In addition to two known staphylococcal enterotoxin-like genes (selj and selr), two novel genes coding for two superantigens, staphylococcal enterotoxins S and T (SES and SET), were identified in plasmid pF5, which is harbored by food poisoning-related Staphylococcus aureus strain Fukuoka 5. This strain was implicated in a food poisoning incident in Fukuoka City, Japan, in 1997. Recombinant SES (rSES) specifically stimulated human T cells in a T-cell receptor V␤9-and V␤16-specific manner in the presence of major histocompatibility complex (MHC) class II ؉ antigen-presenting cells (APC). rSET also stimulated T cells in the presence of MHC class II ؉ APC, although its V␤ skewing was not found in reactive T cells. Subsequently, we examined the emetic activity of SES and SET. We also studied SElR to determine emetic activity in primates. This toxin was identified in previous studies but was not examined in terms of possession of emetic activity for primates. rSES induced emetic reactions in two of four monkeys at a dose of 100 g/kg within 5 h of intragastric administration. In one monkey, rSET induced a delayed reaction (24 h postadministration) at a dose of 100 g/kg, and in the other one, the reaction occurred 5 days postadministration. rSElR induced a reaction in two of six animals within 5 h at 100 g/kg. On this basis, we speculate that the causative toxins of vomiting in the Fukuoka case are SES and SER. Additionally, SES, SER, and SET also induced emesis in house musk shrews as in the monkeys.Staphylococcus aureus produces a variety of superantigenic toxins (SAGTs), which selectively activate a vast number of T cells, depending on V␤ elements in the ␤ chain of a T-cell receptor (TCR), in direct association with major histocompatibility complex (MHC) class II molecules on antigen-presenting cells (APC) (14, 31). Staphylococcal SAGTs can be divided into three large groups and one minor group on the basis of similarity of amino acid sequences (31). Most toxins of the three groups, including staphylococcal enterotoxins A and B (SEA and SEB), exhibit strong emetic activity in primates (4, 16, 25); toxic shock syndrome toxin-1, grouped as the minor group, does not possess emetic activity in primates (14, 31). It is noteworthy that toxins designated SE-like toxins, such as SElP and SElR, which either have not been examined for emetic activity or have been reported not to have emetic activity, have been discovered in S. aureus strains (12,13,20,27). S. aureus strain Fukuoka 5 was isolated from food as the causative microbe in a food poisoning outbreak in Fukuoka City, Japan, in 1997, although this strain did not carry any well-recognized SAGT genes with emetic activity (19). Subsequently, Omoe et al. (19) discovered, using a plaque hybridization with a seg probe, that 2.8 kbp of the EcoRI fragment of plasmid pF5, carried by Fukuoka 5, carries two genes, a novel SAGT gene designated selr and a previously reported gene, selj (33).We undertook research to explore, using a PCR walking technique, whether there were addition...

g Staphylococcal enterotoxins (SEs) are a common causative agent of food poisoning. Recently, many new SE-like (SEl) toxins have been reported, although the role of SEls in food poisoning remains unclear. In this study, the emetic potentials of SElK, SElL, SElM, SElN, SElO, SElP, and SElQ were assessed using a monkey-feeding assay. All the SEls that were tested induced emetic reactions in monkeys at a dose of 100 g/kg, although the numbers of affected monkeys were significantly smaller than the numbers that were affected after consuming SEA or SEB. This result suggests that these new SEs may play some role in staphylococcal food poisoning. Staphylococcal enterotoxins (SEs) are exotoxins that cause staphylococcal food poisoning in humans worldwide (1-4). Several classical types (SEA, SEB, SEC, SED, and SEE) have been characterized (1, 2, 5). Recently, SEG, SEH, SEI, SER, SES, and SET were identified as potential agents of food poisoning (6-10). In addition, new proteins (SE-like toxin J [SElJ], SElK, SElL, SElM, SElN, SElO, SElP, SElQ, SElU, SElV, and SElX) with amino acid sequences that are similar to those of the above-mentioned SEs have been identified (11)(12)(13)(14)(15)(16)(17)(18)(19). SEs are also known to be members of the superantigen family (3,20). During the last few decades, numerous studies have been conducted on the nature of SEs and their superantigenic activities (3,20). However, the emetic activities of these toxins have not been studied. To better understand the etiologic nature of staphylococcal food poisoning, the emetic potentials of SEls should be evaluated in a primate model.We demonstrated the emetic activities of SElK, SElL, SElM, SElN, SElO, SElP, and SElQ using a primate model (cynomolgus monkeys); the number of vomiting events, the time until the first vomiting event (latency period), and behavioral changes were recorded for each animal. We compared the emetic activities of classical and new SEs as well as the activities of three large groups of SEs that were grouped according to the similarity of their amino acid sequences. MATERIALS AND METHODS Preparation of SEls.To construct the SElK, SElL, SElM, SElN, SElO, and SElQ expression plasmids, PCR primers were designed to amplify the gene fragment corresponding to the mature forms of these SEls (Table 1). The selk and selq genes were amplified from genomic DNA of the Staphylococcus aureus S6 strain, which harbors the sea, seb, selk, and selq genes (21). The sell gene was amplified from genomic DNA of the S. aureus bov1117 strain (harboring sea, sec, sell, sed, selj, selr, and tst-1), isolated from cow's milk, and the selm, seln, and selo genes were amplified from genomic DNA of the S. aureus Fukuoka 2 strain (21, 22). The PCR products were digested with BamHI and EcoRI or SalI and then subcloned into a pGEX6P-1 glutathione S-transferase (GST) fusion expression vector. These clones were designated pKKX (including the selk gene), pKLX (including the sell gene), pKMX (including the selm gene), pKNX (including the seln gene), pKOX (includin...

e Staphylococcal enterotoxins (SEs) produced by Staphylococcus aureus have superantigenic and emetic activities, which cause toxic shock syndrome and staphylococcal food poisoning, respectively. Our previous study demonstrated that the sequence of SET has a low level of similarity to the sequences of other SEs and exhibits atypical bioactivities. Hence, we further explored whether there is an additional SET-related gene in S. aureus strains. One SET-like gene was found in the genome of S. aureus isolates that originated from a case of food poisoning, a human nasal swab, and a case of bovine mastitis. The deduced amino acid sequence of the SET-like gene showed 32% identity with the amino acid sequence of SET. The SET-like gene product was designated SElY. In the food poisoning and nasal swab isolates, mRNA encoding SElY was highly expressed in the early log phase of cultivation, whereas a high level of expression of this mRNA was found in the bovine mastitis isolate at the early stationary phase. To estimate whether SElY has both superantigenic and emetic activities, recombinant SElY was prepared. Cell proliferation and cytokine production were examined to assess the superantigenic activity of SElY. SElY exhibited superantigenic activity in human peripheral blood mononuclear cells but not in mouse splenocytes. In addition, SElY exhibited emetic activity in house musk shrews after intraperitoneal and oral administration. However, the stability of SElY against heating and pepsin and trypsin digestion was different from that of SET and SEA. From these results, we identified SElY to be a novel staphylococcal emetic toxin. Staphylococcus aureus produces a variety of exotoxins, including staphylococcal enterotoxins (SEs) and toxic shock syndrome toxin 1 (TSST-1) (1). These toxins are superantigens, which have the ability to stimulate a large repertoire of the V␤ elements of T cell receptor (TCR)-bearing T cells. SEs are also emetic toxins causing staphylococcal food poisoning in humans, although the mechanism of SE-induced emesis has not been entirely verified (2). Due to these properties, SEs are assumed to be a menace to public health. Five major serological types (SEA to SEE) have been characterized (2), and new types of SE-related toxins (SEG to SEI, SElJ, SEK to SET, SElU, SElV, and SElX) have recently been reported (2-8). Moreover, superantigen-related genes, such as staphylococcal superantigen-like protein (SSL) genes (ssl1 to ssl26), were discovered during determination of the complete genome sequences of several S. aureus strains (9-11). It has been recognized that the superantigen genes are associated with mobile genetic elements (MGEs), such as pathogenicity islands, prophages, or plasmids (5,(11)(12)(13)(14). This fact implies that these superantigen genes move among S. aureus strains by horizontal transfer and that such MGEs play an important role in the evolution of S. aureus as a pathogen.We have reported that SET shows mitogenicity to human T cells and requires major histocompatibility complex (MHC) cla...

e Molecular characterization of isolates from staphylococcal food poisoning (SFP) outbreaks in Japan showed that the dominant lineage causing SFP outbreaks is clonal complex 81 (CC81), a single-locus variant of sequence type 1, coagulase type VII, positive for sea and/or seb, and positive for seh. Among various CC lineages producing staphylococcal enterotoxin A, CC81 showed the highest toxin productivity. Staphylococcal food poisoning (SFP), one of the most common food-borne diseases, results from the consumption of foods containing toxic amounts of staphylococcal enterotoxins (SEs) (1-4). SFP is associated with toxigenic Staphylococcus aureus strains that produce one or more members of a family of genes encoding heat-stable SEs. Recently, a superfamily of more than 23 different SEs and SE-like toxins (SEls) was studied for their biological activities (4-8). These bacterial toxins are also known as pyrogenic superantigens that stimulate polyclonal T-cell proliferation and can potentially cause toxic shock syndrome (1-4). The genes for SEs and SEls are harbored by various mobile genetic elements and/or genomic islands, including prophages, plasmids, S. aureus pathogenicity islands (SaPIs), and Sa␤. To date, in addition to the five classical types of SEs (SEA through SEE), 18 new types of SEs and SEls (SEG through SElX) have been described (4-8). Our recent study confirmed the emetic potential of SElK, SElL, SElM, SElN, SElO, SElP, and SElQ in the monkey, and these SEls were renamed SEK, SEL, SEM, SEN, SEO, SEP, and SEQ, respectively (9). Comparing SEs and SEls, SEA is considered the most important SE causing SFP. S. aureus is a common commensal bacterium of the skin and mucosal surfaces of humans, with estimates of 20% persistent and 60% intermittent colonization (10). Food handlers carrying enterotoxin-producing S. aureus in their nasal cavities or on their skin are important sources of food contamination during the cooking process (3). Contamination with S. aureus is believed to be associated primarily with improper handling of cooked or processed foods with improper storage under conditions that allow the growth of S. aureus and the production of SEs. Reports concerning the molecular epidemiology and genetic diversity of the isolates from SFP outbreaks are limited when clinical isolates are compared. In this investigation, we ex-

Staphylococcus aureus pathogenicity islands (SaPIs) form a growing family of mobile genetic elements (MGEs) in Staphylococci. Horizontal genetic transfer by MGEs plays an important role in the evolution of S. aureus. Several SaPIs carry staphylococcal enterotoxin and SE-like toxin genes. To comprehensively investigate the diversity of SaPIs, a series of primers corresponding to sequences flanking six SaPI insertion sites in S. aureus genome were designed and a long and accurate (LA)-PCR analysis method established. LA-PCR products of 13-17 kbp were observed in strains with seb, selk or selq genes. Restriction fragment length polymorphism (RFLP) analysis showed that the products have different RFLP characteristics than do previously described SaPIs; they were therefore predicted to include new SaPIs. Nucleotide sequencing analysis revealed seven novel SaPIs: seb-harboring SaPIivm10, SaPishikawa11, SaPIivm60, SaPIno10 and SaPIhirosaki4, selk and selq-harboring SaPIj11 and non-superantigen-harboring SaPIhhms2. These SaPIs have mosaic structures containing components of known SaPIs and other unknown genes. Strains carrying different SaPIs were found to have significantly different production of superantigen toxins. The present results show that the LA-PCR approach can comprehensively identify SaPI diversity and is useful for investigating the evolution of S. aureus pathogenicity.

Staphylococcal superantigen (SAg) toxins are the most notable virulence factors associated with Staphylococcus aureus, which is a pathogen associated with serious community and hospital acquired infections in humans and various diseases in animals. Recently, SAg toxins have become a superfamily with 29 types, including staphylococcal enterotoxins (SEs) with emetic activity, SE-like toxins (SEls) that do not induce emesis in primate models or have yet not  been tested, and toxic shock syndrome toxin-1 (TSST-1). SEs and SEls can be subdivided into classical types (SEA to SEE) and novel types (SEG to SElY, SE01, SE02, SEl26 and SEl27). The genes of SAg toxins are located in diverse accessory genetic elements and share certain structural and biological properties. SAg toxins are heat-stable proteins that exhibit pyrogenicity, superantigenicity and capacity to induce lethal hypersensitivity to endotoxin in humans and animals. They have multiple pathogenicities that can interfere with normal immune function of host, increase the chances of survival and transmission of pathogenic bacteria in host, consequently contribute to the occurrence and development of various infections, persistent infections or food poisoning. This review focuses on the following aspects of SAg toxins: (1) superfamily members of classic and novelty discovered staphylococcal SAgs; (2) diversity of gene locations and molecular structural characteristics; (3) biological characteristics and activities; (4) multi-pathogenicity of SAgs in animal and human diseases, including bovine mastitis, swine sepsis, abscesses and skin edema in pig, arthritis and septicemia in poultry, and nosocomial infections and food-borne diseases in humans.

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Staphylococcal enterotoxin A (SEA) is a leading causative toxin of staphylococcal food poisoning. However, it remains unclear how this toxin induces emesis in humans, primates, and certain experimental animals. To understand the mechanism of SEA-induced emesis, we investigated the behavior of SEA in the gastrointestinal (GI) tract in vivo using the house musk shrew (Suncus murinus). Immunofluorescence of GI sections showed that perorally administered SEA translocated from the lumen to the interior tissues of the GI tract and rapidly accumulated in certain submucosa cells. These SEA-binding cells in the submucosa were both tryptase- and FcεRIα-positive, suggesting these SEA-binding cells were mast cells. These SEA-binding mast cells were 5-hydroxytryptamine (5-HT)-positive, but the intensity of the 5-HT signal decreased over time compared to that of mast cells in the negative control. Furthermore, toluidine blue staining showed the number of metachromatic mast cells was decreased in the duodenal submucosa, suggesting that SEA binding induced degranulation and release of 5-HT from submucosal mast cells. These observations suggest that the target cells of SEA are submucosal mast cells in the GI tract and that 5-HT released from submucosal mast cells plays an important role in SEA-induced emesis.