Heat stability and species range of purified staphylococcal alpha-toxin

Cooper, Louis Z. (New England Medical Center Hospital, Boston, Mass.), Morton A. Madoff, and Louis Weinstein. Heat stability and species range of purified staphylococcal alpha-toxin. J. Bacteriol. 91:1686-1692. 1966.-Heating of high-titer purified staphylococcal alpha-toxin at 60 and 80 C resulted in a double-sloped curve of inactivation of the hemolytic effect on rabbit erythrocytes. Early inactivation was less at the lower temperature, but activity persisted for a longer time at 80 C. Toxin inactivated at 60 C showed renewed activity when heated briefly at 80 C. A precipitate which formed during heating of alpha-toxin at 60 or 80 C yielded hemolytic activity when resuspended and heated at 80 but not at 60 C. Supernatant fluid of heat-precipitated toxin was heat-labile and did not regain activity when heated at 80 C. The results indicate that the "paradoxical effect" of heating of staphylococcal alpha-toxin is not due to a thermolabile inhibitor, but results from alteration of the toxin molecule to a heat-stable active form. Demonstration of renewed activity by 80 C heating of purified toxin requires potent toxin preparations and brief heating periods. Hemolysis of erythrocytes of several animal species by purified alpha-toxin was generally similar to that produced by impure toxin. Rabbit cells were most susceptible. Human and horse erythrocytes hemolyzed to less than 0.1% of the extent of rabbit cells. Blood cells of other species were intermediate in their response to the lytic effect of alpha-toxin.     

Staphylococcus aureus alpha-toxin. Dual mechanism of binding to target cells

Staphylococcal alpha-toxin was radiolabeled to high specific radioactivity (1,500-3,000 Ci/mmol) under retention of its hemolytic activity. Binding studies with susceptible rabbit erythrocytes and highly resistant human erythrocytes revealed that binding of alpha-toxin to target cells can occur via two different mechanisms. Binding of alpha-toxin to rabbit erythrocytes initially involves specific binding sites and occurs at low concentrations, with half-maximal binding at 1-2 nM. In contrast, toxin binding to human erythrocytes is absorptive and nonspecific, in this case, significant binding as well as hemolysis occur only at alpha-toxin concentrations exceeding 1 microM. Autoradiographic analyses of membrane-associated alpha-toxin from either cell species proved that hemolysis was inevitably associated with the formation of toxin hexamers. Our data indicate that the high susceptibility of certain target cells toward alpha-toxin is caused by the presence of specific binding sites. However, membrane damage of both susceptible and nonsusceptible target cells occurs via a common mechanism involving toxin oligomerization and pore formation.     

Iodination of a tyrosyl residue in staphylococcal alpha-toxin.

Iodination of staphylococcal alpha-toxin by the lactoperoxidase method resulted in the maximal incorporation of about 2.5 atoms of iodine per molecule of alpha-toxin. The iodination primarily involved a single tyrosine residue as shown by analysis of both cyanogen bromide and tryptic peptides. Iodination at a level of 1.2 iodine atoms per alpha-toxin molecule led to a dramatic decrease in the hemolytic and lethal activities, although no decrease in the binding of iodinated toxin to rabbit erythrocytes was observed (Cassidy and Harshman (1976), Biochemistry, the following paper in this issue). Monoiodinated alpha-toxin was found to have 15% of the specific hemolytic activity of native alpha-toxin. Incubation of rabbit erythrocytes with iodinated alpha-toxin led to a significant protection from the hemolytic activity of native alpha-toxin added later. The results show the modification of a single unique tyrosyl residue in alpha-toxin permits the resolution of alpha-toxin's biological activities from its cell binding activity.     

Isolation and characteristics of staphylococcal alpha-toxin

A method for isolation of staphylococcal alpha-toxin preparations has been elaborated. Characteristics of the toxin isolated by the method are as follows: mol. mass = 35 Kd; HU = 0.1 microgram; DnD= 0.1 microgram; LD50 = 2 micrograms. It is for the first time that alpha-toxin was fragmented by papain and digested by alpha, gamma-chemotrypsin. The papain fragments (18.5 and 15 Kd) retained lethal activity but lost hemolytic and dermonecrotic activities. Alpha, gamma-chemotryptic digested fragments (18 and 15 Kd) retained hemolytic and lethal effects, but lost their dermonecrotic activity.     

Studies on the binding of staphylococcal 125I-labeled alpha-toxin to rabbit erythrocytes.

Staphylococcal alpha-toxin, a hemolytic exotoxin, can be iodinated using the lactoperoxidase method. 125 I-Labeled alpha-toxin binds to rabbit erythrocytes in an apparently irreversible and highly specific manner. The binding of 125 I-labeled alpha-toxin to erythrocytes of rabbit and human reflects the species specificity of native alpha-toxin. Binding of 125I-labeled alpha-toxin is blocked by the presence of native alpha-toxin, 127I-labeled alpha-toxin, or anti-alpha-toxin antibody. Simultaneous assays of 125I-labeled alpha-toxin binding and leakage of intracellular 86Rb+ suggest that toxin binding and membrane damage are separate, sequential functions. Both the rate and extent of binding are temperature dependent. Rabbit erythrocytes possess 5 X 10(3) binding sites/cell, while human erythrocytes possess no detectable binding sites. Treatment of rabbit erythrocytes with 125I-labeled alpha-toxin appears to decrease the number of unoccupied binding sites. Chaotropic ions can inhibit 125I-labeled alpha-toxin binding and cause bound 125I-labeled alpha-toxin to dissociate from rabbit erythrocyte membranes. Treatment of intact rabbit erythrocytes with pronase reduces both the binding capacity of the cells for 125I-labeled alpha-toxin, and the cells' sensitivity to hemolysis by native alpha-toxin. It is proposed that the primary binding site for alpha-toxin in biomembranes is a surface membrane protein.     

Hemolytic toxin produced by Porphyromonas gingivalis

Abstract Porphyromonas gingivalis no. 381 and ATCC 33277 produced an extracellular hemolytic toxin which was heat-labile, trypsin-sensitive, and lytic to human, horse, sheep and rabbit erythrocytes. The hemolytic toxin is a 'hot-cold', thiol-independent toxin. The production of the hemolytic toxin was greatly enhanced by addition of hemoglobin to the culture medium.     

Effects of Lectins on the Hemolysis of Rabbit Erythrocytes by Staphylococcal Alpha Toxin

When concanavalin A (1 μg/ml) or wheat germ agglutinin (2 μg/ml) was preincubated with a suspension of 2% rabbit erythrocytes for 5 min at 20 C, the binding of [¹²⁵I]-labeled staphylococcal alpha toxin to these erythrocytes was greatly inhibited and the hemolytic action of alpha toxin was decreased. The inhibitory effect of concanavalin A on hemolysis by alpha toxin was completely reversed in the presence of 0.1 M α-methyl-D -glucoside or α-methyl-D -mannoside. Phytohemagglutinin-P from Phaseolus vulgaris and soybean agglutinin inhibited hemolysis by the toxin at concentrations exceeding 20 μg/ml. The effect of concanavalin A on alpha-toxin hemolysis was studied further to ascertain the nature of the inhibition. Double reciprocal plots were made of hemolysis against alpha toxin concentrations, and the data suggested that inhibition of the initial rate of the hemolysis by concanavalin A is competitive in nature. This was probably due to an interaction with the alpha toxin binding sites on the cell membrane surface.     

Physical states of staphylococcal alpha-toxin

At least three different forms of staphylococcal alpha-toxin have been shown to exist: soluble active alpha-toxin (alpha 3S), soluble inactive alpha-toxin (alpha(12s)), and insoluble inactive aggregate. Aggregation to the insoluble, biologically inactive form could be induced by brief heating to 60 C. The aggregate was dissociated by treatment with 8 m urea with reappearance of biological activity. Subsequent removal of urea by dialysis resulted in some spontaneous reaggregation to the insoluble state. The supernatant fluid obtained after dialysis contained soluble active alpha-toxin of high specific activity, possessing physical, toxic, and immunological properties closely resembling those of native toxin. The soluble biologically inert component (alpha(12s)) was identified as a third physical state. Negatively stained preparations of this material, when examined in the electron microscope, showed rings of approximately 100 A outside diameter containing 6 +/- 1 subunits.     

Purification of alpha-toxin from Staphylococcus aureus and application to cell permeabilization

Crude alpha-toxin was produced by Staphylococcus aureus, strain Wood 46. The amount of exotoxin was monitored during growth and all subsequent purification steps by determination of its hemolytic activity against rabbit erythrocytes. The culture supernatant was treated with ammonium sulfate (75% saturation). The resulting precipitate was dialyzed and subjected to cation-exchange chromatography. The fractions containing the hemolytic activity were further purified by gel chromatography. The final product was enriched by a factor of 8.5 compared to the crude toxin. In sodium dodecyl sulfate-polyacrylamide gel electrophoresis the purified toxin exhibited one major band. It caused the release of 86Rb+ and ATP from rat insulinoma (RIN A2) as well as pheochromocytoma cells (PC12) in culture, indicating efficient permeabilization of their plasma membranes for small molecules.     

Lysosomal disruption by bacterial toxins

Bernheimer, Alan W. (New York University School of Medicine, New York), and Lois L. Schwartz. Lysosomal disruption by bacterial toxins. J. Bacteriol. 87:1100-1104. 1964.-Seventeen bacterial toxins were examined for capacity (i) to disrupt rabbit leukocyte lysosomes as indicated by decrease in turbidity of lysosomal suspensions, and (ii) to alter rabbit liver lysosomes as measured by release of beta-glucuronidase and acid phosphatase. Staphylococcal alpha-toxin, Clostridium perfringens alpha-toxin, and streptolysins O and S affected lysosomes in both systems. Staphylococcal beta-toxin, leucocidin and enterotoxin, Shiga neurotoxin, Serratia endotoxin, diphtheria toxin, tetanus neurotoxin, C. botulinum type A toxin, and C. perfringens epsilon-toxin were not active in either system. Staphylococcal delta-toxin, C. histolyticum collagenase, crude C. perfringens beta-toxin, and crude anthrax toxin caused lysosomal damage in only one of the test systems. There is a substantial correlation between the hemolytic property of a toxin and its capacity to disrupt lysosomes, lending support to the concept that erythrocytes and lysosomes are bounded by similar membranes.     

Effect of streptolysin O on erythrocyte membranes, liposomes, and lipid dispersions. A protein-cholesterol interaction

The effect of the bacterial cytolytic toxin, streptolysin O (SLO), on rabbit erythrocyte membranes, liposomes, and lipid dispersions was examined. SLO produced no gross alterations in the major erythrocyte membrane proteins or lipids. However, when erythrocytes were treated with SLO and examined by electron microscopy, rings and "C"-shaped structures were observed in the cell membrane. The rings had an electron-dense center, 24 nm in diameter, and the overall diameter of the structure was 38 nm. Ring formation also occurred when erythrocyte membranes were fixed with glutaraldehyde and OsO4 before the addition of toxin. In contrast, rings were not seen when erythrocytes were treated with toxin at 0 degrees C, indicating that adsorption of SLO to the membrane is not sufficient for ring formation since toxin is known to bind to erythrocytes at that temperature. The ring structures were present on lecithin-cholesterol-dicetylphosphate liposomes after SLO treatment, but there was no release of the trapped, internal markers, K2CrO4 or glucose. The crucial role of cholesterol in the formation of rings and C's was demonstrated by the fact that these structures were present in toxin-treated cholesterol dispersions, but not in lecithin-dicetylphosphate dispersions nor in the SLO preparations alone. The importance of cholesterol was also shown by the finding that no rings were present in membranes or cholesterol dispersions which had been treated with digitonin before SLO was added. Although rings do not appear to be "holes" in the membrane, a model is proposed which suggests that cholesterol molecules are sequestered during ring and C-structure formation, and that this process plays a role in SLO-induced hemolysis.     

Lytic Effects of Staphylococcal α-Toxin and δ-Hemolysin

Several preparations of staphylococcal alpha-toxin and delta-lysin were studied in order to compare hemolytic activity with capacity to lyse bacterial protoplasts. delta-Lysin in relatively low concentration lysed protoplasts of Sarcina lutea, protoplasts of Streptococcus faecalis, and spheroplasts of Escherichia coli. Lysis of bacterial protoplasts by preparations of alpha-toxin appeared to be due to contamination of the preparations with delta-lysin. Data comparing the protoplast-lysing activity of various lytic agents are presented.     

Influence of membrane fluidity on the assembly of Staphylococcus aureus alpha-toxin, a channel-forming protein, in liposome membrane.

By use of multilamellar phosphatidylcholine (PC) liposomes of different acyl composition and cholesterol content as model membranes, we studied whether or not membrane fluidity affects the assembly process of Staphylococcus aureus alpha-toxin. Under conditions using fluid and solid membranes, we assayed accessibility (or hemolytic activity) of liposome-bound alpha-toxin to rabbit erythrocytes added, hexamerization of membrane-bound toxin using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under nondenaturating conditions, and susceptibility of liposome-bound toxin to trypsin digestion. Our data indicated 1) that alpha-toxin bound to PC membrane as a hemolytically active monomer (or reversibly bound state); 2) that when the membrane was fluidized either by phase transition of PC or by inclusion of cholesterol over 20 mol %, the hemolytically active monomer of the toxin was irreversibly converted to nonhemolytic monomer (and/or unstable oligomer) in a first-order kinetics with a t1/2 of about 1 min, and thereafter hexamerization of the toxin gradually proceeded in the following 60-90 min; 3) that alpha-toxin might have different topology and/or conformation in PC membrane, depending on the presence or absence of cholesterol in the PC membrane; and 4) that coexistence of unsaturated acyl chain-carrying PC and cholesterol was a prerequisite for efficient hexamerization of alpha-toxin in membrane. Thus, increase in membrane fluidity promoted the assembly process of S. aureus alpha-toxin.     

The structure of Staphylococcus aureus α-toxin: Effects of trypsin treatment

Staphylococcus aureus alpha-toxin was treated with trypsin, which inactivates the toxin. Two-dimensional crystals of the modified protein were produced on preformed lipid layers. The projection structure obtained by electron crystallographic analysis of a large number of crystals showed tetragonal p4 symmetry and a resolution of approximately 12 A. The fragments of the toxin, 17 and 18 kDa large, were arranged in a way resembling those observed earlier for the native protein (Olofsson et al., J. Mol. Biol. 214, 299-306, 1990). However, after trypsin treatment the stain-deficient region corresponding to one alpha-toxin monomer shows two separated subdomains of similar size. This separation is probably related to the inability of the modified toxin to undergo the conformational change thought to be essential for the membrane-damaging effect.     

Role of the C-domain in the biological activities of Clostridium perfringens alpha-toxin

Clostridium perfringens alpha-toxin (370 residues) possesses hemolytic and lethal activities as well as the enzymatic activity of phospholipase C (PLC). In this study we examined the role of the C-domain (251-370 residues; CP251- 370) in biological activities of the toxin. The N-domain (1-250 residues; CP1- 250) of the alpha-toxin as well as the Bacillus cereus phospholipase C (BcPLC) possessed PLC activity, but did not bind to rabbit erythrocytes and lyse them. A hybrid protein (BC-CP251-370) consisting of BcPLC and CP251- 370 bound to the red cells and lysed them. Incubation of CP1-250 with CP251-370 completely complemented hemolytic and PLC activities. CP251-370 also conferred hemolytic activity on BcPLC. CP251-340 (251-340 residues) significantly stimulated PLC activity of CP1-250), but did not confer hemolytic activity on CP1-250. Kinetic analysis suggested that CP251-370 increased affinity toward the substrate of CP1-250. The results suggested that CP251-370 plays an important role in binding to erythrocytes and the hemolytic and enzymatic activities of CP1-250. Acrylodan-labeled CP251-370 variants (S263C and S365C) bound to liposomes and exhibited a marked blue shift, and in addition, an N,N'-dimethyl-N-(iodoacetyl)-N'-(7-nitrobenz-2-oxa-1,3-diazolyl)ethylene diamine (NBD)-labeled CP251-370 (S365C) variant also bound to liposomes and the fluorescence intensity significantly increased, suggesting movement of CP251-370 to a hydrophobic environment. These observations suggest that interaction of CP251-370 of alpha-toxin with fatty acyl residues of phosphatidylcholine plays an important role in the biological activities of CP1-250.     

Hemolysis induced by Bacillus cereus sphingomyelinase

Bacillus cereus sphingomyelinase (Bc-SMase) induces hemolysis of sheep erythrocytes which contain large amounts of sphingomyelin. We investigated the mechanism of this hemolysis in comparison to that induced by Clostridium perfringens alpha-toxin. Pertussis toxin, a Gi-specific inhibitor, N-oleoylethernolamine, a ceramidase inhibitor, and dihydrosphingosine, a sphingosine kinase inhibitor, did not inhibit the hemolysis by Bc-SMase, but did inhibit that by alpha-toxin. Bc-SMase broadly bound to whole membranes, and alpha-toxin specifically bound to the detergent-resistant membrane fractions, lipid rafts. The level of ceramide production induced by Bc-SMase in sheep erythrocytes was 6- to 15-fold that induced by alpha-toxin, when the extent of the hemolysis by Bc-SMase was the same as that by the toxin. However, the level of ceramide production induced by Bc-SMase in SM-liposomes was equal to that triggered by the toxin, when the carboxyl fluorescein-release from liposomes induced by Bc-SMase was the same as that induced by alpha-toxin. Confocal laser microscopy showed that treatment of the cells with Bc-SMase resulted in the formation of ceramide-rich domains. A photobleaching analysis suggested that treatment of the cells with Bc-SMase leads to a reduction in membrane fluidity. These results show that Bc-SMase-induced hemolysis of sheep erythrocytes is related to the formation of interface between ceramide-rich domains and ceramide-poor domains through production of ceramide from SM.     

The binding of fluorescein-labelled stapbylococcal alpha toxoid to erythrocytes

Erythrocytes of different animal species have variable hemolytic sensitivity to staphylococcal alpha toxin. Specific and non-specific binding of toxin was measured using fluorescein-labelled toxoid. These studies indicate that toxoid binding to erythrocytes increases with concentration for all species tested. Scatchard plot analyses of 35 animals representing seven species indicate that rabbit, pig, cow, and chicken erythrocytes possess 125 980, 103 920, 82 500, and 41 200 receptors per cell, respectively. The number of receptors remains constant over a period of at least 10 days. No detectable receptors were found for human, rat, and guinea pig erythrocytes. A correlation coefficient of 0.992 exists between receptor number and hemolytic sensitivity for those species having receptors. Variation in hemolytic sensitivity is governed by receptor number and not by variation in the dissociation constant. A threshold sensitivity of 37 000 receptors per cell has been calculated. Since species lacking detectable receptors have considerable sensitivity to hemolysis, it is proposed that two binding mechanisms, specific and non-specific, exist which prepare erythrocytes for destruction.     

Clostridium perfringens alpha-toxin activates the sphingomyelin metabolism system in sheep erythrocytes

Clostridium perfringens alpha-toxin induces hemolysis of rabbit erythrocytes through the activation of glycerophospholipid metabolism. Sheep erythrocytes contain large amounts of sphingomyelin (SM) but not phosphatidylcholine. We investigated the relationship between the toxin-induced hemolysis and SM metabolic system in sheep erythrocytes. Alpha-toxin simultaneously induced hemolysis and a reduction in the levels of SM and formation of ceramide and sphingosine 1-phosphate (S1P). N-Oleoylethanolamine, a ceramidase inhibitor, inhibited the toxin-induced hemolysis and caused ceramide to accumulate in the toxin-treated cells. Furthermore, dl-threo-dihydrosphingosine and B-5354c, isolated from a novel marine bacterium, both sphingosine kinase inhibitors, blocked the toxin-induced hemolysis and production of S1P and caused sphingosine to accumulate. These observations suggest that the toxin-induced activation of the SM metabolic system is closely related to hemolysis. S1P potentiated the toxin-induced hemolysis of saponin-permeabilized erythrocytes but had no effect on that of intact cells. Preincubation of lysated sheep erythrocytes with pertussis toxin blocked the alpha-toxin-induced formation of ceramide from SM. In addition, incubation of C. botulinum C3 exoenzyme-treated lysates of sheep erythrocytes with alpha-toxin caused an accumulation of sphingosine and inhibition of the formation of S1P. These observations suggest that the alpha-toxin-induced hemolysis of sheep erythrocytes is dependent on the activation of the SM metabolic system through GTP-binding proteins, especially the formation of S1P.     

On the mechanism of membrane damage by Staphylococcus aureus alpha- toxin

Rabbit or human erythrocytes lysed with Staphylococcus aureus alpha-toxin were solubilized with Triton X-100, and the toxin was subsequently isolated by gel chromatography, sucrose density gradient centrifugation, and reincorporation into liposomes. In the presence of Triton X-100, the toxin exhibited a sedimentation coefficient of 11S and eluted at a position between those of IgG and alpha 2-macroglobulin in gel chromatography. A single polypeptide subunit of 34,000 mol wt was found in SDS PAGE. In the electron microscope, ring-shaped or cylindrical structures were observed, 8.5-10 nm in diameter, harboring central pits or channels 2-3 nm in diameter. An amphiphilic nature of these structures was evident from their capacity to bind lipid and detergent, aggregation in the absence of detergents, and low elutability from biological and artificial membranes through ionic manipulations. In contrast to the membrane-derived form of alpha-toxin, native toxin was a water-soluble, 34,000 mol wt, 3S molecule, devoid of an annular structure. Because studies on the release of radioactive markers from resealed erythrocyte ghosts indicated the presence of circumscribed lesions of approximately 3-nm effective diameter in toxin-treated membranes, the possibility is raised that native alpha-toxin oligomerizes on and in the membrane to form an amphiphilic annular complex that, through its partial embedment within the lipid bilayer, generates a discrete transmembrane channel.     

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.