Skip to main content
  • ASM
    • Antimicrobial Agents and Chemotherapy
    • Applied and Environmental Microbiology
    • Clinical Microbiology Reviews
    • Clinical and Vaccine Immunology
    • EcoSal Plus
    • Eukaryotic Cell
    • Infection and Immunity
    • Journal of Bacteriology
    • Journal of Clinical Microbiology
    • Journal of Microbiology & Biology Education
    • Journal of Virology
    • mBio
    • Microbiology and Molecular Biology Reviews
    • Microbiology Resource Announcements
    • Microbiology Spectrum
    • Molecular and Cellular Biology
    • mSphere
    • mSystems
  • Log in
  • My alerts
  • My Cart

Main menu

  • Home
  • Articles
    • Current Issue
    • Accepted Manuscripts
    • Archive
    • Minireviews
  • For Authors
    • Submit a Manuscript
    • Scope
    • Editorial Policy
    • Submission, Review, & Publication Processes
    • Organization and Format
    • Errata, Author Corrections, Retractions
    • Illustrations and Tables
    • Nomenclature
    • Abbreviations and Conventions
    • Publication Fees
    • Ethics Resources and Policies
  • About the Journal
    • About IAI
    • Editor in Chief
    • Editorial Board
    • For Reviewers
    • For the Media
    • For Librarians
    • For Advertisers
    • Alerts
    • RSS
    • FAQ
  • Subscribe
    • Members
    • Institutions
  • ASM
    • Antimicrobial Agents and Chemotherapy
    • Applied and Environmental Microbiology
    • Clinical Microbiology Reviews
    • Clinical and Vaccine Immunology
    • EcoSal Plus
    • Eukaryotic Cell
    • Infection and Immunity
    • Journal of Bacteriology
    • Journal of Clinical Microbiology
    • Journal of Microbiology & Biology Education
    • Journal of Virology
    • mBio
    • Microbiology and Molecular Biology Reviews
    • Microbiology Resource Announcements
    • Microbiology Spectrum
    • Molecular and Cellular Biology
    • mSphere
    • mSystems

User menu

  • Log in
  • My alerts
  • My Cart

Search

  • Advanced search
Infection and Immunity
publisher-logosite-logo

Advanced Search

  • Home
  • Articles
    • Current Issue
    • Accepted Manuscripts
    • Archive
    • Minireviews
  • For Authors
    • Submit a Manuscript
    • Scope
    • Editorial Policy
    • Submission, Review, & Publication Processes
    • Organization and Format
    • Errata, Author Corrections, Retractions
    • Illustrations and Tables
    • Nomenclature
    • Abbreviations and Conventions
    • Publication Fees
    • Ethics Resources and Policies
  • About the Journal
    • About IAI
    • Editor in Chief
    • Editorial Board
    • For Reviewers
    • For the Media
    • For Librarians
    • For Advertisers
    • Alerts
    • RSS
    • FAQ
  • Subscribe
    • Members
    • Institutions
Bacterial Infections

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

Hisaaki Sato, Takao Watanabe, Yasuko Murata, Ayumi Ohtake, Mayumi Nakamura, Chikara Aizawa, Hiroshi Saito, Nobutoshi Maehara
Hisaaki Sato
Department of Veterinary Microbiology, School of Veterinary Medicine and Animal Sciences, Kitasato University, Towada, Aomori 034, and
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Takao Watanabe
Department of Veterinary Microbiology, School of Veterinary Medicine and Animal Sciences, Kitasato University, Towada, Aomori 034, and
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Yasuko Murata
Department of Veterinary Microbiology, School of Veterinary Medicine and Animal Sciences, Kitasato University, Towada, Aomori 034, and
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Ayumi Ohtake
Department of Veterinary Microbiology, School of Veterinary Medicine and Animal Sciences, Kitasato University, Towada, Aomori 034, and
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Mayumi Nakamura
Department of Veterinary Microbiology, School of Veterinary Medicine and Animal Sciences, Kitasato University, Towada, Aomori 034, and
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Chikara Aizawa
Department of Virology, The Kitasato Institute, Minato-ku, Tokyo 108, Japan
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Hiroshi Saito
Department of Virology, The Kitasato Institute, Minato-ku, Tokyo 108, Japan
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Nobutoshi Maehara
Department of Veterinary Microbiology, School of Veterinary Medicine and Animal Sciences, Kitasato University, Towada, Aomori 034, and
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
DOI: 10.1128/IAI.67.8.4014-4018.1999
  • Article
  • Figures & Data
  • Info & Metrics
  • PDF
Loading

ABSTRACT

A new serotype of Staphylococcus hyicus exfoliative toxin (SHET), serotype B, was isolated from the culture filtrate of a plasmid-carrying strain of S. hyicus. The new SHET was purified by precipitation with 70% saturated ammonium sulfate, gel filtration on a Sephadex G-75 column, column chromatography on DEAE–Cellulofine A-500, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The new SHET caused exfoliation of the epidermis as determined by the so-called Nikolsky sign when inoculated into 1-day-old chickens. The new SHET was serologically different fromStaphylococcus aureus exfoliative toxins (ETs) (ETA, ETB, and ETC) and from the SHET from the plasmidless strain but showed the same molecular weight as the other serotypes of toxins on SDS-PAGE. It was thermolabile and lost its toxicity after being heated at 60°C for 30 min. We propose that the new SHET be designated SHETB and that the SHET produced by the plasmidless strain be designated SHETA.

Staphylococcus hyicus is the causative agent of exudative epidermitis (EE) in pigs (22). EE is a generalized infection of the skin characterized by greasy exudation, exfoliation, and vesicle formation (5, 11). Sato et al. (19) have isolated an exotoxin from the culture supernatant of S. hyicus P-1 and designated it S. hyicus exfoliative toxin (SHET). The molecular mass of SHET has been estimated to be 27 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and it differs from Staphylococcus aureus exfoliative toxins (ETs) ETA, ETB, and ETC in its antigenicity and in the susceptible animal species (18, 24). Recently, we have confirmed that SHET-producing strains result in clinical signs of EE, whereas non-SHET-producing strains do not cause any clinical signs of EE (25). These findings suggest that SHET, with a molecular mass of 27 kDa, is a causative factor of EE and causes intraepidermal splitting in the granular layer of the epidermis, similar to the case for ETA, ETB, and ETC. However, it differs from ETA, ETB, and ETC in its antigenicity and in the susceptible animal species.

We have recently identified a new serotype of SHET, serotype B (SHETB), from field isolates of S. hyicus (20, 25). All of the SHETB-producing strains possess large plasmids as well as the 42-kb plasmid of the ETB-producing strains, and their plasmid-cured substrains have lost their SHET-producing ability (20). In the present paper we describe the antigenicity and some properties of purified SHETB.

MATERIALS AND METHODS

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.

RESULTS

Partial purification of SHETB.SHET activity could not be detected in the fraction precipitated with ammonium sulfate at a saturation of 50% or less. The maximum SHET activity was obtained from the fraction precipitated by ammonium sulfate at 70% saturation, and its score was 15 exfoliation units/ml. Figure1 shows the results of the Sephadex G-75 gel filtration of the toxic fraction precipitated with ammonium sulfate at 70% saturation. Three protein peaks (S-1, S-2, and S-3) were obtained. Fractions of each protein peak were pooled and concentrated to 1 mg/ml. When 10 μg of each preparation was injected subcutaneously into each of two chickens, exfoliative activity was found only with the S-2 preparation (Table1). In the chickens injected with the S-2 preparation, typical Nikolsky signs appeared at the site of injection within 1 h (Fig. 2). Figure3 shows the results of the DEAE-Cellulofine column chromatography of the S-2 preparation. Two major peaks (D-1 and D-3) and one minor peak (D-2) were obtained. The fractions for each protein peak were pooled and concentrated to 1 mg/ml. Figure 4 shows the SDS-PAGE pattern for each preparation (D-1, D-2, and D-3). The D-2 preparation gave four major protein bands of 40, 29, 27, and 22 kDa. The 27-kDa protein was detectable in the D-2 preparation but not in the D-1 and D-3 preparations. When 10 μg of each preparation was injected subcutaneously into each of two chickens, exfoliative activity was found only with the D-2 preparation (Table 1).

Fig. 1.
  • Open in new tab
  • Download powerpoint
Fig. 1.

Sephadex G-75 gel filtration of the 70% saturated ammonium sulfate fraction of culture filtrate of strain P-23.

View this table:
  • View inline
  • View popup
Table 1.

Purification of SHETB

Fig. 2.
  • Open in new tab
  • Download powerpoint
Fig. 2.

One-day-old chicken inoculation test of each fraction on Sephadex G-75 gel filtration. Chicks were inoculated with S-1 (left), S-2 (center), or S-3 (right).

Fig. 3.
  • Open in new tab
  • Download powerpoint
Fig. 3.

DEAE–Cellulofine A-500 chromatography of the S-2 preparation. Solid line, protein concentration; dashed line, NaCl concentration.

Fig. 4.
  • Open in new tab
  • Download powerpoint
Fig. 4.

SDS-PAGE of each peak fraction on DEAE–Cellulofine A-500 chromatography. Lane M, marker proteins (78, 66, 43, 26, and 14 kDa); lanes 1 and 2, D-1 fraction; lanes 3 and 4, D-3 fraction; lanes 5 and 6, D-2 fraction.

Purification of SHETB.The 40-, 29-, 27-, and 22-kDa proteins were extracted from an SDS-polyacrylamide gel. When 10 μg of each extract was injected subcutaneously into each of two chickens, exfoliative activity was found only with the 27-kDa protein (Table 1). Figure 5 shows the SDS-PAGE pattern for this extract, confirming that the extract consists of the 27-kDa protein alone. Based on these results, we considered the 27-kDa protein to be SHETB.

Fig. 5.
  • Open in new tab
  • Download powerpoint
Fig. 5.

SDS-PAGE of purified SHETB. Lane M, marker proteins (78, 66, 43, 26, and 14 kDa).

Partial characterization of SHETB.Table2 shows the animal susceptibilities of five ETs. In the suckling-mouse inoculation test, the Nikolsky sign was observed in each of two mice inoculated with ETA, ETB, and ETC, whereas no sign was observed in the mice inoculated with SHETA and SHETB. In the 1-day-old chicken inoculation test, the Nikolsky sign was observed in each of two chickens inoculated with SHETA, SHETB, and ETC, whereas no sign was observed in the chickens inoculated with ETA and ETB. Table3 shows the heat-stabilities of five ETs. The toxic activities of SHETB, ETB, and ETC were eliminated after treatment at 60°C for 30 min. The activity of SHETA was eliminated after treatment at 60°C for 15 min. ETA was stable after being heated at 100°C for 20 min. Figure 6 shows the results of the Western blot analysis of the ETs. The anti-SHETB antibody reacted with the 27-kDa protein band of only SHETB. Similarly, the anti-ETA, anti-ETB, and anti-SHETA antibodies reacted with the 27-kDa protein bands of their homologous toxins.

View this table:
  • View inline
  • View popup
Table 2.

Animal species susceptible to SHETB

View this table:
  • View inline
  • View popup
Table 3.

Heat stability of SHETB

Fig. 6.
  • Open in new tab
  • Download powerpoint
Fig. 6.

Western blot analysis of SHETB. Lane M, marker proteins (78, 66, 43, 26, and 14 kDa); lane 1, anti-ETA antibody; lane 2, anti-ETB antibody; lane 3, anti-SHETA antibody; lane 4, anti-SHETB antibody.

DISCUSSION

S. hyicus is a causative agent of EE in pigs (22). Amtsberg (1) has suggested that SHET is one of the virulence factors of S. hyicus; at the time of Amtsberg’s report, however, the isolation and purification of SHET had not yet been achieved. In 1991, we reported the isolation of SHET from culture filtrates of S. hyicus and the production of exfoliation in piglets injected with SHET (19). We also stated that the molecular mass of SHET seemed to be 27 kDa (24) and that the susceptible animal species were piglets and chickens (17). We have recently clarified that SHET can be divided into at least two serotypes (SHETA and SHETB) and that SHET-producing strains of S. hyicus cause EE in piglets, whereas non-SHET-producing strains do not (20, 25). Similar findings have been reported by Andresen (2), Andresen et al. (3), and Wegener et al. (28). Moreover, we have reported that the production of SHETA and SHETB is genetically controlled by chromosomal DNA and a 42-kb plasmid (pKUH-1) (20).

Several investigators have isolated two serotypes of ETs (ETA and ETB) from S. aureus strains derived from patients with staphylococcal scalded-skin syndrome (7-9, 29). ETA is a heat-stable toxin and its production is genetically controlled by chromosomal DNA (7, 14, 16), while ETB is a heat-labile toxin and its production is controlled by a 42-kb plasmid (8, 13, 15). However, both toxins have the same molecular mass (27 kDa), the same susceptible animal species (humans and suckling mice), and the same toxic activities (formation of an intraepidermal cleavage plane). The SHETA produced by the plasmidless strain of S. hyicushas been determined to be a heat-labile toxin, since after heat treatment at 60°C for 15 min it loses its exfoliative activity (19) and can no longer cross-react with antibodies to ETA and ETB (24). However, the physicochemical, biological, and antigenic characteristics of SHETB produced by the plasmid-carrying strain of S. hyicus are not fully understood. We therefore attempted to purify SHETB in this study.

In the purification of SHETA (24, 27), SHETA activity was highest in the fraction precipitated with a 90% saturation of ammonium sulfate, and the SHETA activity could be detected in the second peak on Sephadex G-75 gel filtration and in the first peak (at 0 to 0.05 M NaCl) on ion-exchange chromatography. The SHETB activity was highest in the fraction precipitated with a 70% saturation of ammonium sulfate, and the SHETB activity could be detected in the second peak on gel filtration and the second peak (at 0.12 to 0.15 M NaCl) on ion-exchange chromatography. Such differences in the purification steps for the SHETs seem to reflect differences in composition between the SHETA and SHETB molecules. Differences in the optimal concentrations of ammonium sulfate and different elution profiles on ion-exchange chromatography were also seen in the purification steps for ETA and ETB (7, 8).

SHETA is a heat-labile toxin, since its toxicity is lost after heating at 60°C for 15 min, and its molecular weight is approximately 27,000 (19, 24). SHETB is also a heat-labile toxin with a molecular weight of 27,000, but it retains its toxicity longer, finally losing its toxic activity after heating at 60°C for 30 min. The differences in heat stability between SHETA and SHETB also seem to reflect the composition differences between SHETA and shETB molecules.

In the Western blotting analysis, antibody to SHETB reacted only with SHETB and not with ETA, ETB, and SHETA. Similarly, each toxin reacted with antibody to the same toxin. These results suggest that the SHETB obtained from the plasmid-carrying strain (P-23) of S. hyicus is a new serotype of SHET.

ACKNOWLEDGMENTS

This research was supported by grants-in-aid for scientific research (no. 06660391 and no. 089660372) from the Ministry of Education, Science, and Culture, Japan.

Notes

Editor: V. A. Fischetti

FOOTNOTES

    • Received 14 October 1998.
    • Returned for modification 9 December 1998.
    • Accepted 9 April 1999.
  • Copyright © 1999 American Society for Microbiology

REFERENCES

  1. 1.↵
    1. Amtsberg G.
    Nachweis von Exofoliation auslösenden Substanzen in Kulturen von Staphylococcus hyicus des Schweines und Staphylococcus epidermidis Biotyp 2 des Rindes.Zentralbl. Veterinaermed. Reihe B261979257272
    OpenUrlPubMed
  2. 2.↵
    1. Andresen L. O.
    Differentiation and distribution of three types of exfoliative toxin produced by Staphylococcus hyicus from pigs with exudative epidermitis.FEMS Immun. Med. Microbiol.201998301310
    OpenUrlCrossRefPubMed
  3. 3.↵
    1. Andresen L. O.,
    2. Bille-Hansen V.,
    3. Wegener H. C.
    Staphylococcus hyicus exfoliative toxin: purification and demonstration of antigenic diversity among toxins from virulent strains.Microb. Pathog.221997113122
    OpenUrlCrossRefPubMed
  4. 4.↵
    1. Fairbanks G.,
    2. Steck T. L.,
    3. Wallach D. F. H.
    Electrophoretic analysis of the major polypeptides of the human erythrocyte membrane.Vet. Med.56197195103
    OpenUrl
  5. 5.↵
    1. Jones L. D.
    Observation of exudative epidermitis.Vet. Med.14196110281033
    OpenUrl
  6. 6.↵
    1. Kapral F. A.,
    2. Miller M. M.
    Product of Staphylococcus aureus responsible for the scalded-skin syndrome.Infect. Immun.41971541545
    OpenUrlAbstract/FREE Full Text
  7. 7.↵
    1. Kondo I.,
    2. Sakurai S.,
    3. Sarai Y.
    Purification of exfoliatin produced by Staphylococcus aureus of bacteriophage group 2 and its physicochemical properties.Infect. Immun.81973156164
    OpenUrlAbstract/FREE Full Text
  8. 8.↵
    1. Kondo I.,
    2. Sakurai S.,
    3. Sarai Y.
    New type of exfoliatin obtained from staphylococcal strains, belonging to phage groups other than group 2, isolated from patients with impetigo and Ritter’s disease.Infect. Immun.101974851861
    OpenUrlAbstract/FREE Full Text
  9. 9.↵
    1. Kondo I.,
    2. Sakurai S.,
    3. Sarai Y.,
    4. Futaki S.
    Two serotypes of exfoliatin and their distribution in staphylococcal strains isolated from patients with scalded skin syndrome.J. Clin. Microbiol.11975397400
    OpenUrlAbstract/FREE Full Text
  10. 10.↵
    1. Laemmli U. K.
    Cleavage of structural proteins during the assembly of the head of bacteriophage T4.Nature (London)2271970680685
    OpenUrlCrossRefPubMedWeb of Science
  11. 11.↵
    1. L’Ecuyer C.
    Exudative epidermitis in pigs. Clinical studies and preliminary transmission trials.Can. J. Comp. Med. Vet. Sci.301966916
    OpenUrlPubMed
  12. 12.↵
    1. Melish M. E.,
    2. Glasgow L. A.
    The staphylococcal scalded skin syndrome: development of an experimental model.N. Engl. J. Med.282197011141119
    OpenUrlCrossRefPubMedWeb of Science
  13. 13.↵
    1. O’Reilly M.,
    2. Dougan G.,
    3. Foster J.,
    4. Arbuthnott J. P.
    Plasmid in epidermolytic strain of Staphylococcus aureus.J. Gen. Microbiol.124198199107
    OpenUrlCrossRefPubMedWeb of Science
  14. 14.↵
    1. O’Toole P. W.,
    2. Foster T. J.
    Molecular cloning and expression of the epidermolytic toxin A gene of Staphylococcus aureus.Microb. Pathog.11986583594
    OpenUrlCrossRefPubMed
  15. 15.↵
    1. O’Toole P. W.,
    2. Foster T. J.
    Epidermolytic toxin serotype B of Staphylococcus aureus is plasmid encoded.FEMS Microbiol. Lett.361986311314
    OpenUrlCrossRef
  16. 16.↵
    1. Sakurai S.,
    2. Suzuki H.,
    3. Kondo I.
    DNA sequencing of the eta gene coding for staphylococcal exfoliative toxin serotype A.J. Gen. Microbiol.1341988711717
    OpenUrlCrossRefPubMed
  17. 17.↵
    1. Sato H.,
    2. Kuramoto M.,
    3. Tanabe T.,
    4. Saito H.
    Susceptibility of various animals and cultured cells to exfoliative toxin produced by Staphylococcus hyicus subsp. hyicus.Vet. Microbiol.281991157169
    OpenUrlCrossRefPubMed
  18. 18.↵
    1. Sato H.,
    2. Matsumori Y.,
    3. Tanabe T.,
    4. Saito H.,
    5. Shimizu A.,
    6. Kawano J.
    A new serotype of staphylococcal exfoliative toxin from a Staphylococcus aureus strain isolated from a horse with phlegmon.Infect. Immun.62199437803785
    OpenUrlAbstract/FREE Full Text
  19. 19.↵
    1. Sato H.,
    2. Tanabe T.,
    3. Kuramoto M.,
    4. Tanaka K.,
    5. Hashimoto T.,
    6. Saito H.
    Isolation of exfoliative toxin from Staphylococcus hyicus subsp. hyicus and its exfoliative activity in the piglet.Vet. Microbiol.271991263275
    OpenUrlCrossRefPubMed
  20. 20.↵
    1. Sato H.,
    2. Tanabe T.,
    3. Watanabe T.,
    4. Teruya K.,
    5. Ohtake A.,
    6. Saito H.,
    7. Maehara N.
    Chromosomal and extrachromosomal synthesis of exfoliative toxin from Staphylococcus hyicus Proceedings of the 14th IPVS Congress, Bologna, Italy 1996 339
  21. 21.↵
    1. Smith P. K.,
    2. Krohm R. I.,
    3. Hermanson G. T.,
    4. Mallia A. K.,
    5. Gartner F. H.,
    6. Provenzano M. D.,
    7. Fujimoto E. K.,
    8. Goeke N. M.,
    9. Olson B. J.,
    10. Klenk D. C.
    Measurement of protein using bicinchoninic acid.Anal. Biochem.15019857685
    OpenUrlCrossRefPubMedWeb of Science
  22. 22.↵
    1. Sompolinsky D.
    De l’impetigo contagiosa suis et du Micrococcus hyicus n. sp.Schweiz. Arch. Tierhilkt.951953302309
    OpenUrl
  23. 23.↵
    1. Sorensen K.,
    2. Brodbeck U.
    A sensitive protein assay method using microtiter plates.Experientica421986161162
    OpenUrl
  24. 24.↵
    1. Tanabe T.,
    2. Sato H.,
    3. Kuramoto M.,
    4. Saito H.
    Purification of exfoliative toxin produced by Staphylococcus hyicus and its antigenicity.Infect. Immun.61199329732977
    OpenUrlAbstract/FREE Full Text
  25. 25.↵
    1. Tanabe T.,
    2. Sato H.,
    3. Sato H.,
    4. Watanabe K.,
    5. Hirano M.,
    6. Hirose K.,
    7. Kurokawa S.,
    8. Nakano K.,
    9. Saito H.,
    10. Maehara N.
    Correlation between occurrence of exudative epidermitis and exfoliative toxin-producing ability of Staphylococcus hyicus.Vet. Microbiol.481996917
    OpenUrlCrossRefPubMed
  26. 26.↵
    1. Towbin H.,
    2. Staehelin T.,
    3. Gordon J.
    Electrophoretic transfer of protein from polyacrylamide gels to nitrocellulose sheets: procedure and some applications.Proc. Natl. Acad. Sci. USA76197943504354
    OpenUrlAbstract/FREE Full Text
  27. 27.↵
    1. Watanabe T.,
    2. Tanabe T.,
    3. Sato H.,
    4. Fuse Y.,
    5. Ueda K.,
    6. Nakano K.,
    7. Saito H.,
    8. Maehara N.
    Optimum culture conditions for production of exfoliative toxin by Staphylococcus hyicus.Microbiol. Immunol.391996369377
    OpenUrl
  28. 28.↵
    1. Wegener H. C.,
    2. Andresen L. O.,
    3. Bille-Hansen V.
    Staphylococcus hyicus virulence in relation to exudative epidermitis in pigs.Can. J. Vet. Res.571993119125
    OpenUrlPubMed
  29. 29.↵
    1. Wiley B. B.,
    2. Rogolsky M.
    Molecular and serological differentiation of staphylococcal exfoliative toxin synthesized under chromosomal and plasmid control.Infect. Immun.181977487494
    OpenUrlAbstract/FREE Full Text
PreviousNext
Back to top
Download PDF
Citation Tools
New Exfoliative Toxin Produced by a Plasmid-Carrying Strain of Staphylococcus hyicus
Hisaaki Sato, Takao Watanabe, Yasuko Murata, Ayumi Ohtake, Mayumi Nakamura, Chikara Aizawa, Hiroshi Saito, Nobutoshi Maehara
Infection and Immunity Aug 1999, 67 (8) 4014-4018; DOI: 10.1128/IAI.67.8.4014-4018.1999

Citation Manager Formats

  • BibTeX
  • Bookends
  • EasyBib
  • EndNote (tagged)
  • EndNote 8 (xml)
  • Medlars
  • Mendeley
  • Papers
  • RefWorks Tagged
  • Ref Manager
  • RIS
  • Zotero
Print

Alerts
Sign In to Email Alerts with your Email Address
Email

Thank you for sharing this Infection and Immunity article.

NOTE: We request your email address only to inform the recipient that it was you who recommended this article, and that it is not junk mail. We do not retain these email addresses.

Enter multiple addresses on separate lines or separate them with commas.
New Exfoliative Toxin Produced by a Plasmid-Carrying Strain of Staphylococcus hyicus
(Your Name) has forwarded a page to you from Infection and Immunity
(Your Name) thought you would be interested in this article in Infection and Immunity.
CAPTCHA
This question is for testing whether or not you are a human visitor and to prevent automated spam submissions.
Share
New Exfoliative Toxin Produced by a Plasmid-Carrying Strain of Staphylococcus hyicus
Hisaaki Sato, Takao Watanabe, Yasuko Murata, Ayumi Ohtake, Mayumi Nakamura, Chikara Aizawa, Hiroshi Saito, Nobutoshi Maehara
Infection and Immunity Aug 1999, 67 (8) 4014-4018; DOI: 10.1128/IAI.67.8.4014-4018.1999
del.icio.us logo Digg logo Reddit logo Twitter logo CiteULike logo Facebook logo Google logo Mendeley logo
  • Top
  • Article
    • ABSTRACT
    • MATERIALS AND METHODS
    • RESULTS
    • DISCUSSION
    • ACKNOWLEDGMENTS
    • Notes
    • FOOTNOTES
    • REFERENCES
  • Figures & Data
  • Info & Metrics
  • PDF

KEYWORDS

Exfoliatins
plasmids
Staphylococcus

Related Articles

Cited By...

About

  • About IAI
  • Editor in Chief
  • Editorial Board
  • Policies
  • For Reviewers
  • For the Media
  • For Librarians
  • For Advertisers
  • Alerts
  • RSS
  • FAQ
  • Permissions
  • Journal Announcements

Authors

  • ASM Author Center
  • Submit a Manuscript
  • Article Types
  • Ethics
  • Contact Us

Follow #IAIjournal

@ASMicrobiology

       

ASM Journals

ASM journals are the most prominent publications in the field, delivering up-to-date and authoritative coverage of both basic and clinical microbiology.

About ASM | Contact Us | Press Room

 

ASM is a member of

Scientific Society Publisher Alliance

 

American Society for Microbiology
1752 N St. NW
Washington, DC 20036
Phone: (202) 737-3600

Copyright © 2021 American Society for Microbiology | Privacy Policy | Website feedback

Print ISSN: 0019-9567; Online ISSN: 1098-5522