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.

FIG 1

Phylogenetic tree of staphylococcal enterotoxins, including SElY and TSST-1. This phylogenetic tree was constructed using ClustalW. The tree shows that SElY belongs to the SET/SElY subgroup, and that subgroup is related to a subgroup consisting of SElX and TSST-1.

FIG 2

Detection of sely mRNA by real-time quantitative PCR. (A) The growth of the S. aureus isolates was determined by measurement of the optical density (O.D.) at 660 nm. Total RNA was prepared from S. aureus cells collected at 3, 6, 9, and 12 h after inoculation. (B) Expression of sely mRNA was determined by normalization to gyrB mRNA expression. Data are expressed as the mean ± standard deviation for four samples. These data were reproducible in three experiments. Statistical analysis was performed using ANOVA followed by the Tukey test. A P value of less than 0.05 was considered statistically significant. *, P < 0.05.

FIG 3

Superantigenic activity of SElY in mouse splenocytes. Mouse splenocytes were incubated with various concentrations of SEA, SET, and SElY for 72 h. (A) The uptake of [³H]thymidine was determined to assess lymphocyte proliferation. (B and C) The production of IFN-γ (Β) and IL-2 (C) was determined by sandwich ELISAs. Mock, mock treatment with no toxin. Each bar represents the mean ± standard deviation for triplicate wells from a representative experiment. These data were reproducible in three experiments.

FIG 4

Superantigenic activity of SElY in human PBMCs. Human PBMCs were incubated with various concentrations of SEA, SET, and SElY for 72 h. (A) The uptake of [³H]thymidine was determined to assess lymphocyte proliferation. (Β) The production of IFN-γ was determined by sandwich ELISAs. Each bar represents the mean ± standard deviation for triplicate wells from a representative experiment. These data were reproducible in three experiments.

FIG 5

Effects of heat and digestive enzyme treatments on SEs. (A) SEs and BSA were maintained at 100°C. After the heat treatment, each sample was analyzed by SDS-PAGE. (B) Each protein (100 μg/ml) was incubated with pepsin (10 μg/ml) and analyzed by SDS-PAGE. (C) Each protein (100 μg/ml) was incubated with trypsin (100 μg/ml) and analyzed by SDS-PAGE. NT, no treatment; P, pepsin; Tr, trypsin.