Purification and physical properties of group C streptococcal phage-associated lysin.

A purification procedure for the Group C phage-associated lysin is described utilizing tetrathionate to protect the enzyme's -SH group(s) from thiol-inactivating agents. A 652-fold purification has been accomplished yielding a solution in which the enzyme activity corresponds to essentially a single band on polyacrylamide gel which accounts for 70% of the total protein in the preparation. A molecular weight of 101,000 and frictional ratio of 1.526 was determined for the lysin utilizing experimentally determined values for its Stokes radius and sedimentation coefficient.

Staphylococcal alpha-toxin: effects on artificial lipid spherules.

Staphylococcal alpha-toxin induces the release of previously sequestered anions or glucose from artificial phospholipid spherules, an effect abolished by specific antitoxin. Alphatoxin resembles streptolysin S in releasing anions or glucose from spherules prepared without cholesterol, and can be distinguished from the membrane-active polyene amphotericin B, which preferentially disrupts spherules containing cholesterol. It may affect biological structures by a similiar interaction with membrane phospholipids.

Comparative toxinology of Loxosceles reclusa and Corynebacterium pseudotuberculosis.

In contrast to other kinds of phospholipases, phospholipases D that are toxic for humans and animals are not commonly encountered as constituents of venoms or as products of pathogenic microorganisms. Toxic phospholipases D are present, however, in the venom of the brown recluse spider (Loxosceles reclusa) and in supernatants or filtrates of cultures of Corynebacterium pseudotuberculosis. Although the two enzyme toxins are derived from phylogenetically disparate entities, they are similar in molecular weight, charge, substrate specificity, and in several biological activities. They are immunologically distinguishable.

A cytolytic protein from the edible mushroom, Pleurotus ostreatus.

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

Effect on sphingomyelin-containing liposomes of phospholipase D from Corynebacterium ovis and the cytolysin from Stoichactis helianthus.

The toxic, sphingomyelin-specific phospholipase D (phosphatidylcholine phosphatidohydrolase EC 3.1.4.4) from Corynebacterium ovis was purified to near homogeneity. It has a molecular weight of 31 000 and a pI of approx. 9.8. Although not cytolytic itself, it protected red cells from hemolysis by staphylococcal sphingomyelinase (beta-hemolysin) and helianthus toxin. The apparently non-enzymatic cytolysin (helianthus toxin) from the sea anemone Stoichactis helianthus also interacts with membrane sphingomyelin. C. ovis and helianthus toxins were compared with regard to their effects on liposome model membranes, and they were found both to produce changes analogous to those in erythrocytes. Only helianthus toxin caused release of trapped glucose marker, but liposomes could be protected from release by pretreatment with C. ovis toxin. Both toxins demonstrated binding to sphingomyelin-containing liposomes, but only the bacterial sphingomyelinase catalyzed the release of choline from these vesicles.

Interactions between membranes and cytolytic peptides.

The physico-chemical and biological properties of cytolytic peptides derived from diverse living entities have been discussed. The principal sources of these agents are bacteria, higher fungi, cnidarians (coelenterates) and the venoms of snakes, insects and other arthropods. Attention has been directed to instances in which cytolytic peptides obtained from phylogenetically remote as well as from related sources show similarities in nature and/or mode of action (congeneric lysins). The manner in which cytolytic peptides interact with plasma membranes of eukaryotic cells, particularly the membranes of erythrocytes, has been discussed with emphasis on melittin, thiolactivated lysins and staphylococcal alpha-toxin. These and other lytic peptides are characterized in Table III. They can be broadly categorized into: (a) those which alter permeability to allow passage of ions, this process eventuating in colloid osmotic lysis, signs of which are a pre-lytic induction or latent period, pre-lytic leakage of potassium ions, cell swelling and inhibition of lysis by sucrose. Examples of lysins in which this mechanism is involved are staphylococcal alpha-toxin, streptolysin S and aerolysin; (b) phospholipases causing enzymic degradation of bilayer phospholipids as exemplified by phospholipases C of Cl. perfringens and certain other bacteria; (c) channel-forming agents such as helianthin, gramicidin and (probably) staphylococcal delta-toxin in which toxin molecules are thought to embed themselves in the membrane to form oligomeric transmembrane channels.

Interaction between sphingomyelin and a cytolysin from the sea anemone Stoichactis helianthus.

The cytolytic toxin from the sea anemone Stoichactis helianthus was inhibited up to 90--95% by suspensions of sphingomyelin but not by phosphatidylcholine or other membrane lipids. When the toxin was incubated with sphingomyelin and the mixture fractionated either by isoelectric focusing or Sephadex gel filtration, the residual hemolytic units migrated together with the lipid and not as free toxin. Incubation with phosphatidylcholine, however, did not shift the toxin peak in either type of column. A toxin-ferritin conjugate retaining hemolytic activity was observed by negative staining to bind to liposomes prepared with sphingomyelin but not with liposomes containing phosphatidylcholine. The results provide evidence that the membrane binding site of the toxin is sphingomyelin.

Alteration by cereolysin of the structure of cholesterol-containing membranes.

When erythrocyte membranes were treated with cereolysin, negatively stained and examined by electron microscopy, ring and arc-shaped structures were observed in the membrane. The outside diameter of the rings varied from 33 to 50 nm with a border thickness of 6.7 to 8.3 nm. The arcs varied in length from 33 to 170 nm with a border thickness of also 6.7 to 8.3 min. When right-side-out erythrocyte ghosts which had been treated with cereolysin were examined by electron microscopy after freeze-fracture, structures with a diameter of 31 to 63 nm were seen in the fracture face of the exoplasmic half of the membrane, but no alterations were visible in the fracture face of the protoplasmic half of the membrane bilayer. Thus the ring structures did not appear to form holes through the membrane. At cereolysin concentrations above 6 microgram/ml rings and arcs were seen when purified toxin alone was examined. At or below 6 microgram/ml toxin rings and arcs were seen only if toxin was incubated with free or membrane-bound cholesterol. Our interpretation is that cereolysin tends to aggregate into ring and arc-shaped structures, and that the tendency to aggregate is increased by cholesterol. Rings and arcs were not seen when erythrocyte ghosts were treated with low, but lytic amounts of cereolysin that significantly altered the premeability of the ghosts.

Surface properties of membrane systems. Transport of staphylococcal delta-toxin from aqueous to membrane phase.

Hemolytic delta-toxin from Staphylococcus aureus was soluble in either water, methanol or chloroform/methanol (2 : 1, v/v). The toxin spread readily from distilled water into films with pressures (pi) of 10 dynes/cm on water and 30 dynes/cm on 6 M urea; from chloroform/methanol it produced 40 dynes/cm pressure on distilled water. The toxin adsorbed barely from water (pi = 1 dyne/ cm) but it did rapidly from 6 M urea (pi = 35 dynes/cm). The protein films had unusually high surface potentials, which increased with the film pressure and decreased with increasing both pH and urea concentration in the aqueous phase. The fluorescence of 1-aniline 8-naphthalene sulfonate with delta-toxin was much greater than that with RNAase and dipalmitoyl phosphatidylcholine itself, indicating probably a marked lipid-binding character of the toxin. By circular dichroism the alpha-helix content of delta-toxin was 42% in water, 45% in methanol, 24% in 6 M urea. Infrared spectroscopy showed predominant alpha-helix in both 2H2O and deuterated chloroform/methanol as well as in films spread from either solvent on 2H2O. In spreading from 6 M [2H]urea, in which the major infrared absorption was that of [2H]urea with peaks at 1600 and 1480 cm(-1), the delta-toxin film showed prevalently non-alpha-helix structures with major peak intensities at 1633 cm(-1) > 1680 cm(-1), indicating the appearance of new beta-aggregated and beta-antiparallel pleated sheet structures in the film. The data prove that (1) high pressure protein films can consist of alpha-helix as well as non-alpha-helix structures and, differently from another cytolytic protein, melittin, delta-toxin does not resume the alpha-helix conformation in going into the film phase from the extended chain in 6 M urea; (2) conformational changes are important in the transport of proteins from aqueous to lipid or membrane phase; (3) delta-toxin is by far more versatile in structural dynamics and more surface active than alpha-toxin.

Some properties of flammutoxin from the edible mushroom Flammulina velutipes.

A cytolytic toxin from the basidiocarps of the edible mushroom Flammulina velutipes was purified to homogeneity. The lysin, flammutoxin, is a single polypeptide chain of Mr 32,000 and pK about 5.4. It contains unusually large amounts of tryptophan, serine and glycine, and few or none of the sulfur-containing amino acids. Erythrocytes of rat, rabbit, guinea pig, man, mouse, cat and dog were sensitive to lysis, in that order, whereas erythrocytes of sheep, ox, goat, swine and horse were largely or completely resistant to lysis. The toxin appears not to be a phospholipase and it was not inhibitable by any of a variety of lipids. Hemolysis probably involves alteration of the erythrocyte membrane, with formation of submicroscopic ion channels, and it appears to be of the osmotic type. In some respects flammutoxin resembles phallolysin, a cytolytic toxin obtained from the mushroom Amanita phalloides.

Properties of a cytolytic toxin from the sea anemone, Stoichactis kenti.

A cytolytic toxin (kentin) from the Indo-Pacific sea anemone, Stoichactis kenti, was purified to near homogeneity. The toxin is a basic polypeptide of molecular weight approximately 18,000. It broadly resembles cytotoxins from Stoichactis helianthus (helianthin), as well as similar toxins from a number of other anemones, namely Condylactis, Epiactis, Actinia, Pseudactinia, Tealia, Anthopleura, Radianthus and Gyrostoma. The amino acid composition of kentin shows considerable resemblance to that of helianthin, but there are also several significant differences. Neutralization tests indicate that kentin and helianthin are immunologically related but distinguishable. In contrast, no immunological relatedness was found between helianthin and cytolytic toxins from Condylactis gigantea and Epiactis prolifera.

Action of bacterial cytotoxins on normal mammalian cells and cells with altered membrane lipid composition.

Cytotoxic proteins produced by a number of bacteria, as well as one from a marine invertebrate, were tested for their ability to disrupt the permeability barrier of mammalian cells. Agents were tested individually and in combination shown to have synergistic disruptive actions on erythrocytes. Toxins included the lipid-hydrolyzing enzymes sphingomyelinases C and D and cholesterol oxidase, as well as the non-enzymatic agents, helianthus toxin, streptolysin O and saponin. Cells treated included cultured human skin fibroblasts, normal human erythrocytes and erythrocytes enhanced and depleted in membrane cholesterol. Fibroblasts were disrupted by helianthus toxin and by the combination of sphingomyelinase C and cholesterol oxidase. Thin layer chromatographic analysis of the treated cells confirmed the enzymatic alteration of membrane lipids by the lipid hydroxylases. Human erythrocytes having an increased content of membrane cholesterol were more sensitive than normal cells to agents which interact with membrane sterol. Conversely, cholesterol-depleted cells were more resistant to these as well as other agents. Results are discussed in relation to biochemical mechanisms of action of the agents tested, and to their possible significance in bacterial pathogenesis.

Isoelectric analysis of some Australian elapid snake venoms with special reference to phospholipase B and hemolysis.

Venoms of the Australian elapid snakes Austrelaps superbus and Pseudechis colletti were analyzed in an electrofocusing column. A. superbus venom, little studied in the past, was found to have a mouse i.p. lethal potency of 0.48 mg/kg and to contain at least four lethal components. Venoms of both species had relatively high direct hemolytic activity for washed rabbit erythrocytes, as contrasted with venoms from 23 other species of snakes that were not hemolytic under the conditions used. Among venoms of the same 25 species, those of A. superbus and P. colletti produced turbidity in diluted egg yolk, those of Bungarus caeruleus and Bungarus multicinctus were quantitatively less active on egg yolk, whereas venoms of the 21 remaining species were negative. The component of the venoms responsible for egg yolk reactivity was partially purified and the preparations obtained were strongly active when tested with diluted egg yolk or with erythrocytes. Thin layer and paper chromatographic studies showed that these preparations possessed phospholipase B activity for phosphatidylcholine, lysophosphatidylcholine, phosphatidylethanolamine and phosphatidylserine, but sphingomyelin was not degraded. The results suggest that hydrolysis of phosphatidylcholine is responsible for both egg yolk reactivity and hemolysis.

Isolation and characterization of a phospholipase B from venom of Collett's snake, Pseudechis colletti.

Phospholipase B in the venom of the Australian elapid snake, Pseudechis colletti, was purified to near homogeneity. By means of gel filtration it had an Mr of about 35,000, and by SDS-polyacrylamide gel electrophoresis an Mr of about 16,500. These presumably are dimeric and monomeric forms of the enzyme. It was isoelectric at pH 6.2 as compared to 7.8 for phospholipase A2 from which it was readily separated. It was relatively thermostable. As determined by release of water-soluble phosphorous, it degraded phosphatidylcholine and phosphatidylethanolamine, but did not degrade other phospholipids tested. The purified enzyme was strongly hemolytic in vitro for rabbit and human erythrocytes, but not for bovine or ovine erythrocytes. Hemolysis of rabbit erythrocytes gave rise to membranes showing ultrastructural changes that may be unique for this enzyme. The protein was highly active in producing turbidity in dilute solutions of egg yolk. It was cytotoxic for cultured rhabdomyosarcoma cells and was lethal for mice in which death was preceded by massive myoglobinuria.

Kinetics of hemolysis induced by a toxin from Bacillus thuringiensis israelensis.

The kinetics of hemolysis resulting from the action on rabbit erythrocytes of a highly purified cytolytic toxin (26,000 mol. wt) isolated from a spore-crystal mixture of Bacillus thuringiensis israelensis was studied. Course of hemolysis, as determined by release of hemoglobin, yielded sigmoid curves whose maximum slopes were taken as a measure of the rate of lysis. Hemolysis occurred without an induction period, and the rate of lysis was a linear function of toxin concentration. Rate of hemolysis as a function of temperature yielded an Arrhenius constant of 9300 calories per mole. The toxin was active between pH 4.5 and 8.0. Lysis was strongly inhibited by Cu2+, Fe2+ and Zn2+ in concentrations as low as 0.025 M. Phosphatidylserine, phosphatidylinositol, phosphatidylcholine and sphingomyelin inhibited lysis, whereas phosphatidylethanolamine, cerebroside, cholesterol and major integral erythrocyte membrane proteins caused little or no inhibition. Inhibition of lysis by sucrose indicates that hemolysis is of the colloid-osmotic type.

Isolation of a hemolysin from a spore-crystal mixture of Bacillus thuringiensis israelensis (serotype H-14).

A hemolytic toxin from Bacillus thuringiensis israelensis was obtained by alkaline extraction and fast protein liquid chromatography (chromatofocusing followed by gel filtration). The toxin displayed a pI of 4.6-4.8 and an Mr of 26,000. Amino acid analysis demonstrated large amounts of serine, glycine and glutamic acid. The toxin was strongly lytic for rabbit, human and non-human primate erythrocytes, and was weakly lytic toward equine, feline, canine, bovine, amphibian and reptilian erythrocytes. It was weakly entomocidal as indicated by a lethal potency (LC50) of 25 micrograms/ml for second instar larvae of Anopheles stephansi. Direct micro-ELISA indicated that the toxin lacked antigenic identity with numerous eukaryotic and prokaryotic toxins and venoms.

Characterization and amino acid sequences of two lethal peptides isolated from venom of Wagler's pit viper, Trimeresurus wagleri.

Two lethal toxins were isolated from Trimeresurus wagleri venom by fast protein liquid chromatography (molecular sieve) and high performance liquid chromatography (reverse phase). The toxins (termed peptide I and II) had mol. wt of 2504 and 2530, respectively, pIs of 9.6-9.9 and lacked phospholipase A, proteolytic, and hemolytic activity. Lethal peptide I had a murine i.p. LD50 of 0.369 mg/kg, while lethal II had a murine i.p. LD50 of 0.583 mg/kg. Peptide I retained full toxicity after autoclaving at 121 degrees C for 40 min. The lethal activity was found to represent less than 1% of the total venom protein, which was only 62-65% of crude venom. The amino acid sequence of peptide I revealed a proline-rich (over 30% of total sequence) sequence unique among snake venom toxins. Lethal peptide II showed the same sequence except for a second tyrosine in the position of histidine (residue No. 10) in peptide I. The toxin lacked antigenic identity with a number of representative neurotoxins and myotoxins. The crude venom shared at least one antigen with Crotalus scutulatus scutulatus venom. This antigen was not Mojave toxin. The toxin appears symptomatologically suggestive of a vasoactive peptide or neurotoxin.

Purification and properties of a toxin from the South African sea anemone, Pseudactinia varia.

A comparison was made of the hemolytic potency of aqueous extracts prepared from five species of intertidal sea anemones from the coast of South Africa. The active agent in an extract of Pseudactinia varia was purified by ammonium sulfate precipitation, gel permeation chromatography and isoelectric focusing. The hemolytic toxin, termed variolysin, is a protein having a molecular weight of 19,500 and an isoelectric pH of 9.8. It retained appreciable activity after heating to 70 degrees for 40 min. Amino acid analysis revealed that it lacked methionine and cysteine. Its hemolytic activity was inhibited by sphingomyelin. The properties of variolysin show that it is broadly similar to cytolytic toxins isolated from a number of other anthozoans.

Oxidation of macrophage membrane cholesterol by intracellular Rhodococcus equi.

Phagocytic uptake by cultured mouse macrophages (PD388D1) of a virulent strain (ATCC 33701) of Rhodococcus equi producing substantial cholesterol oxidase was accompanied by intracellular survival of the bacteria, and enzymatic oxidation of macrophage membrane cholesterol. A non-virulent strain (4219) lacking cholesterol oxidase was largely eliminated from the macrophages and did not bring about oxidation of membrane cholesterol. When R. equi 33701 was co-phagocytosed with Corynebacterium pseudotuberculosis there was a significant enhancement (10-fold) in the amount of oxidation product (4-cholesten-3-one) generated. R. equi and C. pseudotuberculosis are cooperative partners in the hemolysis of sheep erythrocytes, traceable to the cholesterol oxidase of the former, and phospholipase D of the latter. Results are discussed relative to the role of cooperative cytotoxins in damage to host tissue by bacterial pathogens.