Interaction of Clostridium perfringens theta-haemolysin, a contaminant of commercial phospholipase C, with erythrocyte ghost membranes and lipid dispersions. A morphological study.

Commercially available preparations of phospholipase C from Clostridium perfringens are commonly contaminated with theta haemolysin, one of a group of bacterial haemolysins called oxygen labile (O-labile) haemolysins. Treatment of erythrocyte ghosts and a mixed lipid dispersion containing cholesterol with commercially available phospholipase C in the absence of Ca-2+ and the presence of phosphate buffer and/or EDTA resulted in the formation and release of ring or arc-shaped structures. Highly purified phospholipase C, free of theta-haemolysin, produced no changes in the morphology of erythrocyte ghosts or lipid dispersions in the presence of phosphate or EDTA, but caused the formation of typical diglyceride droplets in the presence of Ca-2+ in the absence of these inhibitors. Ring structures, identical to those caused by commercial phospholipase C, were formed on addition of highly purified theta-haemolysin to erythrocyte ghost membranes, lipid dispersions containing cholesterol and cholesterol dispersions, but not on treatment of membranes from Micrococcus lysodeikticus. Heat-inactivated O-haemolysin (60 degrees C for 10 min) produced no such effects. The dimensions of rings and arcs displayed heterogeneity. The outside diameters in various preparations varied from approx. 27-58 nm with border thickness of 4.1-7.8 nm.

Evaluation of different methods for the detection of outer membrane proteins and lipopolysaccharides of Pasteurella haemolytica by immunoblotting.

The optimal conditions for the detection of outer membrane proteins (OMPs) and lipopolysaccharide (LPS) of Pasteurella haemolytica by immunoblotting were evaluated. The variables examined included the equilibration time of the gels before transfer, composition of the transfer buffer, type of blotting membrane, blocking agent, effect of the zwitterionic detergent Empigen BB on protein renaturation, and the development reagent. The composition of the transfer buffer and time of gel equilibration significantly affected the efficiency of transfer of both OMPs and LPS. However, the optimal conditions for the transfer of OMPs were not the same as those for LPS. Thus, optimal transfer of OMPs occurred in Tris-glycine buffer, with prior equilibration of the gels to allow for expansion, whereas optimal transfer of LPS was achieved in Tris-glycine-methanol buffer with no equilibration of the gels. In Tris-glycine-methanol buffer, gel equilibration resulted in a significantly reduced transfer of both OMPs and LPS, probably due to the removal of SDS from these components. The use of Zeta-Probe blotting membrane which, unlike nitrocellulose, does not require methanol for optimal protein binding, did not result in improved binding of OMPs or LPS in the absence of methanol and, even after prolonged blocking (> 2 h), gave higher background staining than did nitrocellulose. Effective blocking of nitrocellulose was achieved with 3% (w/v) gelatin, 2.5% (w/v) skimmed milk or 0.3% (v/v) Tween 20, whereas increased background staining occurred with 1% (w/v) bovine serum albumin or 1% (w/v) ovalbumin. The incorporation of Empigen BB in the primary antibody buffer did not improve antibody recognition of proteins as a result of their renaturation. For the horseradish-peroxidase enzyme development system, the substrate 3,3'-diaminobenzidine tetrahydrochloride was more sensitive, and developed more quickly, than 4-chloro-1-naphthol, but faded more rapidly after drying of the membrane. 4-chloro-1-naphthol was more suitable for identifying OMPs because less background staining occurred, whereas 3,3'-diaminobenzidine tetrahydrochloride was more suitable for the detection of LPS due to its greater sensitivity.

Optimal conditions for the analysis of Pasteurella haemolytica lipopolysaccharide by sodium dodecyl sulphate-polyacrylamide gel electrophoresis.

The optimal conditions for the analysis of the lipopolysaccharide (LPS) of two serotype A1 isolates and a serotype A2 isolate of Pasteurella haemolytica by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and silver staining were determined. The LPS of the A1 isolates possessed O side chains, consisting of high molecular mass bands with the appearance of a ladder-like pattern, as well as a low molecular mass core-oligosaccharide region; the LPS of the A2 isolate consisted only of the core-oligosaccharide region. Furthermore, the LPS of the two A1 isolates differed in the core-oligosaccharide region. Optimal resolution of low molecular mass LPS components was obtained in a 15% acrylamide resolving gel containing 4 M urea whereas optimal resolution of high molecular mass components was obtained when urea was omitted. Conventional silver staining resulted in excellent visualisation of LPS bands, whereas a modified staining method did not detect additional bands, as has been demonstrated with the LPS of Pseudomonas aeruginosa. Proteinase K digestion of outer membranes gave more clearly defined LPS profiles than did similar digestions of whole cells, and more closely resembled the profiles of purified LPS. With the exception of slight variation in the average molecular mass of a group of O side chains between logarithmic and stationary phases there were no differences in LPS profiles at various stages of the growth cycle; freezing and thawing of LPS samples had no effect on the profiles.

Accumulation of a precursor of subunit S1 of pertussis toxin in cell envelopes of Bordetella pertussis in response to the membrane perturbant phenethyl alcohol.

Exponential cultures of Bordetella pertussis strain 18334 were treated with the membrane-perturbing agent phenethyl alcohol which, at a concentration of 0.075% v/v, blocked the synthesis of mature subunit S1 of pertussis toxin as revealed by Western blotting. It also caused the accumulation of a precursor, pS1, with an estimated mol. wt of 32 X 10(3), that was located in the cytoplasmic membrane. These findings suggested that subunit S1 of pertussis toxin was exported in a signal peptide-dependent manner.

Binding of 125I-alpha toxin of Staphylococcus aureus to erythrocytes.

Alpha toxin purified from Staphylococcus aureus strain Wood 46 and radioiodinated by the lactoperoxidase method retained full haemolytic activity and was used to study factors affecting binding to rabbit and horse erythrocytes. A relatively fixed percentage of added toxin bound to both cell types; the percentage bound was independent of temperature, pH, cell concentration and toxin concentration. Neither a 50-fold excess of native toxin nor Concanavalin A inhibited the binding of iodinated toxin to erythrocytes. The results suggest that differences in the sensitivity of erythrocytes to haemolysis do not reflect the abundance of high affinity toxin receptors on sensitive cells, but are more probably the result of differences in the intrinsic stability of the membrane and its sensitivity to perturbation by amphiphilic agents.

Inhibition of secretion of staphylococcal alpha toxin by cerulenin.

Secretion of alpha toxin by Staphylococcus aureus strain Wood 46 was preferentially inhibited by cerulenin, an antibiotic that stops fatty-acid synthesis by inhibiting beta-keto acyl acyl carrier-protein synthetase. At the concentrations used, cerulenin had a negligible effect on cell growth and total protein synthesis, but reduced lipid synthesis by 50%. Extracellular and membrane-associated alpha toxin was absent in cultures treated with cerulenin, but toxin formation was resumed after either removal of the antibiotic or addition of exogenous fatty acids. The apparent absence of toxin precursor in membranes of inhibited cells favours inhibition at an earlier stage in toxin synthesis.

Assay of Bordetella pertussis heat-labile toxin with human embryonic lung cells.

An assay has been developed for Bordetella pertussis heat-labile toxin (HLT) based on morphological alterations in certain human embryonic lung (HEL) cell lines. Eighteen cell lines from human and other sources were tested but only two, MRC-5 and HELu2, were responsive to HLT. Confluent monolayers of the cells contracted within 24 h of exposure to the toxin, but without loss of viability during incubation for a further 3 days. The effect of HLT was quantitated by scoring the extent of morphological change, and by the decrease in Giemsa staining of the cell monolayers, as measured on an ELISA plate reader. This cell culture assay for HLT was more sensitive than lethality titration in mice but the dose-response curve had a lower slope. The specificity of the response was established by comparing unheated HLT with HLT heated at 56 degrees C, and with extracts from transposon-insertion mutants of B. pertussis which were deficient in HLT. Purified preparations of pertussis toxin and B. pertussis lipopolysaccharide gave no morphological response even at high doses.

Heat-labile and heat-stable haemolysins of Campylobacter jejuni.

During studies on the virulence mechanisms of Campylobacter jejuni clinical isolates it became apparent that some strains produced one or more haemolysins and some did not. There was no great difference between Group C (cholera-like) strains and Group D (dysentery-like) strains. The protein haemolysin(s) showed a spectrum of activity against erythrocytes from different animals; with maximum activity against rabbit and minimal activity against chicken erythrocytes. The results suggested a two-stage activation mechanism for haemolysis which involved a multi-hit lytic activity. It was concluded that the C. jejuni haemolysins were not identical to those described in other organisms and they may be involved in iron acquisition in vivo.

Cytolytic toxins and surface activity.

Surface-active properties of alpha-toxin and delta-lysin, two cytolytic protein exotoxins of the bacterium Staphylococcus aureus are summarised. The relevance of differences in surface charge density on membranes is discussed in relation to possible mechanisms of binding and membrane penetration by alpha-toxin. An hypothesis for the mechanism of membrane disruption by delta-lysin involving the formation of hydrophilic transmembrane pores is proposed.

A colourimetric, microplate assay for the leucotoxin of Pasteurella haemolytica.

Culture supernates of Pasteurella haemolytica, which contain leucotoxin, inhibited the reduction of nitroblue tetrazolium (NBT) by bovine and ovine but not rabbit leucocytes in response to phorbol 12-myristate 13-acetate (PMA). Culture supernates of P. multocida, which contain no leucotoxin, had no inhibitory effect on the response of leucocytes from any species. The inhibition of NBT reduction was assessed visually or spectrophotometrically in the wells of microplates and used as a simple assay for leucotoxin. It was as sensitive as the trypan blue dye-exclusion method and did not require the use of radioisotopes. In addition, sera from P. haemolytica-infected calves inhibited leucotoxin activity in the microplate assay. Thus, inhibition of NBT reduction after stimulation of ruminant leucocytes with PMA can be used as a simple, specific assay for leucotoxin and for anti-leucotoxin antibodies.

Isolation and properties of mesosomal membrane fractions from Micrococcus lysodeikticus.

1. A method is described for the isolation of pure mesosomal membrane fractions from Micrococcus lysodeikticus. 2. Plasmolysis of cells, before wall digestion, was necessary for effective mesosome release. 3. The effect of mild shearing forces, temperature and time upon the release of mesosomal membrane from protoplasts was investigated. 4. The optimum yield of mesosomal membranes from stable protoplasts was achieved at 10mm-Mg(2+). 5. Mesosomal membrane vesicle fractions prepared at differing Mg(2+) concentrations above 10mm were similar in chemical composition. 6. Comparison of the properties of peripheral and mesosomal membrane fractions revealed major differences in the distribution of protein components, membrane phosphorus, mannose and dehydrogenase activities between the two fractions. 7. Only cytochrome b(556) was detected in mesosomal membranes, whereas peripheral membranes contained a full complement of cytochromes. 8. Preliminary investigations suggested the localization of an autolytic enzyme(s) in the mesosomal vesicles. 9. The anatomy of mesosomal and peripheral membrane have been compared by the negative-staining and freeze-fracture technique. 10. The results are discussed in relation to a plausible role for the mesosome.

Factors influencing the activity of succinate dehydrogenase in membrane preparations from Micrococcus lysodeikticus.

1. Some properties of succinate dehydrogenase [succinate-(acceptor) oxidoreductase, EC 1.3.99.1] in membrane preparations from Micrococcus lysodeikticus (N.C.T.C. 2665) were investigated. 2. In the spectrophotometric assay system adopted the reaction velocity was shown to be proportional to the amount of membrane added. Dichlorophenol-indophenol, reduced photochemically in the presence of phenazine methosulphate, or enzymically by the membrane-bound enzyme, was shown to undergo reoxidation in the dark. 3. The membrane-bound enzyme was found to be inactivated at temperatures above 10 degrees C. 4. The specific activity of membrane-bound succinate dehydrogenase was found to increase between two- and three-fold in diluted membrane preparations equilibrated at 0 degrees C for 6h. Membranes treated with sodium deoxycholate showed no enzyme activation on dilution but displayed maximal activity, all activity being sedimentable at 103000g. The increase in specific activity observed on dilution could be partially inhibited by fixation with glutaraldehyde, or by the presence of bovine serum albumin. 5. The addition of Mg(2+) or Ca(2+) ions to membrane suspensions caused an overall depression of enzyme activity. 6. The results suggest the presence of an ;inhibitor' that affects the expression of membrane bound succinate dehydrogenase activity.

Inhibition of Sindbis virus plaque formation by extracts of Escherichia coli.

Vilcek, Jan (New York University School of Medicine, New York, N.Y.), and John H. Freer. Inhibition of Sindbis virus plaque formation by extracts of Escherichia coli. J. Bacteriol. 92:1716-1722. 1966.-Extracts prepared from washed cells of Escherichia coli B by sonic treatment and subsequent filtration through a 0.45-mu membrane filter significantly inhibited plaque formation with Sindbis virus in cultures or primary chick embryo cells up to a dilution of 1:20,000. The inhibitor acted on the cells rather than directly on the virus. The inhibiting substance was nondialyzable. Treatment of crude extracts with nucleases, trypsin, chymotrypsin, pepsin, or ether had no effect on the activity. Treatment with pronase destroyed the virus-inhibiting effect. Extracts prepared from two strains of E. coli B and one strain of E. coli K-12 all showed inhibitory activity against Sindbis virus. The inhibitor was present in the cytoplasmic fraction of bacteria. It was also active against Sindbis virus in human cells and showed some activity against vesicular stomatitis and vaccinia viruses in different types of cells. Interferon was not shown to be involved in the inhibition, although actinomycin D partially reversed the inhibitory activity of the extracts.

Fine structure of Bacillus megaterium during microcycle sporogenesis.

Ultrathin sections were prepared from cultures of Bacillus megaterium QM B1551 undergoing microcycle sporogenesis (initial spore to primary cell to second-stage spore without intervening cell division) on a chemically defined medium. The cytoplasmic core of the dormant spore was surrounded by plasma membrane, cell-wall primordium, cortex, outer cortical layer, and spore coats. Early in the cycle, the coat opened at the germinal groove, the cortex swelled, ribosomes and a chromatinic area associated with large mesosomes (which may later be incorporated into the expanding plasma membrane) appeared in the core, and the cell wall became defined at the site of the cell wall primordium. Poly-beta-hydroxybutyrate granules began to appear in the primary cell at about 3 hr. By 7 hr, the forespore of the second-stage spore was delineated by typical double membranes. Between 7 and 12 hr, second-stage cell-wall primordium and cortex developed between the separating forespore membranes. The inner membrane became the plasma membrane of the second-stage spore, and the outer membrane eventually disintegrated within the second-stage spore cortex. A densely staining double layer (spore-coat primordium) developed external to the outer forespore membrane. The inner spore coat and the outer cortical layer of the second-stage spore developed from this primordium. The outer part of the spore coat, probably of sporangial origin, was laid down on the external surface of the inner spore coat. By 12 hr, second-stage spores were almost mature. By 20 hr, the mature endospores, with a thickened outer coat, were often still enclosed by degenerate primary cell wall and by the outer cortical layer and spore coat of the initial spore.

Location of binding sites on common type 1 fimbriae from Escherichia coli.

Common type 1 fimbriae were isolated from Escherichia coli and their length distribution profile was determined before and after treatment with ultrasound. As fimbriae were shortened, so their haemagglutinating capacity decreased, but their ability to bind to erythrocytes did not decrease to the same extent. Isolated fimbriae did not agglutinate inside-out vesicles prepared from horse erythrocytes or liposomes, suggesting that the binding mechanism was not based on non-specific hydrophobic interactions. The results support a lateral rather than a terminal location for the fimbrial binding site responsible for haemagglutination.