INFECTION AND IMMUNITY, Sept. 1976, p. 767-775 Copyright ©D 1976 American Society for Microbiology
Vol. 14, No. 3 Printed in U.S.A.
Further Purification of Group A Streptococcal Pyrogenic Exotoxin and Characterization of the Purified Toxin CAROLYN M. CUNNINGHAM, EDWARD L. BARSUMIAN, AND DENNIS W. WATSON*
Department of Microbiology, Medical School, University of Minnesota, Minneapolis, Minnesota 55455
Received for publication 21 May 1976
Streptococcal pyrogenic exotoxin (SPE) isolated from culture filtrates of strain NY-5 (type 10), and separated from other extracellular enzymes 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, immunosuppression, 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 homogeneous 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 that NY5 produces pyrogenic exotoxins A and B in vitro as well as in vivo.
Streptococcal pyrogenic exotoxins (SPE) are extracellular proteins produced by a number of strains of group A streptococci growing in vivo or in vitro. Three antigenically distinct pyrogenic exotoxins have been described and designated SPE A, B, and C (7, 16). These toxins, in addition to their pyrogenic activity in rabbits, are associated with scarlet fever erythrogenic toxins and can induce erythema when injected intradermally in humans and sensitized animals (16). A number of other manifestations of the toxicity of SPE have been described, including enhancement of susceptibility to lethal endotoxin shock in rabbits, mice, and monkeys (9), cytotoxicity for splenic macrophages (9) and blockage of reticuloendothelial clearance of colloidal carbon in rabbits (4), alteration of the antibody response to sheep erythrocytes in rabbits and mice (3, 5, 6), and mitogenic activity for human and rabbit lymphocytes (8; E. L. Barsumian and D. W. Watson, unpublished observations). The production of SPE in vivo by all group A streptococci tested and their striking effects on several aspects of the immune system suggest a role for these toxins in streptococcal infections and their nonsuppurative sequelae. The study of streptococcal toxins is complicated by the large number of products of group
A streptococci that exhibit various biological activities. For this reason the use of highly purified SPE is necessary before the activities attributed to SPE can be assigned with certainty to a single- streptococcal extracellular product. Most of the earlier work on the activities of SPE was done using toxin purified from culture filtrates of NY-5 (type 10) by differential solubility in ethanol and acetate-buffered saline. Whereas this material was free of many other streptococcal enzymatic activities (9), it has been shown by thin-layer isoelectric focusing (IEF) and double immunodiffusion in agar to consist of several different proteins. The present report describes the further purification of SPE by ion exchange chromatography and characterization of the active fractions chemically and biologically. (Results of this study' were presented in part at the 76th Annual Meeting of the American Society for Microbiology, Atlantic City, N.J., 27 May 1976.) MATERIALS AND METHODS All glassware and reagents used for purification and biological assays of the toxin were maintained pyrogen-free. Production of SPE. The NY-5 (type 10) strain of group A hemolytic streptococcus (Streptococcus py-
767
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CUNNINGHAM, BARSUMIAN, AND WATSON
ogenes), maintained as described previously (9), was used to produce SPE. To make SPE, autoclaved dialyzed beef heart medium (16) was inoculated with 5%, by volume, of an exponentially growing culture of NY-5. The cultures were incubated with stirring at 37°C for 5 to 6 h until the end of logarithmic growth. The culture supernatant was collected on ice by continuous-flow centrifugation using a Beckman model J-21 centrifuge (Beckman Instruments, Inc., Fullerton, Calif.) and filtered through a 0.22Am sterile membrane filter (Millipore Corp., Bedford, Mass.). The toxin was partially purified from cell-free culture filtrates by differential solubility in ethanol and acetate-buffered saline. This procedure has been described in detail (9). Toxin which had been precipitated two times was dialyzed and lyophilized. This material, referred to as NY-5 EtOH-2, was used for further purification by ion exchange chromatography. Ion exchange chromatography. QAE-Sephadex A-50 (Pharmacia Fine Chemicals, Uppsala, Sweden) in jacketed columns (2.6 by 40 cm) was used for separation of protein components of the reprecipitated toxin. The buffers used sequentially were: (i) imidazole-acetic acid, 0.1 M (pH 7.0); (ii) imidazoleacetic acid, 0.1 M (pH 5.0) plus 0.1 M NaCl; (iii) sodium acetate-acetic acid, 0.1 M (pH 4.0) plus 1 M NaCl. A 50- to 60-mg portion of NY-5 EtOH-2 was loaded on the column in 10 to 15 ml of the first buffer. At least two column volumes of each buffer were used for elution. Absorbance at 280 nm was measured and recorded (Uvicord, LKB-Produkter, Stockholm, Sweden) as 5-ml fractions were collected in a refrigerated fraction collector. Peaks of absorbance were combined, dialyzed against double-distilled water, and lyophilized. Physicochemical analyses. Hyaluronic acid content of the toxin and fractions was determined by the carbazole reaction (2). After hyaluronidase (Nutritional Biochemicals Corp., Cleveland, Ohio) treatment of the toxin, hyaluronic acid was assayed by increased turbidity with an acid albumin solution (17). Amino acid compositions were determined in a Beckman 120B automatic analyzer after hydrolyzing the toxin in 6 N HCl at 100°C in vacuum-sealed vials. The molecular weights of NY-5 EtOH-2 and the QAE fractions at concentrations of 0.2 to 1.0 mg/ ml in phosphate-buffered saline were determined by the high-speed sedimentation equilibrium meniscus depletion method of Yphantis (18). Amino acid compositions were used to estimate the partial specific volumes of the proteins (13). Thin-layer polyacrylamide gel IEF using pH 3.5 to 10 ampholyte solution (Ampholine; LKB-Produkter, Stockholm, Sweden) was carried out using the LKB Multiphor apparatus. Isoelectric points were determined by comparing the position of stained protein bands to the pH gradient determined by measuring the pH of eluted portions of the gel. NY-5 EtOH-2 preparations were also tested for proteinase activity (10), ribonuclease activity (15), and the presence of rhamnose (1), an indication of the group-specific carbohydrate. Biological assays. The solutions of SPE were pre-
INFECT. IMMUN.
pared in sterile pyrogen-free phosphate-buffered saline (0.005 M phosphate buffer, pH 7.0, plus 0.15 M NaCl) for all biological assays. American Dutch rabbits, 1.0 to 1.5 kg, were used to determine pyrogenicity, lethality within 48 h, and enhanced susceptibility to endotoxin shock (9). Pyrogenicity was quantitated as the minimal pyrogenic dose causing a 0.55°C (1°F) (MPD-4) rise of temperature 4 h after intravenous injection as described previously (9). Enhanced susceptibility to endotoxin shock was measured as the mean lethal dose of SPE when 25 Lg of Salmonella typhimurium lipopolysaccharide per kg was given 5 h after the SPE. Old New Zealand rabbits were used to test skin reactivity at 24 h to intradermally injected toxin preparations. Immunizations. Pyrogenic immunity to SPE was developed as described previously (9). For hyperimmunization, American Dutch rabbits were given a mixture of 50 ,l. of SPE (2 to 4 mg/ml) and 50 ul of Freund incomplete adjuvant subcutaneously on days 1, 14, 28, and 42 and every 4 weeks thereafter. Rabbits were bled by cardiac puncture 1 week after booster injections, and the serum was used for double immunodiffusion in agar. RESULTS
Composition of partially purified SPE. SPE made from culture filtrates of NY-5 by two consecutive precipitations (EtOH-2) consisted of protein and hyaluronic acid. The hyaluronic acid content varied between different cultures of NY-5 from 23 to 43% by weight. Removal of the hyaluronic acid by hyaluronidase treatment of the toxin did not alter its pyrogenic activity (Fig. 1). The partially purified material
1.5.~o 14~r W.
/
G.s HOURS
FIG. 1. Effect of hyaluronidase treatment on fever response in rabbits to NY-5 EtOH-2. Samples of NY5 EtOH-2 (10 pg/ml), NY-5 EtOH-2 (10 pgIml) plus hyaluronidase (1 pg/ml), and hyaluronidase (1 pgl ml) were incubated 20 min at 37°C. Five rabbits in each group received 1 ml/kg at time 0. The untreated NY-5 EtOH-2 contained 25% hyaluronic acid, and the enzyme-treated NY-5 EtOH-2 contained 1 % hyaluronic acid. Symbols: (0) NY-5 EtOH-2; (O) NY-5 EtOH-2 hyaluronidase treated; (A) hyaluronidase.
VOL. 14, 1976
PURIFICATION OF STREPTOCOCCAL EXOTOXIN
from NY-5, previously shown to be free of mucopeptide, streptolysin 0, streptolysin S, nicotinamide adenine dinucleotide nucleosidase, and deoxyribonuclease (9), was also found to contain no detectable proteinase, ribonuclease, or rhamnose. Thin-layer polyacrylamide gel IEF of the EtOH-2 material revealed the presence of several proteins with different charge properties, as shown in Fig. 2. Neither the removal of the hyaluronic acid by hyaluronidase treatment nor continuation of purification to six precipitations altered the pattern found with IEF. Purification by ion exchange chromatography. The availability of pyrogen-free ion exchange gels has ma'de possible the further separation of the protein components of NY-5 EtOH-2. (Preliminary investigation of the use of QAE-Sephadex to separate the protein fractions of SPE was carried out by K. W. Brunson [Ph.D. thesis, Univ. of Minnesota, Minneapolis, 1973].) The method was developed using the toxin produced by NY-5 but has been applied
769
successfully to toxins of other strains. Several types of column chromatography were used in initial attempts at fractionation. It was found that mild anion exchange using Ecteola-cellulose (pH 7) or mild cation exchange using carboxymethyl-cellulose (pH 4-7) resulted in separation of the protein from the hyaluronic acid, indicating that the two moieties were not covalently linked. However, the various protein fractions were separated only when a stronger anion exchange gel, QAE-Sephadex, was used. The results of a typical QAE-Sephadex column are shown in Fig. 3. The physicochemical properties of the four fractions isolated by this method are given in Table 1 and the IEF patterns in Fig. 2. Fraction 2 shows a degree of homogeneity in IEF which is comparable to that obtained with recrystallized proteins. Fractions 1 and 3, however, appear to contain minor contaminating bands. The results of double immunodiffusion in agar using antisera raised in rabbits against NY-5 EtOH-2 indicate that fractions 2 and 3 are antigenically identi-
pH 9 8 7 6 5 4 N Y-S
fl
f2
f3
FIG. 2. Thin-layer polyacrylamide gel isoelectric focusing ofNY-5 EtOH-2 and QAE-Sephadex fractions 1, 2, and 3. Stained with Coomassie blue.
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CUNNINGHAM, BARSUMIAN, AND WATSON
TABLE 1. Physicochemical properties ofNY-5 EtOH2 and its QAE-Sephadex fractions Fraction 1 2 3 4 NY-5 EtOH-2
% Hyaluronic acid (wt/wt) 4 4 8 85 25
Isoelectric point of proteins 8.5-9.5 4.5-5.5 4.5-5.5 _b
Mol wta
21,900 8,000 5,500
NDC
21,000
Partial specific volumes determined to be 0.07 (13). b-, Not detected. ND, Not determined.
a c
1.0
cal and that fraction 1 is distinct from the others (Fig. 4). Amino acid analyses of the fractions revealed a surprising similarity in amino acid composition of the fractions with the previously reported composition of NY-5 EtOH-2 (9) and with each other (Table 2). Fractions 1, 2, and 3 were all high in the dicarboxylic acids such as aspartic and glutamic acids and relatively low in the basic amino acids such as lysine and arginine. A large quantity of ammonia was found in the hydrolysates, suggesting that ami-
f4 hanr hnnea
.75 280 nm
-50S f2
fl
bufferI
pH7
0.1 M Imidozole-
Acetic Acid
buffter 2 pH 5 O.IM Imidozole-Acetic Acid 0.1 M Na CI
f3
buffer 3
pH4
O.IM Sodium Acetate-Acetic Acid
IM NoCd
FIG. 3. Fractionation of NY-5 EtOH-2 on QAE-Sephadex A-50.
FIG. 4. Double immunodiffusion in Noble agar. Center well contains antiserum to NY-5 EtOH-2. fl, t2, 3, are QAE-Sephadex fractions of NY-5 EtOH-2. NY-5 is NY-5 EtOH-2.
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PURIFICATION OF STREPTOCOCCAL EXOTOXIN
771
TABLE 2. Amino acid composition of NY-5 EtOH-2 and its QAE-Sephadex fractions Amino acid
NY-5 EtOH-2
Fraction 1
Fraction 2
Fraction 3
Cysteic acid Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Half-cystine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Lysine Histidine Arginine Total amino acids
None 17.08a 3.94 9.20 16.42 3.28 11.99 7.88 1.97 2.96 0.98 1.48 4.10 2.96 2.79 8.54 1.64 2.79 100
None 13.80 3.39 8.15 8.57 6.90 10.47 6.90 0.36 3.33 None 2.62 4.70 8.81 11.60 5.23 2.14 3.03 100
None
3.23 18.22 5.03 8.62 15.17 4.94 10.59 5.12 2.96 2.87 None 2.06 4.04 1.80 2.42 7.54 2.15 3.23 100
23.42 2.38 None None
34.07 6.66 None None
Ammonia 20.29 Glucosamine 11.74 Diaminopimelic acid None Muramic acid None a Moles per 100 moles of total amino acids.
dation of the dicarboxylic acids might contribute to the high isoelectric point of fraction 1. Biological activities of the purified fractions. The QAE fraction of NY-5 EtOH-2 was tested in rabbits for pyrogenicity, skin test reactivity, and ability to enhance the susceptibility to lethal endotoxin shock. Fraction 2 showed the greatest activity by weight in each assay, whereas fraction 4, which was mainly hyaluronic acid, showed minimal activity (Table 3). Rabbits can be made immune to the pyrogenic effects of the toxin by injection of small doses (10 MPD-4) of toxin every other day for 2 weeks. Rabbits immunized in this way with fraction 1 showed no cross immunity to fractions 2 or 3, whereas rabbits immunized with fraction 2 were immune to fraction 3 but not to fraction 1 (Fig. 5). Identification of the fractions. It appears that fractions 2 and 3 are both exotoxin A, because they are eluted from QAE-Sephadex in the same fractions as the SPE of T-28, a strain that produces only exotoxin A (Fig. 6). In addition, fractions 2 and 3 react with a commercial antitoxin A (American Cyanamid Co., Lederle Laboratories Div., Pearl River, N.Y.), whereas fraction 1 does not (Fig. 7). Pyrogen cross testing of fractions 2 and 3 in T-28 EtOH-2 immune rabbits gave inconclusive results, possibly because complete pyrogenic immunity to the T-28 toxin was difficult to achieve.
16.20 6.34 7.57 16.11 5.37 8.10 4.49 1.85 3.79
None 2.55 6.43 4.23 4.14 7.57 2.73 2.55 100
16.99 10.59
None None
TABLE 3. Biological activities of NY-5 EtOH-2 and its QAE-Sephade-x fractions Fraction
Pyrogenicity MPD-4 (ILg/ kg)a
Enhanced susceptibility to endotoxin LD50
STD
(g)
( tg/kg)b
1 2 3 4
NY-5 EtOH-2
3.2 0.02 11.2 46.4 0.3
25 14 15 63 5
0.1 0.1 0.1 1 0.1
a MPD-4, Minimal pyrogenic dose causing a 0.55°C (1°F) rise of temperature 4 h after intravenous injection. b Mean lethal dose (LDN) of SPE with 25 ,ug of S. typhimurium lipopolysaccharide (LPS) per kg given 5 h later. The LD50 of NY-5 EtOH-2 alone was 2,500 The LD50 of LPS alone was 535 ,ug/kg. Ag/kg. c STD, Intradermal dose of SPE required for a skin reaction of an area greater than 6 by 6 mm at 24
to 48 h.
Fraction 1 has an IEF pattern like the exotoxin B produced by strain T-19 (Fig. 8), and antiserum prepared against fraction 1 will react with T-19 EtOH-2, forming a line of identity (Fig. 9). In pyrogen cross testing, rabbits immunized with T-19 EtOH-2 had significantly less fever at 4 h than control rabbits when injected with fraction 1, and rabbits immunized with fraction 1 showed significant immunity to
772
CUNNINGHAM, BARSUMIAN, AND WATSON
the corresponding QAE fraction of T-19 (Fig. 19).
DISCUSSION SPE prepared from NY-5 culture filtrates by alcohol precipitation has in the present study been separated by ion exchange chromatography on QAE-Sephadex into three protein fractions with the characteristic biological activities of SPE and a fourth carbohydrate-rich fraction that is relatively inert. Although this method of purification separates the toxin into several distinct components, the yield of the active fractions per column is rather low. Preliminary experiments suggest that prior treatment of the toxin with hyaluronidase may increase the efficiency of the ion exchange procedure. Affinity chromatography using antisera made monospecific by absorption with the appropriate purified QAE fractions and thin-layer IEF in Sephadex gels (12) are also being explored as methods of purification. Although NY-5 ethanol-precipitated SPE had previously been identified as exotoxin A (9), the three fractions of NY-5 EtOH-2 from QAE-Sephadex were found to represent two immunologically distinct toxins, as shown both by immunodiffusion and pyrogen cross testing. However, in vivo the NY-5 strain produces exotoxin B as well as exotoxin A (7, 16), and the high activity of fraction 2 may have obscured the other activity in the partially purified material. We have identified both fraction 2 and fraction 3 as exotoxin A. Whereas these fractions are eluted from QAE-Sephadex with the same buffer and seem to be identical by IEF and
INFECT. IMMUN.
immunodiffusion, they differ in hyaluronic acid content and in molecular weight. Since fraction 3 has a lower molecule weight than fraction 2 SPE Fo, Im...ilatio.
SPE Tesed
(1o MPO-4 /kg)
FEVER AT 4 HOURS A c O5 O
°r
NSY- 5 Frectlon 2
Ny- 5 F,oction
NIY- 5
NY-5 F"Pction
NY-5 Froct,oo 3
NY-5
NY- 5 Froction
F,octio. 2
F,actio.
It L_
A SPE For Im....iZatio.
SPE Tesled 0 0MPD -4 /kg
FEVER AT 4 HOURS *C 0I
05
NY-S F,octioo 2
NY-5
NY-5 FAoctAo 2
NY- 5 FACAtA0 S
NY-5 FRaction 2
NY-S F,act.on 2
B FIG. 5. Pyrogen cross tests of QAE fractions of NY-5 EtOH-2. All immunizing and challenge doses of SPE were 10 minimal pyrogenic doses per kg. (A) Fraction 1 of NY-5 used as SPE for immunization. (B) Fraction 2 of NY-5 used as SPE for immunization. Bars indicate ± standard error of the mean.
obsorbonce 280 nm
0
I buffer pH 7 01M Imidazole-
buffer 2
Acetic Acid
OIM NaCI
pH
5
FRactio-
1.0. .25
10
NY-5 Froetion
.75 .50
5
5
01 M Imidazole -
FIG. 6. Fractionation of T-28 EtOH-2
I
buffer 3 pH 4 01 M Sodum Acetote-Acetic Acid M NoCI
on
QAE-Sephadex A-50.
Vol. 14, No. 3 Printed in U.S.A.
Further Purification of Group A Streptococcal Pyrogenic Exotoxin and Characterization of the Purified Toxin CAROLYN M. CUNNINGHAM, EDWARD L. BARSUMIAN, AND DENNIS W. WATSON*
Department of Microbiology, Medical School, University of Minnesota, Minneapolis, Minnesota 55455
Received for publication 21 May 1976
Streptococcal pyrogenic exotoxin (SPE) isolated from culture filtrates of strain NY-5 (type 10), and separated from other extracellular enzymes 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, immunosuppression, 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 homogeneous 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 that NY5 produces pyrogenic exotoxins A and B in vitro as well as in vivo.
Streptococcal pyrogenic exotoxins (SPE) are extracellular proteins produced by a number of strains of group A streptococci growing in vivo or in vitro. Three antigenically distinct pyrogenic exotoxins have been described and designated SPE A, B, and C (7, 16). These toxins, in addition to their pyrogenic activity in rabbits, are associated with scarlet fever erythrogenic toxins and can induce erythema when injected intradermally in humans and sensitized animals (16). A number of other manifestations of the toxicity of SPE have been described, including enhancement of susceptibility to lethal endotoxin shock in rabbits, mice, and monkeys (9), cytotoxicity for splenic macrophages (9) and blockage of reticuloendothelial clearance of colloidal carbon in rabbits (4), alteration of the antibody response to sheep erythrocytes in rabbits and mice (3, 5, 6), and mitogenic activity for human and rabbit lymphocytes (8; E. L. Barsumian and D. W. Watson, unpublished observations). The production of SPE in vivo by all group A streptococci tested and their striking effects on several aspects of the immune system suggest a role for these toxins in streptococcal infections and their nonsuppurative sequelae. The study of streptococcal toxins is complicated by the large number of products of group
A streptococci that exhibit various biological activities. For this reason the use of highly purified SPE is necessary before the activities attributed to SPE can be assigned with certainty to a single- streptococcal extracellular product. Most of the earlier work on the activities of SPE was done using toxin purified from culture filtrates of NY-5 (type 10) by differential solubility in ethanol and acetate-buffered saline. Whereas this material was free of many other streptococcal enzymatic activities (9), it has been shown by thin-layer isoelectric focusing (IEF) and double immunodiffusion in agar to consist of several different proteins. The present report describes the further purification of SPE by ion exchange chromatography and characterization of the active fractions chemically and biologically. (Results of this study' were presented in part at the 76th Annual Meeting of the American Society for Microbiology, Atlantic City, N.J., 27 May 1976.) MATERIALS AND METHODS All glassware and reagents used for purification and biological assays of the toxin were maintained pyrogen-free. Production of SPE. The NY-5 (type 10) strain of group A hemolytic streptococcus (Streptococcus py-
767
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CUNNINGHAM, BARSUMIAN, AND WATSON
ogenes), maintained as described previously (9), was used to produce SPE. To make SPE, autoclaved dialyzed beef heart medium (16) was inoculated with 5%, by volume, of an exponentially growing culture of NY-5. The cultures were incubated with stirring at 37°C for 5 to 6 h until the end of logarithmic growth. The culture supernatant was collected on ice by continuous-flow centrifugation using a Beckman model J-21 centrifuge (Beckman Instruments, Inc., Fullerton, Calif.) and filtered through a 0.22Am sterile membrane filter (Millipore Corp., Bedford, Mass.). The toxin was partially purified from cell-free culture filtrates by differential solubility in ethanol and acetate-buffered saline. This procedure has been described in detail (9). Toxin which had been precipitated two times was dialyzed and lyophilized. This material, referred to as NY-5 EtOH-2, was used for further purification by ion exchange chromatography. Ion exchange chromatography. QAE-Sephadex A-50 (Pharmacia Fine Chemicals, Uppsala, Sweden) in jacketed columns (2.6 by 40 cm) was used for separation of protein components of the reprecipitated toxin. The buffers used sequentially were: (i) imidazole-acetic acid, 0.1 M (pH 7.0); (ii) imidazoleacetic acid, 0.1 M (pH 5.0) plus 0.1 M NaCl; (iii) sodium acetate-acetic acid, 0.1 M (pH 4.0) plus 1 M NaCl. A 50- to 60-mg portion of NY-5 EtOH-2 was loaded on the column in 10 to 15 ml of the first buffer. At least two column volumes of each buffer were used for elution. Absorbance at 280 nm was measured and recorded (Uvicord, LKB-Produkter, Stockholm, Sweden) as 5-ml fractions were collected in a refrigerated fraction collector. Peaks of absorbance were combined, dialyzed against double-distilled water, and lyophilized. Physicochemical analyses. Hyaluronic acid content of the toxin and fractions was determined by the carbazole reaction (2). After hyaluronidase (Nutritional Biochemicals Corp., Cleveland, Ohio) treatment of the toxin, hyaluronic acid was assayed by increased turbidity with an acid albumin solution (17). Amino acid compositions were determined in a Beckman 120B automatic analyzer after hydrolyzing the toxin in 6 N HCl at 100°C in vacuum-sealed vials. The molecular weights of NY-5 EtOH-2 and the QAE fractions at concentrations of 0.2 to 1.0 mg/ ml in phosphate-buffered saline were determined by the high-speed sedimentation equilibrium meniscus depletion method of Yphantis (18). Amino acid compositions were used to estimate the partial specific volumes of the proteins (13). Thin-layer polyacrylamide gel IEF using pH 3.5 to 10 ampholyte solution (Ampholine; LKB-Produkter, Stockholm, Sweden) was carried out using the LKB Multiphor apparatus. Isoelectric points were determined by comparing the position of stained protein bands to the pH gradient determined by measuring the pH of eluted portions of the gel. NY-5 EtOH-2 preparations were also tested for proteinase activity (10), ribonuclease activity (15), and the presence of rhamnose (1), an indication of the group-specific carbohydrate. Biological assays. The solutions of SPE were pre-
INFECT. IMMUN.
pared in sterile pyrogen-free phosphate-buffered saline (0.005 M phosphate buffer, pH 7.0, plus 0.15 M NaCl) for all biological assays. American Dutch rabbits, 1.0 to 1.5 kg, were used to determine pyrogenicity, lethality within 48 h, and enhanced susceptibility to endotoxin shock (9). Pyrogenicity was quantitated as the minimal pyrogenic dose causing a 0.55°C (1°F) (MPD-4) rise of temperature 4 h after intravenous injection as described previously (9). Enhanced susceptibility to endotoxin shock was measured as the mean lethal dose of SPE when 25 Lg of Salmonella typhimurium lipopolysaccharide per kg was given 5 h after the SPE. Old New Zealand rabbits were used to test skin reactivity at 24 h to intradermally injected toxin preparations. Immunizations. Pyrogenic immunity to SPE was developed as described previously (9). For hyperimmunization, American Dutch rabbits were given a mixture of 50 ,l. of SPE (2 to 4 mg/ml) and 50 ul of Freund incomplete adjuvant subcutaneously on days 1, 14, 28, and 42 and every 4 weeks thereafter. Rabbits were bled by cardiac puncture 1 week after booster injections, and the serum was used for double immunodiffusion in agar. RESULTS
Composition of partially purified SPE. SPE made from culture filtrates of NY-5 by two consecutive precipitations (EtOH-2) consisted of protein and hyaluronic acid. The hyaluronic acid content varied between different cultures of NY-5 from 23 to 43% by weight. Removal of the hyaluronic acid by hyaluronidase treatment of the toxin did not alter its pyrogenic activity (Fig. 1). The partially purified material
1.5.~o 14~r W.
/
G.s HOURS
FIG. 1. Effect of hyaluronidase treatment on fever response in rabbits to NY-5 EtOH-2. Samples of NY5 EtOH-2 (10 pg/ml), NY-5 EtOH-2 (10 pgIml) plus hyaluronidase (1 pg/ml), and hyaluronidase (1 pgl ml) were incubated 20 min at 37°C. Five rabbits in each group received 1 ml/kg at time 0. The untreated NY-5 EtOH-2 contained 25% hyaluronic acid, and the enzyme-treated NY-5 EtOH-2 contained 1 % hyaluronic acid. Symbols: (0) NY-5 EtOH-2; (O) NY-5 EtOH-2 hyaluronidase treated; (A) hyaluronidase.
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PURIFICATION OF STREPTOCOCCAL EXOTOXIN
from NY-5, previously shown to be free of mucopeptide, streptolysin 0, streptolysin S, nicotinamide adenine dinucleotide nucleosidase, and deoxyribonuclease (9), was also found to contain no detectable proteinase, ribonuclease, or rhamnose. Thin-layer polyacrylamide gel IEF of the EtOH-2 material revealed the presence of several proteins with different charge properties, as shown in Fig. 2. Neither the removal of the hyaluronic acid by hyaluronidase treatment nor continuation of purification to six precipitations altered the pattern found with IEF. Purification by ion exchange chromatography. The availability of pyrogen-free ion exchange gels has ma'de possible the further separation of the protein components of NY-5 EtOH-2. (Preliminary investigation of the use of QAE-Sephadex to separate the protein fractions of SPE was carried out by K. W. Brunson [Ph.D. thesis, Univ. of Minnesota, Minneapolis, 1973].) The method was developed using the toxin produced by NY-5 but has been applied
769
successfully to toxins of other strains. Several types of column chromatography were used in initial attempts at fractionation. It was found that mild anion exchange using Ecteola-cellulose (pH 7) or mild cation exchange using carboxymethyl-cellulose (pH 4-7) resulted in separation of the protein from the hyaluronic acid, indicating that the two moieties were not covalently linked. However, the various protein fractions were separated only when a stronger anion exchange gel, QAE-Sephadex, was used. The results of a typical QAE-Sephadex column are shown in Fig. 3. The physicochemical properties of the four fractions isolated by this method are given in Table 1 and the IEF patterns in Fig. 2. Fraction 2 shows a degree of homogeneity in IEF which is comparable to that obtained with recrystallized proteins. Fractions 1 and 3, however, appear to contain minor contaminating bands. The results of double immunodiffusion in agar using antisera raised in rabbits against NY-5 EtOH-2 indicate that fractions 2 and 3 are antigenically identi-
pH 9 8 7 6 5 4 N Y-S
fl
f2
f3
FIG. 2. Thin-layer polyacrylamide gel isoelectric focusing ofNY-5 EtOH-2 and QAE-Sephadex fractions 1, 2, and 3. Stained with Coomassie blue.
770
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CUNNINGHAM, BARSUMIAN, AND WATSON
TABLE 1. Physicochemical properties ofNY-5 EtOH2 and its QAE-Sephadex fractions Fraction 1 2 3 4 NY-5 EtOH-2
% Hyaluronic acid (wt/wt) 4 4 8 85 25
Isoelectric point of proteins 8.5-9.5 4.5-5.5 4.5-5.5 _b
Mol wta
21,900 8,000 5,500
NDC
21,000
Partial specific volumes determined to be 0.07 (13). b-, Not detected. ND, Not determined.
a c
1.0
cal and that fraction 1 is distinct from the others (Fig. 4). Amino acid analyses of the fractions revealed a surprising similarity in amino acid composition of the fractions with the previously reported composition of NY-5 EtOH-2 (9) and with each other (Table 2). Fractions 1, 2, and 3 were all high in the dicarboxylic acids such as aspartic and glutamic acids and relatively low in the basic amino acids such as lysine and arginine. A large quantity of ammonia was found in the hydrolysates, suggesting that ami-
f4 hanr hnnea
.75 280 nm
-50S f2
fl
bufferI
pH7
0.1 M Imidozole-
Acetic Acid
buffter 2 pH 5 O.IM Imidozole-Acetic Acid 0.1 M Na CI
f3
buffer 3
pH4
O.IM Sodium Acetate-Acetic Acid
IM NoCd
FIG. 3. Fractionation of NY-5 EtOH-2 on QAE-Sephadex A-50.
FIG. 4. Double immunodiffusion in Noble agar. Center well contains antiserum to NY-5 EtOH-2. fl, t2, 3, are QAE-Sephadex fractions of NY-5 EtOH-2. NY-5 is NY-5 EtOH-2.
VOL. 14, 1976
PURIFICATION OF STREPTOCOCCAL EXOTOXIN
771
TABLE 2. Amino acid composition of NY-5 EtOH-2 and its QAE-Sephadex fractions Amino acid
NY-5 EtOH-2
Fraction 1
Fraction 2
Fraction 3
Cysteic acid Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Half-cystine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Lysine Histidine Arginine Total amino acids
None 17.08a 3.94 9.20 16.42 3.28 11.99 7.88 1.97 2.96 0.98 1.48 4.10 2.96 2.79 8.54 1.64 2.79 100
None 13.80 3.39 8.15 8.57 6.90 10.47 6.90 0.36 3.33 None 2.62 4.70 8.81 11.60 5.23 2.14 3.03 100
None
3.23 18.22 5.03 8.62 15.17 4.94 10.59 5.12 2.96 2.87 None 2.06 4.04 1.80 2.42 7.54 2.15 3.23 100
23.42 2.38 None None
34.07 6.66 None None
Ammonia 20.29 Glucosamine 11.74 Diaminopimelic acid None Muramic acid None a Moles per 100 moles of total amino acids.
dation of the dicarboxylic acids might contribute to the high isoelectric point of fraction 1. Biological activities of the purified fractions. The QAE fraction of NY-5 EtOH-2 was tested in rabbits for pyrogenicity, skin test reactivity, and ability to enhance the susceptibility to lethal endotoxin shock. Fraction 2 showed the greatest activity by weight in each assay, whereas fraction 4, which was mainly hyaluronic acid, showed minimal activity (Table 3). Rabbits can be made immune to the pyrogenic effects of the toxin by injection of small doses (10 MPD-4) of toxin every other day for 2 weeks. Rabbits immunized in this way with fraction 1 showed no cross immunity to fractions 2 or 3, whereas rabbits immunized with fraction 2 were immune to fraction 3 but not to fraction 1 (Fig. 5). Identification of the fractions. It appears that fractions 2 and 3 are both exotoxin A, because they are eluted from QAE-Sephadex in the same fractions as the SPE of T-28, a strain that produces only exotoxin A (Fig. 6). In addition, fractions 2 and 3 react with a commercial antitoxin A (American Cyanamid Co., Lederle Laboratories Div., Pearl River, N.Y.), whereas fraction 1 does not (Fig. 7). Pyrogen cross testing of fractions 2 and 3 in T-28 EtOH-2 immune rabbits gave inconclusive results, possibly because complete pyrogenic immunity to the T-28 toxin was difficult to achieve.
16.20 6.34 7.57 16.11 5.37 8.10 4.49 1.85 3.79
None 2.55 6.43 4.23 4.14 7.57 2.73 2.55 100
16.99 10.59
None None
TABLE 3. Biological activities of NY-5 EtOH-2 and its QAE-Sephade-x fractions Fraction
Pyrogenicity MPD-4 (ILg/ kg)a
Enhanced susceptibility to endotoxin LD50
STD
(g)
( tg/kg)b
1 2 3 4
NY-5 EtOH-2
3.2 0.02 11.2 46.4 0.3
25 14 15 63 5
0.1 0.1 0.1 1 0.1
a MPD-4, Minimal pyrogenic dose causing a 0.55°C (1°F) rise of temperature 4 h after intravenous injection. b Mean lethal dose (LDN) of SPE with 25 ,ug of S. typhimurium lipopolysaccharide (LPS) per kg given 5 h later. The LD50 of NY-5 EtOH-2 alone was 2,500 The LD50 of LPS alone was 535 ,ug/kg. Ag/kg. c STD, Intradermal dose of SPE required for a skin reaction of an area greater than 6 by 6 mm at 24
to 48 h.
Fraction 1 has an IEF pattern like the exotoxin B produced by strain T-19 (Fig. 8), and antiserum prepared against fraction 1 will react with T-19 EtOH-2, forming a line of identity (Fig. 9). In pyrogen cross testing, rabbits immunized with T-19 EtOH-2 had significantly less fever at 4 h than control rabbits when injected with fraction 1, and rabbits immunized with fraction 1 showed significant immunity to
772
CUNNINGHAM, BARSUMIAN, AND WATSON
the corresponding QAE fraction of T-19 (Fig. 19).
DISCUSSION SPE prepared from NY-5 culture filtrates by alcohol precipitation has in the present study been separated by ion exchange chromatography on QAE-Sephadex into three protein fractions with the characteristic biological activities of SPE and a fourth carbohydrate-rich fraction that is relatively inert. Although this method of purification separates the toxin into several distinct components, the yield of the active fractions per column is rather low. Preliminary experiments suggest that prior treatment of the toxin with hyaluronidase may increase the efficiency of the ion exchange procedure. Affinity chromatography using antisera made monospecific by absorption with the appropriate purified QAE fractions and thin-layer IEF in Sephadex gels (12) are also being explored as methods of purification. Although NY-5 ethanol-precipitated SPE had previously been identified as exotoxin A (9), the three fractions of NY-5 EtOH-2 from QAE-Sephadex were found to represent two immunologically distinct toxins, as shown both by immunodiffusion and pyrogen cross testing. However, in vivo the NY-5 strain produces exotoxin B as well as exotoxin A (7, 16), and the high activity of fraction 2 may have obscured the other activity in the partially purified material. We have identified both fraction 2 and fraction 3 as exotoxin A. Whereas these fractions are eluted from QAE-Sephadex with the same buffer and seem to be identical by IEF and
INFECT. IMMUN.
immunodiffusion, they differ in hyaluronic acid content and in molecular weight. Since fraction 3 has a lower molecule weight than fraction 2 SPE Fo, Im...ilatio.
SPE Tesed
(1o MPO-4 /kg)
FEVER AT 4 HOURS A c O5 O
°r
NSY- 5 Frectlon 2
Ny- 5 F,oction
NIY- 5
NY-5 F"Pction
NY-5 Froct,oo 3
NY-5
NY- 5 Froction
F,octio. 2
F,actio.
It L_
A SPE For Im....iZatio.
SPE Tesled 0 0MPD -4 /kg
FEVER AT 4 HOURS *C 0I
05
NY-S F,octioo 2
NY-5
NY-5 FAoctAo 2
NY- 5 FACAtA0 S
NY-5 FRaction 2
NY-S F,act.on 2
B FIG. 5. Pyrogen cross tests of QAE fractions of NY-5 EtOH-2. All immunizing and challenge doses of SPE were 10 minimal pyrogenic doses per kg. (A) Fraction 1 of NY-5 used as SPE for immunization. (B) Fraction 2 of NY-5 used as SPE for immunization. Bars indicate ± standard error of the mean.
obsorbonce 280 nm
0
I buffer pH 7 01M Imidazole-
buffer 2
Acetic Acid
OIM NaCI
pH
5
FRactio-
1.0. .25
10
NY-5 Froetion
.75 .50
5
5
01 M Imidazole -
FIG. 6. Fractionation of T-28 EtOH-2
I
buffer 3 pH 4 01 M Sodum Acetote-Acetic Acid M NoCI
on
QAE-Sephadex A-50.
