Outer Membrane Of Cell Wall Research Articles

Effect of parenteral endotoxin administration on the immuno-haematological responses of catfish, Heteropneustes fossilis

Endotoxin, the outer cell wall membrane lipopolysaccharide component of the Gram-negative bacteria is a factor responsible for a number of complications/disorders and plays important role in the associated with pathophysiological complications and pathogenesis of many diseases in animals. Unlike higher animals which are extremely sensitive to endotoxin, fish are found to be resistant to endotoxic shock and earlier studies though limited have demonstrated the patho-physiological, immuno-endocrinological and immuno-neurological effects of LPS/endotoxin in aquatic animals including fish. Herein in the present investigation, the effect of pure endotoxin on immuno-haematological parameters of stinging catfish, Heteropneustes fossilis ranging from 50–60 g was studied by intraperitoneally injecting 0.1, 0.05 and 0.01 mg endotoxin per fish. H. fossilis yearlings were found to resist the endotoxin concentration up to 0.1 mg without any mortality. While, no change in immune parameters was recorded in stinging catfish injected with low dose of endotoxin (0.01 mg), most of the immune parameters were found to be significantly elevated in catfish injected with 0.05 mg endotoxin. Different serum and immune parameters like protein, globulin, lysozyme, respiratory burst activity, myeloperoxidase activity, natural agglutination titre were found to be significantly high (p < 0.01) at a dose of 0.05 mg endotoxin per fish. On the contrary, most of these parameters were decreased at high dose i.e., 0.1 mg endotoxin per fish, thereby indicating the immuno-suppressive effect of the endotoxin. The findings of the modulation of innate immunity also corroborated with the results of Aeromonas hydrophila pathogen challenge study with highest percent of mortality in group injected with 0.1 mg endotoxin per fish and least percentage in group injected with 0.05 mg endotoxin per fish.

Enolase-like protein present on the outer membrane of Pseudomonas aeruginosa binds plasminogen

Pseudomonas aeruginosa is one of the pathogenic bacteria which utilize binding of the host plasminogen (Plg) to promote their invasion throughout the host tissues. In the present study, we confirmed that P. aeruginosa exhibits binding affinity for human plasminogen. Furthermore, we showed that the protein detected on the cell wall of P. aeruginosa and binding human plasminogen is an enolase-like protein. The hypothesis that alpha-enolase, a cytoplasmatic glycolytic enzyme, resides also on the cell surface of the bacterium was supported by electron microscopy analysis. The plasminogen-binding activity of bacterial cell wall outer membrane enolase-like protein was examined by immunoblotting assay.

Gonococcal Infections

Gonococcal Infections

Gonococcal Infections

Gonococcal Infections

Capsular polysaccharide of the bacterium Azospirillum lipoferum Sp59b: Structure and antigenic specificity

Antigenic differences were revealed between the cell wall outer membrane lipopolysaccharides and the capsular high molecular weight bioglycans for a typical strain of the nitrogen-fixing rhizobacterium Azospirillum lipoferum Sp59b using antibodies prepared against the homologous lipopolysaccharide and lipopolysaccharide-protein complex. From the capsular lipopolysaccharide-protein and polysaccharide-lipid complexes of A. lipoferum Sp59b, polysaccharides were isolated and their structure was for the first time established in Azospirillum by monosaccharide analysis which included determination of the absolute configurations, methylation, O-deacetylation, and one- and two-dimensional NMR spectroscopy. The polysaccharides of the capsular complexes were shown to have identical structure of the branched tetrasaccharide repeating unit, which differs from the structure of the O-specific polysaccharide within the outer membrane lipopolysaccharide of this strain.

Superior bactericidal activity of SDS capped silver nanoparticles: Synthesis and characterization

Silver nanoparticles were synthesized through UV photo-reduction of silver nitrate aqueous solution, containing ethanol and sodium dodecyl sulfate (SDS) using an UV digester equipped with high pressure mercury lamp of 500 W. The synthesized nanoparticles were characterized by UV–vis spectroscopy (UV–vis), transmission electron microscopy (TEM), X-ray diffraction (XRD) and X-ray photoelectron spectroscopy (XPS). The formation of silver nanoparticles was confirmed from the appearance of surface plasmon absorption maxima at 418 nm. TEM showed the spherical nanoparticles with size in 23–67 nm (average 45 ± 10 nm). The silver nanoparticles were stable for more than 8 months. The antibacterial activity of these SDS capped silver nanoparticles was tested using Pseudomonas aeruginosa as a model strain for gram-negative bacteria. SDS capped silver nanoparticles exhibit a much higher bactericidal activity compared to silver nanoparticles capped with other capping agents. Even at a low silver nanoparticle concentration of 5 μg/ml, complete inhibition of 10 7 colony forming units (CFU) was achieved with SDS capped silver nanoparticles. This concentration is much lower than the values reported by other authors. This enhanced bactericidal activity is attributed to much efficient transport of silver nanoparticles by SDS to the outer membrane of cell wall compared to the other capping agents and have a better interaction of nanoparticles with the cell.

Laser interferometric and cultivation methods for measurement of colistin/ampicilin and saponin interactions with smooth and rough of Proteus mirabilis lipopolysaccharides and cells

Laser interferometry is commonly used in permeability studies of soluble substances. In this study a modification that allowed testing partially insoluble mixtures is presented. The modification relies on the measurement of diffusion from 1% agarose gel. As a model for this study, two Proteus mirabilis strains were used that differ in polysaccharide content: smooth P. mirabilis S1959 strain and its Re-type mutant, strain R45. By laser interferometry and precipitation it is shown that R45 lipopolysaccharide is more effective in binding colistin. It has been shown with the laser interferometric method that saponins, which are detergent-like substances of plant origin, partially enhance the interaction of colistin with the S and Re types of P. mirabilis. These results were confirmed with whole cell Proteus studies. The saponin partially inhibited the growth of the S and Re P. mirabilis strains at doses of 31–500 μg/ml. A sub-inhibitory dose — 15 μg/ml of saponins alone do not reduced the numbers of P. mirabilis S1959 and R45 cells. However, the presence of colistin or amipicillin and 15 μg/ml of saponins reduced the amount of P. mirabilis S1959 and R45 cells. The saponins enhanced sensitivities of S and R P. mirabilis cells towards colistin and amipicillin. One may proposed that saponins binds to lipid A part of LPS may resulted on an increase in bacterial cell wall outer-membrane permeabilities and by that facilitated antibiotics penetration into the bacterial cells. In conclusion, the laser interferometric method is a useful tool for studies of lipopolysaccharide–antibiotic interactions even if the tested substances are not fully soluble in water.

Effect of endotoxin on the immunity of Indian major carp, Labeo rohita

Endotoxin, a lipopolysaccharide component of outer cell wall membrane of the Gram-negative bacteria is a factor responsible for a number of biological effects including immunostimulatory activities in different animal species including fish. In this study, L. rohita yearlings of weight ranging from 80 to 100 g were injected intraperitoneally with 0.5, 1, 2, 5, 10 and 20 EU/fish dose of endotoxin to find out its effect on the immunity. The L. rohita yearlings were found to resist the endotoxin dose up to 20 EU/fish and at the lower doses, i.e., at 1 and 2 EU/fish; it acted as an immune potentiator. Different serum and immune parameters like protein, globulin, lysozyme, respiratory burst activity, myeloperoxidase activity, natural agglutination titre were found to be significantly high ( p < 0.01) at a dose of 1 EU/fish. While at 10 and 20 EU/fish, most of these parameters were lower thereby indicating the immuno-suppressive nature of the endotoxin at these higher doses.

Antibodies against human muscle enolase recognize a 45-kDa bacterial cell wall outer membrane enolase-like protein

Enolase, is a glycolytic enzyme ubiquitous in higher organisms, where it forms tissue specific dimers of isoforms, also found in the cytoplasm of fermentative bacteria. The aim of this work was to identify enolase-like proteins in the cell wall of some Gram-negative bacteria using antibodies against human β-enolase, an isoenzyme specific to skeletal and heart muscles. Cell wall outer membrane protein (OMP) preparations were obtained from 9 strains of Enterobacteriaceae and one of Pseudomonas aeruginosa. Specific enzymatic enolase activity was detected in the supernatant fractions of cytosolic and inner membrane material, but not in purified OMP preparations. Rabbit polyclonal antibodies specific against human β-enolase were prepared and purified using immobilized human β-enolase in affinity chromatography. In SDS–polyacrylamide gel electrophoresis and immunoblotting assay of purified OMP preparations, rabbit anti-enolase antibody interacted specifically with a few OMPs, of which a 45-kDa band also interacted with human sera of patients presenting Buerger disease and atherosclerosis. The most distinct interaction of human sera was observed with a 45-kDa OMP of Klebsiella pneumoniae. This protein was further isolated from K. pneumoniae cell mass in two ways, namely preparative SDS–polyacrylamide gel electrophoresis and specific affinity chromatography using immobilized affinity-purified rabbit antibody raised against human β-enolase. The data obtained from tandem mass spectrometry tryptic peptide analysis and sequence comparison of human and bacterial enolases using protein databases, could reveal the similarity in the epitopes between membrane enolase-like protein from Klebsiella and human β-enolase. The results show that the protein present in all studied strains has a common epitope on human β-enolase. These data raise the question whether such a bacterial protein might be a marker for detecting and monitoring damage to skeletal and heart muscles.

Ultrastructural Localization of Cell Surface Immunospecificity in Anabaena azollae Using Indirect Fluorescent Antibody Staining

Antisera prepared against whole cells were used to differentiate between freshly isolated and cultured cyanobacteria from the nitrogen-fixing fern Azolla filiculoides using both light and transmission electron microscopy (TEM). TEM suggested that the differentiating antigens are evenly distributed on the cell wall outer membrane of the cyanobacteria. The fluorescein isothiocyanate (FITC)-conjugated secondary antibody accumulation was sufficiently electron-dense to be detected without the need for either gold or ferritin labelling. Relative amounts of fluorescence which occurred on specimens viewed with fluorescent light microscopy were comparable to the thickness of electron dense material on the surfaces of these cells. Thus, it is possible to use the same immunological procedure to prepare cells for both light and TEM study.

ULTRASTRUCTURAL LOCALIZATION OF CELL SURFACE IMMUNOSPECIFICITY IN ANABAENA AZOLLAE USING INDIRECT FLUORESCENT ANTIBODY STAINING

Antisera prepared against whole cells were used to differentiate between freshly isolated and cultured cyanobacteria from the nitrogen‐fixing fern Azolla filiculoides using both light and transmission electron microscopy (TEM). TEM suggested that the differentiating antigens are evenly distributed on the cell wall outer membrane of the cyanobacteria. The fluorescein isothiocyanate (FITC)‐conjugated secondary antibody accumulation was sufficiently electron‐dense to be detected without the need for either gold or ferritin labelling. Relative amounts of fluorescence which occurred on specimens viewed with fluorescent light microscopy were comparable to the thickness of electron dense material on the surfaces of these cells. Thus, it is possible to use the same immunological procedure to prepare cells for both light and TEM study.

Chroococcus S24 and Chroococcus N41 (cyanobacteria): morphological, biochemical and genetic characterization and effects of water stress on ultrastructure

Two strains of desiccation-tolerant coccoid cyanobacteria, Chroococcus S24, a marine form, and Chroococcus N41, a cryptoendolith isolated from a hot-desert rock, have been characterized. The mol % DNA base compositions of the strains are 47.1 and 48.9% respectively. Plasmid DNA was not detected in either strain. The pigment contents and nutritional characteristics of the strains are identical. Both lack phycoerythrinoid pigments and, in culture, behave as slow-growing halotolerant marine forms with elevated requirements for Na+, Cl−, Mg2+ and Ca2+. Sucrose was the only carbon source of those tested that supported photoheterotrophic growth. Each strain synthesizes nitrogenase under anaerobic conditions but not in air. Morphologically the two strains are indistinguishable. They are considered to be independent isolates of the same cyanobacterial species. Chroococcus N41 was studied in detail with the electron microscope. When brought to equilibrium at matric water potentials of-168 MPa and lower (to-673 MPa=c0.12a w) the protoplast shrinks, but the cells maintain the same size and diameter as those at-2,156 kPa (MN medium; control); the sheath expands and remains attached to the cell wall outer membrane by fibrils. The cell wall, cell membrane, thylakoid membranes, cyanophycin granules and carboxysomes appeared intact in desiccated cells.

Binding of a bacteriophage to wall‐membrane adhesion in Proteus mirabilis

AbstractA bacteriophage was shown to adsorb to plasmolysed non‐swarming cells of Proteus mirabilis preferentially at the sites of adhesion between the inner membrane and outer cell wall membrane; 75% of phage particles were adsorbed at these sites, while 25% were not. Differences in outer membrane composition between swarming and non‐swarming cells were reflected in altered phage‐binding properties, with only 33% of phage absorbed at these adhesion sites in swarming cells. On the basis of their phage distribution, cross‐sections of swarm cells could be distinguished from sections of short non‐swarming cells.

Binding of a bacteriophage to wall-membrane adhesion in proteus mirabilis.

A bacteriophage was shown to adsorb to plasmolyzed non-swarming cells of Proteus mirabilis preferentially at the sites of adhesion between the inner membrane and outer cell wall membrane; 75% of phage particles were adsorbed at these sites, while 25% were not. Differences in outer membrane composition between swarming and non-swarming cells were reflected in altered phage-binding properties, with only 33% of phage absorbed at these adhesion sites in swarming cells. On the basis of their phage distribution, cross-sections of swarm cells could be distinguished from sections of short non-swarming cells.

Bacterial sheathed flagella and the rotary motor model for the mechanism of bacterial motility

The occurrence of bacterial flagella sheathed with an extension of the Gram-negative outer cell wall membrane poses problems for universal application of the rotary motor model for the bacterial flagellum mechanism. Two mechanisms are proposed which attempt to reconcile the existence of sheathed flagella with the rotary motor model, one of which involves rotation of both flagellar sheath and core, the other of which involves rotation of the core only, within a flexible wave-propagating sheath.

Studies on gonococcus infection. XVIII. 125I-labeled peptide mapping of the major protein of the gonococcal cell wall outer membrane

The major outer membrane proteins from 10 gonococcal strains were examined after 125I-labeling of the proteins as single bands resolved by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. These 125I-proteins were then treated with either trypsin or alpha-chymotrypsin, and the resultant 125I-peptides were visualized by autoradiography after two-dimensional electrophoretic and chromatographic separation on thin-layer cellulose sheets. Several 125I-peptides were present in all the major outer membrane proteins examined. The presence and absence of additional 125I-peptides segregated the major proteins into two pattern groups. One group consisted of major outer membranes with molecular weights of 34,000 or 33,000; major proteins with molecular weights of 32,000 constituted the other group. Two beta-lactamase-producing gonococcal isolates were examined. Their major outer membrane proteins were identical in apparent molecular weights and alpha-chymotryptic 125I-peptide fingerprints; these proteins contained 125I-peptides not found in other gonococcal major proteins. No 125I-peptide differences were found among the major outer membrane proteins of strain F62 gonococci that exhibited differences in piliation and/or colony opacity characteristics.

Ultrastructural and chemical alteration of the cell envelope of Pseudomonas aeruginosa, associated with resistance to ethylenediaminetetraacetate resulting from growth in a Mg2+-deficient medium.

Cells of Pseudomonas aeruginosa became resistant to the lytic effect of ethylenediametetraacetate (EDTA) when grown in a Mg(2+)-deficient medium. To correlate ultrastructural changes in the cell wall associated with the shift to EDTA-resistance, a freeze-etch study was performed. Upon fracturing, the outer cell wall membrane split down the hydrophobic center to reveal the outer (concave) and inner (convex) layers. The concave cell wall layer of EDTA-sensitive cells grown in Mg(2+)-sufficient medium contained spherical units resting on an underlying smooth support layer. Upon EDTA treatment, approximately one-half of these spherical units were extracted. Cells grown in Mg(2+)-deficient medium were resistant to EDTA. The concave cell wall layer of EDTA-resistant cells had increased numbers of highly compacted spherical units, giving this layer a disorganized appearance. The highly compacted appearance of this layer was unaltered by EDTA treatment. Thus, growth in Mg(2+)-deficient medium resulted in cells which were resistant to EDTA and which possessed an ultrastructurally altered outer layer of the outer cell wall membrane. Cell envelopes from EDTA-resistant cells were found to possess 18% less phosphorus, 16.4% more total carbohydrate, and 13.3% more 2-keto-3-deoxyoctonate than cell envelopes from EDTA-sensitive cells. There were also qualitative, but not quantitative, differences in the protein content of cell envelopes from EDTA-resistant and EDTA-sensitive cells.

Proteins released from cell envelopes of Pseudomonas aeruginosa on exposure to ethylenediaminetetraacetate: comparison with dimethylformamide-extractable proteins.

Isolated cell envelopes of Pseudomonas aeruginosa were treated with N,N'-dimethylformamide (DMF) or with ethylenediaminetetraacetate (EDTA). DMF solubilized 73% of the dry weight of the cell envelope, 76% of the protein, 78% of the carbohydrate, and 76% of the phosphorus. Electron microscopy showed that DMF caused extensive alterations in the appearance of the cell envelope with blebs and bleblike vesicles predominating. After incubation with EDTA, the cell envelopes appeared to have lost material, but still retained the cell-like morphology. Analysis of DMF-solubilized proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed 16 protein bands. There were three major proteins that predominated, however, with molecular masses of 43,000 (protein A), 16,500 (protein B), and 72,000 daltons (protein C). Evidence is presented that protein A and protein B are glycoproteins. Gel electrophoresis of EDTA-solubilized material revealed that a number of proteins were released from the cell envelope. However, electrophoresis of an isolated protein-lipopolysaccharide complex released by EDTA showed that protein A and protein B were the major protein components of this complex. These data suggest that protein A and protein B are components of the outer cell wall membrane of P. aeruginosa. There is suggestive evidence that these proteins may play a role in maintaining the structural integrity of the cell envelope. Whether these proteins also have enzymatic activity could not be discerned from the present study, although it is possible that they may be associated with the terminal stages of lipopolysaccharide synthesis.

Freeze-etch study of Pseudomonas aeruginosa: localization within the cell wall of an ethylenediaminetetraacetate-extractable.

A freeze-etch study of normal cells of Pseudomonas aeruginosa and of cells after incubation with ethylenediaminetetraacetate (EDTA) and tris(hydroxymethyl)aminomethane (Tris) was performed. When cells were freeze-etched without a cryoprotective agent, a smooth outer cell wall layer, which showed a regular array of subunits, and the presence of flagella and pili were observed. These features were not observed in cells freeze-etched after cryoprotection with glycerol. Four fracture surfaces, which resulted from splitting down the center of the outer wall membrane and of the inner cytoplasmic membrane, were revealed in freeze-etched glycerol-protected cells. The murein layer was seen in profile between the outer cell wall membrane and the cytoplasmic membrane. Spherical units and small rods composed of the spherical units were observed in the inner layer of the outer cell wall membrane. These spherical units appeared to be attached to, or embedded in, the inner face of the outer layer of the outer cell wall membrane. These spherical units were removed from cells on exposure to EDTA-Tris, resulting in cells that were osmotically fragile. The spherical units were detected via electron microscopy of negatively stained preparations in the supernatant fluid of cellular suspensions treated with EDTA-Tris. Upon addition of Mg(2+), the spherical units were reaggregated into the inner layer of the outer cell wall membrane and the cells were restored to osmotic stability. The spherical units were shown to consist primarily of protein. These data are thought to represent the first ultrastructural demonstration of reaggregation of cell wall components within a living cell system.

The serum bactericidal system: ultrastructural changes in Neisseria meningitidis exposed to normal rat serum.

Exposure of meningococci to the bactericidal system of normal rat serum initiates a series of ultrastructural changes that accompany death of the organism. These morphological alterations consist of complement-dependent holes in the cell wall outer membrane, edema of the periplasmic space and cytoplasm, and accumulation of fibrillar material in and on the cell wall. Dissolution of the dense line and rupture of the cytoplasmic membrane also occur in meningococci exposed to normal rate serum. These latter two changes, however, are not seen in meningococci that are killed by bentonite-absorbed (lysozyme-deficient) serum, nor are they invariably present when other serum-susceptible organisms (N. catarrhalis and Herellea sp.) are treated with normal rat serum. The pattern of development of edema in serum-treated meningococci suggests that the cell wall outer membrane, mucopeptide layer, and cytoplasmic membrane act synergistically to maintain osmotic equilibrium of the bacterium. Death of the bacterium seems related to alteration of permeability following injury to the outer membrane. Holes are demonstrable, by negative straining, in the outer membrane of the meningococcal cell wall after exposure to the bactericidal effects of rat serum. The lesions are 110 A average greatest diameter and depend on the presence of antibody and complement for their formation. In addition, 82 A diameter holes are present in the walls of normal, untreated meningococci. The relation of complement-dependent holes to the smaller, naturally occurring holes is not known.