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Purification of Staphylococcal Exfoliative Toxin by High Pressure Liquid Chromatography
Zbl. Bakt. 273, 5-11 (1990) © Gustav Fischer Verlag, StuttgartlNew York
Purification of Staphylococcal Exfoliative Toxin by High Pressure Liquid Chromatography MOTOYUKI SUGAI, SHINGO INOUE, TAKAMUNE HINO, MASAO KUWABARAl, YEONG-MAN HONG 2 , YOICHIRO MIYAKE, and HIDEKAZU SUGINAKA Department of Microbiology, Hiroshima University, School of Dentistry, Hiroshima, Japan 1 The 3rd Department of Internal Medicine, Hiroshima Prefectural Hospital, Hiroshima 734,Japan 2 Cellular Technology Institute, Ohtsuka Pharmaceutical Co., Ltd. Tokushima 771-01, Japan
With 3 Figures· Received June 16, 1989 . Accepted in revised form September 20, 1989
Abstract Exfoliative toxin (ET) isolated from a clinical strain of Staphylococcus aureus was purified to homogeneiety, using a 3-step HPLC system. NHr terminal20 amino residues of purified ET was found to be identical with ETA of S. aureus TA (7), S. aureus TC16 (9) and S. aureus ZM (10), but stability of purified ET was completely different from that of ETA. This purification system gave a high yield of pure ET, which exhibited higher purity than specimens purified by more complicated and time-consuming procedures. It is useful for small-scale purification for the comparative study of ET and easy to scale up for preparative purification. Zusammenfassung Aus einem klinischen Isolat von Staphylococcous aureus isoliertes exfoliatives Toxin (ET) wurde in einem 3stufigen HPLC-Verfahren gereinigt. Die aminoterminalen Aminosauren des gereinigten ET sind identisch mit ETA von S. aureus TA (7), S. aureus TC 16 (9) und S. aureus ZM (10), aber die Stabilitat des gereinigten ET unterschied sich deutlich von der des ETA. Das Reinigungsverfahren lieferte eine hohe Ausbeute an reinerem ET als mit komplizierteren und zeitaufwendigeren Methoden gewonnene Proben. Es eignet sich fur die Reinigung kleiner Mengen, kann aber auch im praparativen Magstab eingesetzt werden.
Introduction Staphylococcal exfoliative toxin (ET) is an exotoxin which may cause an exfoliation of the epidermis in infected infants (8). The toxin has been purified in a number of laboratories (1, 2, 4, 5, 6, 8) and several reports have dealt with differences among
6
M. Sugai et al.
types of ET. It has been shown that at least two serologically distinct toxins, ETA and ETB, exist (3, 6). They differ in amino acid composition, heat sensitivity and metal ion dependence although they share the same toxic activity. Reported protocols for ET purification require highly productive strains (2, 3, 5, 6) because of low recovery yields; these procedures were time-consuming. The purification procedures described to date have yielded a maximum of 34% of the homogeneous biologically active ET (2). These procedures seem to be inadequate to purify ET from clinically isolated st~ains including low ET producing strains for comparative study. In recent years, highperformance liquid chromatography (HPLC) techniques have become accepted procedures for biochemical problems, but a classical reversed-phase HPLC technique is of limited use in biological applications due to extreme pH values and the use of organic solvents. We have designed a purification scheme for ET utilizing HPLC systems under biological conditions, e. g. normal water buffer systems. In this study, we describe an improved method based on the principles described previously for a rapid and highyield purification of ET from a clinical strain and characterize the purified ET preparation.
Material and Methods
Bacterial strain. Staphylococcus au reus E-1 was isolated from skin lesions of impetigo patients (11). It is capable of causing a general exfoliation of the epidermis when inoculated subcutaneously into newborn mice. It produces alpha-hemolysin and coagulase. Growth conditions. S. aureus E-1 exponentially growing in TY medium (10 g yeast extract, 17 g T rypticase, 5 g NaCl, and 2.5 g K2HP0 4 per liter of distilled water) was inoculated into 2 liters of the same fresh medium and incubated with continuous agitation for 24 h in a constant atmosphere of 30% CO 2 at 37°C. ET bioassay. ET activities were assayed in a newborn mouse bioassay system (8). 1-2day-old CD-I mice were utilized. Test solutions diluted in O.OlM phosphate-buffered saline (PBS, pH 7.1) were administered to groups of 3 mice each by s.c. injection (0.03 ml). In the purification steps, each fraction was assayed and fractions causing a positive Nicholsky sign within 2 h were designated as ET fractions. Samples known to contain hemolytic activity were mixed with the appropriate amount of antiserum (Wellcome Research Laboratories, Beckenham, England) to neutralize the hemolysin prior to testing. One unit was defined as the amount of toxin causing a positive Nicholsky sign (peeling of the skin upon slight rubbing) in 100% of the test animals at 2 h. Assay for hemolysin. Hemolytic activity of test solutions was assayed using a microtiter plate method as previously described (3). Preparation of concentrated culture filtrate (CCF). Bacterial cultures were centrifuged at 10000 g for 20 min at 2°C. The supernatant fluid was passed through a membrane filter which had a pore size of 0.22 !-tm (Millipore Corp., Bedford, MA, USA) and concentrated by using a Pellicon Lab Cassette (Millipore Corp.) with 4 packets of PTGC OLC 05 membranes (Millipore Corp.) at 4°C. After a 50-fold concentration, the material was dialyzed against 0.05 M phosphate buffer (pH 5.0) and stored at -20°C until use. The stored CCF did not lose its exfoliative activity within one year, while purified toxin rapidly lost its activity at -20°C. Purification procedure. HPLC was performed on a TOYOSODA liquid chromatography system equipped with MODEL HLC-803D solvent delivery pumps, a MODEL GE4 automated gradient programmer and UV8 model II absorbance detector (Toso, Tokyo, Japan) to allow the detection of wavelengths in the range 214-436 nm. The cation exchange-HPLC column was a TSKgel SP 5PW (7.5 mm i. d. X 75 mm long) (Toso), the hydroxyapatiteHPLC column was a PENTAX PEC101 (7.5 mm i.d. X 75 mm long) (PENTAX, Tokyo,
Purification of an Exfoliative Toxin by HPLC
7
Japan) and gel permeation-HPLC column was TSKgel G3000 SW (7.5 mm i. d. x 600 mm long) (Toso). The CCF was subjected to cation exchange-HPLC to collect active fractions, which were then applied to hydroxyapatite-HPLC. The bioactive fraction eluted from the hydroxyapatite column was collected and subsequently applied to gel permeation-HPLC. Protein determination. Protein was determined by a dye binding method (Bio-Rad Protein Assay, Bio-Rad Lab, Richmond, CA USA) using bovine serum albumin as a standard. Antisera. Purified toxin (490 f!g) in I ml of PBS emulsified with an equal volume of Freund's complete adjuvant (Difco Laboratories, Detroit, Mich USA) was injected into the footpads of 2 kg rabbits. 2 and 4 weeks after the first injection, the rabbits were injected intravenously with 100 f!g of the purified toxin in PBS. Antiserum was obtained 5 weeks after the first injection for use in the immunoblotting analysis. SDS-PAGE and immunoblot analysis. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed in a 10°/r, gel by the method of Weber and Osborn (13). Protein samples were denatured by heating at 100°C for 1 min in a solution of 1 % SDS and 0.1 % ~-mercaptoethanol. After electrophoresis, the gel was stained with a silver stain kit (WAKO, Osaka, Japan). The molecular weight of the ET was estimated by use of molecular weight markers (Bio Rad Lab.). For immunoblotting, the purified toxin preparations were transferred electrophoretic-ally to a nitrocellulose sheet (12). After the blotting, the sheet was soaked in PBS containing 2 'X, BSA for 2 h. The sheet was then transferred to an anti-ET antiserum solution diluted with PBS to approximately 1 mg/ml and incubated for 1 hat 3rc. The sheet was washed with PBS containing 0.05% Tween 20 and then treated for 1 h with peroxidase-conjugated ann-rabbit IgG (Fah)'2 diluted lOOO-fold with PBS. After the sheet had been washed, the hound peroxidase was allowed to react on4-chloro-naphtol (0.5 mg/ml)-0.003% H 2 0 2 III Tris-HCl buffer (pH 7.S). Histological examination. Sections of the epidermis from the control and toxin-injected mice were examined by light microscopy. Tissues were embedded in paraffin, sectioned (6 f!m thick), and stained WIth hematoxylin and eosin according to a conventional method. Amino acid sequence analysis. Samples were degraded with an automated gas-phase protein sequencer (model 470A; Applted Biosystems, Foster City, CA, USA) according to operation program 02RPTH provided for the sequencer. PTH amino acids liberated were identified by HPLC on an Ultrasphere ODS column (2 x 250 mm, Beckman Instruments, Palo Alto, CA, USA) at 49 "(: with a 0.3 mUmin flow. Solvent A contained 9% acetonitrile, while solvent B contained SOD/" acetonitrile. Solvents were composed of 0.1 % TFA adjusted to pH 4.9 with 20 mM sodium acetate. The gradient programme used was as follows; O%B for 0.5 min, from O%B to 35'Yc,B in 1.5 min, from 35%B to 78%B in 4 min, 78%B for 14 min, from 78%B to 100%B III I min, I OO%B for I min, from 100%B in 1 min, O%B for 12 min to get initial conditions. The eluates were monitored simultaneously at 269 and 322 nm.
Results
Ion-exchange HPLC CCF was loaded onto a TSKgel SP 5PW cation exchange column equilibrated with 50 mM phosphate buffer, pH 5.0. The column was washed with the starting buffer until the major portions of the unbound proteins had passed. ET activity bound to the column was then eluted by a linear gradient from the starting buffer to 50 mM phosphate buffer, pH 5.0 containing 0.5 M NaCI. Fractions with exfoliative activities (0.2-0.35 M NaCl) were pooled and dialyzed against 10 mM phosphate buffer, pH 6.8. Analysis of the fractions from the cation exchange column indicated that there was almost no contaminating hemolytic activity in the pooled fractions.
8
M. Sugai et al.
Hydroxyapatite HPLC The dialysate was loaded onto PENTAX PEC101 equilibrated with 10 mM phosphate buffer, pH 6.8. Exfoliative activity bound to this column was eluted by a linear gradient from 10 mM to 500 mM phosphate buffer. The exfoliative activity was eluted in the first major peak (Fig. 1). Trace amounts of contaminating hemolytic activity were completely removed at this purification step.
Gel permeation HPLC The active peak fractions from PENTAX PEC101 were loaded onto a TSKgel G3000 SW column. Elution was made with 10 mM phosphate buffer, pH 7.0 containing O.lM Na l S04 at a flow rate of 0.5 ml/min. A pattern of gel-permeation HPLC showed a single symmetrical peak of ET activity (Fig. 2). The ET from this peak was found to be homogeneous in SDS-PAGE since only one protein band stained with silver (Fig. 3a). The homogeneiety of this preparation was also confirmed by the immunoblotting analysis, and a single peroxidase-positive band with a mobility corresponding to the purified ET was recognized (Fig. 3b).
Sequence analysis The results of the automated sequence of the ET from the main peak of GPC-HPLC suggested the following N-terminal sequence: Glul-VaI2-Ser3-Ala4-Glu5-Glu6-Ile7-Lys8Lys9-His 10-Glu 11_Glu 12_ Lys 13 _Trpl4-Asn 15 _Lysl6Tyrl7 _Tyr18 _Glyl9 _VallO.
Properties The activity of the purified ET was completely lost within 1 week in PBS at -20°C. Purified ET lost its toxic activity when heated at 60°C for 30 min.
0.5
UJ
U
z
co a:
Cl U)
co
o
10
20
30
FRACTION NUMBER
40
Fig. 1. Chromatography of ET by hydroxyapatite HPLC. Active fractions from TSKgel SP 5PW were pooled and applied to a hydroxyapatite column. Column: PENTAX PEClOl run at room temperature at 60 mllh. Solvent A: 10 mM phosphate buffer (pH 6.9); Solvent B: 500 mM phosphate buffer (pH 6.9). Gradient: 0--100% Bin 60 min. The horizontal bar indicates the pooled ET fraction.
Purification of an Exfoliative Toxin by HPLC
9
0.08 E
c:
C)
co N
ti' LU
U
Z
<{
co 0::
o
en co
«
l~l~ o 10 20 FRACTION NUMBER
30
Fig. 2. Chromatography of ET by GPC-HPLC. Pooled fractions from PENTAX PEC101 were applied in sequential runs to the column for gel permeation chromatography. Column: TSKgel G3000 SW run at room temperature at 30 mllh. Solvent: 10 mM phosphate buffer containing 100 mM Na2S04 (pH 7.0).
97.4--66.242.7-
31
---+
21.5 ---+
(I) ") Fig. 3. SDS-polyacrylamide gel electrophoresis of purified ET (a) stained with silver stain kit, (b)transferred to nitrocellulose and immunologically detected with anti-ET antiserum (see Materials and Methods). Standard Mr values are indicated by the arrows (x10 3 ).
Histological findings Sections of the control showed no pathological lesions. Sections of the skin injected with the purified ET showed typical subcorneal splitting in the epidermis. These findings were first evident at 2 h after injection of 0.4 Ilg of purified ET.
10
M. Sugai et al. Discussion
In the present report, an improved scheme for the purification of staphylococcal ET from clinically isolated S. aureus is described. We have applied HPLC methods including ion exchange-HPLC, hydroxyapatite-HPLC and GPC-HPLC for the purification of ET. With these methods, ET can be purified very efficiently and with a high degree of both purity and recovery (Table 1). Table 1 shows that the toxic activity recovered was higher than that in the CCF. The exact reason of this is unknown, but it might be possible that some inhibitor(s) contained in the CCF had been eliminated through purification steps. The ET purified from gel permeation HPLC was found to be homogeneous by the following criteria: (i) single symmetrical peak by size exclusion in HPLC (Fig. 2), (ii) single band by SDS-PAGE analysis (Fig. 3a) with silver staining and immunoblotting analysis (Fig. 3b), (iii) single amino acid sequence (described below) and (iv) high specific activity in ET bioassay (Table 1) and histological examination. The molecular weight of purified ET was determined to be 32000 by SDS-PAGE analysis (Fig. 3). NHrterminal amino acid sequences were identical to those of ETA from strains TA, TC16 and ZM. However, the purified toxin has different properties of heat stability and stability under freezing. Our purified toxin was labile at -20°C and -80 0c. This lability was not improved when stored in PBS containing 50% glycerol. These properties are somewhat similar to those of ETB (6). Earlier purification studies were performed with high-ET-producing staphylococci (2, 5, 6). This has prompted many investigators to purify ET from normaVlow ET producing clinical strains with low biological activity. By using the protocol described in this report, highly purified ET could be obtained from normal ET producing strains without difficulty. The availability of these techniques will permit the rapid purification of ET from clinical strains and is useful for comparative studies of ET.
Table 1. Summary of purification Sample
Volume (ml)
Protein ([.tg/ml)
Specific activity
% Recovery Purification
Factor
(Uf!.tg)
Crude (CCF) SP 5PW PECI0l G3000
36.5 18 12 6
190 187 118 58.7
0.132 1.064 1.695 3.450
100 394.2 263.2 131.1
1.00 8.06 12.84 26.14
References
1. Arbuthnott, j. R., J. Kent, A. Lyell, and C. G. Gemmell: Toxic epidermal necrolysis produced by an extracellular product of Staphylococcus aureus. Brit. J. Dermatol. 85 (1971) 145-149 2. Johnson, A. D., j. F. Metzger, and L. Spero: Production, purification and chemical characterization of Staphylococcus aureus exfoliative toxin. Infect. Immun. 12 (1975) 1206-1210
Purification of an Exfoliative Toxin by HPLC
11
3. Johnson, A. D., L. Spero, S. C. Jeffrey, and B. T. Decocco: Purification and characterization of different types of exfoliative toxin from Staphylococcus aureus. Infect. Immun. 24 (1979) 679-684 4. Kapral, F. A. and M. M. Miller: Product of Staphylococcus au reus responsible for the scalded skin syndrome. Infect. Immun. 4 (1971) 541-545 5. Kondo, 1., S. Sakurai, and Y. Sarai: Purification of exfoliatin produced by Staphylococcus aureus of bacteriophage group 2 and its physicochemical properties. Infect. Immun. 8 (1973) 156-164 6. Kondo, l., S. Sakurai, and Y. Sarai: New type of exfoliatin obtained from staphylococal
strains belonging to phage groups other than group II, isolated from patients with impetigo and Ritter's disease. Infect. Immun. 10 (1974) 851-861 7. Lee, C. Y.,J..J. Schmidt, A. D. Johnson- Winegar, L. Spero, and.J..J. landolo: Sequence determination and comparison of the exfoliative toxin A and toxin B genes from Staphylococcus aureus. J. Bact. 169 1987) 3904--3909 8. Melish, M. E. and L. A. Glasgow: The staphylococcal scalded skin syndrome: development of an experimental model. New Engl. J. Med. 282 (1970) 1114--1119. 9. O'Toole, P. W. and T.f. /-oster: Nucleotide sequence of the epidermolytic toxin A gene of Staphylococcus aureus. J. Bact. 169 (1987) 3910-3915 10. Sakurai, S., H. Suzuki, and I. Kondo: DNA sequencing of the eta gene coding for staphylococcal exfoliative toxin serotype A. J. Gen. Microbiol. 134 (1988) 711-717 11. Sugai, M., T. Enomoto, Y. MIyake, and H. Suginaka: Extracellular products of Staphylococcus aureus reversiblv ll1hibit the terminal differentiation of cultured mouse epidermal cells. Cell Struct. Funet. 12 i 1987) 395-399 12. Towbin, H., T. Stahelin, and J. Gordon: Electrophoretic transfer of proteins from polyacrylamide gels on nitrocellulose sheets: procedure and some applications. Proc. Nat!' Acad. Sci. USA 76 (1979) 4350-4354 13. Weber, K. and M. Osborn: The reliability of molecular weight determinations by dodecyl sulfate polvacrylamide gel electrophoresis. J. BioI. Chern. 244 (1969)
4406-4412
Motoyuki Sugai, Department of Microbiology, Hiroshima University, School of Dentistry, 1-2-3 Kasumi, Minami-ku o Hiroshima 734, Japan
Purification of Staphylococcal Exfoliative Toxin by High Pressure Liquid Chromatography MOTOYUKI SUGAI, SHINGO INOUE, TAKAMUNE HINO, MASAO KUWABARAl, YEONG-MAN HONG 2 , YOICHIRO MIYAKE, and HIDEKAZU SUGINAKA Department of Microbiology, Hiroshima University, School of Dentistry, Hiroshima, Japan 1 The 3rd Department of Internal Medicine, Hiroshima Prefectural Hospital, Hiroshima 734,Japan 2 Cellular Technology Institute, Ohtsuka Pharmaceutical Co., Ltd. Tokushima 771-01, Japan
With 3 Figures· Received June 16, 1989 . Accepted in revised form September 20, 1989
Abstract Exfoliative toxin (ET) isolated from a clinical strain of Staphylococcus aureus was purified to homogeneiety, using a 3-step HPLC system. NHr terminal20 amino residues of purified ET was found to be identical with ETA of S. aureus TA (7), S. aureus TC16 (9) and S. aureus ZM (10), but stability of purified ET was completely different from that of ETA. This purification system gave a high yield of pure ET, which exhibited higher purity than specimens purified by more complicated and time-consuming procedures. It is useful for small-scale purification for the comparative study of ET and easy to scale up for preparative purification. Zusammenfassung Aus einem klinischen Isolat von Staphylococcous aureus isoliertes exfoliatives Toxin (ET) wurde in einem 3stufigen HPLC-Verfahren gereinigt. Die aminoterminalen Aminosauren des gereinigten ET sind identisch mit ETA von S. aureus TA (7), S. aureus TC 16 (9) und S. aureus ZM (10), aber die Stabilitat des gereinigten ET unterschied sich deutlich von der des ETA. Das Reinigungsverfahren lieferte eine hohe Ausbeute an reinerem ET als mit komplizierteren und zeitaufwendigeren Methoden gewonnene Proben. Es eignet sich fur die Reinigung kleiner Mengen, kann aber auch im praparativen Magstab eingesetzt werden.
Introduction Staphylococcal exfoliative toxin (ET) is an exotoxin which may cause an exfoliation of the epidermis in infected infants (8). The toxin has been purified in a number of laboratories (1, 2, 4, 5, 6, 8) and several reports have dealt with differences among
6
M. Sugai et al.
types of ET. It has been shown that at least two serologically distinct toxins, ETA and ETB, exist (3, 6). They differ in amino acid composition, heat sensitivity and metal ion dependence although they share the same toxic activity. Reported protocols for ET purification require highly productive strains (2, 3, 5, 6) because of low recovery yields; these procedures were time-consuming. The purification procedures described to date have yielded a maximum of 34% of the homogeneous biologically active ET (2). These procedures seem to be inadequate to purify ET from clinically isolated st~ains including low ET producing strains for comparative study. In recent years, highperformance liquid chromatography (HPLC) techniques have become accepted procedures for biochemical problems, but a classical reversed-phase HPLC technique is of limited use in biological applications due to extreme pH values and the use of organic solvents. We have designed a purification scheme for ET utilizing HPLC systems under biological conditions, e. g. normal water buffer systems. In this study, we describe an improved method based on the principles described previously for a rapid and highyield purification of ET from a clinical strain and characterize the purified ET preparation.
Material and Methods
Bacterial strain. Staphylococcus au reus E-1 was isolated from skin lesions of impetigo patients (11). It is capable of causing a general exfoliation of the epidermis when inoculated subcutaneously into newborn mice. It produces alpha-hemolysin and coagulase. Growth conditions. S. aureus E-1 exponentially growing in TY medium (10 g yeast extract, 17 g T rypticase, 5 g NaCl, and 2.5 g K2HP0 4 per liter of distilled water) was inoculated into 2 liters of the same fresh medium and incubated with continuous agitation for 24 h in a constant atmosphere of 30% CO 2 at 37°C. ET bioassay. ET activities were assayed in a newborn mouse bioassay system (8). 1-2day-old CD-I mice were utilized. Test solutions diluted in O.OlM phosphate-buffered saline (PBS, pH 7.1) were administered to groups of 3 mice each by s.c. injection (0.03 ml). In the purification steps, each fraction was assayed and fractions causing a positive Nicholsky sign within 2 h were designated as ET fractions. Samples known to contain hemolytic activity were mixed with the appropriate amount of antiserum (Wellcome Research Laboratories, Beckenham, England) to neutralize the hemolysin prior to testing. One unit was defined as the amount of toxin causing a positive Nicholsky sign (peeling of the skin upon slight rubbing) in 100% of the test animals at 2 h. Assay for hemolysin. Hemolytic activity of test solutions was assayed using a microtiter plate method as previously described (3). Preparation of concentrated culture filtrate (CCF). Bacterial cultures were centrifuged at 10000 g for 20 min at 2°C. The supernatant fluid was passed through a membrane filter which had a pore size of 0.22 !-tm (Millipore Corp., Bedford, MA, USA) and concentrated by using a Pellicon Lab Cassette (Millipore Corp.) with 4 packets of PTGC OLC 05 membranes (Millipore Corp.) at 4°C. After a 50-fold concentration, the material was dialyzed against 0.05 M phosphate buffer (pH 5.0) and stored at -20°C until use. The stored CCF did not lose its exfoliative activity within one year, while purified toxin rapidly lost its activity at -20°C. Purification procedure. HPLC was performed on a TOYOSODA liquid chromatography system equipped with MODEL HLC-803D solvent delivery pumps, a MODEL GE4 automated gradient programmer and UV8 model II absorbance detector (Toso, Tokyo, Japan) to allow the detection of wavelengths in the range 214-436 nm. The cation exchange-HPLC column was a TSKgel SP 5PW (7.5 mm i. d. X 75 mm long) (Toso), the hydroxyapatiteHPLC column was a PENTAX PEC101 (7.5 mm i.d. X 75 mm long) (PENTAX, Tokyo,
Purification of an Exfoliative Toxin by HPLC
7
Japan) and gel permeation-HPLC column was TSKgel G3000 SW (7.5 mm i. d. x 600 mm long) (Toso). The CCF was subjected to cation exchange-HPLC to collect active fractions, which were then applied to hydroxyapatite-HPLC. The bioactive fraction eluted from the hydroxyapatite column was collected and subsequently applied to gel permeation-HPLC. Protein determination. Protein was determined by a dye binding method (Bio-Rad Protein Assay, Bio-Rad Lab, Richmond, CA USA) using bovine serum albumin as a standard. Antisera. Purified toxin (490 f!g) in I ml of PBS emulsified with an equal volume of Freund's complete adjuvant (Difco Laboratories, Detroit, Mich USA) was injected into the footpads of 2 kg rabbits. 2 and 4 weeks after the first injection, the rabbits were injected intravenously with 100 f!g of the purified toxin in PBS. Antiserum was obtained 5 weeks after the first injection for use in the immunoblotting analysis. SDS-PAGE and immunoblot analysis. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed in a 10°/r, gel by the method of Weber and Osborn (13). Protein samples were denatured by heating at 100°C for 1 min in a solution of 1 % SDS and 0.1 % ~-mercaptoethanol. After electrophoresis, the gel was stained with a silver stain kit (WAKO, Osaka, Japan). The molecular weight of the ET was estimated by use of molecular weight markers (Bio Rad Lab.). For immunoblotting, the purified toxin preparations were transferred electrophoretic-ally to a nitrocellulose sheet (12). After the blotting, the sheet was soaked in PBS containing 2 'X, BSA for 2 h. The sheet was then transferred to an anti-ET antiserum solution diluted with PBS to approximately 1 mg/ml and incubated for 1 hat 3rc. The sheet was washed with PBS containing 0.05% Tween 20 and then treated for 1 h with peroxidase-conjugated ann-rabbit IgG (Fah)'2 diluted lOOO-fold with PBS. After the sheet had been washed, the hound peroxidase was allowed to react on4-chloro-naphtol (0.5 mg/ml)-0.003% H 2 0 2 III Tris-HCl buffer (pH 7.S). Histological examination. Sections of the epidermis from the control and toxin-injected mice were examined by light microscopy. Tissues were embedded in paraffin, sectioned (6 f!m thick), and stained WIth hematoxylin and eosin according to a conventional method. Amino acid sequence analysis. Samples were degraded with an automated gas-phase protein sequencer (model 470A; Applted Biosystems, Foster City, CA, USA) according to operation program 02RPTH provided for the sequencer. PTH amino acids liberated were identified by HPLC on an Ultrasphere ODS column (2 x 250 mm, Beckman Instruments, Palo Alto, CA, USA) at 49 "(: with a 0.3 mUmin flow. Solvent A contained 9% acetonitrile, while solvent B contained SOD/" acetonitrile. Solvents were composed of 0.1 % TFA adjusted to pH 4.9 with 20 mM sodium acetate. The gradient programme used was as follows; O%B for 0.5 min, from O%B to 35'Yc,B in 1.5 min, from 35%B to 78%B in 4 min, 78%B for 14 min, from 78%B to 100%B III I min, I OO%B for I min, from 100%B in 1 min, O%B for 12 min to get initial conditions. The eluates were monitored simultaneously at 269 and 322 nm.
Results
Ion-exchange HPLC CCF was loaded onto a TSKgel SP 5PW cation exchange column equilibrated with 50 mM phosphate buffer, pH 5.0. The column was washed with the starting buffer until the major portions of the unbound proteins had passed. ET activity bound to the column was then eluted by a linear gradient from the starting buffer to 50 mM phosphate buffer, pH 5.0 containing 0.5 M NaCI. Fractions with exfoliative activities (0.2-0.35 M NaCl) were pooled and dialyzed against 10 mM phosphate buffer, pH 6.8. Analysis of the fractions from the cation exchange column indicated that there was almost no contaminating hemolytic activity in the pooled fractions.
8
M. Sugai et al.
Hydroxyapatite HPLC The dialysate was loaded onto PENTAX PEC101 equilibrated with 10 mM phosphate buffer, pH 6.8. Exfoliative activity bound to this column was eluted by a linear gradient from 10 mM to 500 mM phosphate buffer. The exfoliative activity was eluted in the first major peak (Fig. 1). Trace amounts of contaminating hemolytic activity were completely removed at this purification step.
Gel permeation HPLC The active peak fractions from PENTAX PEC101 were loaded onto a TSKgel G3000 SW column. Elution was made with 10 mM phosphate buffer, pH 7.0 containing O.lM Na l S04 at a flow rate of 0.5 ml/min. A pattern of gel-permeation HPLC showed a single symmetrical peak of ET activity (Fig. 2). The ET from this peak was found to be homogeneous in SDS-PAGE since only one protein band stained with silver (Fig. 3a). The homogeneiety of this preparation was also confirmed by the immunoblotting analysis, and a single peroxidase-positive band with a mobility corresponding to the purified ET was recognized (Fig. 3b).
Sequence analysis The results of the automated sequence of the ET from the main peak of GPC-HPLC suggested the following N-terminal sequence: Glul-VaI2-Ser3-Ala4-Glu5-Glu6-Ile7-Lys8Lys9-His 10-Glu 11_Glu 12_ Lys 13 _Trpl4-Asn 15 _Lysl6Tyrl7 _Tyr18 _Glyl9 _VallO.
Properties The activity of the purified ET was completely lost within 1 week in PBS at -20°C. Purified ET lost its toxic activity when heated at 60°C for 30 min.
0.5
UJ
U
z
co a:
Cl U)
co
o
10
20
30
FRACTION NUMBER
40
Fig. 1. Chromatography of ET by hydroxyapatite HPLC. Active fractions from TSKgel SP 5PW were pooled and applied to a hydroxyapatite column. Column: PENTAX PEClOl run at room temperature at 60 mllh. Solvent A: 10 mM phosphate buffer (pH 6.9); Solvent B: 500 mM phosphate buffer (pH 6.9). Gradient: 0--100% Bin 60 min. The horizontal bar indicates the pooled ET fraction.
Purification of an Exfoliative Toxin by HPLC
9
0.08 E
c:
C)
co N
ti' LU
U
Z
<{
co 0::
o
en co
«
l~l~ o 10 20 FRACTION NUMBER
30
Fig. 2. Chromatography of ET by GPC-HPLC. Pooled fractions from PENTAX PEC101 were applied in sequential runs to the column for gel permeation chromatography. Column: TSKgel G3000 SW run at room temperature at 30 mllh. Solvent: 10 mM phosphate buffer containing 100 mM Na2S04 (pH 7.0).
97.4--66.242.7-
31
---+
21.5 ---+
(I) ") Fig. 3. SDS-polyacrylamide gel electrophoresis of purified ET (a) stained with silver stain kit, (b)transferred to nitrocellulose and immunologically detected with anti-ET antiserum (see Materials and Methods). Standard Mr values are indicated by the arrows (x10 3 ).
Histological findings Sections of the control showed no pathological lesions. Sections of the skin injected with the purified ET showed typical subcorneal splitting in the epidermis. These findings were first evident at 2 h after injection of 0.4 Ilg of purified ET.
10
M. Sugai et al. Discussion
In the present report, an improved scheme for the purification of staphylococcal ET from clinically isolated S. aureus is described. We have applied HPLC methods including ion exchange-HPLC, hydroxyapatite-HPLC and GPC-HPLC for the purification of ET. With these methods, ET can be purified very efficiently and with a high degree of both purity and recovery (Table 1). Table 1 shows that the toxic activity recovered was higher than that in the CCF. The exact reason of this is unknown, but it might be possible that some inhibitor(s) contained in the CCF had been eliminated through purification steps. The ET purified from gel permeation HPLC was found to be homogeneous by the following criteria: (i) single symmetrical peak by size exclusion in HPLC (Fig. 2), (ii) single band by SDS-PAGE analysis (Fig. 3a) with silver staining and immunoblotting analysis (Fig. 3b), (iii) single amino acid sequence (described below) and (iv) high specific activity in ET bioassay (Table 1) and histological examination. The molecular weight of purified ET was determined to be 32000 by SDS-PAGE analysis (Fig. 3). NHrterminal amino acid sequences were identical to those of ETA from strains TA, TC16 and ZM. However, the purified toxin has different properties of heat stability and stability under freezing. Our purified toxin was labile at -20°C and -80 0c. This lability was not improved when stored in PBS containing 50% glycerol. These properties are somewhat similar to those of ETB (6). Earlier purification studies were performed with high-ET-producing staphylococci (2, 5, 6). This has prompted many investigators to purify ET from normaVlow ET producing clinical strains with low biological activity. By using the protocol described in this report, highly purified ET could be obtained from normal ET producing strains without difficulty. The availability of these techniques will permit the rapid purification of ET from clinical strains and is useful for comparative studies of ET.
Table 1. Summary of purification Sample
Volume (ml)
Protein ([.tg/ml)
Specific activity
% Recovery Purification
Factor
(Uf!.tg)
Crude (CCF) SP 5PW PECI0l G3000
36.5 18 12 6
190 187 118 58.7
0.132 1.064 1.695 3.450
100 394.2 263.2 131.1
1.00 8.06 12.84 26.14
References
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Purification of an Exfoliative Toxin by HPLC
11
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Motoyuki Sugai, Department of Microbiology, Hiroshima University, School of Dentistry, 1-2-3 Kasumi, Minami-ku o Hiroshima 734, Japan