Purification and physicochemical and biological characterization of a staphylococcal pyrogenic exotoxin.

A staphylococcal pyrogenic exotoxin was purified and characterized biochemically and biologically. The organism producing the toxin was a group I Staphylococcus aureus strain which was isolated from a vaginal infection of a patient with mucocutaneous lymph node syndrome (Kawasaki's disease). The possible association of the toxin with the disease syndrome is discussed. The toxin was purified from cell-free culture supernatant fluids by means of differential precipitation with ethanol and resolubilization in pyrogen-free distilled water followed by preparative thin-layer isoelectric focusing. The pyrogenic exotoxin produced fevers in both rabbits and mice and enhanced host susceptibility to lethal shock and myocardial and liver damage by endotoxin. Also, the toxin was a potent nonspecific lymphocyte mitogen, stimulating rabbit spleen cells and human cord blood lymphocytes to proliferate. The toxin migrated as a homogeneous protein when tested with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (molecular weight, 12,000) and reisoelectric focusing (pI 5.3). Hyperimmune antisera raised against the purified toxin reacted with ethanol-precipitated toxin, using immunodiffusion to form a single precipitin arc. The toxin was distinguished from other staphylococcal toxins by a variety of methods. The amino acid composition was determined.     

Purification and characterization of a staphylococcal epidermolytic toxin.

A staphylococcal exotoxin that causes epidermolysis when injected into the skin of the newborn mouse and man was highly purified by coventional biochemical techniques. With Staphylococcus aureus EV, the epidermolytic toxin was a major protein component of supernatant culture fluids. The initial step in purification was zone electrophoresis in Pevikon carried out at pH 9.0, the isoelectric point of alpha-hemolytic toxin, which remained near the origin. Fractions containing the epidermolytic toxin, but free of alpha-toxin, were then subjected to cation exchange chromatography on carboxymethyl-Sephadex C-50 to remove trace contaminants. A major highly purified epidermolytic toxin migrated as a single band in polyacrylamide gel electrophoresis, sedimented as a single component in the analytical ultracentrifuge, and elicited a single precipitating antibody after injection into rabbits. A smaller amount of a second epidermolytic toxin, identical in molecular weight and antigenicity but differing in electrophoretic behavior from the major molecular species, was also identified. The epidermolytic factor had a molecular weight of 28,600 +/- 400 by sodium dodecyl sulfate-acrylamide electrophoresis and 32,500 +/- 120 by approach to sedimentation equilibrium.     

Production of staphylococcal enterotoxin F and pyrogenic exotoxin C by Staphylococcus aureus isolates from toxic shock syndrome-associated sources.

A total of 136 isolates of Staphylococcus aureus were tested for production of staphylococcal enterotoxin F (SEF) and pyrogenic exotoxin C (PEC), both of which have been identified as reliable indicators of toxic shock syndrome (TSS)-associated strains. SEF and PEC production by isolates from TSS-associated and other sources was tested independently in two laboratories, after which the two sets of data were compared. A 100% concordance between SEF and PEC production was obtained. The TSS toxin candidates were produced by 30 of 136 isolates, and in all instances SEF and PEC were made concurrently by the same strains; in no case was one toxin made and not the other. In the five groups of S. aureus tested, toxins were detected as follows: 23 of 25 (92%) acute TSS isolates, 2 of 48 (4.2%) genital non-TSS isolates, 2 of 16 (12.5%) recovered TSS isolates, 1 of 23 (4.3%) clinical nongenital isolates, and 2 of 24 (8.3%) enterotoxigenic food outbreak isolates. Comparison of purified SEF and purified PEC by immunological and biochemical criteria by immunodiffusion, isoelectric focusing, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and Western blot analysis show that the toxins are immunologically identical and strongly suggest that the two nominal TSS toxins are in fact a single protein.     

Presence of toxic shock toxin in toxic shock and other clinical strains of Staphylococcus aureus

Toxic shock toxin (TST), also known as pyrogenic exotoxin C (Schlievert et al., J. Infect. Dis. 143:509-516, 1981) and staphylococcal enterotoxin F (Bergdoll et al., Lancet i:1017-1021, 1981), was purified from toxic shock strains of Staphylococcus aureus by preparative isoelectric focusing and by chromatofocusing. Neither method produced an absolutely pure protein as determined by silver staining of sodium dodecyl sulfate-acrylamide gels, although chromatofocusing was the better method of the two. Three molecular weight variants of the protein were found in the two toxic shock syndrome strains that were studied, regardless of the purification method that was used. An isoelectric point of 7.15 and molecular weights of 21,400, 22,100, and 23,200 were determined for the different forms of the protein from electrophoresis data. A sedimentation coefficient of 2.3S was determined by sucrose gradient centrifugation, and a Stokes radius of 2 X 10(-7) cm was determined by gel filtration. An average molecular weight of 18,900 for all of the TST forms was calculated from these data by the Stokes-Einstein equation. A survey for TST in 32 control and 46 toxic shock strains of S. aureus by isoelectric focusing and by agarose gel double immunodiffusion with specific rabbit antiserum revealed that the isoelectric focusing method tends to overestimate the number of TST-positive strains because of the detection of non-TST, neutral staphylococcal proteins. Based on immunodiffusion data, the association of TST with toxic shock strains was found to be 100% in vaginal isolates and 62% in non-vaginal isolates. In the control strains, TST was found in 16% of the vaginal strains and 23% of the non-vaginal strains. The value of this toxin as a marker for toxic shock and its relationship to the pathogenesis of this disease are discussed.     

Toxicity of recombinant toxic shock syndrome toxin 1 and mutant toxins produced by Staphylococcus aureus in a rabbit infection model of toxic shock syndrome.

Menstrually associated toxic shock syndrome (TSS) is attributed primarily to the effects of staphylococcal exotoxin toxic shock syndrome toxin 1 (TSST-1). A region of the 194-amino-acid toxin spanning residues 115 through 144 constitutes a biologically active site. Several point mutations in the TSST-1 gene in that region result in gene products with reduced mitogenic activity for murine T cells. In this study we evaluated the toxicity of recombinant TSST-1 and several mutants of TSST-1 made by transformed Staphylococcus aureus during in vivo growth in a rabbit infection model of TSS. The toxicities of the transformed strains of S. aureus for rabbits correlated with the mitogenic activities of the recombinant toxins. An isolate originally obtained from a patient with a confirmed case of TSS (S. aureus 587) implanted in a subcutaneous chamber served as a positive control. TSST-1 produced in vivo led to lethal shock within 48 h, and a TSST-1-neutralizing antibody (monoclonal antibody 8-5-7) administered to rabbits challenged with S. aureus 587 prevented fatal illness. Rabbits infected with transformed S. aureus RN4220 expressing wild-type toxin (p17) or mutant toxins retaining mitogenic activity for T cells succumbed within a similar time frame. Blood chemistries of samples obtained from infected animals before death indicated abnormalities in renal and hepatic functions similar to those induced by parenteral injection of purified staphylococcal TSST-1. Mutant toxin 135 (histidine modified to alanine at residue 135) possessed only 5 to 10% of the mitogenic activity of wild-type toxin. Rabbits challenged with transformed S. aureus RN4220 expressing mutant toxin 135 exhibited only mild transient illness. Mutant toxin 135 retained reactivity with monoclonal antibody 8-5-7 and by several criteria was conformationally intact. Toxin from a double mutant, 141.144, with alanine substitutions at residues 141 (histidine) and 144 (tyrosine), also was devoid of mitogenic activity. In this case, antibody recognition was lost. Mutant toxins 115 and 141 were found to possess approximately half-maximal mitogenic activity. Rabbits challenged with S. aureus RN4220 expressing either 115 or 141 toxin succumbed to lethal shock. We conclude that the ability of TSST-1 to activate murine T cells in vitro and its expression of toxicity leading to lethal shock in rabbits are related phenomena.     

Heterogeneity of group A streptococcal pyrogenic exotoxin type B.

Streptococcal pyrogenic exotoxin type B purified from culture filtrates of either the NY-5 or T-19 strain of group A streptococcus was found to be heterogeneous in charge. Three protein fractions with isoelectric points of 8.0, 8.4, and 9.0 were isolated by differential solubility in ethanol and acetate-buffered saline followed by isoelectric focusing and shown to be antigenically identical to streptococcal pyrogenic exotoxin type B. The molecular weights of all three fractions were approximately 17,500, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, with aggregates forming in the presence of hyaluronic acid. Only the pI 8.4 fraction showed the characteristic activities of streptococcal pyrogenic exotoxin in rabbits: pyrogenicity and ability to enhance susceptibility to lethal endotoxin shock. The pI 8.0 and pI 9.0 fractions were not pyrogenic, but could be used to immunize against pyrogenicity. These two fractions failed either to enhance lethal endotoxin shock or to immunize against enhancement activity. When the isolated fractions were electrofocused again they appeared heterogeneous, suggesting an instability of the B toxin molecular forms.     

Production and characterization of monoclonal antibodies to exotoxin A from Pseudomonas aeruginosa

Hybridomas secreting monoclonal antibodies specific for exotoxin A from Pseudomonas aeruginosa strain PA103 were derived from the fusion of spleen cells from mice immunized with: (i) purified exotoxin A, (ii) Formalin-treated exotoxin A, (iii) exotoxin A covalently coupled to Sepharose 4B, or (iv) P. aeruginosa-infected mice. All hybridomas were screened and selected by using an enzyme-linked immuno-adsorbent assay. All antibody isotypes were represented (immunoglobulins G, A, and M) as determined by enzyme-linked immunoadsorbent assay. The most productive fusions resulted from immunization with antigens coupled to an insoluble matrix, such as Sepharose 4B, or by infection of mice. Several hybridomas were selected and cloned by limiting dilution. The specificity of the monoclonal antibodies for exotoxin A was demonstrated by indirect immunoprecipitation of 125I-labeled exotoxin A followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis and by the immunoblotting technique. The protective ability of certain monoclonal antibodies was demonstrated in vitro by toxin neutralization in tissue culture and in vivo by prolonged survival time in the burned mouse infection model, after passive immunization. One monoclonal antitoxin displayed specificity for PA103-derived exotoxin yet failed to react with exotoxin purified from PAO-PR1 or PAO1, suggesting that structural differences exist between these exotoxins.     

Purification and characterization of group A streptococcal pyrogenic exotoxin type C.

Group A streptococcal pyrogenic exotoxin (SPE) type C was partially purified by differential solubility in ethanol and acetate-buffered saline. Toxin prepared in this way consisted of protein and hyaluronic acid. After removal of hyaluronic acid, the toxin remained pyrogenic, enhanced susceptibility of rabbits to letahl endotoxin shock, was stable when treated with acid, base, or pepsin, but was inactivated by heat. Toxin further purified by thin-layer isoelectric focusing was pyrogenic and enhanced the susceptibility of rabbits to lethal endotoxin shock. Purified type C toxin appeared homogeneous when tested by Ouchterlony immunodiffusion and migrated as a single protein band in isoelectric focusing polyacrylamide gels (isoelectric point, 6.7) and sodium dodecyl sulfate-polyacrylamide gels (molecular weight, 13,200). The purified toxin was antigenically distinct from A and B SPE, and antisera raised against the purified toxin neutralized pyrogenic activity. The amino acid composition was determined.     

Survey of Staphylococcal Enterotoxin Genes, Exfoliative Toxin Genes, and Toxic Shock Syndrome Toxin 1 Gene in Non-Staphylococcus aureus Species

The aim of this study was to analyze the presence of staphylococcal exotoxin genes in staphylococci other than Staphylococcus aureus. Whereas for Staphylococcus aureus a large spectrum of different exotoxins responsible for toxin-mediated diseases has been described, only few and conflicting data are available regarding toxin production in all other staphylococcal species. A collection of clinical non-Staphylococcus aureus staphylococcal isolates were systematically screened for the presence of genes for toxic shock syndrome toxin 1, exfoliative toxins, and classical enterotoxins by using multiplex polymerase chain reaction enzyme immunoassays. In a total of 461 isolates of 18 different (sub)species derived from clinical specimens, no toxin gene sequences were amplified. The results indicate that the occurrence of toxin genes involved in staphylococcal toxin-mediated diseases is at least very rare in human-derived isolates of non-Staphylococcus aureus species.     

Expression of the cloned toxic shock syndrome toxin 1 gene (tst) in vivo with a rabbit uterine model.

Toxic shock syndrome (TSS) toxin 1 (TSST1) is produced by strains of Staphylococcus aureus associated with TSS. Purified TSST1 induces in rabbits a shock-like illness with many features similar to TSS in humans. These symptoms were also induced by TSST1-producing bacteria in diffusion chambers implanted in the rabbit uterus. Naturally occurring TSST1+ strains and a TSST1- strain harboring a pE194-derived plasmid carrying the cloned TSST1 determinant tst gave the same symptoms. TSST1- strains and a TSST1- strain carrying a pE194-tst plasmid with a deletion of the tst gene had no effect in rabbits. The results with the plasmid-carrying TSST1+ and TSST1- strains, which were isogenic apart from tst, show that the toxin is responsible for the illness in rabbits and suggest that it is a major factor in the pathogenesis of TSS.     

Purification of staphylococcal exfoliative toxin by high pressure liquid chromatography

Exfoliative toxin (ET) isolated from a clinical strain of Staphylococcus aureus was purified to homogeneity, using a 3-step HPLC system. NH2-terminal 20 amino residues of purified ET was found to be identical with ETA of S. aureus TA (7), S. aureus TC16 (9) and S. aureus ZM (10), but stability of purified ET was completely different from that of ETA. This purification system gave a high yield of pure ET, which exhibited higher purity than specimens purified by more complicated and time-consuming procedures. It is useful for small-scale purification for the comparative study of ET and easy to scale up for preparative purification.     

Natural phosphorylation of group A streptococcal pyrogenic exotoxin type C.

Group A streptococcal pyrogenic exotoxin (SPE) type C, produced by strain T18P grown in the presence of 32P, was separated from culture supernatant fluids by using alcohol precipitation. The resulting toxin (EtOH-1) contained 3 X 10(6) to 5 X 10(6) cpm of 32P per milligram of protein. The radiolabel migrated with SPE C during isoelectric focusing in polyacrylamide gels (pI 6.7) and double immunodiffusion, in which the toxin formed a line of identity with highly purified SPE C when reacted with hyperimmune antisera raised against SPE C. The EtOH-1 radiolabeled toxin was pyrogenic and had the capacity to enhance host susceptibility to lethal endotoxin shock. EtOH-1 toxin lost both radiolabel and biological activity after being treated with alkaline phosphatase. The nonspecific lymphocyte mitogenicity of purified unlabeled SPE C was stimulated by adenosine monophosphate but not adenosine, adenosine diphosphate, or adenosine triphosphate. Adenosine monophosphate may function as a cofactor of SPE C and contribute the phosphate group required for biological activity.     

Purification and characterization of an immunologically distinct delta-hemolysin from a canine strain of Staphylococcus aureus.

Culture supernatants of 26 strains of Staphylococcus aureus possessing delta-hemolytic activity have been tested by immunodiffusion against a serum raised against purified delta-hemolysin from S. aureus CN 4108 (Newman D2). Supernatants from 14 strains of canine origin gave a reaction of partial identity with delta-hemolysin from CN 4108, whereas supernatants from all other strains had full identity. Delta hemolysin from one of these canine strains, CN 7450, was purified by ammonium sulfate fractionation and precipitation at pH 4.5. The physical, chemical, and biological properties of this toxin were compared with those of delta-hemolysin from CN 4108. Differences in molecular weight (as judged by Sepharose 6B chromatography), isoelectric point, and amino acid composition were found. Both toxins caused dermonecrosis in rabbits, lysed erythrocytes from several different species, and were inhibited by normal sera and phospholipids. Unlike delta-hemolysin from CN 4108, the hemolytic activity of delta-hemolysin from CN 7450 was found to be dependent on the incubation temperature over the range of 25 to 37 degrees C. Immunodiffusion results obtained with antisera raised against purified delta-hemolysin from CN 7450 indicated that delta-hemolysins from the canine strains were probably identical and confirmed that these differ immunologically from delta-hemolysin from CN 4108.     

Activation of murine T-suppressor lymphocytes by group A streptococcal and staphylococcal pyurogenic exotoxins.

The effect of group A streptococcal pyrogenic exotoxin (PE) type C and staphylococcal PE on the in vitro antibody response to sheep erythrocytes was studied in cultures of mouse spleen cells. Both exotoxins suppressed the day 4 direct plaque-forming cell response when added to the cultures. The maximum suppression was obtained with 1.0 or 0.1 ng of toxin per culture, and the suppressive effect was reversed by addition of gangliosides to the cultures at the same time as the exotoxins. Preincubation of T lymphocytes for 4 days with either exotoxin resulted in the generation of a suppressor cell population, which produced dose-dependent suppression of the direct plaque-forming cell response when added to fresh sheep eyrthrocyte-activated splenocytes. The suppression obtained was not reversed by gangliosides indicating toxin carry-over was not responsible for the effect. B cells, preincubated with exotoxin, failed to suppress the direct plaque-forming cell response of fresh erythrocyte-activated spleen cells.     

Immunologic cross-reactivity of type A streptococcal exotoxin (erythrogenic toxin) and staphylococcal enterotoxins B and C1.

Immunologic cross-reactivity between Streptococcus pyogenes type A exotoxin (erythrogenic toxin) and Staphylococcus aureus enterotoxins B and C1 was demonstrated by Ouchterlony double diffusion, Western immunoblot, and immunodot analyses. Specific antiserum to type A streptococcal exotoxin reacted more strongly with staphylococcal enterotoxin B than with enterotoxin C1. The reactivity of type A streptococcal exotoxin with antiserum to staphylococcal enterotoxin B was greater than that of antiserum to enterotoxin C1. These results suggest that a conserved domain is present in the three exotoxins, which most likely originated from a common evolutionary ancestor.     

Further purification of group A streptococcal pyrogenic exotoxin and characterization of the purified toxin.

Streptococcal pyrogenic exotoxin (SPE) isolated from culture filtrates of strain NY-5 (type 10), and separated from other extracellular by differential solubility in ethanol and acetate-buffered saline, has previously been shown to exhibit a wide range of biological activities including erythrogenic activity, pyrogenicity, enhancement of susceptibility to endotoxin shock, blockage of the reticuloendothelial system immmunosuppression, and lymphocyte mitogenicity. Toxin prepared in this way was found to consist of hyaluronic acid and several proteins which could be distinguished by thin-layer polyacrylamide isoelectric focusing (IEF), SPE has been further purified by ion exchange chromatography on QAE-Sephadex columns. One of the fractions isolated from QAE-Sephadex, and shown to be a homogenous protein by thin-layer IEF and Ouchterlony with hyperimmune serum, was highly active erythrogenically, pyrogenically, and in enhancing susceptibility to endotoxin. This fraction was identified as exotoxin A. A second, less active fraction identified as SPE B showed similar activities, but differed from the other fraction antigenically and in net charge and molecular weight. These findings indicate that a single highly purified protein can mediate at least three of the biological activities attributed to SPE and NY-5 produces pyrogenic exotoxins A and B in vitro as well as in vivo.     

Purification and some properties of a non-o1 Vibrio cholerae enterotoxin that is identical to cholera enterotoxin.

Cholera-like enterotoxin was isolated and purified from the culture supernatant of a non-O1 strain of Vibrio cholerae, E8498, isolated from the environment. Enterotoxin was purified by aluminum hydroxide absorption and elution and successive gel filtrations on Sephadex G-100, Bio-Gel A-5m, and Sephadex G-75. Purified enterotoxin gave a single stained band on polyacrylamide gel disc electrophoresis, and the mobility was the same as that of cholera enterotoxin. The specific biological activity of the purified enterotoxin was almost the same as that of cholera enterotoxin in the Chinese hamster ovary cell assay, fluid accumulation in mouse ligated intestine, increase in vascular permeability in rabbit skin, and passive immune hemolysis. Sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis showed that the purified enterotoxin consisted of subunits A and B, identical to those of cholera enterotoxin, and Ouchterlony double gel diffusion tests indicated that the two toxins were immunologically identical. Enterotoxins prepared from several non-O1 strains isolated from human patients were also immunologically identical to cholera enterotoxin.     

Fluid replacement protection of rabbits challenged subcutaneous with toxic shock syndrome toxins.

Toxic shock syndrome toxin 1 (TSST-1) and streptococcal pyrogenic exotoxin A (SPE A) belong to a family of pyrogenic toxins produced by Staphylococcus aureus and Streptococcus pyogenes, respectively. Both toxins are responsible for causing toxic shock syndrome (TSS) and related illnesses, clinically characterized by multiorgan involvement. The most severe TSS symptom is acute hypotension and shock after the initial febrile response. In this study, we examined possible mechanisms of shock development in TSS, particularly the role of T-cell proliferation, endotoxin enhancement by toxins, and capillary leakage. American Dutch belted rabbits, with subcutaneously implanted miniosmotic pumps filled with either TSST-1 or SPE A, served as the animal model. For both TSST-1 and SPE A-treated rabbits, administration of cyclosporin A prevented toxin-induced T-cell proliferation but failed to protect the rabbits. Polymyxin B treatment of rabbits, to neutralize endogenous endotoxin, partially protected rabbits from challenge with either exotoxin; two of six rabbits survived on day 2 when treated with only TSST-1, whereas six of six animals survived after challenge with TSST-1 and polymyxin B. Similarly, with SPE A-treated rabbits, only 1 of 10 animals without polymyxin B treatment survived on day 8, but 4 of 6 rabbits survived on day 8 when given polymyxin B. Fluid replacement was successful in preventing lethality. Twelve of 14 rabbits survived when given TSST-1 with fluid, and all rabbits treated with SPE A and fluid survived. Finally, by using miniosmotic pumps, staphylococcal exfoliative toxin A and concanavalin A were administered to rabbits in an attempt to induce lethality. These two T-cell mitogens caused T-cell proliferation but failed to induce lethality in rabbits. The data suggest that toxin interactions causing vascular leakage and to some extent endotoxin enhancement are of major importance in development of hypotension and shock in TSS. It appears that T-cell proliferation may not contribute significantly to the induction of shock and death.     

Purification and Characterization of Pseudomonas aeruginosa Exotoxin

A trypsin-sensitive, heat-labile exotoxin of Pseudomonas aeruginosa strain P-A-103 has been purified by a procedure that includes membrane ultrafiltration, hydroxylapatite chromatography, ion-exchange cellulose chromatography, and gel filtration chromatography. The procedure resulted in the recovery of 48% of the exotoxin with a 40-fold increase in specific activity (micrograms of protein per median lethal dose [LD(50)]). The mean lethal dose of the purified toxin administered intravenously into mice weighing 20 g was approximately 6 mug of protein. The toxin contained virtually no nucleic acid, detectable pigment, or lipopolysaccharide. When subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis, the toxin separated into at least six protein components which appeared to have similar molecular weights. The estimated molecular weight of the toxin is 54,000, and its isoelectric point is 5.0     

Purification and characterization of different types of exfoliative toxin from Staphylococcus aureus.

Exfoliative toxin was isolated from strain DI of Staphylococcus aureus using carboxymethyl cellulose and hydroxylapatite chromatography. This purified toxin was compared with that produced by strain TA. The specific biological activity of the two toxins was the same, but they were serologically distinct. These strains have different loci (plasmid or chromosomal) for toxin production; differences were seen in molecular weight and amino acid composition. N-terminal amino acid sequences from the two strains showed significant homology using a single unit alignment shift.