New Exfoliative Toxin Produced by a Plasmid-Carrying Strain of Staphylococcus hyicus

Bacterial strains. S. hyicus P-23 (20, 25), isolated from a pig affected with EE, was used in this study. The strain was lyophilized and stored at 4°C. The lyophilized bacteria were suspended in heart infusion broth (Difco Laboratories Inc., Detroit, Mich.) and cultured on heart infusion agar (Difco) at 37°C for 18 h. The bacteria were then suspended in 20% glycerol and stored at −80°C.

ETs.SHETA from S. hyicus P-1, ETA from S. aureus ZM, ETB from S. aureus J-sETB-8, and ETC fromS. aureus Horse-1 were isolated and purified by a method described previously (7, 8, 18, 24). These toxins were used as controls in the suckling-mouse inoculation test, the 1-day-old chicken inoculation test, Western blotting, and the heat stability test.

Bacterial culture for isolation of SHETB.For the isolation of SHETB, S. hyicus was cultured under the optimum conditions described by Watanabe et al. (27). S. hyicus cultured on heart infusion agar was suspended in Dulbecco’s phosphate-buffered saline without CaCl₂ and MgCl₂ (PBS) at a concentration of 10⁹ CFU/ml. A 2-ml portion of this suspension was inoculated in 200 ml of TY broth (6) and cultured at 37°C for 18 h with shaking at 75 oscillations per min. The bacterial culture was centrifuged at 10,000 × g for 20 min at 4°C, and the culture supernatant was passed through a membrane filter (0.45-μm pore size; Toyo Roshi Inc., Tokyo, Japan).

Determination of optimum concentration of ammonium sulfate.Ammonium sulfate powder was added to each culture filtrate (100 ml each) at various concentrations (10, 20, 30, 40, 50, 60, 70, 80, 90, and 100% saturation). After overnight incubation at 4°C, each of these suspensions was centrifuged at 10,000 × g for 30 min. Each of the precipitates was dissolved in a small amount of 10 mM Tris-HCl buffer (pH 7.5) and dialyzed against the same buffer at 4°C for 48 h. Each dialysate was then concentrated to 5 ml with a UP-20 ultrafilter (Toyo Roshi) and twofold diluted serially with the same buffer. Each diluted solution was inoculated subcutaneously in each of two 1-day-old chickens. The exfoliative activity (exfoliation units) of each fraction is shown as the reciprocal of the maximum dilution causing the exfoliation (Nikolsky sign).

Partial purification of SHETB.Ten milliliters of concentrated dialysate was loaded on a Sephadex G-75 (Pharmacia Fine Chemicals Inc., Uppsala, Sweden) column (2.6 by 90 cm) equilibrated with 10 mM Tris-HCl buffer (pH 7.5) and then eluted with the same buffer. Fractions of the effluent yielding a high concentration of protein were pooled and concentrated to 5 ml by ultrafiltration with a UP-20 ultrafilter. This concentrated solution was placed on a DEAE–Cellulofine A-500 column (1 by 20 cm; Chisso Corp., Tokyo, Japan) previously equilibrated with Tris-HCl buffer and then eluted with a linear gradient of from 0 to 0.2 M NaCl. Fractions of the effluent yielding a high concentration of protein were pooled and concentrated by ultrafiltration to 2 ml with a UP-20 ultrafilter.

SDS-PAGE.SDS-PAGE was performed at room temperature by the methods of Laemmli (10) and Sato et al. (19). A mixture of 0.05 ml of 500 mM Tris-HCl (pH 6.8), 0.08 ml of 10% SDS, 0.02 ml of 2-mercaptoethanol (Bio-Rad Laboratories, Richmond, Calif.), and 0.05 ml of 0.02% bromothymol blue in 80% glycerol was added to 0.2 ml of protein solution and then incubated at 37°C for 2 h. The cooled sample solution was layered on SDS–12.5% polyacrylamide gel slabs and run at 30 mA per gel. For purification of the SHETB, the proteins in gel slabs were stained with Coomassie brilliant blue R-250 (E. Merck AG Inc., Dermstadt, Germany) and decolorized with 7% acetic acid by the method of Fairbanks et al. (4).

Extraction of SHETB from SDS-polyacrylamide gels.After electrophoresis, the gels corresponding to the protein bands were cut out and cut into small pieces. The pieces of these gels were suspended in 10 volumes of 1% SDS in 20 mM Tris-HCl (pH 8.0) and incubated at room temperature overnight with gentle shaking. The suspension was dispensed in an inner tube with a membrane filter (0.45-μm pore size) in a test tube and centrifuged at 400 × g for 15 min. The filtrates were concentrated to 2 ml with a UP-20 ultrafilter, and 250 μl of this filtrate was dispensed in an Eppendorf tube. One milliliter of cold acetone was added to the filtrate (250 μl) and stored at −80°C for 1 h. After centrifugation at 10,000 × g for 15 min, the precipitates were dissolved in 50 mM Tris-HCl (pH 7.5).

Protein determination.The protein concentration was determined by the bicinchoninic acid protein assay (21, 23) with bovine serum albumin (Sigma Chemical Co., St. Louis, Mo.) as the standard. The bicinchoninic acid protein assay kit was purchased from Pierce Chemical, Inc., Rockford, Ill.

Animals.Ten inbred specific-pathogen-free (SPF) female BALB/c mice, 1 to 3 days old (Japan SLC Inc., Hamamatsu, Japan), were used for the suckling-mouse inoculation test for each ET. Two mice were used for each toxin sample. Four adult female SPF BALB/c mice (more than 8 weeks old; Japan SLC) were used for the preparation of antibodies to SHETB. Nine 1-day-old conventional White Leghorn chickens were used for the 1-day-old chicken inoculation test of each protein fraction and each toxin sample.

Inoculation tests. (i) Suckling-mouse inoculation test.Each purified toxin (SHETA, SHETB, ETA, ETB, and ETC; 10 μg each) was injected subcutaneously into 1- to 3-day-old BALB/c mice. The exfoliative activity was regarded as positive when the Nikolsky sign (peeling off of the skin surface easily caused by slight rubbing with fingertip) (7, 12) was identifiable within 3 h of injection.

(ii) One-day-old chicken inoculation test.Each protein fraction and each purified toxin (SHETA, SHETB, ETA, ETB, and ETC; 10 μg) were injected subcutaneously into 1-day-old chickens. The exfoliative activity was regarded as positive when the Nikolsky sign was identifiable within 3 h of injection.

Antibodies.The purified SHETB was converted to a toxoid by treatment with 0.8% formalin at 37°C for 50 h. The SHETB toxoid was inoculated subcutaneously into adult SPF mice once a week for 4 weeks. The inoculum was a mixture of 50 μg of toxoid and the same volume of Freund’s incomplete adjuvant (Difco). At 4 days after the fourth injection, sarcoma 180 cells (10⁶ cells/0.5 ml) were inoculated intraperitoneally. After 3 days, the toxoid was inoculated into the mice in the same manner. Most of the mice had distended abdomens within 10 to 15 days after the inoculation with sarcoma cells. At that time, the ascitic fluid was withdrawn by paracentesis through an 18-gauge needle into a 10-ml syringe. The ascitic fluids were pooled and centrifuged at 1,500 × g for 20 min. The blood was obtained by cardiac puncture from each of the immunized mice, and the serum was prepared in the same manner as the ascitic fluid. The ascitic fluid and serum were then pooled and designated anti-SHETB antibody. Anti-SHETA, anti-ETA, and anti-ETB antibodies were also prepared from the purified toxins.

ELISA.Enzyme-linked immunosorbent assay (ELISA) was performed by the method of Tanabe et al. (24). A 100-μl portion of each purified toxin (SHETA, SHETB, ETA, ETB, and ETC; 0.5 μg/ml) in 50 mM carbonate-bicarbonate buffer (pH 9.6) was dispensed into each well of a 96-well microplate (Greiner Labortechnik Inc., Frickenhausen, Germany) and incubated at 4°C for 18 h. The plate was then washed twice with PBS supplemented with 0.05% Tween 20 (T-PBS), and subsequently treated with 25% Block Ace (Yukijirushi Milking Co., Ltd., Tokyo, Japan) at 4°C overnight. After washing with T-PBS, a 100-μl portion of antibody to each toxin was dispensed into the wells, and the plate was held at 37°C for 1 h. After another washing, 100 μl of peroxidase-conjugated anti-mouse immunoglobulin G (1:20,000; lot 286005; Jackson Immuno Research Laboratories, Inc., West Grove, Pa.) was dispensed into the wells, and the plate was held at 37°C for 1 h. The substrate solution (0.04%o-phenylenediamine and 0.03% H₂O₂in 0.2 M phosphate–0.1 M citrate buffer, pH 4.8) was then dispensed into each well, the plate was allowed to stand at 37°C for 30 min. Then, 100 μl of 3 N H₂SO₄ was dispensed into each well. The optical density at 490 nm was read with an enzyme immunoassay reader (model 3544; Bio-Rad). The cutoff point was set as the sum of the average optical densities of sera from nonimmunized mice and three times the standard deviation. The ELISA antibody titer of each sample was taken as the reciprocal of the highest serum dilution for which the optical density was above the cutoff point. The ELISA titers of the anti-SHETA, anti-SHETB, anti-ETA, and anti-ETB antibodies were 25,600, 12,800, 204,800, and 102,400, respectively.

Western blotting.Western blotting was carried out by the methods of Towbin et al. (26) and Tanabe et al. (24). After electrophoresis of each toxin (SHETA, SHETB, ETA, and ETB), proteins in the SDS-polyacrylamide gel were transferred onto a polyvinylidene difluoride membrane (Atto Corp. Inc., Tokyo, Japan). A portion of the membrane was stained with 0.05% Coomassie brilliant blue R-250 and decolorized with a 7% acetic acid solution. The remaining membrane was then cut into 1-cm strips and incubated in 25% Block Ace at room temperature for 1 h. The anti-SHETA and anti-SHETB antibodies at a 1:2,000 dilution in 10% Block Ace and the anti-ETA and anti-ETB antibodies at a 1:10,000 dilution in 10% Block Ace were mounted onto each strip and incubated at room temperature for 1 h. After washing with T-PBS, 1:2,000-diluted peroxidase-conjugated anti-mouse immunoglobulin G was mounted onto each strip and incubated at room temperature for 1 h. After washing with T-PBS, the substrate solution (0.05% 3,3′-diaminobenzidine and 0.01% H₂O₂ in 50 mM Tris-HCl, pH 7.7) was mounted on each strip and incubated at room temperature. When the color reaction reached a maximum, each strip was washed with tap water to stop the reaction.

Heat stability of SHETB.Each purified toxin (SHETA, SHETB, ETA, ETB, and ETC) was heated at 100°C for 20 and 40 min and at 60°C for 15 and 30 min. After the heat treatment, 10 μg of each toxin was injected subcutaneously into each of two chickens and two mice, respectively. As a control, the same dose of nontreated toxin was injected subcutaneously into chickens and mice.