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Infection and Immunity, September 2005, p. 5540-5546, Vol. 73, No. 9
0019-9567/05/$08.00+0     doi:10.1128/IAI.73.9.5540-5546.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Characterization of Novel Staphylococcal Enterotoxin-Like Toxin Type P

Katsuhiko Omoe,^1* Ken'ichi Imanishi,² Dong-Liang Hu,³ Hidehito Kato,² Yoshitaku Fugane,¹ Yohei Abe,¹ Shoji Hamaoka,¹ Yutaka Watanabe,¹ Akio Nakane,³ Takehiko Uchiyama,² and Kunihiro Shinagawa¹

Department of Veterinary Medicine, 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, 8-1 Kawada-cho, Shinjuku-ku, Tokyo 162-8666,² Department of Bacteriology, Hirosaki University School of Medicine, 5 Zaifu-cho, Hirosaki 036-8562, Japan³

Received 1 February 2005/ Returned for modification 20 March 2005/ Accepted 16 May 2005

Top Abstract Introduction Materials and Methods Results Discussion References

 
We investigated the biological properties of a novel staphylococcal enterotoxin (SE)-like toxin type P (SElP). SElP induced a substantial proliferative response and the production of cytokines interleukin-2, gamma interferon, tumor necrosis factor alpha, and interleukin-4 from human T cells when administered at a concentration of 0.4 pM (0.01 ng/ml) or more. The expression of major histocompatibility complex class II molecules on accessory cells was required for T-cell stimulation by SElP. SElP selectively stimulated a vast number of human T cells bearing receptors Vß 5.1, 6, 8, 16, 18, and 21.3. These results indicated that SElP acts as a superantigen. SElP proved to be emetic in the house musk shrew emetic assay, although at a relatively high dose (50 to 150 µg/animal). A quantitative assay of SElP production with 30 Staphylococcus aureus strains harboring selp showed that 60% of these strains produced significant amounts of SElP in vitro. All 10 strains carrying seb and selp produced SEB but not SElP, suggesting the inactivation of the selp locus in S. aureus strains with a particular se gene constitution.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Staphylococcal enterotoxins (SEs) are extracellular protein toxins with superantigen (SAg) activity produced by a variety of Staphylococcus aureus strains. Staphylococcal SAgs are composed of a large family of SEs and toxic shock syndrome toxin 1 (TSST-1) and can specifically stimulate a large number of T cells bearing particular Vß elements in their T-cell receptor (TCR) beta chain (19). Some bacterial SAgs are known to act as pathogenic toxins through their strong T-cell stimulation. For example, T-cell stimulation with TSST-1 leads to the massive proliferation of Vß2⁺ T cells and the release of proinflammatory cytokines by these T cells, causing life-threatening toxic shock syndrome (TSS) (14, 18, 29, 32). Different types of SEs (SEA through SEE) had been known prior to the proposal of the superantigen concept in 1989 (3). These SEs are known to be emetic toxins and are the causative agents of staphylococcal food poisoning in humans. Recently, many new types of SE or SE-related toxins have been reported, based on their sequence similarity with classical SEs (15, 16, 19, 23, 24, 26). The International Nomenclature Committee for Staphylococcal Superantigens has proposed that only staphylococcal SAgs that induce emesis after oral administration in a primate model should be designated as SE. The committee also recommended that other related toxins that either lack emetic properties in a primate model or have not been tested should be designated as staphylococcal enterotoxin-like toxin type X (17). To clarify the role of the new SEs and SEl toxins in the pathogenicity of S. aureus, the biological properties of these toxins should be studied.

The gene for staphylococcal enterotoxin-like toxin type P (SElP) is a novel SE-related toxin gene originally identified as sep after the full genome sequencing of the S. aureus N315 strain (15). selp (previously named sep) encodes a SElP precursor 260 amino acids in length. In the present study, we analyzed the biological properties of recombinant SElP (rSElP). The rSElP stimulated T cells bearing TCR Vß 5.1, 6, 8, 16, 18, and 21.3 in the presence of accessory cells expressing major histocompatibility complex (MHC) class II molecules, suggesting that SElP acts as an SAg. SElP proved to be emetic in the house musk shrew (Suncus murinus) emetic assay. In addition, we examined SElP production in 30 strains carrying selp and other related se genes in different combinations.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Cloning and expression of SElP gene. The gene (selp) for the SElP was cloned from S. aureus strain Saga 1, isolated from an SE-unidentified food poisoning outbreak in Japan. The genotype of S. aureus Saga 1 was determined by multiplex PCR (25). Genomic DNA from S. aureus Saga 1 was purified by using a QIAGEN genomic tip (QIAGEN GmbH). Using the online signal peptide prediction software SignalP (http://www.cbs.dtu.dk/services/SignalP) (20), we predicted the N-terminal signal peptide sequence of a mature form of SElP based on the nucleotide sequence of selp reported by Kuroda et al. (15). PCR primers were designed to amplify gene fragment corresponding to the mature form of SElP: SElPS (including a 5' BamHI site, 5'-GGGGGATCCAGCGAAGAATAAATGGAAA-3') and SElPAS (including a 5' SalI site, 5'-GGGGTCGACAAATTTCACTCAAGTTGTAT-3'). selp was amplified by PCR by using Pyrobest DNA polymerase (Takara, Kyoto, Japan), and a selp fragment was then subcloned to pGEM3Zf(+) (Promega) and designated pKOP1. The nucleotide sequencing of pKOP1 was determined by using an ABI3100 Avant DNA sequencer (Applied Biosystems). The DNA fragment that codes for the mature form of SElP was digested from pKOP1 with BamHI and SalI and was then subcloned into a pGEX-6P-1 (Amersham Pharmacia Biotech, Piscataway, NJ) glutathione S-transferase (GST) fusion expression vector. The resultant plasmid containing selp was named pKPX1. Escherichia coli BL21 cells harboring pKPX1 were grown in 2xYT broth (Sigma Chemical Co., St. Louis, MO) containing 100 µg of ampicillin/ml at 18°C to an optical density at 600 nm of 0.5. At that time, the expression of the GST fusion protein was induced by adding IPTG (isopropyl-ß-D-thiogalactopyranoside; Amersham) to a final concentration of 0.5 mM. After 16 h of cultivation at 18°C, the cells were harvested and lysed by B-per solution (Pierce, Rockford, IL). The lysate was separated by centrifugation, and the supernatant was subjected to affinity chromatography by using glutathione-Sepharose 4B (Amersham). The GST fusion SElP was eluted with 10 mM glutathione in 50 mM Tris-HCl (pH 8.0); then, the eluted samples were dialyzed against 50 mM Tris-HCl (pH 7.0), 150 mM NaCl, 1 mM EDTA, and 1 mM dithiothreitol. Mature-form toxins were then released by digestion for 4 h at 4°C with PreScission Protease (Amersham), which cleaves at a single site between the GST tag and the mature-form SElP. The protease and the GST tag were removed by passing the samples through glutathione-sepharose4B. The resulting mature rSElP had five additional amino acid residues, GPLGS, at the N terminus.

Analysis for mitogenicity of SElP. To determine the mitogenic activity of SElP, we stimulated human peripheral blood mononuclear cells (PBMC) for 72 h in 96-well flat-bottom microplates (Becton Dickinson, Franklin Lakes, NJ) with various concentrations of SElP or TSST-1 with or without of 100 U of polymyxin B sulfate (Bio West, Nuaile, France)/ml, a lipopolysaccharide (LPS) inhibitor. The cultures were pulsed with 0.5 mCi of [³H]thymidine for the last 16 h of the culture period, and the incorporation of [³H]thymidine was measured (11, 31). The data are presented as the average of triplicate cultures and the standard error of the counts per minute. Three experiments were performed independently using human PBMC from 3 donors.

Analysis of the requirement of MHC class II molecules for the activation of T cells by SElP. Human PBMC were obtained from three healthy donors by Ficoll-Conray density gradient centrifugation. T cells were obtained by the S-2-aminoethylisothiouronium-treated rosette method and were enriched by the removal of CD16-, CD14-, CD19-, and HLA-DR-positive cells by using monoclonal antibodies (MAbs) to those antigens and anti-mouse immunoglobulin-coated magnetic beads (Dynabeads; Dynal, Oslo, Norway) (13). L cells transfected with the DR4 genes (8124 L cells) and control L cells (8400) were prepared by using a previously described method (11). These L cells were treated with mitomycin C and irradiated with an MBR-1404R X-ray generator (Hitachi, Tokyo, Japan) to block proliferation and used as accessory cells (ACs) for T-cell activation in response to 10 ng of rSElP or TSST-1/ml. 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. The data are presented as the units of IL-2 per milliliter (28, 29). The amounts of gamma interferon (IFN-), tumor necrosis factor- (TNF-), and IL-4 in the culture supernatants were determined by using a sandwich enzyme-linked immunosorbent assay (ELISA) and antibody pairs purchased from Pharmingen (San Diego, CA) (13). IL-2 assays were performed in triplicate, and IFN-, TNF-, and IL-4 assays were performed in duplicate, both for the standards and the experimental samples. The data are presented as nanograms of IFN- per milliliter, nanograms of TNF- per milliliter, and picograms of IL-4 per milliliter. The lower limits of detection for IL-2, IFN-, TNF-, and IL-4 in these assays were 0.1 U/ml, 0.03 ng/ml, 0.02 ng/ml, and 6 pg/ml, respectively.

Analysis of the TCR Vß repertoire of SElP-reactive human T cells. rSElP- or anti-CD3-induced T-cell blasts were obtained by stimulating PBMC with 10 ng of rSElP/ml or 1 µg of MAb to CD3 (OKT3)/ml for 3 days and expanding the harvested blasts for 4 days in the presence of 100 U of human rIL-2 (Shionogi, Osaka, Japan)/ml. T-cell blasts induced with rSElP or ant-CD3 MAb were stained with MAbs to TCR Vß elements (IOTest Beta Mark kit; Beckman Coulter, Inc., Hialeah, Fla.) and PC5-conjugated anti-CD3 MAb. Staining was carried out by incubating 10⁶ cells in 100 µl of the appropriate MAb for 30 min at 4°C. The stained cells were analyzed for TCR Vß expression by using an EPICS XL flow cytometer (Beckman Coulter) with FlowJo software (24, 28). The Vß frequencies of the T-cell preparations were expressed as percentages of CD3⁺ T cells. TCR Vß6 expression in SElP- or antiCD3-induced T-cell blasts was analyzed by reverse transcriptase PCR (RT-PCR) (5, 31). Total RNA was extracted from these T-cell blasts by Isogen (Nippon Gene, Tokyo Japan). First-strand cDNA was synthesized by incubating the total RNA with RT (Takara) and then subjecting the samples to 25 cycles of RT-PCR with the 5'Vß6-specific sense primer and 3'Cß-specific antisense primer. As a control, TCR C cDNA was amplified in each reaction mixture by using the 5'C sense primer and the 3'C antisense primer. The PCR products were separated by electrophoresis on 2.5% agarose gels stained with ethidium bromide and were detected by using a Molecular Imager FX (Bio-Rad).

Emetic assay. Emetic assays were performed according to the method of Hu et al. (8, 9) with healthy adult male house musk shrews (Suncus murinus, Nihon Clea, Tokyo, Japan) weighing 55 to 70 g. The house musk shrews were kept in a room at 22 to 25°C. The room was kept lit for 12 h from 7:00 a.m. to 7:00 p.m. Purified rSElP was diluted in 0.01 M phosphate-buffered saline (PBS; pH 7.2). A total of 200 µl of rSElP at an appropriate dilution was injected intraperitoneally into the house musk shrew. The animals were not starved beforehand. The animals were observed for emesis for 3 h after the intraperitoneal injection of rSElP. The number of vomiting episodes, the time of each vomiting episode, the length of time before the first vomiting episode, and any behavioral changes were recorded.

Preparation of polyclonal antibody. Anti-rSElP serum was prepared by immunizing rabbits with purified rSElP according to the method of Shinagawa et al. (27). The antiserum titers were monitored by using ELISA. Rabbit anti-rSElP immunoglobulin G was purified from hyperimmune serum by using an immobilized protein G-Sepharose column (Amersham). Monospecific rabbit anti-rSElP antibody was affinity purified from hyperimmune serum using an rSElP-coupled Sepharose column. One milligram of monospecific antibody was conjugated to EZ-Link plus horseradish peroxidase (Pierce) according to the manufacturer's instructions.

Sandwich ELISA. Sandwich ELISA was performed by using 96-well Nunc microplates (Nalge Nunc International) according to the method of Omoe et al. (22, 24). The concentration of each toxin in the culture supernatants was determined by converting the absorbance values to the corresponding concentrations via the standard curve. A straight-line relationship between the concentrations and optical density values was observed at concentrations of SElP between 0.5 and 10 ng/ml. SElP was not detectable at concentrations of 0.25 ng/ml. No cross-reactivity was observed between 100 ng of purified rSEA, rSED, and rSEE in this sandwich ELISA system. To evaluate the specificity of the sandwich ELISA, culture supernatants from 11 S. aureus SE-genotype determined reference strains, two Staphylococcus epidermidis strains, and two E. coli strains were subjected to sandwich ELISA (Table 1). S. aureus, S. epidermidis, and E. coli strains were cultured in brain heart infusion broth supplemented with 1% yeast extract by shaking for 40 h, and the supernatants were preincubated with 20% (vol/vol) normal rabbit serum at 4°C overnight and then diluted 10- to 100-fold in PBST (PBS containing 0.05% Tween 20) to avoid any nonspecific reaction caused by protein A (7). Further, the SElP productivity of 30 S. aureus strains harboring selp were assessed by sandwich ELISA. These strains included 7 food poisoning isolates (from four outbreaks that occurred in different cities in Japan), 22 human nasal swab isolates isolated in Morioka city and Hirosaki city in Japan, and 1 reference strain (N315). SE genotyping of the S. aureus isolates was performed by multiplex PCR according to the method of Omoe et al. (25). This multiplex PCR system is capable of detecting 17 known kinds of SE and SEl genes (sea to see and seg to selr) and the TSST-1 gene.

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TABLE 1. Reference strains used in this study

Top Abstract Introduction Materials and Methods Results Discussion References

 
Cloning and expression of selp. Using the online signal peptide prediction software SignalP, we predicted the N-terminal amino acid sequence of the mature form of SElP to be SEEINGKDLQ. The mature SElP sequence was predicted to be 230 amino acids in length, with a predicted molecular weight of 26,351. The mature region of selp was cloned and sequenced. The nucleotide sequence of this fragment was completely identical to that of the previously reported selp (DDBJ/GenBank/EMBLE BA000018). Recombinant SElP was successfully expressed in the soluble fraction of E. coli lysate and purified by glutathione-Sepharose 4B chromatography.

Superantigenic activity of SElP. Initially, the mitogenicity of rSElP was examined using human PBMC from three healthy donors. PBMC were stimulated with various concentrations of rSElP and TSST-1 in the presence or absence of polymyxin B, an inhibitor of LPS. Figure 1 shows representative results for the three experiments. Lymphocyte proliferation was achieved at an SElP concentration of 0.4 pM (0.01 ng/ml), and the strength of the mitogenicity of SElP was comparable to that of TSST-1. The addition of polymyxin B did not influence the results (data not shown), indicating that the effect of contaminated LPS was negligible. Second, the requirement of MHC class II molecules on ACs for SElP-induced T-cell activation was examined. Because previous studies have shown that SEB and TSST-1 bind well to HLA DR molecules (12, 30), we used L cells transfected with DR4 genes (cell line 8124) and control L cells (cell line 8400) as the ACs. Purified T cells from human PBMC obtained from three healthy donors were stimulated in vitro with 10 ng of rSElP/ml in the presence or absence of DR4⁺ L cells or control L cells. The effect of the antibody to HLA DR on the T-cell response was examined in parallel. Culture supernatants were examined for IL-2, IFN-, TNF-, and IL-4 production. Table 2 shows representative results for three experiments. The production of all cytokines measured was increased in the presence of DR⁺ L cells but not in the presence of control L cells. Anti-DR MAb, but not anti-mouse MHC class I MAb, markedly inhibited cytokine production in the presence of DR⁺ L cells. These results indicated that SElP is a potent T-cell activator and that this activity requires the expression of MHC class II molecules on ACs.

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FIG. 1. Mitogenicity of rSElP for human lymphocytes. Whole human PBMC were stimulated with various doses of rSElP or TSST-1 for 72 h. Representative results of three independent experiments are shown. Each bar represents the mean ± the standard error of triplicate wells from a single experiment. The data are representative of the results for three different donors.

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TABLE 2. Requirement for the presence of MHC class II molecules in accessory cells for T-cell activation by SElPa

Next, the TCR Vß repertoire of human T cells reactive to rSElP was examined by using flow cytometry with MAb to 24 different Vß elements. In addition, since an antibody to the human Vß6 element used in the flow cytometric analysis was not commercially available and substantial homology with SElP was found in SEA and SEE, which activate Vß6⁺ T cells (5, 10), RT-PCR was applied to analyze Vß6 expression in SElP-reactive human T cells. Human T-cell blasts were obtained by stimulating human PBMC with rSElP or anti-CD3 MAb for 3 days and expanding the resulting lymphoblasts with IL-2 for 4 days. These SElP-reactive T-cell blasts were applied to both flow cytometric and PCR analyses. Figure 2a shows representative results of three flow cytometry analyses. The sum of the percentages of the individual cell fractions expressing 24 different Vß elements was nearly 70% in both the T-cell blasts induced by anti-CD3 MAb and rSElP, indicating that the assay system used failed to cover ca. 30% of the TCR Vß repertoire of human T cells. T cells bearing TCR Vß 5.1, 8, 16, 18, and 21.3 were preferentially expanded by rSElP over the control T-cell blasts. In particular, the expansion of Vß5.1⁺ T cells was remarkable. In the PCR analysis, the amounts of Vß6 cDNA in the rSElP-induced T-cell blasts showed a 2.5-fold increase over the control T-cell blasts (Fig. 2b). Since Vß6⁺ T cells are the largest human T-cell fraction in terms of the TCR Vß repertoire in the PCR analysis (30), these results suggest strongly that Vß6⁺ T cells are also a major SElP-reactive fraction, in addition to Vß5.1⁺ T cells.

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FIG. 2. TCR Vß profile of SElP. (a) Human PBMC were stimulated with either anti-CD3 antibody or SElP. Cells were stained with MAbs and analyzed by flow cytometry. The data are presented as percentages of the T-cell fractions expressing distinct Vß elements in whole T cells. The data are representative of the results for three different donors. (b) Human PBMC obtained from three donors were stimulated with either anti-CD3 antibody or SElP. mRNA was extracted and subjected to RT-PCR analysis with a Vß6-specific primer set.

Emetic activity of rSElP in house musk shrew. The emetic activity of rSElP was assessed by intraperitoneal injection of the toxin in house musk shrews (Table 3). A total of 200 µl of rSElP (10 to 150 µg/animal) or PBS were injected intraperitoneally into male house musk shrews (body weight, 55 to 70 g). The animals were observed for vomiting for 3 h after the injection. Emetic responses were observed in one of four house musk shrews receiving a 50-µg injection (700 to 900 µg/kg) and in two of three house musk shrews receiving a 150-µg injection (2,100 to 2,700 µg/kg). Vomiting occurred within 130 min after the injection. Death and diarrhea were not observed in any of the animals tested. This result strongly suggests that SElP is an emetic toxin. Further study on the emetic activity of this toxin in primates is needed to definitively classify it as an SEP.

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TABLE 3. Emetic activity of rSElP in the house musk shrew (Suncus murinus) after intraperitoneal administration

SElP production of S. aureus isolates harboring selp. To evaluate the specificity of a newly developed sandwich ELISA for the detection of SElP, culture supernatants of 11 S. aureus SE-genotype-determined reference strains, along with 2 S. epidermidis strains and 2 E. coli strains listed in Table 1, were subjected to a sandwich ELISA to determine the amounts of SElP. Culture supernatants were preincubated with 20% (vol/vol) normal rabbit serum at 4°C overnight and then diluted 10- to 100-fold in PBST to avoid any nonspecific reactions caused by protein A. Among the S. aureus strains, only the supernatant from S. aureus N315, which harbors selp, showed a high optical density value in the sandwich ELISA (Fig. 3a). No significant reactions were observed in supernatants from S. aureus strains not harboring selp. All supernatants from the S. epidermidis strains and the E. coli strains were negative for SElP (data not shown).

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FIG. 3. (a) Specificity of sandwich ELISA for detecting SElP. Culture supernatants obtained from the S. aureus-type strains listed in Table 1 were subjected to sandwich ELISA to detect SElP. (b) Frequency distribution of the productivity of SElP in S. aureus isolates harboring selp.

Subsequently, culture supernatants from 30 S. aureus strains harboring selp were subjected to the sandwich ELISA. These strains included 7 food poisoning isolates, 22 human nasal swab isolates, and 1 reference strain (N315); the strains were divided into six groups according to the se genes that they harbored (Table 4). Culture supernatants of these strains were examined for the amount of SElP produced. Figure 3b shows the frequency distribution of SElP production in the S. aureus strains. Of 30 S. aureus strains 18, including 4 food poisoning strains with genotype 4, produced significant levels of SElP (50 to 340 ng/ml), whereas 12 S. aureus strains did not produce a detectable level (5 ng/ml) of SElP. It is noteworthy that of the 12 SElP nonproductive strains, 10 strains were included in the seb⁺ selp⁺ genotype 2. Among these strains, three were isolated from food-poisoning outbreaks, five were isolated from healthy humans in Morioka city, and two were isolated from healthy humans in Hirosaki city. All 10 strains, however, exhibited the production of a significant amount of SEB when examined by using reverse passive latex agglutination (Denka Seiken, Co., Ltd.). Both of the strains in group 3, which also carried seb, produced a significant amount of SElP (Table 4).

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TABLE 4. Relationship between productivity of SElP and SE genotypes in selp-harboring S. aureus strains

Top Abstract Introduction Materials and Methods Results Discussion References

 
SE and SEl proteins have been classified into three phylogenetic groups based on their amino acid sequences (19, 23, 26). Group 1 contains the classical SEA, SED, and SEE and the newly identified SEH and SElJ. Group 2 contains the classical SEB and SEC and the newly identified SEG and SElR. Group 3 is composed of only newly identified SE and SEls, such as SEI, SElK, SElL, and SElQ. We classified SElP into group 1 because the mature form of SElP exhibited a high homology of amino acid sequences with SEA (78.1% identity) and SEE (79.0% identity). Our data indicated that SElP acts as a SAg and stimulates a vast number of human T cells bearing TCR Vß 5.1, 6, 8, 16, 18, and 21.3. SEA and SEE have been shown to selectively stimulate human T cells bearing TCR Vß 1, 5.2, 5.3, 6, 7, and 9, and T cells bearing TCR Vß 5.1, 6, 8, and 18, respectively (5, 10). The high degree of sharing of TCR Vß elements among human T cells reactive to SElP, SEA, and SEE also supports the validity of the grouping of SElP into group 1. SElP is closer to SEE than SEA in TCR Vß usage, although the entire amino acid sequences of SElP and SEA and of SElP and SEE are very similar. We predicted that the SElP region required for binding with the TCR Vß elements would have a much higher degree of similarity with that of SEE than with that of SEA.

House musk shrews are a small experimental animal and provide a suitable model for research on the emetic response to various emetic drugs (4, 21). Hu et al. demonstrated that eight kinds of SEs (SEA to SEE, SEG to SEI) have different emetic activities in house musk shrews injected intraperitoneally (9). In that study, the dose of SEA that induced vomiting was 0.3 µg per animal (4 to 6 µg/kg), whereas that of SEE was 10 µg per animal (140 to 180 µg/kg). The present assay showed that 50 to 150 µg/animal (700 to 2,700 µg/kg) of SElP was required to cause an emetic reaction in house musk shrews. The much higher dose of SElP required to elicit an emetic response in house musk shrews, compared to SEA, suggests that the emetic activity of SElP in humans may be lower than that of SEA. Since no marked differences in the strength of emetic activities between SEA, SEB, SEC, SED, and SEE are thought to exist in humans and primates as exist in house musk shrews (2, 9), we think that the use of primate model is essential to test the emetic activity of SElP in humans.

In the present study, we developed a sandwich ELISA system to detect the SElP protein and assessed the SElP production of S. aureus strains harboring selp. A total of 60% of the selp-harboring S. aureus strains produced 50 to 340 ng of SElP/ml in vitro. Reportedly, the amount of classical SEs produced varies widely. SEA, SED, and SEE are often produced at concentrations of 5,000 ng/ml or less in vitro, whereas SEB and SEC are produced in larger quantities (34). Our previous studies showed that SEH and SElR are produced at concentrations of 250 to 700 ng/ml and 40 to 320 ng/ml, respectively. The production level of SElP resembles those of these newly described SEs. A previous epidemiological study suggested that a total of 144 ng of SEA in food was sufficient to cause human food poisoning (6). These newly described SE and SEls, including SElP, are produced at somewhat lower levels than classical SEs. In light of the lower emetic activity of SElP in house musk shrews compared to that of SEA, if SElP is an emetic toxin in humans a much higher amount than that of SEA may be required to produce emesis in humans. Before we conclude that SElP is a food-poisoning toxin, however, its emetic activity in a primate model should be analyzed. In addition, the T-cell stimulative activity of SElP is comparable to that of TSST-1 (Fig. 1), suggesting the possibility that SElP may also be a potent causative toxin of life-threatening TSS. To assess the involvement of SElP in the generation of the abnormal changes seen in TSS, further studies on the toxicity, stability, and production level of SElP in vivo are needed.

It is noteworthy that none of the tested S. aureus strains with the seb⁺ selp⁺ genotype produced SElP. The lack of SElP production may be a common trait of S. aureus strains with the seb⁺ selp⁺ genotype, since the S. aureus strains that we tested were collected from different areas of Japan. In contrast to this genotype, two strains with the other genotype that carry other several se and sel genes in addition to seb and selp produced significant amounts of SElP. se and sel are known to be encoded by mobile genetic elements, such as genomic islands (pathogenicity islands and prophages) and plasmids (1, 15, 23, 33, 34). selp is encoded by prophage Sa3n (15), and seb, selk, and selq are encoded by a genomic island, Sa1 (SaPI3) (33). We assumed that the seb⁺ selk⁺ selp⁺ selq⁺ genotype, which produced SElP, may result from the coexistence of Sa1 (SaPI3) and Sa3n in the genome of these S. aureus strains, without any interference in toxin production. At present, we have no data explaining the reason why selp alone is inactivated in strains with the seb⁺ selp⁺ genotype. seb and selp may be encoded together by some unidentified pathogenic island in the seb⁺ selp⁺ genotype in which only selp is inactivated. se and sel are thought to be transferred between mobile genetic elements encoding these genes among different staphylococcal strains by horizontal transfer, playing an important role in the evolution of S. aureus as a pathogen (1, 15, 25, 33). Further study on the regulatory region of selp gene in this genotype and the transfer of se and sel genes between mobile genetic elements encoding these genes would clarify this question.

 
This study was partly supported by grants-in-aid for scientific research from the Japan Society for the Promotion of Science (grants 15580272 and 16380205).

We thank Keichi Hiramatsu, Teruyo Ito, and Tadashi Baba of Juntendo University for kindly providing the S. aureus strains used in this study and for advice on the SE nomenclature.

 
* 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.

Editor: J. T. Barbieri

Top Abstract Introduction Materials and Methods Results Discussion References

 

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Infection and Immunity, September 2005, p. 5540-5546, Vol. 73, No. 9
0019-9567/05/$08.00+0     doi:10.1128/IAI.73.9.5540-5546.2005
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