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At least 487 records · Page 27Linked to original sources
PubMed1995

The Neisseria meningitidis outer membrane protein P1 produced in Bacillus subtilis and reconstituted into phospholipid vesicles elicits antibodies to native P1 epitopes.

Class 1 outer membrane protein (P1) of Neisseria meningitidis group B is considered a promising vaccine candidate because P1 subtype-specific antibodies have been shown to be protective in an animal model. We have previously described the production of P1 in the Gram-positive Bacillus subtilis as intracellular inclusion bodies, from which the protein (BacP1) is easily purified (Nurminen et al., Mol. Microbiol., 1992, 2499-2506). We show here that the purified BacP1 can be reconstituted into phospholipid vesicles with the formation of the native immunodominant surface epitopes. The detergent-solubilized, completely denatured BacP1 was fused with phospholipid-detergent micelles during detergent removal by dialysis or gel filtration to yield protein-lipid vesicles (liposomes). When mice were immunized with these liposomes, they produced high titers of antibodies reacting in a P1 subtype-specific manner with meningococcal cells indicating the presence of conformation-dependent P1-specific epitopes in the liposomes. The results suggest that a vaccine candidate for meningococcal disease could be developed from the BacP1-liposomes. They furthermore demonstrate the feasibility of refolding a denatured outer membrane protein, which has never been exposed to lipopolysaccharide, into a native-like conformation.

Amino Acid Sequence↗
PubMed1990

Two substrate sites in the renal Na(+)-D-glucose cotransporter studied by model analysis of phlorizin binding and D-glucose transport measurements.

Time courses of phlorizin binding to the outside of membrane vesicles from porcine renal outer cortex and outer medulla were measured and the obtained families of binding curves were fitted to different binding models. To fit the experimental data a model with two binding sites was required. Optimal fits were obtained if a ratio of low and high affinity phlorizin binding sites of 1:1 was assumed. Na+ increased the affinity of both binding sites. By an inside-negative membrane potential the affinity of the high affinity binding site (measured in the presence of 3 mM Na+) and of the low affinity binding site (measured in the presence of 3 or 90 mM Na+) was increased. Optimal fits were obtained when the rate constants of dissociation were not changed by the membrane potential. In the presence of 90 mM Na+ on both membrane sides and with a clamped membrane potential, KD values of 0.4 and 7.9 microM were calculated for the low and high affinity phlorizin binding sites which were observed in outer cortex and in outer medulla. Apparent low and high affinity transport sites were detected by measuring the substrate dependence of D-glucose uptake in membrane vesicles from outer cortex and outer medulla which is stimulated by an initial gradient of 90 mM Na+ (out greater than in). Low and high affinity transport could be fitted with identical Km values in outer cortex and outer medulla. An inside-negative membrane potential decreased the apparent Km of high affinity transport whereas the apparent Km of low affinity transport was not changed. The data show that in outer cortex and outer medulla of pig high and low affinity Na(+)-D-glucose cotransporters are present which contain low and high affinity phlorizin binding sites, respectively. It has to be elucidated from future experiments whether equal amounts of low and high affinity transporters are expressed in both kidney regions or whether the low and high affinity transporter are parts of the same glucose transport molecule.

Animals↗
PubMed1990

Heterogeneity among membrane vesicles of Escherichia coli: effects of production and fractionation techniques.

We have previously shown that Escherichia coli membrane proteins are associated with subpopulations of membrane vesicles beyond the well-defined inner versus outer membrane classification. To separate these vesicles we used a shallow sucrose gradient which differed in many respects from established procedures. Here we compare this revised technique to the classical sucrose density centrifugation procedure. We found that certain manipulations common to the latter obscured heterogeneity among membrane vesicles. The following treatments degraded vesicle separation: growth in rich medium addition of EDTA to buffers, spheroplasting, sonication, pelleting of membranes prior to sucrose gradient centrifugation, overloading the gradient, and long centrifugation times. Some treatments, such as EDTA addition, affected only selected vesicles. When determining protein localization within bacterial membranes experiments should be designed to avoid or at least minimize these manipulations.

Cell Membrane↗
PubMed1982

DNA-binding vesicles released from the surface of a competence-deficient mutant of Haemophilus influenzae.

Haemophilus influenzae com-51, a mutant deficient in DNA uptake, produces an extracellular DNA-binding activity. The activity was specific for Haemophilus DNA and was isolated from cell-free competence medium after incubation for 100 to 130 min. Initial steps in the purification procedure resulted in the loss of detectable binding activity, but activity was restored by the addition of a nonionic detergent. The active fractions contained vesicles derived from the outer membrane of the cells. The vesicles were produced only under conditions that normally lead to competence development. The lack of competence of com-51 cells was not due to loss of protein synthesis in M-IV competence medium or to competition of extracellular protein for exogenous DNA. Results suggest that the inability of cells to bind DNA was due in part to the loss of DNA receptors that are released into the medium in membrane fragments.

Bacterial Proteins↗
PubMed2006

The interaction of cationic antimicrobial peptides with vesicles containing synthetic glycolipids as models of the outer membrane of gram-negative bacteria.

Two simple lipid A analogues methyl 2,3-di-O-tetradecanoyl-alpha-D-glucopyranoside (GL1) and methyl 2,3-di-O-tetradecanoyl-alpha-D-glucopyranoside 4-O-phosphate (GL2) were synthesized and used for preparing mixed phosphocholine vesicles as models of the outer membrane of gram-negative bacteria. The interaction of these model membranes with magainin 2, a representative of the alpha-helical membrane active peptides, and apidaecin Ib and drosocin, two insect Pro-rich peptides which do not act at the level of the cellular membrane, were studied by CD and dye-releasing experiments. The CD spectra of apidaecin Ib and drosocin in the presence of GL1- or GL2-containing vesicles were consistent with largely unordered structures, whereas, according to the CD spectra, magainin 2 adopted an amphipathic alpha-helical conformation, particularly in the presence of negatively charged bilayers. The ability of the peptides to fold into amphipathic conformations was strictly correlated to their ability to bind and to permeabilize phospholipid as well as glycolipid membranes. Apidaecin Ib and drosocin, which are unable to adopt an amphipathic structure, showed negligible dye-leakage activity even in the presence of GL2-containing vesicles. It is reasonable to suppose that, as for the killing mechanism, the two classes of antimicrobial peptides follow different patterns to cross the bacterial outer membrane.

Anti-Bacterial Agents↗
PubMed1992

Fusion between disk membranes and plasma membrane of bovine photoreceptor cells is calcium dependent.

Disk membranes and plasma membrane vesicles were prepared from bovine retinal rod outer segments (ROS). The plasma membrane vesicles were labeled with the fluorescent probe octadecylrhodamine B chloride (R18) to a level at which the R18 fluorescence was self-quenched. At pH 7.4 and 37 degrees C and in the presence of micromolar calcium, an increase in R18 fluorescence with time was observed when R18-labeled plasma membrane vesicles were introduced to a suspension of disks. This result was interpreted as fusion between the disk membranes and the plasma membranes, the fluorescence dequenching resulting from dilution of the R18 into the unlabeled membranes as a result of lipid mixing during membrane fusion. While the disk membranes exposed exclusively their cytoplasmic surface, plasma membrane vesicles were found with both possible orientations. These vesicles were fractionated into subpopulations with homogeneous orientation. Plasma membrane vesicles that were oriented with the cytoplasmic surface exposed were able to fuse with the disk membranes in a Ca(2+)-dependent manner. Fusion was not detected between disk membranes and plasma membrane vesicles oriented such that the cytoplasmic surface was on the interior of the vesicles. ROS plasma membrane-disk membrane fusion was stimulated by calcium, inhibited by EGTA, and unaffected by magnesium. Rod photoreceptor cells of vertebrate retinas undergo diurnal shedding of disk membranes containing the photopigment rhodopsin. Membrane fusion is required for the shedding process.

Animals↗
PubMed2004

Enterotoxigenic Escherichia coli vesicles target toxin delivery into mammalian cells.

Enterotoxigenic Escherichia coli (ETEC) is a prevalent cause of traveler's diarrhea and infant mortality in third-world countries. Heat-labile enterotoxin (LT) is secreted from ETEC via vesicles composed of outer membrane and periplasm. We investigated the role of ETEC vesicles in pathogenesis by analyzing vesicle association and entry into eukaryotic cells. Fluorescently labeled vesicles from LT-producing and LT-nonproducing strains were compared in their ability to bind adrenal and intestinal epithelial cells. ETEC-derived vesicles, but not control nonpathogen-derived vesicles, associated with cells in a time-, temperature-, and receptor-dependent manner. Vesicles were visualized on the cell surface at 4 degrees C and detected intracellularly at 37 degrees C. ETEC vesicle endocytosis depended on cholesterol-rich lipid rafts. Entering vesicles partially colocalized with caveolin, and the internalized vesicles accumulated in a nonacidified compartment. We conclude that ETEC vesicles serve as specifically targeted transport vehicles that mediate entry of active enterotoxin and other bacterial envelope components into host cells. These data demonstrate a role in virulence for ETEC vesicles.

Animals↗
PubMed1997

Solubilized and cleaved VP7, the outer glycoprotein of rotavirus, induces permeabilization of cell membrane vesicles.

It has been previously shown that rotavirus triple-layered particles induce permeabilization of liposomes and membrane vesicles. These effects were mediated by one or both of the solubilized outer-capsid proteins, VP4 and VP7. Permeabilization was dependent on trypsin treatment of the viral particles, suggesting that VP4 was involved. To analyse the respective roles of the outer-capsid proteins in this permeabilization process, we have used membrane vesicles loaded with carboxyfluorescein and virus-like particles derived from insect cells co-expressing various sets of capsid proteins. Virus-like particles containing VP2, VP6 and VP7 (VLP2/6/7) are as efficient in permeabilizing vesicles as triple-layered particles. As with double-layered particles, virus-like particles made of VP2 and VP6 had no effect on vesicle permeabilization. Permeabilization of membrane vesicles required trypsinization of the VP7 solubilized from VLP2/6/7. These results show that solubilized and trypsinized VP7 is able to induce membrane permeabilization, independently of the presence of VP4.

Antigens, Viral↗
PubMed1985

[Comparative cytologic studies on the effect of cetyltrimethylammonium bromide on bacterial cells].

Growing cultures of Pseudomonas aeruginosa and Bacillus megaterium show after treatment with cetyltrimethylammonium bromide (CTAB) typical concentration-dependent alterations of envelope. In Ps. aeruginosa low doses of the detergent cause perforations and lesions of the cytoplasmic membrane, by 0.016% CTAB the formation of extracellular vesicles of the outer membrane ("blebs") and the intracellular assembly of lamellar structures can be detected. These intracellular lamellar structures are artifacts of the cytoplasmic membrane after detergent application. In this case the conglomeration of bacterial cells can be demonstrated with the scanning electron microscope. The results are discussed in connection with the use of CTAB in inactivation and permeabilization of bacteria.

Anti-Infective Agents, Local↗
PubMed1985

ATP is essential for protein translocation into Escherichia coli membrane vesicles.

The energy requirement for translocation of alkaline phosphatase and the outer membrane protein OmpA into Escherichia coli membrane vesicles was studied under conditions that permit posttranslational translocation and, hence, prior removal of various components necessary for protein synthesis. Translocation could be supported by an ATP-generating system or, less well, by the protonmotive force generated by D-lactate oxidation; the latter might act by generating ATP from residual bound nucleotides. However, when protonmotive force inhibitors were used or when ATP was further depleted by E. coli glycerol kinase, D-lactate no longer supported the translocation. Furthermore, ATP could still support protein translocation in the presence of proton uncouplers or with membranes defective in the F1 fraction of the H+-ATPase. We conclude that ATP is required for protein translocation in this posttranslational system (and probably also in cotranslational translocation); the protonmotive force may contribute but does not appear to be essential.

Adenosine Diphosphate↗
PubMed1992

Herpes simplex virus envelopment and maturation studied by fracture label.

Herpes simplex virus envelopment and maturation were investigated by thin-section fracture label. The distribution of glycoproteins B and D was analyzed by labeling with antibodies; the precursor and mature forms of the glycoproteins were differentiated by labeling with the lectins concanavalin A (ConA) and wheat germ agglutinin (WGA), respectively. We report that the two glycoproteins were readily detected in the intracellular virion, whether located between the inner and outer nuclear membranes or within cytoplasmic membrane-bound vesicles and in the inner and outer nuclear membranes themselves. The enveloped virion between the inner and outer nuclear membranes labeled with ConA but not with WGA. During the transit to the extracellular space the reactivity of the virion membranes with ConA decreased and that with WGA ensued. The results document that herpes simplex viruses acquire at the inner nuclear membrane an envelope carrying the immature forms of the glycoproteins and that during the transit to the extracellular space the envelope glycoproteins become of the fully processed type.

Animals↗
PubMed1991

Transport kinetics of glucose and alanine in renal brush-border membrane vesicles of cadmium-intoxicated rabbits.

Changes in transport kinetics of D-glucose and L-alanine were studied in renal luminal membrane vesicles isolated from cadmium-intoxicated rabbits. Cadmium intoxication was induced by subcutaneous injections of CdCl2 at a dose of 2 mg Cd/kg/day for 2-3 weeks. Brush-border membrane vesicles were prepared from renal outer cortex and outer medulla by Percoll gradient centrifugation. Cadmium intoxication resulted in a marked attenuation of Na(+)-dependent transports of D-glucose and L-alanine in both outer cortical and outer medullary brush-border membrane vesicles, and this was due to reduction in Vmax and not Km. Similar results were obtained in normal vesicles directly exposed to free cadmium. These results suggest that in long-term cadmium-exposed animals free cadmium ions liberated in the proximal tubular cytoplasm may directly impair brush-border membranes thereby reducing capacity of Na(+)-dependent transport systems for glucose and amino acids.

Alanine↗
PubMed1987

DCCD inhibits protein translocation into plasma membrane vesicles from Escherichia coli at two different steps.

In vitro translocation of periplasmic and outer membrane proteins into inverted plasma membrane vesicles from Escherichia coli was completely prevented by the H+-ATPase inhibitor N,N'-dicyclohexylcarbodiimide (DCCD). DCCD was inhibitory to both co- and post-translational translocations, suggesting an involvement of the H+-translocating F1F0-ATPase in either mode of transport. This was verified by (i) the dependence of efficient co-translational translocation upon a low salt, i.e. F1-containing extract from membrane vesicles; (ii) the co-purification of the translocation activity present in this extract and F1-ATPase; (iii) the inability of either vesicles or their low-salt extract, derived from F1F0-ATPase-lacking mutant strains, to support translocation; and (iv) the greatly diminished extent of ATP-dependent, post-translational translocation into F1-deprived vesicles. Membranes devoid of F1 did show, however, residual translocation activity that was also found to be inhibitable by DCCD. These results suggest a dual target for DCCD in bacterial protein export, one being the H+-ATPase and the other an as yet unidentified translocation factor.

Bacterial Outer Membrane Proteins↗
PubMed1984

Incorporation of ion channels from bovine rod outer segments into planar lipid bilayers.

Membranes vesicles, prepared from bovine rod outer segments were fused with planar lipid bilayers. Two different ion channels were identified by recording currents from single channels. Both types of channels were selective for sodium rather than potassium and were impermeable to chloride ions. Unit conductances were 20 and 120 pS, respectively, in 150 mM sodium chloride. The channel with the larger unit conductance was sensitive to the transmembrane potential. This channel rapidly activated within less than 10 ms after a voltage jump to a more negative membrane potential and then inactivated after several seconds. The duration of the active period and the properties of the channel depended on the amplitude of the voltage jump. The channel of smaller unit conductance did not show any voltage-dependent activation or inactivation. Both types of channels were insensitive to light in the planar bilayer system. Channels incorporated into planar bilayers on a Teflon sandwich septum or on the tip of a glass micropipette gave similar results.

Animals↗
PubMed2005

Proteomics-based identification of outer-membrane proteins responsible for import of macromolecules in Sphingomonas sp. A1: alginate-binding flagellin on the cell surface.

A nonmotile gram-negative bacterium, Sphingomonas sp. A1, directly incorporates macromolecules such as alginate through a "super-channel" consisting of a pit formed on the cell surface, alginate-binding proteins in the periplasm, and an ATP-binding cassette transporter in the inner membrane. Here, we demonstrate the proteomics-based identification of cell-surface proteins involved in the formation of the pit and/or import of alginate. Cell-surface proteins were prepared from the outer membrane released as vesicles during the conversion of intact cells to spheroplasts. Seven proteins (p1-p7) with acidic isoelectric points were inducibly expressed in the outer membrane of strain A1 cells grown on alginate and showed significant identity with bacterial cell-surface proteins (p1-p4, TonB-dependent outer-membrane transporter; p5 and p6, flagellin; and p7, lipoprotein). Each mutant with a disruption of the p1-p4 or p6 gene showed significant growth retardation in the alginate medium. Flagellin homologues (p5 and p6) were further analyzed because strain A1 forms no flagellum. p5 was found to be uniformly distributed on the cell surface by immunogold-labeling electron microscopy and to exhibit alginate binding with a nanomolar dissociation constant by a surface plasmon resonance sensor. The cell surface of the p6 gene disruptant differed from that of the wild-type strain A1 in that pit formation was incomplete and cell-surface structures shifted from pleats to networks. These results suggest that, distinct from bacterial flagellins constituting a helical filament of flagella, strain A1 cell-surface flagellin homologues function as receptors for alginate and/or regulators of cell-surface structures.

Alginates↗
PubMed1975

Cytoplasmic membrane vesicles of Escherichia coli. A simple method for preparing the cytoplasmic and outer membranes.

A simple preparative method is described for isolation of the cytoplasmic and outer membranes from E. coli. The characteristics of both membrane fractions were studied chemically, biologically, and morphologically. Spheroplasts of E. coli K-12 strain W3092, prepared by treating cells with EDTA-lysozyme [EC 3.2.1.17], were disrupted in a French press. The crude membrane fraction was washed with 3 mM EDTA-10% (w/v) sucrose, pH 7.2, and the cytoplasmic membranes and outer membranes were separated by sucrose isopycnic density gradient centrifugation. The crude membrane fraction contained approximately 10% of the protein of the whole cells, 0.3% of the DNA, 0.7% of the RNA, 0.3% of the peptidoglycan, and about 30% of the lipopolysaccharide. The cytoplasmic membrane fraction was rich in phospholipid, while the outer membrane fraction contained much lipopolysaccharide and carbohydrate; the relative contents of lipopolysaccharide and carbohydrate per mg protein in the cytoplasmic membrane fraction were 12 and 40%, respectively, of the contents in the outer membrane fraction. Cytochrome b1, NADH oxidase, D-lactate dehydrogenase [EC 1.1.1.28], succinate dehydrogenase [EC 1.3.99.1], ATPase [EC 3.5.1.3], and activity for concentrative uptake of proline were found to be localized mainly in the cytoplasmic membranes; their specific activities in the outer membrane fraction were 1.5 to 3% of those in the cytoplasmic membrane fraction. In contrast, a phospholipase A appeared to be localized mainly in the outer membranes and its specific activity in the cytoplasmic membrane fraction was only 5% of that in the outer membrane fraction. The cytoplasmic and outer membrane fractions both appeared homogeneous in size and shape and show vesicular structures by electron microscopy. The advantages of this method for large scale preparation of the cytoplasmic and outer membrane fractions are discussed.

Adenosine Triphosphatases↗