Vol. 23, No. 3
INFECTION AND IMMUNITY, Mar. 1979, p. 609-617 0019-9567/79/03-0609/09$02.00/0
Purification and Physicochemical and Biological Characterization of a Staphylococcal Pyrogenic Exotoxin PATRICK M. SCHLIEVERT, DIANE J. SCHOETTLE, AND DENNIS W. WATSON* Department of Microbiology, Medical School, University of Minnesota, Minneapolis, Minnesota 55455 Received for publication 11 December 1978
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.
Staphylococcus aureus strains produce a large number of extracellular products, many of them potent toxins, which may contribute to establishment and maintenance of the disease state. It has been shown that even in the presence of antibiotic therapy strains may continue to produce exotoxin (40). This may result from the organisms' remarkable capacities to become antibiotic resistant (22, 33, 40) and to colonize areas of the body not reached easily by antibiotics (22, 33, 40). S. aureus strains also may be carried by many people without producing apparent harmful effects, and if present in the throat they often are not considered pathogens (11, 22). During the course of our studies of the group A streptococcal pyrogenic exotoxins (PE), we were asked to test an S. aureus strain for production of streptococcal PE. The organism was isolated by David Schlossberg, Harrisburg, Pa., from a patient with mucocutaneous lymph node syndrome (Kawasaki's disease) for which the etiological agent has not been elucidated. The disease state is defined on the basis of the following criteria: (i) fever lasting 5 or more days and unresponsive to antibiotics; (ii) bilateral congestion of ocular conjunctivae; (iii) changes in the lips and oral mucosa, including dry, red fissuring of the tongue and diffuse reddening of the palms and soles and indurative edema followed by
membranous desquamation; (iv) polymorphous nonvesicular truncal exanthem; and (v) acute nonsuppurative enlargement of the cervical lymph nodes. The disease may have associated with it meningitis and an incidence of coronary disease of 5% (23, 24, 27, 30-32, 34, 47). Also, the syndrome must be distinguished from scarlet fever. Many of the properties described above may be manifestations of direct or enhanced toxicities due to streptococal PE. The streptococcal PE produce fever in a variety of laboratory animals, including rabbits, mice, and monkeys (5, 25, 36, 43). They enhance host susceptibility to insults by other agents such as endotoxin or streptolysin 0, which may result in either lethal shock or myocardial damage (25, 37, 43). The streptococcal PE also enhance hypersensitivity reactions, which may contribute to generation of a red rash (unpublished data). Lymph node swelling may result from the nonspecific lymphocyte mitogenic activity induced by the toxins (6). In addition, the host may be predisposed to meningitis by alteration of blood-brain barrier permeability (36). At a recent symposium on streptococci, it was suggested that mucocutaneous lymph node syndrome may be caused by antigens associated with group A streptococci (T. Ueno and F. Matsumi, VIIth International Sym609
610
SCHLIEVERT, SCHOETTLE, AND WATSON
INFECT. IMMUN.
formed by the manufacturer's specifications as deon Streptococci and Streptococcal DisSt. Anne's College, Oxford, England, scribed earlier (35). Gels were stained with Coomassie blue R-250. Abstr. no. 54, 1978). We, therefore, examined the brilliant acid composition of the toxin was deteramino The S. aureus strain for production of the known mined by a procedure described previously (35). streptococcal PE types or a related family of Protease activity was measured by the procedure toxins. described in the Worthington Biochemicals Manual This paper describes the purification and (Worthington Enzyme Manual, p. 122-123, Worthingphysicochemical and biological characterization ton Biochemicals Corp., Freehold, N.J.). Biological assays. Pyrogenicity and capacity to of a low-molecular-weight PE produced by the enhance lethal endotoxin shock of the staphylococcal Harrisburg S. aureus strain. PE in rabbits and mice were measured as described MATERIALS AND METHODS for the group A streptococcal PE (25, 36). All reagents and glassware used for production, previously to the pyrogenicity of the toxin was develImmunity purification, and characterization of the staphylococ- oped by giving rabbits intravenous injections of 100 cal PE were maintained pyrogen-free. MPD-4/kg every other day for five injections (25, 35, Bacterium. The staphylococcal strain, designated 43). Rabbits were hyperimmunized with toxin by use David Schlossberg, was isolated by strain, Harrisburg of a procedure described elsewhere (35). Antibody for Polyclinic Medical Center, Harrisburg, Pa. The strain use in Ouchterlony immunodiffusion assays was prewas beta-hemolytic and coagulase positive, and it beby precipitation from hyperimmune antiserum, longed to phase group I (major types 29/52A/79). It pared 33% (final concentration) ammonium sulfate. using with infection of a a patient was isolated from vaginal antibody was dissolved in distilled The precipitated mucocutaneous lymph node syndrome (Kawasaki's
posium eases,
disease). The organism was maintained in the lyophilized state in the presence of whole defibrinated fresh rabbit blood. Animals. American Dutch belted rabbits were obtained from a local source and weighed from 1 to 1.5 kg. BALB/cWAT mice weighing approximately 25 g were obtained from our mouse colony. Young adult cats weighed 1.5 to 2 kg and were accustomed to the laboratory environment before use. Production and purification of exotoxin. The staphylococcal PE was produced by growing the staphylococcal strain in a beef heart dialysate medium (25). Partially purified exotoxin (EtOH-1) was obtained from culture supernatant fluids by means of differential precipitation with -20°C ethanol (final concentration, 75%) and resolubilization in distilled water. Final toxin purification was achieved by preparative thin-layer isoelectric focusing (35, 45) of EtOH-1 toxin (200 mg, dry weight) with a pH 3.5 to 10 ampholyte range (Ampholine; LKB-Produkter, Stockholm, Sweden). The specific activity of the toxin, determined during purification steps, was defined as minimum pyrogenic dose at 4 h (MPD-4) per milligram of protein. One unit of toxin (1 MPD-4) was the minimal dose required to produce a 0.5°C average fever response per kg of rabbit body weight 4 h after intravenous injection. Biochemical tests. Protein was determined by the Bradford assay (9) with bovine serum albumin serving as the standard, ribonucleic acid was measured by the orcinol method (2) with yeast ribonucleic acid as the standard, and deoxyribonucleic acid (DNA) was assessed by use of the diphenylamine reagent (14) with calf thymus DNA serving as the standard. Total carbohydrate was determined by the phenol-sulfuric acid method (15) with glucose as the standard. The molecular weight of the toxin was estimated by use of sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (44). Standards consisted of bovine serum albumin, ovalbumin, pepsin, and lysozyme. Gels were stained with Coomassie brilliant blue R-250 and photographed with the use of indirect lighting. Reisoelectric focusing of staphylococcal PE was per-
water at 10 times the serum concentration.
Ouchterlony immunodiffusion was performed in Noble agar. Antisera to the enterotoxins and staphylococcal enterotoxins used in immunodiffusion tests were kindly provided by M. S. Bergdoll and E. Schantz, Food Research Institute, University of Wisconsin, Madison. Enterotoxic activity of the staphylococcal PE was assessed by injecting three young adult cats intraperitoneally with 100 jig of toxin per kg. Cats were monitored for 5 h for emesis. Positive controls consisted of the same cats receiving 25 jig of staphylococcal enterotoxin B per kg intraperitoneally 2 days later. Assays for hemolytic activity were performed by the procedure of Bernheimer and Schwartz (8). Exfoliative toxin activity was measured by use of an assay described previously (29). Nonspecific mitogenicity of the staphylococcal PE toxin was determined with the use of human cord blood lymphocytes and rabbit spleen cells (6).
RESULTS The methods used to purify the staphylococcal PE were modified from those used for the group A streptococcal PE. Partially purified ethanol-precipitated staphylococcal PE (EtOH1) was pyrogenic in rabbits (1 MPD-4/kg was 1.5 ,ug of protein) and also enhanced lethal endotoxin shock (Fig. 1). The fever maximum was near 4 h after toxin injection. As reported previously for the streptococcal PE (25), the survivors shown in Fig. 1 exhibited considerable myocardial and limited liver necrosis 3 days after receiving toxin. No kidney damage was observed. Final purification of staphylococcal PE was accomplished by means of preparative thin-layer isoelectric focusing with a pH gradient from 3.5 to 10. A representative zymogram print (45) from one electrofocusing run is shown in Fig. 2. The protein with an isoelectric point of 5.3 was
STAPHYLOCOCCAL PYROGENIC EXOTOXIN
VOL. 23, 1979
ENHANCED
OEAD/ TOTAL
D
5/5
)p
4/5
a 0-5
2/5
HOURS
FIG. 1. Pyrogenicity and capacity to enhance k_ thal endotoxin shock by ethanol-]Precipitated staphy lo cocca lpyrogen ic exotox in . Do ses of exotoxin were 100 ug of protein/kg (0), 10 pgkg (0), and 1 pglkg (0) by the intravenous route. All rrabbits (five per group) were given 25 psg of endotoxin (Salmonella typhimnurium, 0.05 LDW9 per kg intravenous)usly at 4 h.
the pyrogeniic exotoxin. A concentrication of 1 MPD-4/kg of the toxin purified by isoelectric focusing was approximately 0.1 ,ug in rabbits (Fig. 3), and the toxin enhanced hosit susceptibility to lethal shock or myocardial and liver damage by endotoxin. The staphyloitcoccal PE produced fever in mice (Fig. 3) com]parable to that produced by the streptococcal P'E and enhanced susceptibility to lethal endoto)xin shock. The higher dose of exotoxin given to mice may have produced shock, contributing tb;o the lowered fever response (36). Rabbits were immunized against the pyrogeni. activity of the staphylococcal toxin and then challenged with homologous toiin or endotoxin to test for ability to develop slpecific immunity (Fig. 4). Rabbits immunized vvith staphylococcal PE were immune to the PD yrogenicity after homologous challenge (20 MPDg -4/kg), but showed the typical biphasic fever re1sponses to heterologous challenge with endot toxin (100 MPD-3/kg). Nonimmunized animaby s exhibited fevers after challenge with the PE (220 MPD-4/ I
,
611
kg). The data indicate that specific immunity to the pyrogenicity of the staphylococcal PE could be developed.
The recovery of pyrogenic activity and degree
of toxin purification are shown in Table 1. A recovery of 34% activity and a final 200-fold purification were achieved. Toxin purity was assessed on the basis of several criteria. The toxin migrated as a homogeneous protein with a molecular weight of approximately 12,000 when tested by SDS-polyacrylamide gel electrophoresis (Fig. 5A), and it was homogeneous when re-electrofocused (Fig. 5B) in polyacrylamide gels (isoelectric point, 5.3). EtOH-1 staphylococcal PE (3 mg of protein/ml) reacted with hyperimmune antiserum raised against the purified toxin, using immunodiffusion to produce a single precipitin arc (Fig. 6A and B). The antiserum did not react either with the known streptococcal PE types (Fig. 6A) or with the staphylococcal enterotoxins (Fig. 6B). Also, antisera raised against either the streptococcal PE or enterotoxins did not react with the purified staphylococcal PE. The PE contained no detectable ribonucleic acid or DNA but did contain 5% carbohydrate. The sugar residues may be covalently linked to the toxin since isoelectric focusing failed to separate them from the protein. As proposed for hyaluronic acid associated with the streptococcal PE, the carbohydrate may facilitate alcohol precipitation during purification and stabilize biological activities of the toxin (5, 36). The amino acid composition of the staphylococcal PE shared characteristics associated with both the streptococcal PE and enterotoxins (Table 2). Like the streptococcal PE, the staphylococcal PE contained low levels of aromatic amino acids (12, 35), but it contained high lysine, as do the enterotoxins (7). Many of the glutamyl and aspartyl residues contained in the staphylococcal PE may be in the acid form, required to offset the lysine positive charge contribution to achieve an isoelectric point of 5.3. At a concentration of 100 ,ug/kg, the staphylococcal PE did not produce emesis in any of three young adult cats, whereas enterotoxin b (25 ug/kg) did so in all three in less than 2 h. The toxin also was not hemolytic, proteolytic, or exfoliative for newborn mice and did not exhibit coagulase activity. It was, however, a potent nonspecific lymphocyte mitogen (Fig. 7 and 8) as measured by incorporation of [3H]thymidine into DNA. The PE induced rabbit spleen cell proliferation at both 1 and 10 ,jg/well, and the maximum response was at 3 to 4 days or later. In contrast, concanavalin A, a potent thymusderived (T) lymphocyte mitogen, induced cells to proliferate, with a maximum response occur-
612
SCHLIEVERT, SCHOETTLE, AND WATSON
INFECT. IMMUN.
TOXIN
APPL .
Jim.
:.
3
4
5
pH
6 GRADIENT
7
8
9
FIG. 2. Zymogram print (45) of staphylococcal pyrogenic exotoxin subjected to preparative thin-layer isoelectric focusing with the use of a pH range of 3.5 to 10. The print was stained with Coomassie brilliant
blue R-250.
ENHANCED DEAD/ TOTAL
ENHANCED
DEAD/TOTAL
RABBIT
2.0
MOUSE
2.0
1/5
4/5 w
W a.
1.5
1.0
5/5
.
w1
L.5, 5
a. 1i0 a
~~~ ~ ~~~~3/'5
W
0/5
4O 3/5 5-
4 0.5-
FI. P . rgnctan caaiytenaclthl edtnsok yiolcrcfouigprfe 0
2 3 HOURS
4
0
2
3
4
HOURS
FIG. 3. Pyrogenicity and capacity to enhance lethal endotoxin shock by isoelectric focusing-purified staphylococcalpyrogenic exotoxin in rabbits and mice. Toxin protein doses in rabbits (five per group) were 10 p~g/kg (U), 1 pg/kg (0), and 0. 1 p~g/kg (0) intravenously. Rabbits were given 25 pig of Salmonella typhimurium endotoxin per kg (0.05 LDIIQ intravenously at 4 h. Toxin doses in mice (five per group) were 50 pg/0. 1 ml (U), 10 pug/0.1 ml (0), and 1 pig/0.1 ml (0) intravenously. Endotoxin (100 pg/mouse) was given at 4 h.
ring at 2 to 3 days. The toxin was mitogenic also for human cord blood lymphocytes (Fig. 8), with counts obtained comparable to those obtained with concanavalin A. Duplicate experiments gave similar results. DISCUSSION The staphylococcal PE isolated in this study was shown to be a potent pyrogen and nonspecific lymphocyte mitogen and had the capacity
to enhance host susceptibility to insults by endotoxin-either lethal shock or myocardial and liver damage. The PE lacked hemolytic, proteolytic, and exfoliative toxin activities and, because of charge and molecular weight differences, was distinguished from other extracellular staphylococcal proteins including: leukocidin (46), protein A (16, 38), deoxyribonuclease (1), ribonuclease (19), hyaluronidase (1), and staphylokinase (3).
VOL. 23, 1979
STAPHYLOCOCCAL PYROGENIC EXOTOXIN
Like the staphylococcal PE, the staphylococcal enterotoxins are pyrogenic (10) and mitogenic (42), and they enhance susceptibility to lethal shock (39), though myocardial and liver damage have not been reported associated with enhancement by the enterotoxins. In addition to being able to distinguish the PE from the enterotoxins on the basis of charge and molecular weight differences (7), the staphylococcal PE did not react in immunodiffusion with antisera to 2.
1.5=
613
the enterotoxins, and antisera to the staphylococcal PE did not react with the enterotoxins. The toxin did not produce emesis in young adult cats as shown previously for the enterotoxins (28) and, therefore, was not considered a new enterotoxin type. Previously, a potent lymphocyte mitogen was identified and partially characterized physicochemically from an S. aureus strain (26, 41). It is possible that this mitogen and the staphylococcal PE are the same protein or belong to a similar family. The mitogen could be purified by ethanol precipitation (70%, final volume) followed by column isoelectric focusing (26, 41). Its molecular weight was estimated to be 14,000, and it had an isoelectric point of 5.5 to 5.7. The phage group ofthe DA352 strain used to produce the mitogen was not listed. It would be of interest if both the mitogen- and staphylococcal PEproducing strains belonged to the same phage group.
Another group of researchers have demonstrated the presence of an extracellular pyrogen in culture supernatant fluids of S. aureus (4).
a
0.5-
0
2
3
4
HOU RS
FIG. 4. Development of specific immunity to the isoelectric focusing-purified staphylococcalpyrogenic exotoxin. Rabbits were made immune to the pyrogenicity of the exotoxin by giving 100 MPD-4/kg intravenously every other day for five injections. The immunized rabbits (five per group) were given either 20 MDP-4 of exotoxin per kg () or 100 MPD-3 of Salmonella Minnesota Re595 endotoxin per kg (U) intravenously. Nonimmunized control rabbits (five per group) received 20 MPD-4 of exotoxin per kg (0).
FIG. 5. Polyacrylamide gel isoelectric focusing (A) and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (B) of staphylococcal pyrogenic exotoxin. In both systems 50 pg of toxin was applied to the gels. Gels were stained with Coomassie brilliant blue R250.
TABLE 1. Purification of staphylococcal pyrogenic exotoxin Fraction
Protein (mg)
Total activity
(UU)
Specific activity (U/mg of protein)
Purification (fold)
Yield activ-
Supernatant fluid EtOH-1 .........
3.8 x 104 2.25 x 103 65
1.9 x 106 1.5 x 10" 6.5 x 105
50 670 1.0 x 104
-
-
13.4 200
79 34
IEFb-purified
.................
a One unit equals 1 MPD-4/kg. b Isoelectric focusing.
ity (%)
614
SCHLIEVERT, SCHOETTLE, AND WATSON
INFECT. IMMUN.
FIG. 6. Ouchterlony immunodiffusion of ethanol-precipitated staphylococcal pyrogenic exotoxin (EtOH-1) reacted with hyperimmune antisera raised against the isoelectric focusing-purified toxin. Frame A: Outer wells contained EtOH-1 exotoxin (TOX) at a concentration of 3 mg ofprotein/ml and streptococcalpyrogenic exotoxin types A, B, and C at concentrations of 2 mg/ml. Frame B: Outer wells contained EtOH-1 exotoxin (3 mg/ml) and staphylococcal enterotoxins A, B, C, D, and E at concentrations of 2 mg/ml. Inner wells in both frames contained antisera. All wells contain 25 ,ul of material.
TABLE 2. Amino acid composition of staphylococcal pyrogenic exotoxin Residue Percent mol Aspartyl ..................... 11.8 Threonine .................... 6.5 Serine ....................... 6.5 Glutamyilb ..................... 14.7 Proline ....................... 5.6 Glycine ...................... 15.2 Alanine ...................... 6.2 1.9 Cysteine ...................... Valine ....................... 4.2 Isoleucine .................... 3.3 Leucine ...................... 4.8 Tyrosine ..................... 1.7 Phenylalanine ................ 1.3 Lysine ....................... 10.4 Histidine ..................... 3.4 Arginine 3.5 Aspartyl includes aspartic acid and asparagine. b Glutamyl includes glutamic acid and glutamine. ......................
a
This pyrogen, although neither purified nor characterized, may be the PE described in this study, which has been purified to a high specific activity based on its pyrogenicity in rabbits. The staphylococcal PE was homogeneous when tested by SDS-polyacrylamide gel electrophoresis or reisoelectric focusing in polyacrylamide gels and when allowed to react against hyperimmune antisera. The staphylococcal PE resembles closely the streptococcal PE, both physicochemically and biologically, though they were shown to be se-
rologically distinct. The staphylococcal PE and the streptococcal PE are relatively low molecular weight (
INFECTION AND IMMUNITY, Mar. 1979, p. 609-617 0019-9567/79/03-0609/09$02.00/0
Purification and Physicochemical and Biological Characterization of a Staphylococcal Pyrogenic Exotoxin PATRICK M. SCHLIEVERT, DIANE J. SCHOETTLE, AND DENNIS W. WATSON* Department of Microbiology, Medical School, University of Minnesota, Minneapolis, Minnesota 55455 Received for publication 11 December 1978
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.
Staphylococcus aureus strains produce a large number of extracellular products, many of them potent toxins, which may contribute to establishment and maintenance of the disease state. It has been shown that even in the presence of antibiotic therapy strains may continue to produce exotoxin (40). This may result from the organisms' remarkable capacities to become antibiotic resistant (22, 33, 40) and to colonize areas of the body not reached easily by antibiotics (22, 33, 40). S. aureus strains also may be carried by many people without producing apparent harmful effects, and if present in the throat they often are not considered pathogens (11, 22). During the course of our studies of the group A streptococcal pyrogenic exotoxins (PE), we were asked to test an S. aureus strain for production of streptococcal PE. The organism was isolated by David Schlossberg, Harrisburg, Pa., from a patient with mucocutaneous lymph node syndrome (Kawasaki's disease) for which the etiological agent has not been elucidated. The disease state is defined on the basis of the following criteria: (i) fever lasting 5 or more days and unresponsive to antibiotics; (ii) bilateral congestion of ocular conjunctivae; (iii) changes in the lips and oral mucosa, including dry, red fissuring of the tongue and diffuse reddening of the palms and soles and indurative edema followed by
membranous desquamation; (iv) polymorphous nonvesicular truncal exanthem; and (v) acute nonsuppurative enlargement of the cervical lymph nodes. The disease may have associated with it meningitis and an incidence of coronary disease of 5% (23, 24, 27, 30-32, 34, 47). Also, the syndrome must be distinguished from scarlet fever. Many of the properties described above may be manifestations of direct or enhanced toxicities due to streptococal PE. The streptococcal PE produce fever in a variety of laboratory animals, including rabbits, mice, and monkeys (5, 25, 36, 43). They enhance host susceptibility to insults by other agents such as endotoxin or streptolysin 0, which may result in either lethal shock or myocardial damage (25, 37, 43). The streptococcal PE also enhance hypersensitivity reactions, which may contribute to generation of a red rash (unpublished data). Lymph node swelling may result from the nonspecific lymphocyte mitogenic activity induced by the toxins (6). In addition, the host may be predisposed to meningitis by alteration of blood-brain barrier permeability (36). At a recent symposium on streptococci, it was suggested that mucocutaneous lymph node syndrome may be caused by antigens associated with group A streptococci (T. Ueno and F. Matsumi, VIIth International Sym609
610
SCHLIEVERT, SCHOETTLE, AND WATSON
INFECT. IMMUN.
formed by the manufacturer's specifications as deon Streptococci and Streptococcal DisSt. Anne's College, Oxford, England, scribed earlier (35). Gels were stained with Coomassie blue R-250. Abstr. no. 54, 1978). We, therefore, examined the brilliant acid composition of the toxin was deteramino The S. aureus strain for production of the known mined by a procedure described previously (35). streptococcal PE types or a related family of Protease activity was measured by the procedure toxins. described in the Worthington Biochemicals Manual This paper describes the purification and (Worthington Enzyme Manual, p. 122-123, Worthingphysicochemical and biological characterization ton Biochemicals Corp., Freehold, N.J.). Biological assays. Pyrogenicity and capacity to of a low-molecular-weight PE produced by the enhance lethal endotoxin shock of the staphylococcal Harrisburg S. aureus strain. PE in rabbits and mice were measured as described MATERIALS AND METHODS for the group A streptococcal PE (25, 36). All reagents and glassware used for production, previously to the pyrogenicity of the toxin was develImmunity purification, and characterization of the staphylococ- oped by giving rabbits intravenous injections of 100 cal PE were maintained pyrogen-free. MPD-4/kg every other day for five injections (25, 35, Bacterium. The staphylococcal strain, designated 43). Rabbits were hyperimmunized with toxin by use David Schlossberg, was isolated by strain, Harrisburg of a procedure described elsewhere (35). Antibody for Polyclinic Medical Center, Harrisburg, Pa. The strain use in Ouchterlony immunodiffusion assays was prewas beta-hemolytic and coagulase positive, and it beby precipitation from hyperimmune antiserum, longed to phase group I (major types 29/52A/79). It pared 33% (final concentration) ammonium sulfate. using with infection of a a patient was isolated from vaginal antibody was dissolved in distilled The precipitated mucocutaneous lymph node syndrome (Kawasaki's
posium eases,
disease). The organism was maintained in the lyophilized state in the presence of whole defibrinated fresh rabbit blood. Animals. American Dutch belted rabbits were obtained from a local source and weighed from 1 to 1.5 kg. BALB/cWAT mice weighing approximately 25 g were obtained from our mouse colony. Young adult cats weighed 1.5 to 2 kg and were accustomed to the laboratory environment before use. Production and purification of exotoxin. The staphylococcal PE was produced by growing the staphylococcal strain in a beef heart dialysate medium (25). Partially purified exotoxin (EtOH-1) was obtained from culture supernatant fluids by means of differential precipitation with -20°C ethanol (final concentration, 75%) and resolubilization in distilled water. Final toxin purification was achieved by preparative thin-layer isoelectric focusing (35, 45) of EtOH-1 toxin (200 mg, dry weight) with a pH 3.5 to 10 ampholyte range (Ampholine; LKB-Produkter, Stockholm, Sweden). The specific activity of the toxin, determined during purification steps, was defined as minimum pyrogenic dose at 4 h (MPD-4) per milligram of protein. One unit of toxin (1 MPD-4) was the minimal dose required to produce a 0.5°C average fever response per kg of rabbit body weight 4 h after intravenous injection. Biochemical tests. Protein was determined by the Bradford assay (9) with bovine serum albumin serving as the standard, ribonucleic acid was measured by the orcinol method (2) with yeast ribonucleic acid as the standard, and deoxyribonucleic acid (DNA) was assessed by use of the diphenylamine reagent (14) with calf thymus DNA serving as the standard. Total carbohydrate was determined by the phenol-sulfuric acid method (15) with glucose as the standard. The molecular weight of the toxin was estimated by use of sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (44). Standards consisted of bovine serum albumin, ovalbumin, pepsin, and lysozyme. Gels were stained with Coomassie brilliant blue R-250 and photographed with the use of indirect lighting. Reisoelectric focusing of staphylococcal PE was per-
water at 10 times the serum concentration.
Ouchterlony immunodiffusion was performed in Noble agar. Antisera to the enterotoxins and staphylococcal enterotoxins used in immunodiffusion tests were kindly provided by M. S. Bergdoll and E. Schantz, Food Research Institute, University of Wisconsin, Madison. Enterotoxic activity of the staphylococcal PE was assessed by injecting three young adult cats intraperitoneally with 100 jig of toxin per kg. Cats were monitored for 5 h for emesis. Positive controls consisted of the same cats receiving 25 jig of staphylococcal enterotoxin B per kg intraperitoneally 2 days later. Assays for hemolytic activity were performed by the procedure of Bernheimer and Schwartz (8). Exfoliative toxin activity was measured by use of an assay described previously (29). Nonspecific mitogenicity of the staphylococcal PE toxin was determined with the use of human cord blood lymphocytes and rabbit spleen cells (6).
RESULTS The methods used to purify the staphylococcal PE were modified from those used for the group A streptococcal PE. Partially purified ethanol-precipitated staphylococcal PE (EtOH1) was pyrogenic in rabbits (1 MPD-4/kg was 1.5 ,ug of protein) and also enhanced lethal endotoxin shock (Fig. 1). The fever maximum was near 4 h after toxin injection. As reported previously for the streptococcal PE (25), the survivors shown in Fig. 1 exhibited considerable myocardial and limited liver necrosis 3 days after receiving toxin. No kidney damage was observed. Final purification of staphylococcal PE was accomplished by means of preparative thin-layer isoelectric focusing with a pH gradient from 3.5 to 10. A representative zymogram print (45) from one electrofocusing run is shown in Fig. 2. The protein with an isoelectric point of 5.3 was
STAPHYLOCOCCAL PYROGENIC EXOTOXIN
VOL. 23, 1979
ENHANCED
OEAD/ TOTAL
D
5/5
)p
4/5
a 0-5
2/5
HOURS
FIG. 1. Pyrogenicity and capacity to enhance k_ thal endotoxin shock by ethanol-]Precipitated staphy lo cocca lpyrogen ic exotox in . Do ses of exotoxin were 100 ug of protein/kg (0), 10 pgkg (0), and 1 pglkg (0) by the intravenous route. All rrabbits (five per group) were given 25 psg of endotoxin (Salmonella typhimnurium, 0.05 LDW9 per kg intravenous)usly at 4 h.
the pyrogeniic exotoxin. A concentrication of 1 MPD-4/kg of the toxin purified by isoelectric focusing was approximately 0.1 ,ug in rabbits (Fig. 3), and the toxin enhanced hosit susceptibility to lethal shock or myocardial and liver damage by endotoxin. The staphyloitcoccal PE produced fever in mice (Fig. 3) com]parable to that produced by the streptococcal P'E and enhanced susceptibility to lethal endoto)xin shock. The higher dose of exotoxin given to mice may have produced shock, contributing tb;o the lowered fever response (36). Rabbits were immunized against the pyrogeni. activity of the staphylococcal toxin and then challenged with homologous toiin or endotoxin to test for ability to develop slpecific immunity (Fig. 4). Rabbits immunized vvith staphylococcal PE were immune to the PD yrogenicity after homologous challenge (20 MPDg -4/kg), but showed the typical biphasic fever re1sponses to heterologous challenge with endot toxin (100 MPD-3/kg). Nonimmunized animaby s exhibited fevers after challenge with the PE (220 MPD-4/ I
,
611
kg). The data indicate that specific immunity to the pyrogenicity of the staphylococcal PE could be developed.
The recovery of pyrogenic activity and degree
of toxin purification are shown in Table 1. A recovery of 34% activity and a final 200-fold purification were achieved. Toxin purity was assessed on the basis of several criteria. The toxin migrated as a homogeneous protein with a molecular weight of approximately 12,000 when tested by SDS-polyacrylamide gel electrophoresis (Fig. 5A), and it was homogeneous when re-electrofocused (Fig. 5B) in polyacrylamide gels (isoelectric point, 5.3). EtOH-1 staphylococcal PE (3 mg of protein/ml) reacted with hyperimmune antiserum raised against the purified toxin, using immunodiffusion to produce a single precipitin arc (Fig. 6A and B). The antiserum did not react either with the known streptococcal PE types (Fig. 6A) or with the staphylococcal enterotoxins (Fig. 6B). Also, antisera raised against either the streptococcal PE or enterotoxins did not react with the purified staphylococcal PE. The PE contained no detectable ribonucleic acid or DNA but did contain 5% carbohydrate. The sugar residues may be covalently linked to the toxin since isoelectric focusing failed to separate them from the protein. As proposed for hyaluronic acid associated with the streptococcal PE, the carbohydrate may facilitate alcohol precipitation during purification and stabilize biological activities of the toxin (5, 36). The amino acid composition of the staphylococcal PE shared characteristics associated with both the streptococcal PE and enterotoxins (Table 2). Like the streptococcal PE, the staphylococcal PE contained low levels of aromatic amino acids (12, 35), but it contained high lysine, as do the enterotoxins (7). Many of the glutamyl and aspartyl residues contained in the staphylococcal PE may be in the acid form, required to offset the lysine positive charge contribution to achieve an isoelectric point of 5.3. At a concentration of 100 ,ug/kg, the staphylococcal PE did not produce emesis in any of three young adult cats, whereas enterotoxin b (25 ug/kg) did so in all three in less than 2 h. The toxin also was not hemolytic, proteolytic, or exfoliative for newborn mice and did not exhibit coagulase activity. It was, however, a potent nonspecific lymphocyte mitogen (Fig. 7 and 8) as measured by incorporation of [3H]thymidine into DNA. The PE induced rabbit spleen cell proliferation at both 1 and 10 ,jg/well, and the maximum response was at 3 to 4 days or later. In contrast, concanavalin A, a potent thymusderived (T) lymphocyte mitogen, induced cells to proliferate, with a maximum response occur-
612
SCHLIEVERT, SCHOETTLE, AND WATSON
INFECT. IMMUN.
TOXIN
APPL .
Jim.
:.
3
4
5
pH
6 GRADIENT
7
8
9
FIG. 2. Zymogram print (45) of staphylococcal pyrogenic exotoxin subjected to preparative thin-layer isoelectric focusing with the use of a pH range of 3.5 to 10. The print was stained with Coomassie brilliant
blue R-250.
ENHANCED DEAD/ TOTAL
ENHANCED
DEAD/TOTAL
RABBIT
2.0
MOUSE
2.0
1/5
4/5 w
W a.
1.5
1.0
5/5
.
w1
L.5, 5
a. 1i0 a
~~~ ~ ~~~~3/'5
W
0/5
4O 3/5 5-
4 0.5-
FI. P . rgnctan caaiytenaclthl edtnsok yiolcrcfouigprfe 0
2 3 HOURS
4
0
2
3
4
HOURS
FIG. 3. Pyrogenicity and capacity to enhance lethal endotoxin shock by isoelectric focusing-purified staphylococcalpyrogenic exotoxin in rabbits and mice. Toxin protein doses in rabbits (five per group) were 10 p~g/kg (U), 1 pg/kg (0), and 0. 1 p~g/kg (0) intravenously. Rabbits were given 25 pig of Salmonella typhimurium endotoxin per kg (0.05 LDIIQ intravenously at 4 h. Toxin doses in mice (five per group) were 50 pg/0. 1 ml (U), 10 pug/0.1 ml (0), and 1 pig/0.1 ml (0) intravenously. Endotoxin (100 pg/mouse) was given at 4 h.
ring at 2 to 3 days. The toxin was mitogenic also for human cord blood lymphocytes (Fig. 8), with counts obtained comparable to those obtained with concanavalin A. Duplicate experiments gave similar results. DISCUSSION The staphylococcal PE isolated in this study was shown to be a potent pyrogen and nonspecific lymphocyte mitogen and had the capacity
to enhance host susceptibility to insults by endotoxin-either lethal shock or myocardial and liver damage. The PE lacked hemolytic, proteolytic, and exfoliative toxin activities and, because of charge and molecular weight differences, was distinguished from other extracellular staphylococcal proteins including: leukocidin (46), protein A (16, 38), deoxyribonuclease (1), ribonuclease (19), hyaluronidase (1), and staphylokinase (3).
VOL. 23, 1979
STAPHYLOCOCCAL PYROGENIC EXOTOXIN
Like the staphylococcal PE, the staphylococcal enterotoxins are pyrogenic (10) and mitogenic (42), and they enhance susceptibility to lethal shock (39), though myocardial and liver damage have not been reported associated with enhancement by the enterotoxins. In addition to being able to distinguish the PE from the enterotoxins on the basis of charge and molecular weight differences (7), the staphylococcal PE did not react in immunodiffusion with antisera to 2.
1.5=
613
the enterotoxins, and antisera to the staphylococcal PE did not react with the enterotoxins. The toxin did not produce emesis in young adult cats as shown previously for the enterotoxins (28) and, therefore, was not considered a new enterotoxin type. Previously, a potent lymphocyte mitogen was identified and partially characterized physicochemically from an S. aureus strain (26, 41). It is possible that this mitogen and the staphylococcal PE are the same protein or belong to a similar family. The mitogen could be purified by ethanol precipitation (70%, final volume) followed by column isoelectric focusing (26, 41). Its molecular weight was estimated to be 14,000, and it had an isoelectric point of 5.5 to 5.7. The phage group ofthe DA352 strain used to produce the mitogen was not listed. It would be of interest if both the mitogen- and staphylococcal PEproducing strains belonged to the same phage group.
Another group of researchers have demonstrated the presence of an extracellular pyrogen in culture supernatant fluids of S. aureus (4).
a
0.5-
0
2
3
4
HOU RS
FIG. 4. Development of specific immunity to the isoelectric focusing-purified staphylococcalpyrogenic exotoxin. Rabbits were made immune to the pyrogenicity of the exotoxin by giving 100 MPD-4/kg intravenously every other day for five injections. The immunized rabbits (five per group) were given either 20 MDP-4 of exotoxin per kg () or 100 MPD-3 of Salmonella Minnesota Re595 endotoxin per kg (U) intravenously. Nonimmunized control rabbits (five per group) received 20 MPD-4 of exotoxin per kg (0).
FIG. 5. Polyacrylamide gel isoelectric focusing (A) and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (B) of staphylococcal pyrogenic exotoxin. In both systems 50 pg of toxin was applied to the gels. Gels were stained with Coomassie brilliant blue R250.
TABLE 1. Purification of staphylococcal pyrogenic exotoxin Fraction
Protein (mg)
Total activity
(UU)
Specific activity (U/mg of protein)
Purification (fold)
Yield activ-
Supernatant fluid EtOH-1 .........
3.8 x 104 2.25 x 103 65
1.9 x 106 1.5 x 10" 6.5 x 105
50 670 1.0 x 104
-
-
13.4 200
79 34
IEFb-purified
.................
a One unit equals 1 MPD-4/kg. b Isoelectric focusing.
ity (%)
614
SCHLIEVERT, SCHOETTLE, AND WATSON
INFECT. IMMUN.
FIG. 6. Ouchterlony immunodiffusion of ethanol-precipitated staphylococcal pyrogenic exotoxin (EtOH-1) reacted with hyperimmune antisera raised against the isoelectric focusing-purified toxin. Frame A: Outer wells contained EtOH-1 exotoxin (TOX) at a concentration of 3 mg ofprotein/ml and streptococcalpyrogenic exotoxin types A, B, and C at concentrations of 2 mg/ml. Frame B: Outer wells contained EtOH-1 exotoxin (3 mg/ml) and staphylococcal enterotoxins A, B, C, D, and E at concentrations of 2 mg/ml. Inner wells in both frames contained antisera. All wells contain 25 ,ul of material.
TABLE 2. Amino acid composition of staphylococcal pyrogenic exotoxin Residue Percent mol Aspartyl ..................... 11.8 Threonine .................... 6.5 Serine ....................... 6.5 Glutamyilb ..................... 14.7 Proline ....................... 5.6 Glycine ...................... 15.2 Alanine ...................... 6.2 1.9 Cysteine ...................... Valine ....................... 4.2 Isoleucine .................... 3.3 Leucine ...................... 4.8 Tyrosine ..................... 1.7 Phenylalanine ................ 1.3 Lysine ....................... 10.4 Histidine ..................... 3.4 Arginine 3.5 Aspartyl includes aspartic acid and asparagine. b Glutamyl includes glutamic acid and glutamine. ......................
a
This pyrogen, although neither purified nor characterized, may be the PE described in this study, which has been purified to a high specific activity based on its pyrogenicity in rabbits. The staphylococcal PE was homogeneous when tested by SDS-polyacrylamide gel electrophoresis or reisoelectric focusing in polyacrylamide gels and when allowed to react against hyperimmune antisera. The staphylococcal PE resembles closely the streptococcal PE, both physicochemically and biologically, though they were shown to be se-
rologically distinct. The staphylococcal PE and the streptococcal PE are relatively low molecular weight (
