of Triton X-100.

Multilamellar liposomes containing pure phosphatidylcholine (PC) or mixtures of PC with cholesterol, cholesteryl palmitate, beta-carotene, cardiolipin, phosphatidylethanolamine or gramicidin A have been treated with the detergent Triton X-100. Solubilization has been monitored as a decrease in turbidity of the liposome suspension, and also by determination of bilayer components in the solubilized fraction. The same solubilization pattern is found for unsaturated (egg yolk) or saturated (dimyristoyl) PC. Similar results are also found when dimyristoyl PC is solubilized above or below its gel-to-fluid transition temperature. Cholesterol solubilizes in parallel with PC; gramicidin A is solubilized preferentially to this phospholipid and the non-polar lipids cholesteryl palmitate or beta-carotene remain insoluble at detergent concentrations producing complete PC solubilization. Addition of cardiolipin or phosphatidylethanolamine does not seem to alter the general pattern of PC solubilization. Phosphatidylethanolamine is less soluble than PC, while cardiolipin solubilizes at the same detergent concentrations than PC. These results are considered in relation to previous studies with natural membranes.

Lipid-protein interactions. The mitochondrial complex III-phosphatidylcholine-water system.

Bovine heart mitochondrial complex III (ubiquinol-cytochrome-c reductase) has been reconstituted into phosphatidylcholine bilayers and the effect of varying lipid/protein ratios on the structure and function of the protein has been examined. Electron microscopy, differential scanning calorimetry and Arrhenius plots of enzyme activity provide evidence that the protein is incorporated in an active conformation into pure phosphatidylcholine bilayers. At low lipid/protein ratios (e.g. 80:1 molar ratio) the protein exists in the form of aggregates. As the lipid proportion is increased, electron microscopy reveals the gradual formation of lipid bilayers; structures with the appearance of closed vesicles are seen at or above 300:1 phospholipid/protein molar ratios. Changes in enzyme activity as a function of lipid contents reveal a progressive increase in activity as more lipid is added, with a tendency to reach a saturation point. From the experimental data, a kinetic model is proposed, according to which the protein has an indefinite number of unspecific, independent and identical binding sites for phospholipids, the latter acting as essential enzyme activators. Varying lipid/protein ratios induce structural changes in complex III; visible spectra indicate changes in the polarity of the heme group environment, while Fourier-transform infrared spectroscopy suggests a change in the secondary structure of the protein as the lipid proportion is increased.

Real-time measurements of chemically-induced membrane fusion in cell monolayers, using a resonance energy transfer method.

Fusion of mouse melanoma cells grown in monolayers has been directly monitored by fluorescence resonance energy transfer between fluorescein and rhodamine probes attached to octadecanoic acid. Various poly(ethylene glycol)s (PEG), either alone or in combination with amphipathic molecules, have been used as fusogens. Fusion starts at a maximum rate as soon as PEG is removed from the medium and reaches a plateau after 20-30 min. Both the initial rate and extent of fusion have been recorded for each experiment. The extent of fusion shows in general a positive correlation with the initial rate, although PEGs with different molar masses appear to induce fusion at different rates, but to a similar extent. A good correlation has been found between the extent of fusion, as measured by fluorescence, and the 'fusion index' computed from cell and nucleus counting; a calibration curve is provided for the interconversion of both parameters. Optimum fusion values are obtained with 50% (w/v) PEG 1500. The effect of pre-treatments with surfactants (Triton X-100, sodium dodecylsulphate) on PEG-induced fusion has also been tested. Sodium dodecylsulphate, but not Triton, enhances considerably both the rate and extent of cell fusion. The in situ generation of the amphipathic molecule diacylglycerol, through the catalytic activity of a phospholipase C, also enhances significantly the fusion parameters. These results are in good agreement with previous studies based on syncytia counting.

Interactions of the HIV-1 fusion peptide with large unilamellar vesicles and monolayers. A cryo-TEM and spectroscopic study.

We have examined the interaction of the human immunodeficiency virustype 1 fusion peptide (23 amino acid residues) and of a Trp-containing analog with vesicles composed of dioleoylphosphatidylcholine, dioleoylphosphatidylethanolamine and cholesterol (molar ratio, 1:1:1). Both the native and the Trp-substituted peptides bound the vesicles to the same extent and induced intervesicular lipid mixing with comparable efficiency. Infrared reflection-absorption spectroscopy data are compatible with the adoption by the peptide of a main beta-sheet structure in a cospread lipid/peptide monolayer. Cryo-transmission electron microscopy observations of peptide-treated vesicles reveal the existence of a peculiar morphology consisting of membrane tubular elongations protruding from single vesicles. Tryptophan fluorescence quenching by brominated phospholipids and by water-soluble acrylamide further indicated that the peptide penetrated into the acyl chain region closer to the interface rather than into the bilayer core. We conclude that the differential partition and shallow penetration of the fusion peptide into the outer monolayer of a surface-constrained bilayer may account for the detected morphological effects. Such single monolayer-restricted interaction and its structural consequences are compatible with specific predictions of current theories on viral fusion.