Staphylococcus aureus of Bacteriophage Group 2

Transcription

1 INFECTION AND IMMUNITY, Aug. 1973, p Copyright 1973 American Society for Microbiology Vol. 8, No. 2 Printed in U.S.A. Purification of Exfoliatin Produced by Staphylococcus aureus of Bacteriophage Group 2 and Its Physicochemical Properties ISAMU KONDO, SUSUMU SAKURAI, AND YASUNAGA SARAI Department of Microbiology, The Jikei University School of Medicine, Tokyo, Japan Received for publication 15 February 1973 An effective method for the isolation and purification of exfoliatin which has been recently reported by Melish and others as the staphylococcal toxin responsible for the scalded skin syndrome and the physicochemical properties of the purified toxin were described. From an active crude toxin produced by one of the clinical isolates of phage group 2, four types of toxic proteins which were all capable of causing the typical Nikolsky sign in neonatal mice were obtained and designated A, B, C, and D toxins. They had a molecular weight of about 24,000 and showed the same serological features in neutralization and precipitation tests, but were different from each other in showing a different single band with their respective mobilities in polyacrylamide disk electrophoresis. They were precipitated between ph 4.0 and 4.5 and lost their exfoliative capabilities. The resulting precipitates, however, could be solubilized in acetate buffer containing 0.5% sodium dodecyl sulfate, restoring their toxicities to almost the same extent as before. They were all stable when heated at 60 C for 60 min and at 100 C for 20 min, but lost their toxicities when heated at 100 C for 40 min. Additionally, the present authors observed that some staphylococcal strains not belonging to the typical phage group 2, isolated from patients with the scalded skin syndrome, were also capable of producing a similar but serologically unrelated exfoliative toxin. Recently several groups of workers have pointed out the close association between Staphylococcus aureus of phage group 2 and the disease of infants known as toxic epidermal necrolysis of the Ritter's type (3, 4, 7-9, 12, 13). An experimental model with newborn mice for the study of the toxic epidermal necrolysis (TEN) has been reported by Melish and Glasgow (10). Subsequently, Arbuthnott et al. (1) and Kapral and Miller (5) reported that culture filtrate of staphylococci belonging to the phage group 2 could cause a wide-spread exfoliation of the epidermis when injected subcutaneously or intraperitoneally to neonatal mice within 5 days of age. On the other hand, Melish and Glasgow (10) proposed that the generalized exfoliation in Ritter's disease and bullous impetigo were different clinical manifestations resulting from the same etiology. More recently, Kapral and Miller (5) demonstrated an extracellular product of staphylococcus responsible for the generalized exfoliation in neonatal mice and named it "exfoliatin." Arbuthnott et al. (1) also reported that epidermal necrolysis could result from the action of a diffusible toxic product of the phage group 2 staphylococci. We have also confirmed that culture filtrates of some staphylococcal strains of phage group 2, especially of phage type 71, could induce a generalized exfoliation of epidermis with the so-called Nikolsky syndrome (peeling off of the skin surface easily caused by a slight rubbing with fingertip) in neonatal mice. Furthermore, we have isolated and purified the extracellular toxic substance corresponding to Melish's exfoliatin from the culture filtrate of one of the clinical isolates which belonged to the typical phage group 2. It was investigated and fractionated into four separate proteins, all of which showed a high specific activity of exfoliatin. They had the same molecular weight (about 24,000) and similar serological features in neutralization tests as well as in Ouchterlony's precipitation tests, but were different from each other with respect to their mobilities in polyacrylamide gel disk electrophoresis. Additionally, we were able to isolate four 156

2 VOL. 8, 1973 PURIFICATION OF S. AUREUS EXFOLIATIN 157 staphylococcal strains which did not belong to the typical phage group 2 but were capable of producing an extracellular toxin similar to the above-mentioned exfoliatin, though possessing different serological features from those of the latter. The present paper will describe the evidences found, especially procedures of isolation and purification of the exfoliatin from staphylococcal strains belonging to the typical phage group 2 and the physicochemical characteristics of the resulting four purified toxic proteins, A, B, C, and D. Another paper will be presented with respect to the exfoliative toxins produced by staphylococcal isolates belonging to the phage groups other than group 2. MATERIALS AND METHODS Bacterial strains employed. Many strains of S. aureus of phage group 2, chiefly belonging to type 71, were used for this study. These are listed in Table 1, including some stock cultures maintained in our department and clinical isolates from patients with impetigo, Ritter's disease, and bacterial toxic epidermal necrolysis. Strain Zm was obtained from the 406th Medical Laboratory through the courtesy of Melvin R. Smith. This strain was chiefly used for the preparation of the purified exfoliatin. It produces coagulase, fibrinolysin, deoxyribonuclease, and a hemolysin, but not f and 6 hemolysin. It is a mannitol fermenter, and its phage type is 55/71. Culture medium. Liquid medium for toxin production contains 10 g of yeast extract, 17 g of Trypticase, TABLE 1. Examination of stock cultures and propagas of group 2 typing phage for exfoliative activity Strain Source Phage type Exfoliative 855 Stock culture 3B/3C/55 Negative 856 Stock culture 3B/3C/55 Negative 959 Stock culture 3C/55/71 Negative 1287 Stock culture 55/71 Negative 1364 Stock culture 3B/3C/55/71 Negative 1381 Stock culture 3B/3C/55/71 Negative 1523 Stock culture 71 Negative 1832 Stock culture 71 Negative 1895 Stock culture 71 Negative PS71 Phage propaga- 3C 71 Negative 3 A Phage propaga- 3A Negative 3 B Phage propaga- 3B/3C Negative 3 C Phage propaga- 3C 71 Negative 55 Phage propaga- 55/71 Negative 5 g of NaCl, and 2.5 g of K2HPO4 per 1 liter of distilled water (TY medium). Microorganisms were cultured in this medium with stirring at 37 C under an atmosphere of 10% CO2. Trypticase soy agar medium was used as the solid medium for maintaining the cultures. Mice. Neonatal mice of the ICR strain were used exclusively. Newborn mice were collected from several parents at birth and separated into groups of 9 to 12 mice per 1 female parent. Antisera. Anti-a toxin horse serum with 320 IU titer was obtained from Y. Sawai, Institute for Medical Science, Tokyo University. Anti-exfoliatin serum was obtained by the immunization of rabbits with the C preparation of exfoliatin. Rabbits weighing 2.0 to 2.5 kg received intramuscular injections of the purified preparation of exfoliatin C mixed with the same volume of Freund incomplete adjuvant (Difco). The first injection was 1.4 mg, the second was 9 mg given 10 days after the first, and the third was 15 mg without the adjuvant 3 weeks after the second immunization. Total bleedings were carried out 8 days after the last injection. In a heutralization test the serum showed a 1: 128 antibody titer. Production of exfoliatin. A fresh culture of the staphylococcal strain under investigation was prepared in the medium mentioned above for 18 h. Ten milliliters of this preculture was added to 500 ml of the same medium placed in a 2-liter Erlenmeyer flask and incubated with continuous agitation using a magnetic stirrer at 37 C for 48 h under a constant atmosphere of 10% CO2. Cultures were centrifuged at 10,000 x g for 20 min at 2 C, and the supematant fluid was removed. Filtrate of the supernatant fluid passed through a membrane filter of 0.45-gm pore size (Millipore Corp.) was assayed for the various toxins. Isolation and purification of exfoliatin. To the supernatant fluid (450-ml volume) of the culture mentioned above a small quantity of powdered ammonium sulfate totaling 153 g was gradually added within 30 min while kept in an ice bath. The resultant precipitate was removed by centrifugation at 10,000 x g for 20 min at 2 C, and 155 g of powdered ammonium sulfate was again added to the supernatant fluid. The mixture was then centrifuged again at 10,000 x g for 20 min at 2 C, and the supernatant fluid was discarded. The second precipitate was dissolved in 15 ml of 0.01 M tris(hydroxymethyl)aminomethane (Tris) -hydrochloride buffer (ph 7.5) (TH) and loaded on a Sephadex G-75 column (4 by 55 cm) equilibrated with TH buffer and eluted with the same buffer. Fractions of the effluent giving a significant optical density value at 280 nm (OD280) were pooled and dialyzed overnight against 2 liters of TH buffer at 4 C. The dialyzed solution, which contained about 20 mg of protein of crude exfoliatin, was placed on a diethylaminoethyl (DEAE)-cellulose column (1 by 20 cm) previously equilibrated with TH buffer and eluted with a linear gradient from 0 to 0.2 M NaCl. Fractions (4 ml) of effluent were collected in small tubes. The optical density of samples from each tube were obtained at 280 nm. Disk electrophoresis. For disk electrophoresis, the method of Davis was employed (11). The vertical tube (0.5 by 7.5 cm) of acrylamide was loaded with 0.3 ml

3 158 KONDO, SAKURAI, AND SARAI of a partially purified sample ( ,g) mixed together with bromophenol blue or dinitrophenollysine as the tracking dye and was run at 3 ma/gel for 1 to 1.5 h. The acrylamide gel was used at a concentration of 7.5%, and M Tris M glycine (ph 8.8) was used as the running buffer. After electrophoresis, the gels were taken out and cut longitudinally into halves along the axis. One of the halves was stained with 1% amido black to reveal the bands of protein. The corresponding portions of the other half responsible for the protein bands in the stained half were sliced out. These gel slices were eluted with distilled water at 0 C, and the eluates were examined for exfoliatin and a toxin activities. Assay for exfoliatin. Samples for testing were serially diluted fivefold with distilled water. Samples (0.05 ml) from each dilution were injected subcutaneously to groups of four to five mice with a syringe and a 27-gauge needle. Exfoliatin activity (EU/mg of protein) was calculated as the reciprocal of the minimal dose (mg of protein) capable of causing a positive Nikolsky phenomenon within 3 h in 100% of the inoculated animals. RESULTS Clinical source and phage type of the strains with exfoliative activity. Results of an examination of various stock and propagating strains of phage 2 group (Table 1) and clinical isolates from so-called scalded skin syndrome and other diseases (Table 2) are recorded for TABLE 2. Production of exfoliatin by various clinical isolates Exfoli- Strain Source Phage type ative activity A G TEN" 71 + I T TEN 6/29/42D/54/70/73 _ O H TEN 42E/47/54/70/79/ (+) 847B Z M Unknown 55/ Osteomyelitis Skin abscess 71 + O K Impetigo 3B/3C/55/71 _ A M Impetigo 29(i)/71 + W A Impetigo 71 + S Z Impetigo 7/29/42B/42E(i)/ (+) 53/54/70/79/ 847B(±+ ) SI Impetigo 71 + S U Impetigo 7/29/42B/52/54( i )/ (±) 79 T A Impetigo 71 + S H Impetigo 7(±i )/42B/52(±i )/79 (±) Y U (H) Impetigo 71 MAF Impetigo 71 + MAR Impetigo 71 + a TEN, Toxic epidermal necrolysis. INFECT. IMMUNITY their exfoliative activities in neonatal mice. As shown in Table 1 all of the old stock cultures of phage 2 group and the standard propagating strains for typing phages of group 2 were nonexfoliative. Among 17 clinical isolates including three strains from toxic epidermal necrolysis, eleven strains from impetigo, one from osteomyelitis, one from a skin abcess, and one unknown, fourteen strains indicated positive results and three were negative. One of the interesting results is that three strains, one isolated from TEN and two from impetigo, were shown to be non-exfoliative while on the contrary two strains, one isolated from a skin abscess and the other from osteomyelitis, were capable of causing generalized exfoliation. Another, more interesting result was that four isolates, one from TEN and three from cases of impetigo, demonstrated miscellaneous phage types, not belonging to phage 2 group, and possessed exfoliative capabilities when tested in neonatal mice. Detailed descriptions of the latter evidence will be reported elsewhere in a separate paper. In the present paper results of the isolation and purification of an exfoliative toxin from the Zm strain of a typical phage 2 group type, 55/71, among the active strains indicated in the table will be reported. On the other hand, no. 17 strain isolated from osteomyelitis and no. 18 strain from a skin abscess, both belonging to phage 71 type, were also positive strains, whereas IT from TEN and OK from impetigo were negative. It is of some interest that the four positive strains not belonging to the typical phage group 2 were all sensitive to phage 79 of phage group 1. Isolation of exfoliatin from the culture filtrate of the Zm strain. Crude material for exfoliatin was prepared by culturing the Zm strain in 500 ml of TY medium with agitation in a CO2 incubator at 37 C for 48 h. The culture was centrifuged and the supernatant fluid was saturated twice with ammonium sulfate as described in the section on the isolation and purification of exfoliatin. The precipitate obtained from the second saturation was dissolved in 15 ml of TH buffer. This solution showed an exfoliative activity of 83 EU per mg of protein which was a 2.6-fold increase over the initial activity of the starting material. The solution was loaded on Sephadex G-75 equilibrated with TH buffer and eluted with the same buffer. Fractions of the effluent giving positive reading at OD280 were pooled and dialyzed against 2 liters of TH buffer at 4 C. The elution profile is shown in Fig. 1. As shown in the figure, three peaks of OD280 were obtained. Exfoliatin activity was found only in the second minor peak,

4 VOL. 8, 1973 PURIFICATION OF S. AUREUS EXFOLIATIN 159 a0 *0 11 A I\ XAI I',\A/ \f Fr,t1m. s0 r \ -60 P S O,ooo~ i FIG. 1. Sephadex G-75 gel filtration of crude exfoliatin precipitated by ammonium sulfate. Sample (70 mg) was applied to Sephadex G-75 column (4 by 55 cm) and was eluted with 0.01 M Tris buffer (ph 7.5) at a flow rate of 20 ml/h. Fraction volume, 5 ml. Symbols: ----, 0D280; 0, a-hemolysin; 0, exfoliatin. together with a high activity of a hemolysin, and was not effectively separated from the latter by this procedure alone. The peak of the a hemolytic activity, however, slightly preceded that of the exfoliatin activity in this peak by three or four fraction tubes, indicating that the molecular size of exfoliatin was smaller than that of a hemolysin. Fractions of this second peak were pooled and loaded on DEAE-cellulose column. Elution of the loaded sample was carried out with a linear gradient from 0 to 0.2 M NaCl. The effluent in each fraction tube was examined at OD280 and both activities of exfoliatin and a hemolysin were tested. The results are shown in Fig. 2. The elution profile is composed of three peaks, one major and two minor at OD280. The activity of exfoliatin (indicated with a solid line and open circle) was distributed to all three peaks, especially in tubes 3 to 8 (peak 1), 13 to 18 (peak 2), and 23 to 28 (peak 3), while the a hemolysin activity (indicated by a dotted line and solid circle) was observed in the first and second peaks, especially in tubes 3 to 8 and 13 to 16, but not in the third peak. Thus, the separation of exfoliatin from a hemolysin was still unsatisfactory in this procedure. Solutions with exfoliative activity in tubes 3 to 8, 13 to 18, and 23 to 28 were pooled separately and then dialyzed against distilled water. The dialyzed samples were designated as peak I, II, and III samples and stored at -20 C before further examination. Polyacrylamide gel disk electrophoresis. Polyacrylamide gel disk electrophoresis of the three peak samples obtained by the above 0 DEAE-cellulose chromatography was performed at ph 8.8. Figure 3 shows the electrophoretic pattems of the three samples. After exposure to the necessary current, each strip of gel was taken out and cut into two longitudinal halves. Protein bands in one half were stained with 1% amido black. Five protein bands were revealed with the sample from peak I, three bands with the peak II sample, and five bands with the peak III sample. Portions in the other half of the gel strips, which corresponded to the protein bands in the stained gel strips, were cut out and extracted with distilled water. All the extracts of the protein bands were examined for the presence or absence of exfoliatin and a hemolysin activities. Results are indicated with white (exfoliatin) and black (a hemolysin) arrows (Fig. 3). As shown in the figure, exfoliatin activity was found in the third, fourth, and fifth bands in the peak I sample (peak I-3, peak I-4 and peak I-5), in the first, second, and third bands in the peak II sample (peak II-1, peak II-2, and peak II-3), and in the first, second, and third bands in the peak III sample (peak HI-1, peak III-2, and peak 111-3). However, a toxin activity was also found in the first, second, and third bands in the peak I sample. It was overlapped with the exfoliatin activity in the third band of the peak I sample. On the other hand, in respect to the mobility, the fourth and fifth bands of the peak I sample seem to correspond to the first and second bands of the peak II sample, and the first, second, and third bands of the peak II sample to FIG. 2. DEAE-cellulose chromatography of partially purified exfoliatin. Peak 2 sample obtained from Sephadex G-75 gel filtration in Fig. 1 (30 mg) was placed on a column (1 by 20 cm) equilibrated with 0.01 M Tris buffer (ph 7.5) and was eluted with a linear gradient from 0 to 0.2 M NaCI at a flow rate of 15 ml/h. Fraction volume, 4 ml. Symbols: ----,OD280; 0, a-hemolysin; 0, exfoliatin.

5 160 KONDO, SAKURAI, AND SARAI INFECT. IMMUNITY F P I P II P III LV FIG. 3. Polyacrylamide disk electrophoresis of the eluate obtained from DEAE-cellulose column chromatography. PI, PII, and PIII show the results of the sample collected from peak 1, peak 2, and peak 3 fractions on DEAE-cellulose column chromatography (Fig. 2). Black arrows indicate protein band having a-hemolytic activity, and white arrows indicate protein band having exfoliative activity. Anode end was at the bottom of the gel columns. the first, second, and third bands of the peak III sample. The identity of the respective pair of bands was confirmed after rerunning the samples isolated from these protein bands. Designations of bands A, B, C, and D were given to the respective bands: A to peak 1-3, B to peak 1-4 and peak II-1, C to peak II-2 and peak III-2, and D to peak II-3 and peak III-3. From the sample of band B, C, and D, the respective exfoliative toxins B, C, and D which were completely devoid of a hemolysin activity could easily be obtained. However, rerunning of the sample obtained from the third band of peak I sample was necessary to separate the band of purified exfoliatin A from that of the contaminating a hemolysin. For the preparation of a larger amount of toxin, the respective peak samples were loaded onto 12 acrylamide gel strips for electrophoresis, and one of them was stained after subjecting them to the necessary current to reveal the protein bands. The remaining strips were laid exactly parallel with this stained strip, and portions corresponding to the stained protein bands were cut out and eluted with distilled water. The eluates were pooled and concentrated into the volume designated for the experiment. Band A and B exfoliatins were thus purified from the sample of peak I, band C exfoliatin from the sample of peak II, and band D exfoliatin from the sample of peak III. Properties of purified exfoliatins. Figure 4

6 VOL. 8, 1973 PURIFICATION OF S. AUREUS EXFOLIATIN 161 \. A B *C D.i_.~ FIG. 4. Polyacrylamide disk electrophoresis of the purified exfoliatin type A, B, C, and D. Anode end was at the bottom of the gel columns. indicates the final result of the polyacrylamide gel disk electrophoresis of these four purified exfoliatins of band A, B, C, and D. These proteins showed respective single bands with different mobilities in the order of A, B, C, and D from slow to fast migration. From the result of examination of their exfoliative activity, the four exfoliatin bands were found to have different specific activities, respectively: A, 625; B, 2,500; C, 3,333; D, 800 exfoliatin units per mg of protein (Table 3). These exfoliatins were chromatographed on a 2.4- by 37-cm column of Sephadex G-50 (Pharmacia, Uppsala, Sweden) at 4 C equilibrated with 0.01 M TH buffer (ph 7.5). As standard markers for the molecular weight, ovalbumin (mol wt 45,000), beef pancreas chymotrypsinogen A (mol wt 25,000), and bacitracin (mol wt 1,450) were also chromatographed under the same conditions using the same columns. The molecular weights were estimated for each of the exfoliatin bands as about 24,000 when calibrated with these standard markers. The activities of these four exfoliatins were all stable for 1 month in TH buffer (ph 7.5) under TABLE 3. Indexes ofpurification for each purification stepa Specific Index Activity Protein activity Purifi- Purifica- (EU/ml) (mg/ml) EUo/mg cation tion step potein) e Culture superna tant Ammonium sulfate- 1, precipitated extract Sephadex G75 5, DEAE-cellulose P-1 12, , P-2 1, , P electrophoresis A B , C , D Disk a Abbreviations: P-1, peak 1; P-2, peak 2; P-3, peak 3; A, exfoliatin A; B, exfoliatin B; C, exfoliatin C; D, exfoliatin D.

7 *:... :; :.: is :.... * ::,;,.,.,. :::. _ :..: 1i :. f... ::. ^; ::.:.;...,... :.: 162 KONDO, SAKURAI, AND SARAI INFECT. IMMUNITY - 20 C. The toxins were irreversibly precipitated at ph 4.0 to 4.5 and lost their exfoliative..f-ii-ie.im.ehmi activities. In our preliminary experiment, however, the precipitates produced at ph 4.0 to 4.5 could be easily solubilized in a small volume of 0.1 M acetate buffer (ph 9.0) containing 0.5% sodium dodecyl sulfate, and the toxic activities.. _S_ were almost entirely restored. Heat stability. The exfoliatin in a crude culture filtrate was stable when heated at 100 C E' o for 90 min but lost its activity after heating at..... _ C for 2 h. Purified band exfoliatins were also.?'a' t. :.: found to be resistant to heat, though to a lesser extent than the former, and all of them lost their toxic activity when heated at 100 C for min, but not for 20 min. Serological natures. Antisera to the purified band C exfoliatin were prepared by immunizing rabbits with this toxin mixed with com- 0 :'. :Y.' plete Freund adjuvant. These antisera could Si neutralize the exfoliative activity of crude culture filtrates of Zm strain and the other exfoliative strains belonging to phage group 2. In addition, the activities of band A, B, and D exfoliatins were also neutralized as well as that of the homologous band C exfoliatin. In the Ouchterlony precipitation reaction with the antisera, rabbit antiserum to exfoliatin C (center well) and four FIG. 5. Ouchterlony precipitation test between the the four toxins produced a single identical line' types of exfoliatins obtained from Zm strain, A (a), B (Fig. 5), and the antisera to a hemolysin did not (b), C (c), and D (d), and the other two exfoliative neutralize the exfoliative activity of these four toxins obtained from the staphylococcal strains, toxins when mixed and tested for their biological SH(e) and SZ (f), both of which do not belong to the phage group 2. Single identical lines are seen between activities. the anti-exfoliatin serum and four types of toxins of On the other hand, preliminary experiments Zm strain A, B, C, and D, but not between the showed that the exfoliative toxins obtained antiserum and the toxins from SH and SZ strains. from the four active strains belonging to phage types other than group 2 were not related to the strains of clinical isolates, 10 of which belonged active strains of phage group 2 in either neutralizing tests or Ouchterlony precipitation reac- of which were of the, other phage groups. to the typical phage group 2 staphylococci but 4 tions. As shown in Table 2, the active strains contained the clinical isolates from Ritter's DISCUSSION The present study confirmed the evidence reported by Melish, Arbuthnott, and Kapral that some strains of S. aureus of phage 2 group produced a new type of toxin capable of causing a generalized exfoliation of epidermis in neonatal mice (5). Kapral and Miller (5) and Arbuthnott et al. (1) were able to isolate the toxin from the culture filtrate and partially purify it, whereas Melish et al. (11) could not find it in the culture filtrate but demonstrated it in the cell-free filtrate of phage group 2 staphylococci grown in dialysis sacs inserted in the peritoneal cavity of rats or rabbits. In our experiment the exfoliative toxin could be found in the culture supernatant fluids of :: :.. :.: _ disease, impetigo, skin abscess, osteomyelitis, and one strain of unknown clinical source. It should be noted that orne strain from osteomyelitis and another from a skin abscess were also capable of producing the active exfoliative toxin. The standard propagas of the typing phages of group 2 and the stock cultures in our department of old clinical isolates belonging to the phage 2 group were all found not to produce this toxin as well as some fresh clinical isolates from TEN. It is of much greater interest that three isolates, one from TEN and three from impetigo, belonging to miscellaneous phage types could produce similar exfoliative toxins which were different from the typical exfoliatin produced by phage group 2 staphylococci.

8 VOL. 8, 1973 PURIFICATION OF S. AUREUS EXFOLIATIN 163 The Zm strain was employed for the purification of exfoliatin due to the nature of this strain which produces a large quantity of this toxin and lacks the ability to produce a 6 hemolysin. Delta toxin has often been reported to be concerned with the manifestation of the scalded skin syndrome (6). One new piece of evidence observed in our study is that four types of proteins with exfoliative activity were isolated and purified from the culture supernatant fluid of the Zm strain. These were tentatively named as bands A, B, C, and D exfoliatins. These exfoliatins were all sensitive to trypsin digestion and lost their exfoliative activities. These proteins showed different mobilities in polyacrylamide gel disk electrophoresis and also different specific activities of exfoliatin. Apart from these two differences, they are all heat stable and have almost the same isoelectric point of ph 4.0 to 4.5. The molecular weight of each of these four band exfoliatins showed almost the same value, 24,000, as far as calibrated from the comparison of the results of column chromatography on Sephadex G-75 of each of the four purified samples with those of three standard markers. A more precise estimation of molecular weight and isoelectric point should be required for the final characterization of these four band exfoliatins. The exfoliative activity of the culture filtrates of Zm and the other active strains were very resistant to heat, not only to heating at 60 C for 30 min but also to heating at 100 C for 1.5 h. The purified band exfoliatins are also resistant but to a lesser degree. They were able to resist heating at 100 C for 20 min, but completely lost their activities after heating at 100 C for 40 min. The heat resistance of the exfoliatins is consistent with the data reported by Kapral and Miller (5) but not with that by Arbuthnott et al. (1). Arbuthnott reported that the exfoliatin he obtained was inactivated by heating at 60 C for 30 min. Other discrepancies between the data of others and our findings are found in the isoelectric point of this toxin and the solubility of the precipitates formed at this isoelectric point. Melish et al. (11) reported that their toxin had an isoelectric point of 7.0, while Kapral and Miller (5) reported that their toxin was irreversibly precipitated at ph 4.0 and lost the toxic activity. However, in our experiment the toxin was precipitated at about ph 4.0 to 4.5 and the resulting precipitate could be solubilized in a small volume of acetate buffer containing sodium dodecyl sulfate after the restoration of its exfoliative activity. Although it is not clear where these discrepancies came from, those that should be considered are the difference in the strains employed and the difference in the methods used for purification of the toxin. As for the latter, at least in the final step of purification, we utilized the polyacrylamide gel disk electrophoresis, Arbuthnott et al. (1) used the treatment with hydroxyapatite, Kapral and Miller (5) used the column chromatography with DEAE Sephadex, and recently Melish et al. (11) used the isoelectric focusing. Successful separation of four band exfoliatins and complete removal of the contamination with a toxin are considered at least to be the merits of the usage of the gel disk electrophoresis. Results of serological and biological experiments indicated that these four exfoliatins were all quite different from a toxin, 6 hemolysin, and enterotoxin. These four band toxins are all neutralized by the specific antisera to the purified band C exfoliatin, but not by anti-a toxin serum. They are all unable to show any lytic reaction to human, rabbit, and sheep red blood cells. On the other hand, the antiserum to the band C exfoliatin could neutralize the exfoliative activity of all our active strains of phage group 2 staphylococci, but not that of the four strains of the miscellaneous phage types. Therefore, the exfoliatins produced by phage group 2 staphylococci seem to be homologous at least in their serological features, and there seem to be other kind(s) of exfoliatins serologically distinguishable from the former, which are produced by staphylococci not belonging to phage group 2. LITERATURE CITED 1. Arbuthnott, J. P., J. Kent, A. Lyell, and C. G. Gemmell Toxic epidermal necrolysis produced by an extracellular product of staphylococcus aureus. Brit. J. Dermatol. 85: Davis, B. J Method and application to human serum proteins. Ann. N. Y. Acad. Sci. 121: Holzel, A., and S. I. Jacobs Toxische epidermale Nekrolyse: Das Verbriings-Syndrom. Schweiz. Med. Wochenschr. 96: Jefferson, J Lyell's toxic epidermal necrolysis: a staphylococcal aetiology? Brit. Med. J. 2: Kapral, F. A., and M. M. Miller Product of staphylococcus aureus responsible for the scalded skin syndrome. Infect. Immunity 4: Kreger, A. S., K. S. Kim, F. Zaboretzky, and A. W. Bernheimer Purification and properties of staphylococcal delta hemolysin. Infect. Immunity 3:

9 164 KONDO, SAKURAI, AND SARAI INFECT. IMMUNITY 7. Lowney, E. D., J. V. Baublis, G. M. Kreye, E. R. Harrell, and A. R. McKenzie The scalded skin syndrome in small children. Arch. Dermatol. 95: Lyell, A A review of toxic epidermal necrolysis in britain. Brit. J. Dermatol. 79: Lyell, A., H. M. Dick, and J. 0. Alexander Outbreak of toxic epidermal necrolysis associated with staphylococci. Lancet 1: Melish, M. E., and L. A. Glasgow The staphylococcal scalded skin syndrome. N. Engi. Med. J. 282: Melish, M. E., L. A. Glasgow, and M. D. Turner The staphylococcal scalded skin syndrome: isolation and partial characterization of the exfoliative toxin. J. Infect. Dis. 125: Samuels, M. J Toxic epidermal necrolysis: a report of 42 cases. Brit. J. Dermatol. 79: Tyson, R. G., S. C. Ushinski. and R. Kisilevskv Toxic epidermal necrolysis (the scalded skin syndrome). Amer. J. Dis. Child. 3: