Insertional inactivation of streptolysin S expression in Streptococcus pyogenes

The inactivation of a genetic determinant critical for streptolysin S production was accomplished by transfer and insertion of the transposon Tn916 into the DNA of a group A streptococcal strain. The group D strain CG110 was able to efficiently transfer Tn916 into the group A strain CS91 when donor and recipient cells were concentrated and incubated together on membrane filters. Among tetracycline-resistant transconjugants, nonhemolytic mutants that no longer produced streptolysin S and retained the capacity to produce streptolysin O were discovered. Hemolytic revertants from these mutants regained tetracycline sensitivity; other revertants still retained a tetracycline resistance phenotype. Hybridization studies employing Tn916 DNA located Tn916 sequences in EcoRI and HindIII fragments of DNA from mutants devoid of streptolysin S; one carried a single copy of Tn916, and the other two carried multiple copies of the transposon.     

Maintenance of motility in mouse sperm permeabilized with streptolysin O

One approach to studying the mechanisms governing sperm motility is to permeabilize sperm and examine the regulation of motility by manipulating the intracellular milieu of the cell. The most common method of sperm permeabilization, detergent treatment, has the disadvantage that the membranes and many proteins are extracted from the cell. To avoid this problem, we have developed a method that uses streptolysin O to create stable pores within the plasma membrane while leaving internal membranes intact. Sperm were permeabilized, preincubated, and then treated with 0.6 U/ml of streptolysin O. Permeabilization was assessed by fluorescent dye technologies and endogenous protein phosphorylation using exogenously added [gamma-32P]ATP. Streptolysin O-induced permeabilization rendered the sperm immotile, and the effect was Ca2+-dependent. When the cells were treated simultaneously with a medium containing ATP, streptolysin O-treated sperm maintained flagellar movement. These results demonstrate that the streptolysin O permeabilization model system is a useful experimental method for studying the mechanisms that regulate sperm motility since it allows the flagellar apparatus to be exposed to various exogenously added molecules.     

Rat mast cells permeabilised with streptolysin O secrete histamine in response to Ca2+ at concentrations buffered in the micromolar range

Rat peritoneal mast cells have been permeabilised by treatment with streptolysin O which generates membrane lesions of macromolecular dimensions. In the presence of Ca2+ buffered at concentrations in the micromolar range, the permeabilised mast cells release histamine, beta-N-acetylglucosaminidase and lactate dehydrogenase. Release of the two secretory components (but not lactate dehydrogenase) has an obligatory requirement for a nucleoside triphosphate and micromolar concentrations of Ca2+. Inosine triphosphate (ITP) supports the release reaction better than ATP does. It is concluded that the secretory materials are released from the cells by an exocytotic mechanism, while lactate dehydrogenase leaks from the cells through the toxin-generated lesions. By initially withholding and then supplying Ca2+ to the permeabilised cells, it is shown that the exocytotic secretory reaction can persist even when the cytosol is depleted of the bulk of soluble proteins. The streptolysin O treated mast cell preparation represents a simplified system with which to study the mechanism of exocytosis.     

Effect of streptolysin S on liposomes Influence of membrane lipid composition on toxin action

The effect of the bacterial cytolytic toxin, streptolysin S, on liposomes composed of various phospholipids was investigated. Large unilamellar vesicles containing [¹⁴C]sucrose were prepared by reverse-phase evaporation, and membrane damage produced by the toxin was measured by following the release of labeled marker. The net charge of the liposomes had little or no effect on their susceptibility to steptolysin S and the toxin was about equally effective on liposomes composed of phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine and phosphatidylglycerol. Experiments with liposomes composed of synthetic phospholipids showed that the ability of the toxin to produce membrane damage depended on the degree of unsaturation of the fatty acyl chains. The order of sensitivity was C18 : 2 phosphatidylcholine > C18 : 1 phosphatidylcholine > C18 : 0 phosphatidylcholine = C16 : 0 phosphatidylcholine. Liposomes containing the latter two phospholipids were virtually unaffected by streptolysin S, and experiments with C18 : 0 phosphatidylcholine suggested that toxin activity does not bind to liposomes composed of phospholipids with saturated fatty acyl chains. The inclusion of 40 mol% cholesterol in C16 : 0 phosphatidylcholine and C18 : 0 phosphatidylcholine liposomes made these vesicles sensitive to streptolysin S. Egg phosphatidylcholine liposomes, which were unaffected at 0°C and 4°C became susceptible to the toxin at these temperatures when cholesterol was included. Liposomes composed of C14 : 0 phosphatidylcholine were unaffected by streptolysin S at temperatures below the chain-melting transition temperature (23°C) of this phospholipid, but became increasingly susceptible above this temperature. The results suggest that the fluidity of the phospholipid hydrocarbon chains in the membrane is important in streptolysin S action.     

Rat pancreatic acini permeabilised with streptolysin O secrete amylase at Ca2+ concentrations in the micromolar range, when provided with ATP and GTP gamma S.

The aim of this study was to define the conditions required for exocytosis in pancreatic acini permeabilised with the bacterial toxin streptolysin O. Treatment of a suspension of acini with streptolysin O caused the release of both the cytoplasmic enzyme lactate dehydrogenase and the zymogen granule enzyme amylase. The release of amylase occurred more quickly than that of lactate dehydrogenase and was smaller in magnitude. In addition, a component of amylase release occurred only in the presence of Ca2+ (at concentrations in the micromolar range), ATP and GTP gamma S. We conclude that this component represents an exocytotic event, but that the release of lactate dehydrogenase occurs through toxin-generated lesions. The concentrations of Ca2+, ATP and GTP gamma S causing half-maximal exocytosis were 0.7 microM, 0.2 mM and 10 microM, respectively. This system should permit a study of the mechanisms underlying regulated exocytosis in this cell type.     

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.     

Effect of streptolysin S on liposomes. Influence of membrane lipid composition on toxin action

The effect of the bacterial cytolytic toxin, streptolysin S, on liposomes composed of various phospholipids was investigated. Large unilamellar vesicles containing [14C]sucrose were prepared by reverse-phase evaporation, and membrane damage produced by the toxin was measured by following the release of labeled marker. The net charge of the liposomes had little or no effect on their susceptibility to steptolysin S and the toxin was about equally effective on liposomes composed of phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine and phosphatidylglycerol. Experiments with liposomes composed of synthetic phospholipids showed that the ability of the toxin to produce membrane damage depended on the degree of unsaturation of the fatty acyl chains. The order of sensitivity was C18 : 2 phosphatidylcholine greater than C18: I phosphatidylcholine greater than C18 : 0 phosphatidylcholine = C16 : 0 phosphatidylcholine. Liposomes containing the latter two phospholipids were virtually unaffected by streptolysin S, and experiments with C18 : 0 phosphatidylcholine suggested that toxin activity does not bind to liposomes composed of phospholipids with saturated fatty acyl chains. The inclusion of 40 mol% cholesterol in C16 : 0 phosphatidylcholine and C18 : 0 phosphatidylcholine liposomes made these vesicles sensitive to streptolysin S. Egg phosphatidylcholine liposomes, which were unaffected at 0 degrees C and 4 degrees C became susceptible to the toxin at these temperatures when cholesterol was included. Liposomes composed of C14 : 0 phosphatidylcholine were unaffected by streptolysin S at temperatures below the chain-melting transition temperature (23 degrees C) of this phospholipid, but became increasingly susceptible above this temperature. The results suggest that the fluidity of the phospholipid hydrocarbon chains in the membrane is important in streptolysin S action.     

Growth of Streptococcus pyogenes and streptolysin O production in complex and synthetic media

A comparative study of the growth of a highly haemolytic group A streptococcal strain in Trypticase-yeast extract medium and in a chemically defined medium was undertaken. Appreciable growth was obtained in the latter medium with the release of significant amounts (120 haemolytic units) of streptolysin O. This indicates that toxinogenic factors present only in the peptones of complex media are not essential for biosynthesis and release of this cytolytic toxin, as is the case for many bacterial toxins. NADase was also released in the synthetic medium. Bacterial cell mass, growth rate, and streptolysin O production were threefold higher in the complex medium. The effects of various carbohydrates on streptolysin O production in complex medium are investigated. No repression of toxin formation by glucose was observed. The relationship between growth and toxinogenesis in streptococci and in other toxinogenic bacteria is discussed.     

Action of streptolysin S, the group D hemolysin, and phospholipase C on whole cells and spheroplasts

Davie, Joseph M. (Indiana University, Bloomington), and Thomas D. Brock. Action of streptolysin S, the group D hemolysin, and phospholipase C on whole cells and spheroplasts. J. Bacteriol. 91:595-600. 1966.-The effect of streptolysin S, the group D hemolysin, and phospholipase C (the alpha toxin of Clostridium perfringens) on whole cells and spheroplasts or protoplasts of three strains of streptococci and Micrococcus lysodeikticus was tested. Viability, C(14)-glycine uptake, and lysis were measured. The group D hemolysin and phospholipase C were active against whole bacteria; streptolysin S was not. All three substances were active on spheroplasts. A partially resistant mutant derived from a strain sensitive to the group D hemolysin was also partially resistant to streptolysin S and phospholipase C. Antimycin A protected spheroplasts from streptolysin S but not from the group D hemolysin.     

Biochemical and ultrastructural study of the disruption of blood platelets by streptolysin O

The membrane-damaging protein toxin, streptolysin O, proved highly lytic on human, guinea-pig and rabbit platelets. About 15 molecules of toxin were sufficient to lyse one cell. Platelet disruption was assessed by electron microscopy, clearing of cell suspensions and assay of lactate dehydrogenase, serotonin, monoamine oxidase and glutathione peroxidase released in the extracellular fluid. This egress reflected the damage of both plasmic and organelle membranes. A quantitative study of lactate dehydrogenase and serotonin liberation taken as respective markers of the cytosol and dense bodies was undertaken as a function of toxin concentration. No platelet aggregation or shape change was elicited by streptolysin O. The ghosts resulting from platelet lysis retained properties of the native membrane such as aggregability and serotonin uptake. Dense bodies were easily separated after gentle disruption of the plasmic membrane by small amounts of toxin. Platelet lysis by streptolysin O proved a useful procedure for the determination of protein content, enzyme activities and serotonin assay on the same lysate in contrast to usual methods.     

Sterol structural requirements for inhibition of streptolysin O activity

Reduced streptolysin O, a toxin produced by certain beta-haemolytic streptococci, lyses human erythrocytes. The reaction is inhibited by cholesterol at concentrations of about 1.0mug/ml. Other sterols inhibit the lysin and there is a specific requirement for a 3beta-hydroxyl group. Inhibition was obtained with 3beta-hydroxychol-5-en-24-oic acid, containing a hydrophilic group at C-24. The mode of inhibition is likely to involve attachment to the fixation site of the lysin which attaches the molecule to cell membranes, probably to membrane cholesterol. A second streptolysin site, concerned in the final haemolytic event, may also be involved. Inhibitors of the latter site have not been characterized, other than antibody with specificity for the site.     

Chemokine production by rat macrophages stimulated with streptolysin O from Streptococcus pyogenes

The contribution of streptolysin O (SLO) from Streptococcus pyogenes to neutrophil infiltration in inflammatory lesions was determined by production of cytokine-induced neutrophil chemoattractant (CINC)-1, -2 and -3, and macrophage inflammatory protein (MIP)-1alpha by rat macrophages stimulated with SLO in culture. Active SLO induced the production of CINCs and MIP-1alpha in dose- and time-dependent manners. These inductions were ascertained by chemokine mRNA expression in macrophages. Streptolysin S was without effect. The SLO-cholesterol complex induced the chemokine production in proportion to the residual hemolytic activity of the complex. In addition, the effects of SLO on the chemokine production were confirmed by the injection of active SLO into the preformed air pouch on the back of rats. The infiltration of neutrophils into the pouch fluid (exudate) increased steadily with a lag phase of about 2 hr. The major chemokine found in exudates was MIP-1alpha but not CINCs. In this study, it became clear that active SLO, but not the inactive one, contributed to the production of MIP-1alpha and CINCs in the conditioned medium and in exudates.     

Resistance to streptolysin O in mammalian cells treated with oxygenated derivatives of cholesterol cholesterol content of resistant cells and recovery of streptolysin O sensitivity

Cultures of L cells and HeLa cells were made resistant to the cytolytic toxin, streptolysin O, by incubating them in the presence of 20α-hydroxycholesterol or 25-hydroxycholesterol. Such cells were also found to be more resistant to the cytotoxic effects of saponin and digitonin, agents known to interact with membrane cholesterol. Sterol synthesis in L cells that had been treated with either of the oxygenated derivatives of cholesterol was reduced by almost 90%, and the free cholesterol content of streptolysin O-resistant HeLa and L cells fell to approx. 50% of control cell levels. Significant recovery of sensitivity to streptolysin O occurred in about 6 h when refractory L cells were incubated in serum or cholesterol. Partial recovery was observed when the cultures were incubated for 24 h in mevalonate or lipid-depleted serum. The results provide further support for the role of membrane cholesterol in the cytotoxic action of streptolysin O on mammalian cells.     

Mutational and comparative analysis of streptolysin O, an oxygen-labile streptococcal hemolysin

The structural gene of streptolysin O was cloned from Streptococcus pyogenes strain Sa and S. equisimilis H46A, and the nucleotide sequences were compared with those of strain Richards. To obtain the minimal active fragment of the toxin and to elucidate structure-function relationships in hemolytic function, streptolysin O mutants deleted in N- and C-terminal regions were constructed. Internal amino acid residues were also replaced by introduction of point mutations. Analyses of these mutants showed that considerable activity was retained even after deletion of the N-terminal 107 residues, but genetic removal of the ultimate C-terminal residue resulted in a marked decrease in hemolytic function. By removal in succession, hemolytic activity declined exponentially, and only 0.002% of the activity remained after deletion of the C-terminal four residues. Nucleotide replacement experiments indicated pivotal roles of I202, V217, D324-L325, V339, and H469 residues in hemolysis.     

A ring-shaped structure with a crown formed by streptolysin O on the erythrocyte membrane.

Streptolysin O (SLO) is a membrane-damaging toxin produced by most strains of group A beta-hemolytic streptococci. We performed ultrastructural analysis of SLO-derived lesions on erythrocyte membranes by examining electron micrographs of negatively stained preparations. SLO formed numerous arc- and ring-shaped structures with or without holes on membranes. Rings formed on intact cell membranes had an inner diameter of ca. 24 nm and had distinct borders of ca. 4.9 nm in width, but the diameter of rings varied from 24 to 30 nm on membranes of erythrocyte ghosts. Image analysis of electron micrographs demonstrated that each ring was composed of an inner and an outer layer. Each layer contained an array of 22 to 24 SLO molecules. On the top of the ring, we found a characteristic crown that projected from the cell membrane. The crown was separated by an electron-dense layer from the basal part of the ring that was embedded in the lipid bilayer of the erythrocyte membrane. Heights of the three parts, namely, the crown (head), the space (neck), and the basal portion (base), were ca. 3.2, 1.6, and 5.0 nm, respectively, and we postulated that these parts are the constituents of a single SLO molecule. The volumes of SLO molecules in the inner and outer layers were calculated to be 77 and 88 nm3. On the basis of a model of the structure of SLO, we propose some new details of the mechanisms of hemolysis by SLO toxin.     

Electron microscopic evaluation of a two-step theory of pore formation by streptolysin O.

The formation of pores by streptolysin O (SLO) was analyzed in erythrocyte membranes and liposomes by immunoelectron microscopy and electron spectroscopic imaging. The binding of SLO molecules to membranes was temperature independent, while the polymerization of SLO molecules was temperature dependent. Our results also suggest that proteins in erythrocyte membranes are not involved in the formation of SLO rings.     

Novel genomic rearrangement that affects expression of the Streptococcus pyogenes streptolysin O (slo) gene

A RecA-independent chromosomal rearrangement in the upstream region of the streptolysin O (slo) gene of Streptococcus pyogenes which affects slo expression was identified. PCR analysis was used to demonstrate that this kind of rearrangement was found in several strains of different lineages. Chromosomal loci involved in the recombination were found to be 746 kb apart on the 1.85-Mb-long chromosome. The primary structure of the splicing region, the reproducibility of the rearrangement, and the fact that reconstructed recombinant molecules fused to erm and lacZ reporter genes affected their expression indicate that this event is not accidental but may play a role in the expression of the slo gene. In addition, the product of the recombining DNAs, including the splicing site, does not follow any example of a known recombination mechanism. The implications of this rearrangement for slo expression are discussed.     

Purification and characterization of streptolysin O from Streptococcus pyogenes.

1. Streptolysin O, an exotoxin produced by group A beta-hemolytic streptococci, has been purified from Streptococcus pyogenes culture supernatants. 2. The isolation and purification procedure involved ammonium sulphate and polyethylene glycol precipitations, and ion-exchange chromatographies on CM-Sepharose and Mono Q. 3. The proposed procedure introduces two ion-exchange chromatography steps making the purification process simpler and, especially, more reproducible than other described protocols. 4. The purified streptolysin O was hemolytically active, had a specific activity of 415,000 HU/mg, an optimum pH of 7.0, a relative molecular mass of 60,100 and an isoelectric pH of 7.5.     

Purification and characterization of a cytolytic pore-forming protein from granules of cloned lymphocytes with natural killer activity

A cytolytic pore-forming protein (PFP, perforin) was purified from isolated granules of cloned NK-like cytolytic cells, which showed an apparent Mr of 70-75 kd (reduced) and 62-66 kd (nonreduced). Cytolysis produced by this protein occurred only in the presence of Ca2+ and was accompanied by the formation of membrane lesions of 160 A diameter. The purified protein depolarized cells and made lipid vesicles leaky to monovalent and divalent ions. This protein formed large, voltage insensitive and nonselective ion channels in planar bilayers that remained preferentially in the open state. The channels were heterogeneous in size distribution averaging 400 pS/U in 0.1 M NaCl. The membrane lesions formed by PFP were morphologically and functionally similar to those formed by intact NK-like cells and their granules. This PFP could be released from granules during cell killing, followed by its polymerization on target membranes to form large transmembrane pores.     

Cytosolic delivery of granzyme B by bacterial toxins: evidence that endosomal disruption, in addition to transmembrane pore formation, is an important function of perforin

Granule-mediated cell killing by cytotoxic lymphocytes requires the combined actions of a membranolytic protein, perforin, and granule-associated granzymes, but the mechanism by which they jointly kill cells is poorly understood. We have tested a series of membrane-disruptive agents including bacterial pore-forming toxins and hemolytic complement for their ability to replace perforin in facilitating granzyme B-mediated cell death. As with perforin, low concentrations of streptolysin O and pneumolysin (causing <10% (51)Cr release) permitted granzyme B-dependent apoptosis of Jurkat and Yac-1 cells, but staphylococcal alpha-toxin and complement were ineffective, regardless of concentration. The ensuing nuclear apoptotic damage was caspase dependent and included cleavage of poly(ADP-ribose) polymerase, suggesting a mode of action similar to that of perforin. The plasma membrane lesions formed at low dose by perforin, pneumolysin, and streptolysin did not permit diffusion of fluorescein-labeled proteins as small as 8 kDa into the cell, indicating that large membrane defects are not necessary for granzymes (32 to 65 kDa) to enter the cytosol and induce apoptosis. The endosomolytic toxin, listeriolysin O, also effected granzyme B-mediated cell death at concentrations which produced no appreciable cell membrane damage. Cells pretreated with inhibitors of endosomal trafficking such as brefeldin A took up granzyme B normally but demonstrated seriously impaired nuclear targeting of granzyme B when perforin was also added, indicating that an important role of perforin is to disrupt vesicular protein trafficking. Surprisingly, cells exposed to granzyme B with perforin concentrations that produced nearly maximal (51)Cr release (1,600 U/ml) also underwent apoptosis despite excluding a 8-kDa fluorescein-labeled protein marker. Only at concentrations of >4,000 U/ml were perforin pores demonstrably large enough to account for transmembrane diffusion of granzyme B. We conclude that pore formation may allow granzyme B direct cytosolic access only when perforin is delivered at very high concentrations, while perforin's ability to disrupt endosomal trafficking may be crucial when it is present at lower concentrations or in killing cells that efficiently repair perforin pores.