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 Vbeta9- and Vbeta16-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 Vbeta 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 microg/kg within 5 h of intragastric administration. In one monkey, rSET induced a delayed reaction (24 h postadministration) at a dose of 100 microg/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 microg/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.

FIG. 1.

Location of the two new superantigen-like genes in the selj and selr flanking regions. An 11,335-bp nucleotide sequence of the selr (ORF3) and selj (ORF4) flanking region was determined, and two new superantigen-like genes, ses (ORF6) and set (ORF5), were identified.

FIG. 2.

Phylogenetic tree of SEs and SEls, including SES and SET. The phylogenetic tree was constructed by the neighbor-joining method based on amino acid sequences. Five distinct groups can be observed. SES is most closely related to SElN and belongs to the SEA group, while SET is distinct from the SEA, SEB, and SEI groups.

FIG. 3.

Mitogenic activity of SES and SET. PBMCs were isolated from human blood samples and incubated with a number of concentrations of rSEA, rSES, and rSET. Means and standard errors for triplicate wells from a single experiment are shown.

FIG. 4.

Representative dot plots of Vβs for CD3-gated events from PBMCs stimulated with SES (A) and anti-CD3 (B). MAbs against Vβ9, Vβ16, and Vβ17 were labeled with phycoerythrin (PE), fluorescein isothiocyanate (FITC), or PE and FITC, respectively. Numbers indicate the percentage of T cells expressing a particular Vβ element.

FIG. 5.

Expression of ses and set. (A) Detection of ses and set mRNAs by RT-PCR. S. aureus Fukuoka 5 was cultured at 37°C and collected for RNA isolation at 0 to 7 h after inoculation. The Fukuoka 5 growth curve was graphed by determining the optical density at 600 nm (O.D.600). (B) Detection of SES and SET in culture supernatants of S. aureus strains harboring plasmid pF5 (Fukuoka 5, Fukuoka 6, and Fukuoka 7) by Western blotting.