This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Omoe, K.
Right arrow Articles by Shinagawa, K.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Omoe, K.
Right arrow Articles by Shinagawa, K.

 Previous Article  |  Next Article 

Infection and Immunity, June 2004, p. 3664-3667, Vol. 72, No. 6
0019-9567/04/$08.00+0     DOI: 10.1128/IAI.72.6.3664-3667.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Biological Properties of Staphylococcal Enterotoxin-Like Toxin Type R

Katsuhiko Omoe,1* Ken'ichi Imanishi,2 Dong-Liang Hu,3 Hidehito Kato,2 Hiromi Takahashi-Omoe,4 Akio Nakane,3 Takehiko Uchiyama,2 and Kunihiro Shinagawa1

Department of Veterinary Microbiology, Faculty of Agriculture, Iwate University, Ueda 3-18-8, Morioka, Iwate 020-8550,1 Department of Microbiology and Immunology, Tokyo Women's Medical University School of Medicine, Shinjuku-ku, Tokyo 162-8666,2 Department of Bacteriology, Hirosaki University School of Medicine, Hirosaki 036-8562,3 Department of Technical Support and Development, National Institute of Radiological Sciences, Anagawa 4-9-1, Inage-ku, Chiba-shi 263-8555, Japan4

Received 4 December 2003/ Returned for modification 5 January 2004/ Accepted 6 March 2004


arrow
ABSTRACT
 
We investigated the biological properties of a novel staphylococcal enterotoxin-like putative toxin, staphylococcal enterotoxin-like toxin type R (SElR). Major histocompatibility complex class II molecules were required for T-cell stimulation by SElR. SElR stimulated T cells bearing receptors Vß 3, 11, 12, 13.2, and 14. These results suggested that SElR acts as a superantigen.


arrow
TEXT
 
Staphylococcus aureus is an important human and animal pathogen. This organism causes a broad range of diseases in humans including septicemia, toxic shock syndrome (TSS), and food poisoning, as well as bovine mastitis. Staphylococcal enterotoxins (SEs) and TSS toxin 1 are members of the superantigenic toxin family and have the ability to stimulate large populations of T cells that have a particular Vß element of the T-cell receptor (TCR). This stimulation subsequently leads to a massive proliferation of T cells and the uncontrolled release of proinflammatory cytokines, which cause life-threatening TSS (3, 12). Five major serological types, SEA through SEE, have been characterized based on their antigenicity (2). These classical SEs are emetic toxins and causative agents in staphylococcal food poisoning. However, in recent years, many new types of SE or SE-like putative toxins have been identified (8, 9, 10, 12, 16, 17, 18, 19, 21). These new SEs and SE-like toxins were designated as members of the SE family based on their sequence similarity with classical SEs. At present, the relationship between these new SEs and human diseases including food poisoning and TSS is not fully understood.

SER is the newest SE-related putative toxin, described by Omoe et al. (16), who showed that the SER amino acid sequence had the highest homology to the SEG sequence and found that SER showed significant T-cell stimulation activity. Here we report the superantigenicity of SER and the SER production levels of S. aureus strains harboring ser gene-carrying plasmids. Based on these findings and the recommendations of the International Nomenclature Committee for Staphylococcal Superantigen Nomenclature, we rename this SE-like putative toxin as staphylococcal enterotoxin-like toxin type R (SElR).

Analysis of the requirement of MHC class II molecules for activation of T cells by SElR. We investigated whether the presence of major histocompatibility complex (MHC) class II molecules on accessory cells (ACs) is required for SElR to activate T cells. Recombinant SElR was expressed and purified according to the work of Omoe et al. (16). Human peripheral blood mononuclear cells (PBMCs) were obtained from healthy donors by Ficoll-Conray density gradient centrifugation. T cells were obtained by the S-2-aminoethylisothiouronium-treated sheep red blood cell rosette method. They were further enriched by removal of CD16-, CD14-, CD19-, and HLA-DR-positive cells with the use of monoclonal antibodies (MAbs) to those antigens and anti-mouse immunoglobulin-coated magnetic beads (Dynabeads; Dynal, Oslo, Norway) (7). L cells transfected with the DR4 gene (8124 L cells) and control L cells (8400) were prepared as described previously (6, 13). These L cells were treated with mitomycin C, irradiated with an MBR-1404R X-ray generator (Hitachi, Tokyo, Japan) to block proliferation, and used as ACs for T-cell activation by SElR. To measure interleukin-2 (IL-2) production from stimulated T cells, IL-2 activity in the culture supernatants was determined with IL-2-dependent CTLL-2 cells. Data are presented as units of IL-2 per milliliter (23). Purified T cells from human PBMCs were stimulated in vitro with 5 or 10 µg of SElR/ml in the presence or absence of L cells transfected with DR genes (8124) or of control L cells (8400). The T cells were then examined for IL-2 production. The effect of the antibody to HLA-DR on the T-cell response was examined in parallel (Table 1). Substantial levels of IL-2 production were seen in the presence of DR-transfected 8124 L cells but not in the presence of control 8400 L cells. Anti-DR MAb markedly inhibited the SElR-induced IL-2 production from T cells in the presence of 8124 L cells. Anti-mouse MHC class I MAb (anti-H-2Kk) did not influence the T-cell response induced by SElR.


View this table:
[in this window]
[in a new window]
  		TABLE 1. Requirement for the presence of MHC class II molecules on ACs in T-cell activation by SElRa

Analysis of the TCR Vß repertoire of SElR-reactive human T cells. SElR- or anti-CD3-induced T-cell blasts were obtained by stimulating PBMCs with 10 µg of SElR/ml or 1 µg of MAb to anti-CD3 (OKT3)/ml for 3 days and expanding harvested blasts for 4 days in the presence of 100 U of recombinant human IL-2 (Shionogi, Osaka, Japan)/ml. T-cell blasts stimulated with SElR or anti-CD3 MAb were stained with MAbs to TCR Vß elements (IOTest Beta Mark kit; Beckman Coulter, Miami, Fla.). Samples were analyzed on an EPICS XL flow cytometer (Beckman Coulter) with FlowJo software, as described previously (22). Figure 1 shows the representative results of three experiments. It was observed that T cells bearing TCR Vß 3, 11, 12, 13.2, and 14 were preferentially activated by SElR. T cells bearing TCR Vß 14 responded with the highest level of expansion (approximately 30% of T cells in three donors). The primary structure of SElR has homology to that of SEG (65.9% of identity) (16). Previously, Jarraud et al. examined the TCR Vß composition of T cells stimulated by recombinant SEG using the immunoscope technique. They showed that T cells bearing TCR Vß 3, 12, 13a, 14, and 15 were selectively expanded by SEG stimulation (8). Furthermore, T cells bearing TCR Vß 14 showed the highest response to both SEG and SElR. These similarities in the TCR Vß skewing of SEG and SElR reflect the similarity of the primary structures of these superantigens.



View larger version (31K):
[in this window]
[in a new window]
  		FIG. 1. TCR Vß profile of SER. Human PBMCs were stimulated with either anti-CD3 antibody or SER. Cells were stained with MAbs and analyzed by flow cytometry. Data are presented as percentages of the T-cell fractions expressing distinct Vß elements in whole T cells. Increases in comparison with anti-CD3 stimulation observed in three donors are indicated with an asterisk.

Development of sandwich enzyme-linked immunosorbent assay (ELISA) for detection of SElR. Rabbit anti-SElR immunoglobulin G was purified from hyperimmune serum (16) by use of an immobilized protein G-Sepharose column (Amersham Pharmacia Biotech Inc., Piscataway, N.J.) and was used as capture antibody. Monospecific rabbit anti-SElR antibody was affinity purified from hyperimmune serum by use of an SElR-coupled Sepharose column. One milligram of monospecific antibody was conjugated to EZ-Link Plus horseradish peroxidase (Pierce, Rockford, Ill.) according to the manufacturer's instructions. ELISA was performed in 96-well Nunc microplates (Nalge Nunc International, Rochester, N.Y.) according to the work of Omoe et al. (15). The concentration of each toxin was determined by converting the absorbance values to the corresponding concentrations via the standard curve. Straight lines were observed at concentrations of SElR between 1.0 and 10 ng/ml (Fig. 2). SElR was not detectable at 0.5 ng/ml. Additionally, 100 ng of purified SEA, SEB, SEC, SED, SEE, SEG, SEH, and SEI was subjected to sandwich ELISA, and no cross-reactivity was observed (data not shown).



View larger version (14K):
[in this window]
[in a new window]
  		FIG. 2. Standard curve for detection of SElR by ELISA. A linear increase was obtained between 1.0 and 10 ng of SElR/ml.

Productivities of SElR in S. aureus strains harboring the selr gene. Ten S. aureus strains harboring the selr gene, four selr-negative S. aureus strains, two Staphylococcus epidermidis strains, and two Escherichia coli strains were cultured in brain heart infusion broth supplemented with 1% yeast extract with shaking for 40 h, and the supernatants were subjected to sandwich ELISA. Culture supernatants were preincubated with 20% (vol/vol) normal rabbit serum at 4°C overnight and then diluted 10-, 100-, and 1,000-fold in phosphate-buffered saline containing 0.05% Tween 20 to avoid any nonspecific reaction caused by protein A (5, 15). Thus, the minimum detectable concentration of SElR in culture supernatant was 10 ng/ml. Nine out of 10 S. aureus isolates produced significant levels of SElR (38.2 to 324.8 ng/ml), while only one human nasal swab isolate did not produce a detectable level (10 ng/ml) of SElR (Table 2). The selr gene exists on two kinds of plasmids, a pIB485-related plasmid and a pF5-related plasmid. The SElR production levels did not differ between S. aureus strains harboring a pIB485-related plasmid and those harboring a pF5-related plasmid (P = 0.3, Mann-Whitney U test). All culture supernatants of selr-negative S. aureus strains, S. epidermidis strains, and E. coli strains were negative for SElR production (Table 2).


View this table:
[in this window]
[in a new window]
  		TABLE 2. Specificity of sandwich ELISA and SElR productivities of S. aureus isolates harboring the selr gene

In conclusion, SElR acts as a superantigen, and the biological activities of SElR most resemble those of SEG. Most S. aureus strains harboring the selr gene produce a significant amount of SElR. We have not tested the emetic activity of SElR using the primate model. The International Nomenclature Committee for Staphylococcal Superantigen Nomenclature has proposed that only staphylococcal superantigens that induce emesis following oral administration in a primate model be designated SEs and that other, related toxins that either lack emetic properties in this model or have not been tested should be designated staphylococcal enterotoxin-like (SEl) superantigens to indicate that their potential role in staphylococcal food poisoning has not been confirmed (personal communication from Keiichi Hiramatsu). Based on this criterion, we rename this staphylococcal enterotoxin-like superantigen as SElR, instead of SER. Further analysis with the primate model is needed to clarify the role of SElR in staphylococcal food poisoning.

As described above, the selr gene is carried by two kinds of plasmids, and the selr gene coexists with sed and sej (pIB485-related plasmid) or sej (pF5-related plasmid). In addition, a recent full genome sequencing analysis (1, 9) and genetic analysis of staphylococcal pathogenicity islands (4, 11, 24) showed that pathogenic factors including SEs and TSS toxin 1 were encoded by mobile genetic elements and several toxin genes linked to particular mobile genetic elements. Orwin et al. suggested that the presence of multiple expressed toxins in a single S. aureus strain makes it less clear whether any single toxin is responsible for diseases and that it is better to assume that these S. aureus strains express multiple toxins with similar activities and that all could contribute to disease (17). In other words, staphylococcal diseases related to superantigenic and emetic toxins, such as food poisoning and TSS, may be assumed to be a complex phenotype caused by these toxins with similar activities. To clarify the nature of SE- and enterotoxin-like toxin-related diseases, it is important to study the biological properties of newly identified SEs and SE-like toxins and to assess the toxin production levels of the S. aureus strains harboring the genes for the new SEs and SE-like toxins in vitro, in vivo, and in foods.


arrow
ACKNOWLEDGMENTS
 
We thank Keiichi Hiramatsu and Tadashi Baba (Department of Bacteriology, Juntendo University School of Medicine) for advice on SE nomenclature.

This work was partly supported by a grant-in-aid for scientific research from the Japan Society for the Promotion of Science (14560259 and 15580272).


arrow
FOOTNOTES
 
* Corresponding author. Mailing address: Department of Veterinary Microbiology, Faculty of Agriculture, Iwate University, Ueda 3-18-8, Morioka, Iwate 020-8550, Japan. Phone: 81-19-621-6221. Fax: 81-19-621-6223. E-mail: omo{at}iwate-u.ac.jp. Back

Editor: J. T. Barbieri


arrow
REFERENCES
 
    1
  1. Baba, T., F. Takeuchi, M. Kuroda, H. Yuzawa, K. Aoki, A. Oguchi, Y. Nagai, N. Iwama, K. Asano, T. Naimi, H. Kuroda, L. Cui, K. Yamamoto, and K. Hiramatsu. 2002. Genome and virulence determinants of high virulence community-acquired MRSA. Lancet 359:1819-1827.[CrossRef][Medline]
    2. 2
  2. Bergdoll, M. S. 1989. Staphylococcus aureus, p. 463-523. In M. P. Doyle (ed.), Foodborne bacterial pathogens. Marcel Dekker, Inc., New York, N.Y.
    3. 3
  3. Dinges, M. M., P. M. Orwin, and P. M. Schlievert. 2000. Exotoxins of Staphylococcus aureus. Clin. Microbiol. Rev. 13:16-34.[Abstract/Free Full Text]
    4. 4
  4. Fitzgerald, J. R., S. R. Monday, T. J. Foster, G. A. Bohach, P. J. Hartigan, W. J. Meaney, and C. J. Smyth. 2001. Characterization of a putative pathogenicity island from bovine Staphylococcus aureus encoding multiple superantigens. J. Bacteriol. 183:63-70.[Abstract/Free Full Text]
    5. 5
  5. Freed, R. C., M. L. Evenson, R. F. Reiser, and M. S. Bergdoll. 1982. Enzyme-linked immunosorbent assay for detection of staphylococcal enterotoxins in foods. Appl. Environ. Microbiol. 44:1349-1355.[Abstract/Free Full Text]
    6. 6
  6. Imanishi, K., H. Igarashi, and T. Uchiyama. 1990. Activation of murine T cells by streptococcal pyrogenic exotoxin type A: requirement for MHC class II molecules on accessory cells and identification of Vß elements in T cell receptor of toxin-reactive T cells. J. Immunol. 145:3170-3176.[Abstract]
    7. 7
  7. Imanishi, K., H. Kato, H. Fujii, and T. Uchiyama. 2003. Maturation of adult peripheral blood CD38+CD4+ T cells demonstrated by cytokine production in response to a superantigen, TSST-1. Cell. Immunol. 222:89-96.[CrossRef][Medline]
    8. 8
  8. Jarraud, S., M. A. Peyrat, A. Lim, A. Tristan, M. Bes, C. Mougel, J. Etienne, F. Vandenesch, M. Bonneville, and G. Lina. 2001. egc, a highly prevalent operon of enterotoxin gene, forms a putative nursery of superantigens in Staphylococcus aureus. J. Immunol. 166:669-677.[Abstract/Free Full Text]
    9. 9
  9. Kuroda, M., T. Ohta, I. Uchiyama, T. Baba, H. Yuzawa, I. Kobayashi, L. Cui, A. Oguchi, K. Aoki, Y. Nagai, J. Lian, T. Ito, M. Kanamori, H. Matsumaru, A. Maruyama, H. Murakami, A. Hosoyama, Y. Mizutani-Ui, N. K. Takahashi, T. Sawano, R. Inoue, C. Kaito, K. Sekimizu, H. Hirakawa, S. Kuhara, S. Goto, J. Yabuzaki, M. Kanehisa, A. Yamashita, K. Oshima, K. Furuya, C. Yoshino, T. Shiba, M. Hattori, N. Ogasawara, H. Hayashi, and K. Hiramatsu. 2001. Whole genome sequencing of methicillin-resistant Staphylococcus aureus. Lancet 357:1225-1240.[CrossRef][Medline]
    10. 10
  10. Letertre, C., S. Perelle, F. Dilasser, and P. Fach. 2003. Identification of a new putative enterotoxin SEU encoded by the egc cluster of Staphylococcus aureus. J. Appl. Microbiol. 95:38-43.[CrossRef][Medline]
    11. 11
  11. Lindsay, J. A., A. Ruzin, H. F. Ross, N. Kurepina, and R. P. Novick. 1998. The gene for toxic shock toxin is carried by a family of mobile pathogenicity islands in Staphylococcus aureus. Mol. Microbiol. 29:527-543.[CrossRef][Medline]
    12. 12
  12. McCormick, J. K., J. M. Yarwood, and P. M. Schlievert. 2001. Toxic shock syndrome and bacterial superantigens: an update. Annu. Rev. Microbiol. 55:77-104.[CrossRef][Medline]
    13. 13
  13. Miyoshi-Akiyama, T., J. Zhao. H. Kato, K. Kikuchi, K. Totsuka, Y. Kataoka, M. Katsumi, and T. Uchiyama. 2003. Streptococcus dysgalactiae-derived mitogen (SDM), a novel bacterial superantigen: characterization of its biological activity and predicted tertiary structure. Mol. Microbiol. 47:1589-1599.[CrossRef][Medline]
    14. 14
  14. Nakane, A., M. Okamoto, M. Asano, M. Kohanawa, and T. Minagawa. 1995. Endogenous gamma interferon, tumor necrosis factor, and interleukin-6 in Staphylococcus aureus infection in mice. Infect. Immun. 63:1165-1172.[Abstract]
    15. 15
  15. Omoe, K., M. Ishikawa, Y. Shimoda, D. L. Hu, S. Ueda, and K. Shinagawa. 2002. Detection of seg, seh, and sei genes in Staphylococcus aureus isolates and determination of the enterotoxin productivities of S. aureus isolates harboring seg, seh, and sei genes. J. Clin. Microbiol. 40:857-862.[Abstract/Free Full Text]
    16. 16
  16. Omoe, K., D. L. Hu, H. Takahashi-Omoe, A. Nakane, and K. Shinagawa. 2003. Identification and characterization of a new staphylococcal enterotoxin-related putative toxin encoded by two kinds of plasmids. Infect. Immun. 71:6088-6094.[Abstract/Free Full Text]
    17. 17
  17. Orwin, P. M., D. Y. M. Leung, H. L. Donahue, R. P. Novick, and P. M. Schlievert. 2001. Biochemical and biological properties of staphylococcal enterotoxin K. Infect. Immun. 69:360-366.[Abstract/Free Full Text]
    18. 18
  18. Orwin, P. M., D. Y. M. Leung, T. J. Tripp, G. A. Bohach, C. A. Earhart, D. H. Ohlendorf, and P. M. Schlievert. 2002. Characterization of a novel staphylococcal enterotoxin-like superantigen, a member of the group V subfamily of pyrogenic toxins. Biochemistry 41:14033-14040.[CrossRef][Medline]
    19. 19
  19. Orwin, P. M., J. R. Fitzgerald, D. Y. M. Leung, J. A. Gutierrez, G. A. Bohach, and P. M. Schlievert. 2003. Characterization of Staphylococcus aureus enterotoxin L. Infect. Immun. 71:2916-2919.[Abstract/Free Full Text]
    20. 20
  20. Sambrook, J., and D. W. Russell. 2001. Molecular cloning: a laboratory manual, 3rd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
    21. 21
  21. Su, Y.-C., and A. C. L. Wong. 1995. Identification and purification of a new staphylococcal enterotoxin, H. Appl. Environ. Microbiol. 61:1438-1443.[Abstract]
    22. 22
  22. Takahashi, N., H. Kato, K. Imanishi, K. Miwa, S. Yamanami, H. Nishida, and T. Uchiyama. 2000. Immunopathophysiological aspects of an emerging neonatal infectious disease induced by a bacterial superantigen. J. Clin. Investig. 106:1409-1415.[Medline]
    23. 23
  23. Uchiyama, T., T. Miyoshi-Akiyama, H. Kato, W. Fujimaki, K. Imanishi, and X. J. Yan. 1993. Superantigenic properties of a novel mitogenic substance produced by Yersinia pseudotuberculosis isolated from patients manifesting acute and systemic symptoms. J. Immunol. 151:4407-4413.[Abstract]
    24. 24
  24. Yarwood, J. M., J. K. McCormick, M. L. Paustian, P. M. Orwin, V. Kapur, and P. M. Schlievert. 2002. Characterization and expression analysis of Staphylococcus aureus pathogenicity island 3. Implications for the evolution of staphylococcal pathogenicity islands. J. Biol. Chem. 277:13138-13147.[Abstract/Free Full Text]

Infection and Immunity, June 2004, p. 3664-3667, Vol. 72, No. 6
0019-9567/04/$08.00+0     DOI: 10.1128/IAI.72.6.3664-3667.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Thomas, D., Dauwalder, O., Brun, V., Badiou, C., Ferry, T., Etienne, J., Vandenesch, F., Lina, G. (2009). Staphylococcus aureus Superantigens Elicit Redundant and Extensive Human V{beta} Patterns. Infect. Immun. 77: 2043-2050 [Abstract] [Full Text]  
  • Ono, H. K., Omoe, K., Imanishi, K., Iwakabe, Y., Hu, D.-L., Kato, H., Saito, N., Nakane, A., Uchiyama, T., Shinagawa, K. (2008). Identification and Characterization of Two Novel Staphylococcal Enterotoxins, Types S and T. Infect. Immun. 76: 4999-5005 [Abstract] [Full Text]  
  • Hu, D.-L., Omoe, K., Inoue, F., Kasai, T., Yasujima, M., Shinagawa, K., Nakane, A. (2008). Comparative prevalence of superantigenic toxin genes in meticillin-resistant and meticillin-susceptible Staphylococcus aureus isolates. J Med Microbiol 57: 1106-1112 [Abstract] [Full Text]  
  • Hsieh, H.-Y., Tseng, C. W., Stewart, G. C. (2008). Regulation of Rot Expression in Staphylococcus aureus. J. Bacteriol. 190: 546-554 [Abstract] [Full Text]  
  • Thomas, D. Y., Jarraud, S., Lemercier, B., Cozon, G., Echasserieau, K., Etienne, J., Gougeon, M.-L., Lina, G., Vandenesch, F. (2006). Staphylococcal Enterotoxin-Like Toxins U2 and V, Two New Staphylococcal Superantigens Arising from Recombination within the Enterotoxin Gene Cluster.. Infect. Immun. 74: 4724-4734 [Abstract] [Full Text]  
  • Omoe, K., Imanishi, K., Hu, D.-L., Kato, H., Fugane, Y., Abe, Y., Hamaoka, S., Watanabe, Y., Nakane, A., Uchiyama, T., Shinagawa, K. (2005). Characterization of Novel Staphylococcal Enterotoxin-Like Toxin Type P. Infect. Immun. 73: 5540-5546 [Abstract] [Full Text]  

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Omoe, K.
Right arrow Articles by Shinagawa, K.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Omoe, K.
Right arrow Articles by Shinagawa, K.

Copyright © 2009 by the American Society for Microbiology.   For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»