Contribution of each of four Superantigens to Streptococcus equi-induced mitogenicity, gamma interferon synthesis, and immunity

Abstract

Streptococcus equi is the causative agent of strangles, the most frequently diagnosed infectious disease of horses worldwide. The disease is characterized by abscessation and swelling of the lymph nodes of the head and neck, which can literally strangle the horse to death. S. equi produces four recently acquired phage-associated bacterial superantigens (sAgs; SeeH, SeeI, SeeL, and SeeM) that share homology with the mitogenic toxins of Streptococcus pyogenes. The aim of this study was to characterize the contribution of each of these S. equi sAgs to mitogenic activity in vitro and quantify the sAg-neutralizing capacity of sera from naturally infected horses in order to better understand their role in pathogenicity. Each of the sAgs was successfully cloned, and soluble proteins were produced in Escherichia coli. SeeI, SeeL, and SeeM induced a dose-dependent proliferative response in equine CD4 T lymphocytes and synthesis of gamma interferon (IFN-gamma). SeeH did not stimulate equine peripheral blood mononuclear cells (PBMC) but induced proliferation of asinine PBMC. Allelic replacement mutants of S. equi strain 4047 with sequential deletion of the superantigen genes were generated. Deletion of seeI, seeL, and seeM completely abrogated the mitogenic activity and synthesis of IFN-gamma, in equine PBMC, of the strain 4047 culture supernatant. Sera from naturally infected convalescent horses had only limited sAg-neutralizing activities. We propose that S. equi sAgs play an important role in S. equi pathogenicity by stimulating an overzealous and inappropriate Th1 response that may interfere with the development of an effective immune response.

FIG. 1.

Cell proliferation induced by S. equi superantigens. (A) Equine PBMC labeled with CFSE and cultured for 3 days in the presence of medium alone (gray) or S. equi sAgs at 0.125 μg/ml (open). A peak in M₁ indicates a fluorescence decrease due to cell division. (B) Dose response. A total of 2 × 10⁵ equine PBMC were cultured for 4 days in triplicate with the indicated concentration of sAg and incubated with ³HT for 16 h before measurement of proliferation. (C) Kinetics of proliferation. A total of 2 × 10⁵ equine PBMC were cultured with 0.125 μg/ml sAg in triplicate for 1 to 5 days, with incorporation of ³HT during the last 16 h of each time period. Results are presented as the stimulation index. (D) Representative cell phenotypes of S. equi sAg-activated PBL. Equine PBMC were cultured with 0.125 μg/ml sAg for 4 days prior to analysis. CD5, CD4, and CD8 lymphoblast staining levels after SeeM stimulation are shown as representative phenotypes of S. equi sAg-activated cells.

FIG. 2.

IFN-γ synthesis induced by S. equi superantigens. (A) A total of 1 × 10⁶ equine PBMC were incubated overnight with medium alone or 0.125 μg/ml sAg. Density plots are representative of at least three experiments. (B) Representative cell phenotypes of IFN-γ⁺ PBL after stimulation with S. equi sAg. Equine PBMC were cultured with 0.125 μg/ml sAg for 4 days prior to analysis. CD5, CD4, and CD8 lymphoblast staining after SeeM stimulation is shown as a representative phenotype of S. equi sAg-activated cells. (C) Dose response of IFN-γ synthesis. A total of 1 × 10⁶ equine PBMC were incubated overnight with the indicated concentrations of sAgs. IFN-γ synthesis was detected by flow cytometry after intracellular staining.

FIG. 3.

IFN-γ synthesis induced by S. equi culture supernatants. (A) Typical density plot of 1 × 10⁶ equine PBMC incubated overnight with medium alone, S. zooepidemicus H70, or S. equi (isolate JKS044) culture supernatants or with 0.125 μg/ml SeeM. IFN-γ synthesis was detected by flow cytometry after intracellular staining. (B) Biparametric histogram of cell proliferation and IFN-γ synthesis induced by culture supernatants (1/20) of 28 S. equi isolates (open circles) and 22 S. zooepidemicus isolates (black circles). For cell proliferation, 2 × 10⁵ equine PBMC were cultured for 4 days in triplicate with culture supernatants and incubated with ³HT for 16 h before measurement of proliferation. For IFN-γ synthesis, 1 × 10⁶ equine PBMC were incubated overnight with culture supernatants. IFN-γ synthesis was detected by flow cytometry after intracellular staining. Dotted lines represent thresholds above which the response was considered positive (>2 for cell proliferation and >3.1 for IFN-γ synthesis).

FIG. 4.

Box-and-whisker plot histograms of cell proliferation (A) and IFN-γ synthesis (B) induced by S. equi culture supernatants after sequential deletion of sAgs. (A) For cell proliferation, 2 × 10⁵ equine PBMC were cultured for 4 days in triplicate with culture supernatants (1/20) and incubated with ³HT for 16 h before measurements of proliferation. Results are expressed as the percentage of proliferation inhibition compared with the mitogenic activity of wild-type S. equi strain 4047. (B) For IFN-γ synthesis, 1 × 10⁶ equine PBMC were incubated overnight with culture supernatants (1/20). IFN-γ synthesis was detected by flow cytometry after intracellular staining. Results are expressed as the percent inhibition of IFN-γ synthesis compared with wild-type strain 4047. Differences between groups are indicated with a letter code (a to d). For example, a and b indicate a statistical difference (P < 0.05) between the two groups, and a group noted with ab is not statistically different from a group noted a or b (P > 0.05). The most relevant P values are indicated. The rectangle represents 50% of the observed response for each group, the horizontal line indicates the median of the group, and the cross indicates the average of the group. Squares indicate outlier results. n indicates the number of samples measured per group.

FIG. 5.

Kinetics of S. equi sAg-specific antibody responses in vivo. The antibody responses to all four S. equi sAgs were quantified by ELISA using sera taken from seven horses during strangles outbreaks. The initial sera (day zero) were taken from healthy horses with no signs of disease. These horses were diagnosed with strangles infection a few days after the initial serum samples were taken. Dotted lines represent the threshold above which the antibody response was considered positive.

FIG. 6.

Quantification of sAg-neutralizing activity of convalescent-phase horse sera (1 to 3 different time points per horse). Equine sera purified from horses numbers 1 to 6 (prior to and after S. equi infection) were incubated for 1 h with recombinant SeeI (A), SeeL (B), or SeeM (C) prior to culture with 2 × 10⁵ equine PBMC. The final concentrations of SeeI, SeeL, and SeeM were 0.5 ng/ml, 1 ng/ml, and 10 pg/ml, respectively. Proliferation levels induced by recombinant sAgs incubated with sera purified prior to S. equi infection and convalescent-phase sera were compared after 4 days of culture. The percentages of proliferation inhibition (open columns) are expressed on the left y axes. Superantigen-specific antibody titers (gray columns) are expressed on the right y axes. Dotted lines represent the thresholds above which the antibody response was considered positive.