Article Figures & Data
Figures
- Fig. 1.
Nucleotide and inferred amino acid sequences ofsek and SEK, respectively, cloned from staphylococcal TSS isolate MN NJ. The putative RBS is in boldface, and putative −10 and −35 promoter sequences are underlined. An asterisk marks the predicted amino terminus of the mature protein after removal of the signal peptide.
- Fig. 2.
Phylogenetic tree diagram of the family of SEs of serotypes A to L. Three distinct subfamilies can be observed. Groupings are an indication of relatedness, but distances are not quantitatively related to evolutionary distance.
- Fig. 3.
Alignment of the mature form of SEK with representative toxins from the major enterotoxin subfamilies (SEA representing the SEA, -D, -E, -H, and -J subfamily; SEB representing the SEB, -C, and -G subfamily; and SEI representing the SEI, -K, and -L subfamily). Residues that are homologous to residues in SEK are highlighted in black. Dashes represent gaps in the aligned sequences.
- Fig. 4.
SDS-PAGE analysis (15% polyacrylamide gel) of rSEK. The gel was stained with Coomassie brilliant blue R250. Lane 1, molecular weight (MW) standards with sizes given to the left in thousands; lane 2, 10 μg of rSEK. The apparent size of rSEK was 30,000.
- Fig. 5.
Western immunoblot of concentrated supernatant fluid from TSS isolate MN JA compared to purified rSEK. Samples were electrophoresed by SDS-PAGE (15% polyacrylamide) and transferred to a nitrocellulose membrane. The blot was developed with hyperimmune rabbit polyclonal antiserum to rSEK. Bound antibody was detected with a secondary antibody to rabbit immunoglobulin G conjugated to alkaline phosphatase followed by substrate (4). Lane 1, 10 μl of crude extract of MN JA supernatant; lane 2, 10 μg of rSEK. MW, sizes in thousands as determined by SDS-PAGE.
- Fig. 6.
Superantigenicity assay of SEK (□) versus TSST-1 (■) as assessed by measuring proliferation of rabbit splenocytes (2 × 105/well/200 μl). Splenocytes in complete RPMI were incubated for 4 days in quadruplicate samples in 96-well microtiter plates in the presence of rSEK or TSST-1 used as a control at the designated concentrations added in 20-μl volumes. Negative control wells contained 20 μl of PBS rather than toxin. [3H]thymidine (1 μCi/well) was added to all wells after 3 days, and splenocyte proliferation was measured by determining cpm of radiolabel incorporation into DNA. Results are the average cpm of quadruplicate wells. Values represent the mean ± standard error of the mean.
- Fig. 7.
Pyrogenicity of rSEK in rabbits and the ability of the toxin to enhance host susceptibility to lethal endotoxin shock. rSEK was injected intravenously at doses of 4.5, 0.45, and 0.045 μg/kg/ml in PBS. Fever development was assessed at 0 and 4 h with rectal thermometers. The values presented represent the mean ± standard error of the mean. At the 4-h time point, all rabbits were injected with 10 μg of LPS from S. enterica serovar Typhimurium. Lethality was assessed during the first 48 h postinjection; lethality at 4.5 and 0.45 μg/kg was significantly different from that at 0.045 μg/kg at P = 0.05 , as determined by Fisher's exact test.
- Fig. 8.
TCR Vβ profile of rSEK. Three patients' PBMCs were stimulated with either anti-CD3 antibody (white bars), which stimulates all T cells, or rSEK (black bars), which selectively stimulates T cells dependent on the variable part of the β chain (Vβ) of the TCR. Cells were stained with monoclonal antibodies against the listed TCR Vβ chains (V-beta type), and the results were evaluated by flow cytometry. The percentages of T cells expressing the listed TCR Vβ are shown. P values were determined by the paired Student'st test (∗∗, P < 0.05 ; ∗, P = 0.055 ). Error bars represent the standard error of the mean for each data set.
