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.

Some aspects of the history of membrane-damaging toxins.

In this review the following topics are considered: (a) the character of biomedical research and how it has changed during the last six decades; (b) the early history of membrane-damaging toxins; (c) comparative toxinology as illustrated by similarity of the toxins of the brown recluse spider and the bacterial agent of peudotuberculosis, and by similarity of a toxin of a sea anemone and the thiol-activated membrane-damaging agents of bacteria; (d) examples of the diversity of membrane-damaging toxins; (e) cooperative or synergistic lytic systems; and (f) the outlook for the future.

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.

Cytotoxicity of cholesterol oxidase to cells of hypercholesterolemic guinea pigs.

1. A high cholesterol diet caused guinea pig erythrocytes to become sensitive to lysis by cholesterol oxidase (CO), a protein not hemolytic to normal cells. 2. Lysis was associated with conversion of membrane cholesterol to its oxidation product (delta-4-cholesten-3-one). 3. Intravenous injection of CO to hypercholesterolemic guinea pigs produced a reduction in serum cholesterol, but was not lethal as it was in rabbits. 4. Homogenized spleen, liver and kidney from the hyperlipidemic animals were sensitive to in vitro cholesterol oxidation while tissues from non-lipemic animals were resistant to modification.

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.

Phospholipase D activity of Vibrio damsela cytolysin and its interaction with sheep erythrocytes.

Exposure of sheep erythrocytes to sublytic amounts of Vibrio damsela cytolysin markedly reduced their membrane sphingomyelin content and their sensitivity to lysis by the sphingomyelin-dependent cytolysins staphylococcal sphingomyelinase C (beta-toxin) and helianthin. The toxin was found to be a phospholipase D active against sphingomyelin.

Toxicity of enzymically-oxidized low-density lipoprotein.

Intravenous injection of cholesterol oxidase into hyperlipidemic rabbits in which aortic atheromatous lesions have been induced by dietary means is lethal within hours, whereas injection of the same enzyme into normal rabbits has no visible adverse effect. The lethal effect of the enzyme is explicable by the finding that injection of cholesterol-oxidase treated low-density lipoprotein kills normal rabbits, in contrast to untreated low-density lipoprotein which does not. Enzymically oxidized low-density lipoprotein was also found to be cytotoxic for two human cell lines and for cultured bovine aortic endothelial cells. We suggest that in vivo enzymic conversion of low-density lipoprotein cholesterol to low-density lipoprotein cholestenone may possibly play a role in the initiation of atheromatous lesions in humans.

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.

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.

Copurification of Leptospira interrogans serovar pomona hemolysin and sphingomyelinase C.

The hemolytic and sphingomyelinase C activities of supernatants of cultures of Leptospira interrogans serovar pomona tended to copurify when isoelectric fractionation was carried out. Both activities focused primarily at pH 8.1. Considered in conjunction with other circumstantial evidence, the results led to the conclusion that sphingomyelinase C is responsible for hemolysis.

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.

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.

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.

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.

Cross-reactions of polysaccharides of fungi, molds, and yeasts in anti-pneumococcal and other antisera.

Cross-reactions of a number of glycans and polysaccharides isolated from fungi, molds, and yeasts in anti-pneumococcal and other antisera are tabulated and discussed insofar as possible with relation to the chemical structures of the cross-precipitating substances and the polysaccharidic antigens giving rise to the reactive antibodies. For example, the slime of Physarum polycephalum precipitated antisera to pneumococcal type 4 and Klebsiella type K1. The polysaccharides of type 4 and K1 contain pyruvic acid in acetal linkage on hydroxyls 2 and 3 of sugars. Therefore, pyruvic acid was looked for and found in Physarum slime. Numerous other relations between chemical structure and immunological specificity are given.

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.

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.