Hisaya K. Ono

Identification and Characterization of Two Novel Staphylococcal Enterotoxins, Types S and T

ABSTRACT 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 Vβ9- and Vβ16-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 Vβ 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 μg/kg within 5 h of intragastric administration. In one monkey, rSET induced a delayed reaction (24 h postadministration) at a dose of 100 μg/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 μg/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.

Emetic Potentials of Newly Identified Staphylococcal Enterotoxin-Like Toxins

ABSTRACT 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 μg/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.

Identification and Characterization of a Novel Staphylococcal Emetic Toxin.

ABSTRACT 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.

A novel comprehensive analysis method for Staphylococcus aureus pathogenicity islands.

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.

Molecular Epidemiology and Identification of a Staphylococcus aureus Clone Causing Food Poisoning Outbreaks in Japan

ABSTRACT 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.

Molecular epidemiological characterization of Staphylococcus aureus isolates originating from food poisoning outbreaks that occurred in Tokyo, Japan

Staphylococcal food poisoning (SFP), one of the commonest food‐borne diseases, results from the ingestion of one or more staphylococcal enterotoxins (SEs) produced in foods by Staphylococcus aureus. In the present study, 203 S. aureus strains originating from 83 outbreaks that had occurred in Tokyo were examined for their coagulase type and genotype of SEs to analyze their molecular epidemiological characteristics. The representative subsets of the 83 S. aureus isolates were analyzed by multilocus sequence typing (MLST) and S. aureus pathogenicity island (SaPI) scanning. The isolates were integrated into eight specific clonal complexes (CC) s; CC81, CC8, CC6, CC5, CC508, CC59, CC20 and CC30. The profiles of the coagulase type, SE/SEl genotype and the suspected type of enterotoxin‐encoding mobile genetic element (MGE) indicated a correlation with each CC. SaPI scanning showed fixed regularity between the distributions of genomic islands, including SaPIs, and the phylogenetic lineage based on MLST. These results indicate that the S. aureus isolates, which classified into eight CCs, have distinguishable properties concerning specific coagulase type, enterotoxin genotype and MGE type. Strains of S. aureus harboring these particular elements possess the potential to cause SFP.

Submucosal mast cells in the gastrointestinal tract are a target of staphylococcal enterotoxin type A

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.

Detection of the staphylococcal enterotoxin D-like gene from staphylococcal food poisoning isolates over the last two decades in Tokyo

The plasmid is a very well-known mobile genetic element that participates in the acquisition of virulence genes, such as staphylococcal enterotoxins (SEs), via horizontal transfer. SEs are emetic toxins and causative agents in staphylococcal food poisoning (SFP). We herein identified the types of plasmids harbored by seven SFP isolates and examined their production of plasmid-related SE/SEl to determine whether the new types of plasmid-related SE or SE-like (SEl) toxins (i.e. SElJ and SER) were involved in SFP. These isolates harbored pIB485-like plasmids, and all, except for one isolate, produced SElJ and SER. The amount of SER produced by each isolate accounted for the highest or second highest percentage of the total amount of SE/SEl produced. These new types of plasmid-related SE/SEls as well as classical SE may play a role in SFP. The seven isolates were classified into two SED-production types; a high SED-production type (>500 ng/ml) and no SED-production type. A nucleotide sequencing analysis revealed that three plasmids harbored by the SED-non-producing isolates had a single-base deletion in the sed gene with a resulting stop codon (from 233 amino acids of the intact SED to 154 amino acids of the mutant SED (mSED)). A real-time reverse transcription-PCR analysis showed that the mRNA of the msed gene was transcribed in the isolates. If the msed gene was translated as a protein, mSED may act as an emetic toxin instead of intact SED.

Acidic environments induce differentiation of Proteus mirabilis into swarmer morphotypes

Although swarmer morphotypes of Proteus mirabilis have long been considered to result from surfaced‐induced differentiation, the present findings show that, in broth medium containing urea, acidic conditions transform some swimmer cells into elongated swarmer cells. This study has also demonstrates that P. mirabilis cells grown in acidic broth medium containing urea enhance virulence factors such as flagella production and cytotoxicity to human bladder carcinoma cell line T24, though no significant difference in urease activity under different pH conditions was found. Since there is little published data on the behavior of P. mirabilis at various hydrogen‐ion concentrations, the present study may clarify aspects of cellular differentiation of P. mirabilis in patients at risk of struvite formation due to infection with urease‐producing bacteria, as well as in some animals with acidic or alkaline urine.

High Affinity of Interaction between Superantigen and T Cell Receptor Vβ Molecules Induces a High Level and Prolonged Expansion of Superantigen-reactive CD4+ T Cells

In mice implanted with an osmotic pump filled with the superantigen (SAG) staphylococcal enterotoxin A (SEA), the Vβ3+CD4+ T cells exhibited a high level of expansion whereas the Vβ11+CD4+ T cells exhibited a mild level of expansion. In contrast, in mice implanted with an osmotic pump filled with SE-like type P (SElP, 78.1% homologous with SEA), the Vβ11+CD4+ T cells exhibited a high level of expansion while the Vβ3+CD4+ T cells exhibited a low level of expansion, suggesting that the level of the SAG-induced response is determined by the affinities between the TCR Vβ molecules and SAG. Analyses using several hybrids of SEA and SElP showed that residue 206 of SEA determines the response levels of Vβ3+CD4+ and Vβ11+CD4+ T cells both in vitro and in vivo. Analyses using the above-mentioned hybrids showed that the binding affinities between SEA and the Vβ3/Vβ11 β chains and between SEA-MHC class II-molecule complex and Vβ3+/Vβ11+ CD4+ T cells determines the response levels of the SAG-reactive T cells both in vitro and in vivo.