Exacerbated type II interferon response drives hypervirulence and toxic shock by an emergent epidemic strain of Streptococcus suis

Abstract

Streptococcus suis, a major porcine pathogen, can be transmitted to humans and cause severe symptoms. A large human outbreak associated with an unusual streptococcal toxic shock-like syndrome (STSLS) was described in China. Albeit an early burst of proinflammatory cytokines following Chinese S. suis infection was suggested to be responsible for STSLS case severity, the mechanisms involved are still poorly understood. Using a mouse model, the host response to S. suis infection with a North American intermediately pathogenic strain, a European highly pathogenic strain, and the Chinese epidemic strain was investigated by a whole-genome microarray approach. Proinflammatory genes were expressed at higher levels in mice infected with the Chinese strain than those infected with the European strain. The Chinese strain induced a fast and strong gamma interferon (IFN-γ) response by natural killer (NK) cells. In fact, IFN-γ-knockout mice infected with the Chinese strain showed significantly better survival than wild-type mice. Conversely, infection with the less virulent North American strain resulted in an IFN-β-subjugated, low inflammatory response that might be beneficial for the host to clear the infection. Overall, our data suggest that a highly virulent epidemic strain has evolved to massively activate IFN-γ production, mainly by NK cells, leading to a rapid and lethal STSLS.

Fig 1

(A) A patient infected with the epidemic Chinese strain of S. suis presenting clinical signs of STSLS, featuring subcutaneous hemorrhage (purpura) in the leg. (B) MLST relationship of S. suis serotype 2 North American intermediately pathogenic strain 89-1591 (ST25), European highly pathogenic strain P1/7 (ST1), and Chinese epidemic strain SC84 (ST7). The whole S. suis MLST database (288 STs in total) is shown as a population snapshot obtained by eBURST (version 3). Each ST was represented by a single strain in the input population profile data. The linked clusters within the diagram represent clonal complexes. Primary founders and subgroup founders of these linked clusters are colored blue and yellow, respectively. Other unlinked individual STs are colored black. The three most important STs, ST7, ST1, and ST25, are labeled with red text to emphasize their significance.

Fig 2

The S. suis epidemic Chinese strain is more virulent than the highly pathogenic European strain and the intermediately pathogenic North American strain. (A) Survival curves for C57BL/6 mice intraperitoneally infected with 1 × 10⁷ CFU of S. suis strains originating from North America (89-1591), Europe (P1/7), or China (SC84). As controls (mock infected), animals were injected with the same broth that was used to grow the bacterial strains (Todd-Hewitt broth) (n = 14 for mice infected with each of S. suis strains; n = 5 for mock-infected mice). *, significantly different (P ≤0.05) compared to P1/7-, 89-1591-, or mock-infected mice, as determined by the log-rank (Mantel-Cox) test. (B) Blood bacteremia of C57BL/6 mice infected with the North American (89-1591), the European (P1/7), or the Chinese (SC84) strain of S. suis. C57BL/6 mice (n = 7 per group) were intraperitoneally infected as described for panel A. At 3 h and 6 h postinfection, mice were euthanized, bacteria from blood were plated, and colonies were counted and expressed as the number of CFU/ml. *, significantly different (P ≤0.05) compared to mice infected for 3 h with the European P1/7 or the Chinese SC84 strain of S. suis, as determined by one-way ANOVA.

Fig 3

Host genes are modified in greater numbers in C57BL/6 mice infected with the epidemic Chinese strain of S. suis than mice infected with either the highly pathogenic European or intermediately pathogenic North American strains. (A to D) Venn diagrams showing the total number of genes modified in C57BL/6 mice (n = 4 per group) infected for 3 and 6 h with different strains of S. suis (North American [89-1591], European [P1/7], or Chinese [SC84]) compared to their expression in mock-infected mice, as determined by the Illumina microarray study. Differentially expressed genes were defined by a fold change greater than 3-fold (upregulation or downregulation) with an accompanying P value of ≤0.05.

Fig 4

Proinflammatory cytokines and chemokines are highly expressed in mice infected with the epidemic Chinese strain of S. suis. Plasma levels of CXCL1 (A), CXCL2 (B), CCL2 (C), CCL3 (D), CCL4 (E), IL-1β (F), IL-6 (G), and TNF (H) proteins in C57BL/6 mice (n = 7 per group) infected with 1 × 10⁷ CFU of the intermediately pathogenic North American (89-1591), highly pathogenic European (P1/7), or epidemic Chinese (SC84) S. suis strain for 6 h, as quantified by Luminex assay, are shown. Data represent mean values (in pg/ml) ± SEMs. Groups that are significantly different (P ≤0.05), as determined by one-way ANOVA, are indicated by letters (a, b, c, and d).

Fig 5

Type I and II IFN pathways are differently expressed in C57BL/6 mice infected with the intermediately pathogenic North American, the highly pathogenic European, or the Chinese epidemic strain of S. suis. (A, B, and D to G) Quantitative PCR analysis of expression of Ifnγ, Ifnβ, and related genes in C57BL/6 mice (n = 7 per group) infected with 1 × 10⁷ CFU of the North American (89-1591), the European (P1/7), or the Chinese (SC84) strain of S. suis. Total RNA was isolated from spleen samples at 6 h postinfection. Data represent mean values ± SEMs of the relative fold expression in infected groups compared to that in the reference mock-infected group. (C, H, and I) Plasma levels of IFN-γ (C), CXCL10 (H), and CXCL9 (I) proteins in C57BL/6 mice infected for 6 h with 1 × 10⁷ CFU of the North American, the European, or the Chinese strain of S. suis (n = 8 per group). Data represent mean values (in pg/ml) ± SEMs. Groups that are significantly different (P ≤ 0.05), as determined by one-way ANOVA, are indicated by letters (a, b, c, and d).

Fig 6

IFN-γ exacerbates the host response following an infection by the epidemic Chinese strain of S. suis: role of NK cells. (A) Survival curves for C57BL/6 or B6.129S7-Ifng^tm1Ts/J (IFN-γ-KO) mice (n = 10 per group) intraperitoneally infected with 1 × 10⁷ CFU of the highly pathogenic European or the epidemic Chinese S. suis strain. *, significantly different (P ≤0.05) compared to IFN-γ-KO mice infected with the Chinese strain of S. suis, as determined by the log-rank (Mantel-Cox) test. (B) IFN-γ levels were measured in DC and NK cell cocultures (ratio, 1:5) infected in vitro with either the epidemic Chinese S. suis strain (SC84) or the highly pathogenic European S. suis strain (P1/7) (2.5 × 10⁵ CFU). After a bacterium-cell contact incubation time of 6 h, gentamicin was added to kill the bacteria and prevent cell toxicity. Supernatants were collected at 14 h of incubation, and IFN-γ levels were measured by ELISA. Nonstimulated cells (medium alone) or cells stimulated with a combination of CpG and LPS served as negative (C−) and positive (C+) controls, respectively. In addition, single-DC cultures were also included as controls. Data represent mean values (in pg/ml) ± SEMs of eight distinct experiments. Groups that are significantly different (P ≤0.05), as determined by one-way ANOVA, are indicated by letters (a, b, and c).