Identification and characterization of a new staphylococcal enterotoxin-related putative toxin encoded by two kinds of plasmids

Abstract

We identified and characterized a novel staphylococcal enterotoxin-like putative toxin, which is named SER. Nucleotide sequencing analysis of the ser gene revealed that ser was most closely related to the seg gene. The ser gene product, SER, was successfully expressed as a recombinant protein in an Escherichia coli expression system, and recombinant SER (rSER) showed significant T-cell stimulation activity. The SER production in ser-harboring Staphylococcus aureus strains was confirmed by Western blot analysis using anti-rSER antibody. Moreover, ser was seen to be encoded by at least two types of plasmids. In particular, one kind of plasmid encoding the ser gene has been known as a sed- and sej-carrying pIB485-related plasmid.

FIG. 1.

Southern blot analysis of SE-unidentified S. aureus Fukuoka 5 isolate under the low-stringency condition. HindIII (H)-, XbaI (X)-, and EcoRI (E)-digested total DNA of the S. aureus Fukuoka 5 strain (F5, food poisoning outbreak-related strain, SE unidentified) and the Fukuoka 1 strain (F1, positive control [PC]; seg and sei positive) were subjected to Southern hybridization using a seg probe.

FIG. 2.

Diagram of cloned DNA containing SE genes. Subclone pKO311 contained 2,751 bp of insert. This insert contained two kinds of SE genes, sej and ser. The directions of transcription of the genes are shown by the arrows. The letters refer to sites cut by the restriction endonucleases: B, BglII; C, ClaI; E, EcoRI; H, HindIII; N, NdeI; S, StuI; SB, SnaBI; X, XbaI.

FIG. 3.

Nucleotide sequence of ser. Nucleotide sequence corresponding to positions 541 to 1440 of 2.8-kbp inset of pKO311 is shown. The putative ribosomal binding site sequence is in boldface. The putative promoter sequences are indicated by double underline. The deduced amino acid sequence with a putative signal peptide (underlined) and its stop codon (asterisk) are indicated below the nucleotide sequence.

FIG. 4.

Production of IFN-γ, TNF-α, and IL-2 by murine splenocytes in response to stimulation with rSER. Mouse splenocytes isolated from C57BL/6 mice were stimulated by various concentrations of rSER and rTSST-1 (positive control). Production of IFN-γ, TNF-α, and IL-2 was measured by sandwich ELISA. Negative control cells were stimulated with phosphate-buffered saline rather than SAgs.

FIG. 5.

Confirmation of secretion of SER by S. aureus isolates by Western blot analysis. Using anti-rSER antibody, we examined SER productivity of S. aureus isolates from a food poisoning outbreak in Fukuoka. Culture supernatants from Fukuoka 5 (F5), Fukuoka 6 (F6), Fukuoka 7 (F7), and Fukuoka 8 (F8) were subjected to Western blot analysis. An approximately 30-kDa signal was detected by anti-rSER antibody. P1, 25 ng of rSER per well; P2, 50 ng of rSER per well.

FIG. 6.

ser and sej genes are carried by two kinds of staphylococcal plasmid. The pF5-like plasmids and pIB485-like plasmids were purified from S. aureus laboratory strains, food poisoning outbreak isolates, and healthy human isolates. These plasmids were digested by EcoRI and then subjected to Southern blot analysis. Lanes: 196E, p196E; 361, p361; 1151, p1151; 727, p727; 740, p740; I-7, pI7; I-16, pI16; I-46, pI46; F5, pF5; F6, pF6; and F7, pF7.