Mutational analysis of superantigen activity responsible for the induction of skin erythema by streptococcal pyrogenic exotoxin C

Abstract

Streptococcal pyrogenic exotoxin C (SPEC), when injected intradermally, induces erythema in unsensitized rabbits. In the present study, we examined whether this erythema induction is due to the T-cell stimulatory activity of SPEC as a superantigen. Analysis by using single-residue mutant SPECs indicated that mutant SPECs Y15I, A16E, and Y17I, in which tyrosine 15, alanine 16, and tyrosine 17 were replaced with isoleucine, glutamic acid, and isoleucine, respectively, exhibited significantly reduced mitogenic activity for Vbeta2(+) human T cells in vitro, and Y15I showed as much as a 1, 000-fold reduction. Y15I mutant SPEC, however, retained the ability to bind to major histocompatibility complex class II antigen and to form a homodimer, implying that residue 15 is critically important for the interaction of SPEC with T-cell antigen receptor beta chains. When injected intradermally into normal rabbits, wild-type SPEC induced a characteristic erythema after 3 h in a dose-dependent fashion, which was associated with polymorphonuclear and mononuclear cell infiltration. This erythema formation was found to be severely suppressed by systemic pretreatment with cyclosporin A, suggesting the involvement of host T cells. Y15I mutant SPEC exhibited nearly 1, 000-fold less erythema induction in vivo than wild-type SPEC. Altogether, the present results strongly suggest that erythema induction in rabbits by SPEC is attributable mostly to its T-cell stimulatory activity as a superantigen.

FIG. 1

Amino acid substitutions in mutant SPECs. Mutagenesis was introduced on the residues located in the N terminus of SPEC. In each amino acid sequence, dashes indicate identical amino acid residues and the substituted amino acid is shown in the one-letter code.

FIG. 2

Mitogenic activity of wild-type and mutant SPECs. Normal human PBL were incubated with each toxin for 56 h and pulsed with 0.5 μCi of [³H]thymidine for 16 h, and [³H]thymidine incorporation was determined. Values are means of triplicate determinations. (A) ○, wild-type SPEC; •, K3E: ▵, D5V; ▴, L14D. (B) ○, wild-type SPEC; •, Y15I; ▵, A16E; ▴, Y17I; □, T20I; ■, D23V; ▿, Y24L. (C) ○, wild-type SPEC; •, N30R; ▵, S32E; ▴, T33R; □, N38I; ■, D40A.

FIG. 3

Vβ2-specific expansion of T cells. Expansion of Vβ2⁺ T cells on SPEC-stimulated human PBL was analyzed by two-color flow cytometry. PBL (10⁶/ml) from three healthy volunteers were incubated with PBS, wild-type SPEC, or mutant SPEC Y15I (final toxin concentration, 100 pg/ml) for 6 days, double stained with a biotin-conjugated anti-OKT3 MAb and a fluorescein isothiocyanate-conjugated anti-Vβ2 MAb, followed by phycoerythrin-avidin, and analyzed by a FACScan. The flow cytometric profiles of three individual samples of PBL are shown.

FIG. 4

Binding of wild-type and mutant SPECs to MHC class II. The ability of each toxin to bind to MHC class II was examined in a competition assay. Raji cells were preincubated with various concentrations of toxins (wild-type SPEC, 10 and 70 μg/ml; Y15I, 10 and 70 μg/ml; A16E, 2.4 μg/ml; Y17I, 10 μg/ml), washed, incubated with wild-type SPEC (2 μg/ml) conjugated with FluorX reactive dye, and then analyzed by flow cytometry.

FIG. 5

Dimer formation of wild-type SPEC and Y15I mutant SPEC. Wild-type SPEC and Y15I mutant SPEC were incubated in 0.5 M Tris-HCl buffer, boiled in sample buffer, and analyzed by SDS-PAGE. Lanes: 1, 3, and 5, 2 μg of wild-type SPEC; 2, 4, and 6, 2 μg of Y15I mutant SPEC; 1 and 2, 0.5 M Tris-HCl buffer and sample buffer adjusted to pH 8.8; 3 and 4, buffers adjusted to pH 6.8; 5 and 6, buffers adjusted to pH 5.0.

FIG. 6

Erythematous reaction induced in a rabbit by intradermal injection of SPEC. (A) Erythema that developed 4 h after wild-type SPEC (200 ng) was injected intradermally into an unsensitized rabbit. (B) Histology of an erythematous lesion that developed 4 h after wild-type SPEC (200 ng) was injected intradermally into a rabbit that had received an injection of the same dose of wild-type SPEC 1 week previously. Hematoxylin-and-eosin staining. Bar, 50 μm.

FIG. 7

(A) Mitogenic activities of wild-type SPEC and Y15I mutant SPEC. Unsensitized rabbit PBL were incubated with various concentrations of either wild-type SPEC or Y15I mutant SPEC for 95 h, pulsed with 0.5 μCi of [³H]thymidine for 20 h, and harvested. [³H]thymidine incorporation was then determined. Values are means ± standard deviations of triplicate measurements. ○, wild-type SPEC; •, Y15I mutant SPEC. (B) Erythema induced by wild-type SPEC and Y15I mutant SPEC. Various concentrations of either wild-type SPEC or Y15I mutant SPEC were injected intradermally into unsensitized rabbits and 4 h later, diameters of erythematous lesions formed around injection sites were measured. Values are means ± standard deviations for groups of four animals. ○, wild-type SPEC; •, mutant SPEC Y15I; ×, PBS. ∗, P < 0.002 (Student’s t test).