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The Journal of Infectious Diseases logoLink to The Journal of Infectious Diseases
. 2023 Nov 15;230(1):188–197. doi: 10.1093/infdis/jiad454

Streptococcus suis Serotype 2 Type IV Secretion Effector SspA-1 Induces Proinflammatory Cytokine Production via TLR2 Endosomal and Type I Interferon Signaling

Supeng Yin 1,#, Mengmeng Yuan 2,#, Sirui Zhang 3, Hongdan Chen 4, Jing Zhou 5, Tongyu He 6, Gang Li 7, Yanlan Yu 8, Fan Zhang 9, Ming Li 10,✉,3, Yan Zhao 11,✉
PMCID: PMC11272045  PMID: 39052722

Abstract

The subtilisin-like protease-1 (SspA-1) plays an important role in the pathogenesis of a highly virulent strain of Streptococcus suis 2. However, the mechanism of SspA-1–triggered excessive inflammatory response is still unknown. In this study, we demonstrated that activation of type I IFN signaling is required for SspA-1–induced excessive proinflammatory cytokine production. Further experiments showed that the TLR2 endosomal pathway mediates SspA-1–induced type I IFN signaling and the inflammatory response. Finally, we mapped the major signaling components of the related pathway and found that the TIR adaptor proteins Mal, TRAM, and MyD88 and the downstream activation of IRF1 and IRF7 were involved in this pathway. These results explain the molecular mechanism by which SspA-1 triggers an excessive inflammatory response and reveal a novel effect of type I IFN in S. suis 2 infection, possibly providing further insights into the pathogenesis of this highly virulent S. suis 2 strain.

Keywords: Streptococcus suis 2, subtilisin-like protease-1, type I interferon signaling, Toll-like receptor 2

This study revealed that SspA-1 of the highly virulent Streptococcus suis 2 strain induces an excessive inflammatory response via TLR2 endosomal and type I IFN signaling, unveiling a novel role of type I IFN in S. suis infection.

Streptococcus suis serotype 2 (S. suis 2) is a zoonotic pathogen worldwide [1–4]. Infection with S. suis 2 may lead to meningitis, pneumonia, arthritis, and even septic shock in pigs and humans. Among the various sequence types (STs) of S. suis 2, ST7 is a highly virulent strain that caused 2 outbreaks in China, in 1998 and 2005 [5, 6]. In these 2 outbreaks, many patients developed lethal streptococcal toxic shock syndrome (STSS), which was characterized by an excessive host inflammatory response. The emergence of this highly virulent strain poses a serious threat to public health.

The type IV secretion system and its effector subtilisin-like protease-1 (SspA-1) were demonstrated to play an important role in the pathogenesis of the Chinese epidemic ST7 strain 05ZYH33 in our previous studies [7–10]. Upon infection by 05ZYH33, the type IV secretion effector SspA-1 triggered an excessive inflammatory response, which may further promote STSS development, in THP-1–derived macrophages and a mouse infection model. However, the underlying molecular mechanism of SspA-1–mediated excessive inflammation remains entirely unclear.

Transcriptome analysis was used to explore the biological role of SspA-1, and type I interferon (IFN) signaling was found to be activated in SspA-1–stimulated THP-1–derived macrophages. Type I IFN was shown to be critical in the host response to infection with viral, bacterial, and fungal pathogens [11–14]. In contrast to its well-established protective roles in most viral infections, type I IFN may exhibit disparate effects during bacterial infections. In infections with different species of bacteria or even different strains of the same species, type I IFN can play different roles in the regulation of immune and tissue homeostasis, leading to either beneficial or detrimental consequences for the host [15–24]. In S. suis 2 infection, type I IFN has been shown to be involved in the host defense by modulating systemic inflammation during infection with less virulent strains. However, this protective role of type I IFN was not observed in infection with the highly virulent ST7 strain [25]. Thus, we sought to determine whether type I IFN signaling may in turn exacerbate the SspA-1–induced proinflammatory response in hosts infected with the highly virulent strain 05ZYH33.

In the present study, we demonstrated that SspA-1 induces proinflammatory cytokine production via Toll-like receptor 2 (TLR2) endosomal and type I IFN signaling. We further mapped the major signaling components of the related pathway. The results reveal the mechanism by which the type IV secretion effector SspA-1 induces an excessive systemic inflammatory response, suggest a novel effect of type I IFN in S. suis 2 infection, and provide further insights into the pathogenesis of this highly virulent S. suis 2 strain.

METHODS

Cell Culture and Stimulation

The human leukemia monocytic cell line THP-1 (American Type Culture Collection) was cultured in RPMI-1640 medium (Gibco) supplemented with 10% heat-inactivated fetal bovine serum. Macrophage differentiation was induced by treating THP-1 cells with 50 nM phorbol 12-myristate 13-acetate (PMA; Sigma‒Aldrich). Recombinant SspA-1 was obtained as described previously [10]. Wild-type, IFNAR1-knockdown, or TLR2-knockdown THP-1–derived macrophages were seeded in a 24-well plate (5 × 106 cells/well) and treated with 100 μg/mL SspA-1, 5 μM of resiquimod (R848; MedChemExpress) or phosphate-buffered saline (PBS) (control group) for 24 hours. In experiments exploring the TLR2 endosomal pathway, 100 nM bafilomycin A1 (Sigma‒Aldrich) or dimethyl sulfoxide (Sigma‒Aldrich) was added 1 hour before SspA-1 challenge.

RNA-seq Analysis

Cells were collected 24 hours after SspA-1 or PBS stimulation. Total RNA was extracted with TRIzol reagent. Biological triplicates were prepared for each group. RNA sequencing (RNA-seq) was conducted using an Illumina HiSeqTM 2500 sequencing system. The differentially expressed genes (DEGs) were analyzed by the DESeq2 R software package (1.10.1). Gene ontology enrichment analysis and gene set enrichment analysis were performed with the DEGs to explore the potential pathways involved in SspA-1 stimulation. The RNA-seq data were deposited into the NCBI Sequence Read Archive under accession No. PRJNA977828.

Quantitative Reverse Transcriptase Polymerase Chain Reaction

Total RNA and the relevant cDNA were obtained using the RNAprep Pure Cell/Bacteria Kit (TIANGEN) and the RevertAid First Strand cDNA Synthesis Kit (Thermo Scientific), respectively. Quantitative polymerase chain reaction (qPCR) was conducted using GoTaq qPCR Master Mix (Promega) in a ViiATM7 Detection System (ABI). The relative gene expression levels were calculated as the ratio of their threshold cycle value to the reference gene GAPDH.

Western Blotting

Monoclonal antibodies against human TLR2, IFNAR1, MYD88, MyD88 adaptor-like (MAL), Toll/IL-1R (TIR) domain-containing adaptor-inducing IFN-β (TRIF)-related adaptor molecule (TRAM), IRF1, IRF3, IRF7, GAPDH, and β-actin (all purchased from Abcam) and a mouse polyclonal anti–SspA-1 antibody (produced in our laboratory) were used for western blotting.

Cytokine Assays

Cytokines were analyzed by enzyme-linked immunosorbent assay (ELISA) using rat anti-human or rat anti-mouse IFN-β, interleukin 6 (IL-6), and tumor necrosis factor-α (TNF-α) ELISA kits (R&D Systems) according to the manufacturer's instructions.

Coimmunoprecipitation

The cell lysate supernatant from SspA-1–stimulated THP-1–derived macrophages was obtained and precleaned via incubation with protein A/G agarose beads (Sangon Biotech) for 30 minutes at 4°C. Then, the supernatant was incubated with an anti-TLR2 antibody or immunoglobulin G (IgG) at 4°C overnight. Immune complexes were precipitated by protein A/G agarose beads at 4°C overnight. The immunoprecipitates were washed 3 times and subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and western blotting.

Microscale Thermophoresis Assay

The microscale thermophoresis assay was conducted using Monolith NT. 115 (NanoTemper Technologies). Recombinant his-tag SspA-1 protein was labelled with RED-NHS second-generation kit (MO-L011; NanoTemper Technologies). The labelled SspA-1 (40 nM) was mixed with varying concentrations of recombinant glutathione S-transferase (GST)-tagged human TLR2 or GST alone. The dissociation constant (Kd) was determined using MO. Control software.

CRISPR-Cas9–Mediated Knockdown

TLR2-knockdown THP-1 cells were generated using CRISPR-Cas9 as described previously [26, 27]. In brief, recombinant pXPR_001 LentiCRISPR vector (Plasmid No. 164993; Addgene) carrying single-guide RNAs (sgRNAs) targeting human TLR2 were constructed. Two sgRNAs targeting TLR2 were designed (Supplementary Table 1). Viral particles were produced in 293FT cells by cotransfection with the lentiviral vector, the packaging plasmid pSPAX2, and the envelope plasmid pMD2G (4:3:1) by the calcium phosphate method [28]. THP-1 cells were infected with the corresponding lentivirus and then subjected to puromycin selection. A nontargeting sgRNA was used as a negative control. TLR2 knockdown was confirmed by quantitative reverse transcription PCR (qRT-PCR; Supplementary Figure 1).

shRNA-Mediated Knockdown

Recombinant adenoassociated viral vectors (pMAGic 7.1-U6-shRNA-CMV-eGFP, constructed by Shanghai Sunbio Medical Biotechnology Co.) carrying short hairpin RNAs (shRNAs) against human IFNAR1 or a control shRNA were used to produce viral particles. The sequences targeting the ifnar1 gene are listed in Supplementary Table 2. THP-1 cells were infected with the corresponding lentivirus and then subjected to puromycin selection. IFNAR1 knockdown was confirmed by western blotting (Supplementary Figure 2).

siRNA-Mediated Knockdown

Small interference RNA (siRNA)-mediated silencing was performed to knock down the expression of Mal, TRAM, MyD88, IRF1, IRF3, and IRF7. Transient transfection of the corresponding siRNAs was performed using the transfection reagent Lipofectamine 3000 (Invitrogen) according to the manufacturer's instructions. The siRNA sequences are listed in Supplementary Table 3. siRNA-mediated knockdown was confirmed by western blotting (Supplementary Figure 3).

Mouse Studies

Six- to eight-week-old female C57BL/6 mice, TLR2−/− mice, and IFNAR−/− mice were maintained under pathogen-free conditions. A total of 12 mice per group were challenged with 100 μL of SspA-1 (1 mg/mL) by tail vein injection. Blood was collected at 3 hours or 24 hours postinjection and employed for cytokine assays. All animal experiments were performed in accordance with protocols approved by the Laboratory Animal Welfare and Ethics Committee of the Army Medical University and in compliance with Institutional Animal Welfare and Ethical guidelines.

Statistical Analysis

Differences between 2 independent groups of data were evaluated by 2-tailed Student t test using SPSS version 20.0. A value of P < .05 was considered to indicate a significant difference.

RESULTS

SspA-1 Activates Type I IFN Signaling

To explore the biological role of SspA-1 in the induction of the host proinflammatory response, RNA-seq was performed to analyze the transcriptome of SspA-1– or PBS (control)–stimulated THP-1–derived macrophages. The RNA-seq data showed that the SspA-1– and control-stimulated cells had markedly distinct gene expression profiles. A total of 4719 genes were determined to be DEGs in the SspA-1 group compared with the control group, of which 2578 were upregulated and 2141 were downregulated (Figure 1A). The Euclidean distance method was used to visualize patterns in the DEGs among the biological replicates (Figure 1B). Gene ontology analysis showed that the type I IFN signaling pathway, which is most likely correlated with the host inflammatory response, was the primary enriched pathway (Figure 1C). Moreover, gene set enrichment analysis showed that the type I IFN signaling pathway was significantly enriched (P < .001; Figure 1D).

Figure 1.

Figure 1.

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Transcriptome analysis of THP-1–derived macrophages stimulated by SspA-1 or phosphate-buffered saline. A, RNA sequencing analysis showed the DEGs between the SspA-1–stimulated group and the control group. The horizontal dashed line indicates adjusted P (Padj) value = .05. B, Transcriptional profiles for each sample analyzed with the pairwise Euclidean distance method (heatmap). S1, S2, and S3 indicate triplicate samples stimulated by SspA-1. Control 1, 2, and 3 indicate triplicate samples stimulated by PBS. C, Functional classification of DEGs in the Gene Ontology database. D, Gene set enrichment analysis of the type I IFN signaling pathway. Abbreviations: DEG, differentially expressed gene; IFN, interferon; SspA-1, subtilisin-like protease-1; NES, normalized enrichment score.

To verify whether type I IFN signaling and the inflammatory response are activated in SspA-1–stimulated host cells, we performed qRT-PCR to confirm the expression of type I IFN-related genes and selected cytokines. After stimulation with SspA-1, the expression levels of IFN-β, IL-6, TNF-α, some interferon regulatory factors (IRFs), such as IRF5 and IRF7, and some IFN-stimulated genes were significantly increased compared with those in the control group. However, the mRNA level of the cytokines TGF-β1, TGF-β2, and TGF-β3, which are unrelated to type I IFN signaling, did not exhibit any significant changes when stimulated by SspA-1 (Figure 2A). In addition, the level of IFN-β in the supernatant of SspA-1–stimulated THP-1–derived macrophages was dramatically higher than that in the supernatant of control macrophages (Figure 2B). These results indicated that type I IFN signaling was activated during SspA-1 stimulation of host cells.

Figure 2.

Figure 2.

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Type I IFN signaling was activated in SspA-1–stimulated cells. A, The mRNA levels of IFN-β, IL-6, TNF-α, and selected type I IFN-related genes in SspA-1–stimulated and control THP-1–derived macrophages were measured by qRT–PCR. B, IFN-β protein production was measured in the supernatant of SspA-1–stimulated and control THP-1–derived macrophages by ELISA. The data are expressed as the mean (SD) of triplicate samples and are representative of 3 independent experiments. **P < .01, ***P < .001. Abbreviations: ELISA, enzyme-linked immunosorbent assay; IFN, interferon; ns, not significant; PBS, phosphate-buffered saline; qRT-PCR, quantitative reverse transcriptase polymerase chain reaction; SspA-1, subtilisin-like protease-1.

Activation of Type I IFN Signaling Is Required for SspA-1–Induced Proinflammatory Cytokine Production

To determine whether the activation of type I IFN signaling contributes to the SspA-1–induced excessive systemic inflammatory response, IFNAR1-knockdown and negative control THP-1–derived macrophages were challenged with SspA-1, and the mRNA expression levels and protein abundances of proinflammatory cytokines, including IL-6 and TNF-α, were measured by qRT-PCR and ELISA, respectively. The levels of IL-6 and TNF-α were significantly decreased in the IFNAR1-knockdown group compared with the negative control group (Figure 3A and 3B). However, the expression of the control cytokine TGF-β2, showed no significant changes (Supplementary Figure 4). Additionally, when exposed to resiquimod (R848), an activator of TLR7 and TLR8, the mRNA and protein levels of IL-6 and TNF-α were not reduced in IFNAR1-knockdown group compared to the negative control group or wild-type group, suggesting that the IFNAR1-knockdown cell line still retains the ability to produce these cytokines (Supplementary Figure 5). This finding indicates that type I IFN is required for SspA-1–induced proinflammatory cytokine production.

Figure 3.

Figure 3.

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Activation of type I IFN signaling is required for SspA-1–induced proinflammatory cytokine production. A, The mRNA levels of IL-6 and TNF-α in SspA-1–stimulated sh-IFNAR1 and sh-NC THP-1–derived macrophages were measured by qRT-PCR. B, The levels of IL-6 and TNF-α were measured by ELISA in the supernatant of SspA-1–stimulated sh-IFNAR1 and sh-NC THP-1–derived macrophages. The data are expressed as the mean (SD) of triplicate samples and are representative of 3 independent experiments. **P < .01, ***P < .001. Abbreviations: ELISA, enzyme-linked immunosorbent assay; IFN, interferon; IL-6, interleukin 6; NC, negative control; qRT-PCR, quantitative reverse transcription polymerase chain reaction; SspA-1, subtilisin-like protease-1; TNF-α, tumor necrosis factor-α; sh, short hairpin.

The SspA-1–Induced Type I IFN Response and Proinflammatory Cytokine Production Are TLR2 Dependent

TLR2 is considered to be a main immune receptor in the response to S. suis infection [29–31]. In addition, several previous studies showed that TLR2 activation can induce the type I IFN response in some cell types, such as human THP-1 monocytes and murine bone marrow-derived macrophages [32, 33]. We therefore investigated the potential role of TLR2 in the SspA-1–induced type I IFN response and proinflammatory cytokine production. The TLR2-knockdown THP-1 cell line was first generated by CRISPR-Cas9 gene editing. Then, TLR2-knockdown and negative control THP-1–derived macrophages were challenged with SspA-1, and the levels of IFN-β, IL-6, and TNF-α in the supernatant were measured by ELISA. The levels of IFN-β, IL-6, and TNF-α were significantly decreased in the TLR2-knockdown group compared with the negative control group (Figure 4A), while the expression of the control cytokine TGF-β2 remained unchanged (Supplementary Figure 4). These results indicate that the SspA-1–induced type I IFN response and proinflammatory cytokine production were significantly impaired when TLR2 was knocked down. Furthermore, we investigated whether SspA-1 can interact with TLR2. As shown in Figure 4B, the results of coimmunoprecipitation followed by western blotting suggested that recombinant SspA-1 coimmunoprecipitated with native TLR2. Subsequently, a microscale thermophoresis assay was performed to quantify the binding affinity of recombinant GST-tagged human TLR2 or GST alone with SspA-1. The results affirmed the direct interaction between SspA-1 and GST-TLR2, yielding a calculated potent Kd value of 24.286 nM, while GST alone showed no interaction with SspA-1 (Figure 4C). Thus, these results suggest that TLR2 mediates the type I IFN response and proinflammatory cytokine production after recognition of SspA-1.

Figure 4.

Figure 4.

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The SspA-1–induced type I IFN response and proinflammatory cytokine production are TLR2 dependent. A, The levels of IFN-β, IL-6, and TNF-α in the supernatant of SspA-1–stimulated TLR2-knockdown and NC THP-1–derived macrophages were measured by ELISA. The data are expressed as the means (SDs) and are representative of 3 independent experiments. B, Coimmunoprecipitation of recombinant SspA-1 and TLR2 in THP-1–derived macrophages. The immunoprecipitates were subjected to western blotting to detect SspA-1 and TLR2. GAPDH was used as the protein loading control. C, Microscale thermophoresis analysis of the binding affinity between SspA-1 and recombinant human GST-TLR2 or TLR2 alone. The measured Kd value between SspA-1 and GST-TLR2 was 24.286 nM. No binding of SspA-1 to GST was detected. ***P < .001. Abbreviations: ELISA, enzyme-linked immunosorbent assay; IFN-β, interferon-β; IgG, immunoglobulin G; IL-6, interleukin 6; IP, immunoprecipitated; KD, knockdown; NC, negative control; SspA-1, subtilisin-like protease-1; TLR2, Toll-like receptor 2; TNF-α, tumor necrosis factor-α; GST, glutathione S-transferase.

TLR2-Mediated Activation of the Type I IFN and Inflammatory Responses Requires the Endosomal Pathway

In previous studies, TLR2 was shown to signal from the endosome to activate the type I IFN response [32–34]. We therefore investigated whether the SspA-1–induced type I IFN and inflammatory responses require the TLR2 endosomal pathway. After treatment with bafilomycin A1, an inhibitor of the endosomal proton pump, the production of IFN-β, IL-6, and TNF-α, but not the control cytokine TGF-β2, was significantly inhibited, as the levels of these cytokines were significantly decreased in the culture of SspA-1–stimulated THP-1–derived macrophages (Figure 5 and Supplementary Figure 4), implying that the TLR2 endosomal pathway is required for the SspA-1–induced type I IFN and inflammatory responses.

Figure 5.

Figure 5.

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TLR2-mediated activation of the type I IFN and inflammatory responses requires the endosomal pathway. The production of IFN-β, IL-6, and TNF-α was measured in the supernatant of SspA-1–stimulated THP-1–derived macrophages treated with or without the endosomal proton pump inhibitor bafilomycin A1 by ELISA. The data are expressed as the means (SDs) and are representative of 3 independent experiments. *P < .05, ***P < .001. Abbreviations: DMSO, dimethyl sulfoxide; ELISA, enzyme-linked immunosorbent assay; IFN-β, interferon-β; IL-6, interleukin 6; SspA-1, subtilisin-like protease-1; TLR2, Toll-like receptor 2; TNF-α, tumor necrosis factor-α.

Identification of Major Signaling Components of the SspA-1–Induced TLR2 Endosomal and Type I IFN Pathway

To gain further knowledge of this pathway, we mapped some potential major signaling components by using siRNAs to knock down their expression. As shown in Figure 6, after transfection of the siRNAs targeting Mal, TRAM, MyD88, IRF1, and IRF7, SspA-1–induced IFN-β, IL-6, and TNF-α production was severely impaired compared with that in the negative control group. In contrast, transfection of the siRNA targeting IRF3 did not impair SspA-1–induced IFN-β, IL-6, and TNF-α production. This result suggests that the TIR adaptor proteins Mal, TRAM, and MyD88 and the downstream activation of IRF1 and IRF7 were involved in the SspA-1–induced TLR2 endosomal and type I IFN pathway.

Figure 6.

Figure 6.

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Identification of major signaling components of the SspA-1–induced TLR2 endosomal and type I IFN pathway. siRNAs targeting MyD88, Mal, TRAM, IRF1, IRF3, and IRF7 were transiently transfected into THP-1–derived macrophages, which were then stimulated by SspA-1. The levels of IFN-β (A), IL-6 (B), and TNF-α (C) in the cell culture supernatant were measured by ELISA and compared with those in the supernatant of si-NC cells. The data are expressed as the mean (SD) of triplicate samples and are representative of 3 independent experiments. *P < .05. Abbreviations: ELISA, enzyme-linked immunosorbent assay; IFN-β, interferon-β; IL-6, interleukin 6; NC, negative control; ns, not significant; siRNA, small interference RNA; SspA-1, subtilisin-like protease-1; TLR2, Toll-like receptor 2; TNF-α, tumor necrosis factor-α.

Verification of the Effects of TLR2 and IFNAR on SspA-1–Induced Proinflammatory Cytokine Production in a Mouse Model

To determine the effect of the TLR2 endosomal and type I IFN pathway on the SspA-1–stimulated inflammatory response in vivo, TLR2−/− mice, IFNAR−/− mice, and wild-type mice were challenged with SspA-1 by tail vein injection. We found that 3 and 24 hours after injection, the serum levels of IL-6 and TNF-α in mice in the TLR2−/− group and IFNAR−/− group were significantly decreased compared with those in wild-type mice (Figure 7). The results of these in vivo experiments were consistent with the above in vitro results, demonstrating the important role of the TLR2 endosomal and type I IFN pathway in the SspA-1–induced host inflammatory response.

Figure 7.

Figure 7.

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Verification of the effects of TLR2 and IFNAR on SspA-1–induced proinflammatory cytokine production in a mouse model. TLR2−/− mice, IFNAR−/− mice, and wild-type mice were challenged with SspA-1 by tail vein injection. The serum levels of IL-6 and TNF-α in the mice in each group were measured by ELISA 3 hours and 24 hours after injection. The data are representative of 3 independent experiments. *P < .001. Abbreviations: ELISA, enzyme-linked immunosorbent assay; IL-6, interleukin 6; SspA-1, subtilisin-like protease-1; TLR2, Toll-like receptor 2; TNF-α, tumor necrosis factor-α; WT, wild type.

DISCUSSION

The effects of type I IFN on bacterial infections vary across species or strains, and type I IFN may play either a proinflammatory or an anti-inflammatory role under specific conditions [15–24]. It was previously demonstrated that type I IFN signaling induced by S. suis 2 is strain dependent [25]. When activated by the less virulent ST1 and ST25 strains, type I IFN plays a beneficial role in host survival by participating in bacterial clearance. However, after infection with the highly virulent ST7 strain, the blood bacterial burden in IFNAR−/− mice was not increased compared with that in wild-type mice. Moreover, the survival rate of IFNAR−/− mice seemed to be higher than that of wild-type mice, although the difference was not significant. These data imply that type I IFN may play a detrimental role in infections with the highly virulent ST7 strain. This hypothesis was supported by the current study. The results revealed that the type IV secretion effector SspA-1 of the highly virulent ST7 strain 05ZYH33 induces excessive proinflammatory cytokine production through activation of type I IFN signaling in THP-1–derived macrophages. In addition, the serum levels of proinflammatory cytokines in IFNAR−/− mice challenged with SspA-1 were significantly decreased compared with those in wild-type mice.

Activation of type I IFN signaling triggers the production of a variety of cytokines. Most studies have shown that IL-6 is a type I IFN-inducible gene [21, 33], but whether the production of TNF-α requires the induction of type I IFN signaling is still controversial. Dietrich et al and Stack et al found that TNF-α production is independent of type I IFN signaling [33, 34]. However, Martin et al demonstrated that the virulence factor protein A of Staphylococcus aureus can induce type I IFN signaling in airway epithelial cells and subsequently increase the production of IL-6 and TNF-α [21]. Mancuso et al also showed that type I IFNs—specifically IFN-β—play an important role in group B Streptococcus-induced production of TNF-α [35]. Auger et al reported that after infection with different strains of S. suis 2 in IFNAR−/− mice in vivo or dendritic cells isolated from IFNAR−/− mice in vitro, the production of IL-6, TNF-α, and certain other inflammatory mediators decreased significantly or at least showed a decreasing trend compared with that in the wild-type counterpart mice and cells [25]. Consistent with these findings, our results also suggest that activation of type I IFN signaling is required for SspA-1–induced IL-6 and TNF-α production in THP-1–derived macrophages. In previous studies, it was hypothesized that type I IFN can modulate the production of a variety of cytokines through an autocrine pathway consisting of activation of IFNAR and the JAK-STAT cascade [21, 36, 37]. However, the exact mechanisms in different cell types and regarding specific inflammatory mediators, such as TNF-α, remain to be elucidated.

Pattern recognition receptors, such as TLRs, serve as front-line sentinels for the pathogen detection by the immune system [38, 39]. Previous studies have shown that several TLRs, including TLR2, TLR6, and TLR9, are involved in the recognition of S. suis 2 and the inflammatory response in host cells [29–31]. Among these TLRs, TLR2 is considered the main trigger of the release of proinflammatory cytokines such as IL-6 and TNF-α. However, in a recent study, Auger et al found that TLR3, TLR4, TLR7, and TLR9 are partially implicated in the activation of type I IFN signaling and cytokine production in dendritic cells [25]. They also demonstrated that TLR7 and TLR9 are responsible for the recognition of S. suis 2 nucleic acids and play major roles in inducing IFN-β expression. However, S. suis 2 is generally considered an extracellular bacterium, and even though small amounts of intracellular bacteria may stimulate the host inflammatory response through TLR7 and TLR9, only relatively weak cytokine production is likely induced. This observation is not sufficient to explain the excessive inflammatory response during infection with the highly virulent strain 05ZYH33. In the present study, we demonstrated that TLR2 on THP-1–derived macrophages recognized SspA-1 directly and was responsible for the production of IFN-β, IL-6, and TFN-α, which may be the main contributor to STSS caused by 05ZYH33.

Recently, some studies demonstrated that TLR2 can activate type I IFN signaling and IFN-inducible genes via an endosomal pathway, similar to TLR4 [32–34]. Our results showed that this TLR2 endosomal pathway is also employed in SspA-1–induced type I IFN signaling activation. However, many details of this signaling cascade, such as the identities of the TIR adaptor proteins and IRFs involved, remain to be defined. We demonstrated that Mal, TRAM, MyD88, IRF1, and IRF7 were required for the activation of the SspA-1–induced TLR2 endosomal and type I IFN pathway. Considering the results of some previous studies, the major molecules implicated in this pathway differ among the studies, an inconsistency that may be attributed to the various host cell types, bacterial strains, and virulence factors used in these studies [40, 41].

Taken together, the results of the current study reveal that SspA-1, a type IV secretion effector of the highly virulent S. suis 2 strain 05ZYH33, triggers excessive cytokine production via TLR2 endosomal and type I IFN signaling. The underlying mechanism shows a novel effect of type I IFN in S. suis 2 infection, providing further insights into the pathogenesis of this highly virulent S. suis 2 strain.

Supplementary Data

Supplementary materials are available at The Journal of Infectious Diseases online (http://jid.oxfordjournals.org/). Supplementary materials consist of data provided by the author that are published to benefit the reader. The posted materials are not copyedited. The contents of all supplementary data are the sole responsibility of the authors. Questions or messages regarding errors should be addressed to the author.

Supplementary Material

jiad454_Supplementary_Data
jiad454_supplementary_data.zip (651.5KB, zip)

Contributor Information

Supeng Yin, Department of Breast and Thyroid Surgery, Chongqing General Hospital, Chongqing, China.

Mengmeng Yuan, Department of Microbiology, College of Basic Medical Sciences, Key Laboratory of Microbial Engineering Under the Educational Committee in Chongqing, Army Medical University, Chongqing, China.

Sirui Zhang, The First Clinical Medical School, Guangzhou University of Chinese Medicine, Guangzhou, China.

Hongdan Chen, Department of Breast and Thyroid Surgery, Chongqing General Hospital, Chongqing, China.

Jing Zhou, Department of Microbiology, College of Basic Medical Sciences, Key Laboratory of Microbial Engineering Under the Educational Committee in Chongqing, Army Medical University, Chongqing, China.

Tongyu He, Department of Microbiology, College of Basic Medical Sciences, Key Laboratory of Microbial Engineering Under the Educational Committee in Chongqing, Army Medical University, Chongqing, China.

Gang Li, Department of Microbiology, College of Basic Medical Sciences, Key Laboratory of Microbial Engineering Under the Educational Committee in Chongqing, Army Medical University, Chongqing, China.

Yanlan Yu, Department of Microbiology, College of Basic Medical Sciences, Key Laboratory of Microbial Engineering Under the Educational Committee in Chongqing, Army Medical University, Chongqing, China.

Fan Zhang, Department of Breast and Thyroid Surgery, Chongqing General Hospital, Chongqing, China.

Ming Li, Department of Microbiology, College of Basic Medical Sciences, Key Laboratory of Microbial Engineering Under the Educational Committee in Chongqing, Army Medical University, Chongqing, China.

Yan Zhao, Department of Microbiology, College of Basic Medical Sciences, Key Laboratory of Microbial Engineering Under the Educational Committee in Chongqing, Army Medical University, Chongqing, China.

Notes

Author contributions. Y. Z., M. L., and F. Z. designed the study. S. Y., M. Y., S. Z., H. C., T. H., J. Z., G. L., and Y. Y. performed the experiments. Y. Z. and M. L. analyzed the data. S. Y. and Y. Z. wrote the paper.

Acknowledgments. We cordially thank Dr Yuzhang Wu and Dr Xiao Yu for providing the C57BL/6 TLR2−/− mice and C57BL/6 IFNAR1−/− mice, respectively. We thank LetPub (www.letpub.com) for linguistic assistance and presubmission expert review.

Financial support. This work was supported by the National Natural Science Foundation of China (grant number 82002119 to S. P. Y.); and the Natural Science Foundation of Chongqing Municipality, China (grant number cstc2019jcyj-msxmX0142 to Y. Z.).

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