In Table 1, we showed the information of bacterial strains and plasmids used in this study. SS2 strain SC19 was selected as the wild-type (WT) strain, which “was isolated from the brain of a dead pig during the epidemic outbreak in the Sichuan Province of China in 2005” (26). SC19 is highly pathogenic to mice and pigs and can cause STSLS (27). SC19, Δhp1330, and CΔhp1330 were cultured in tryptic soy broth (TSB) or on tryptic soy agar (TSA) plates (Difco, MI, USA) with 10% newborn bovine serum (Sijiqing Biological Engineering Materials Co., Ltd., Hangzhou, China) at 37°C (28).

The HP1330 protein was prepared according to published methods (30). Briefly, the hp1330 gene was amplified by PCR using the primers listed in Table 2, and then inserted into the pET28a vector. After the recombinant vector was transformed into E. coli BL21 (DE3) cells, 0.5 mM isopropyl-b-d-thiogalactopyranoside was added to induce expression. Then HP1330 was purified by ultrasonication and Ni-NTA agarose chromatography. Before being used to stimulate RAW264.7 macrophages, HP1330 was confirmed to contain low levels of endotoxin, using the Endotoxin Removal Kit (Genmed Scientifics Inc., USA) and Quantitative Chromogenic Tachypleus Amebocyte Lysate for Endotoxin Detection Kit (Chinese Horseshoe Crab Reagent Manufactory Co., Ltd., Xiamen, China) (31). After passage through a 0.22-µm filter, the HP1330 protein was stored at −80°C.

“RAW 264.7 macrophages were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum (Gibco, USA) at 37°C in a 5% CO₂ atmosphere” (32). “Primary mouse macrophages were prepared as described previously” (18). Toll-like receptor 2 (TLR2)-deficient, TLR4-deficient, and WT mice were injected intraperitoneally (i.p.) with 4% thioglycolate (TLR2-deficient and TLR4-deficient mice were obtained from the Collaborative Innovation Center of Model Animal, Wuhan University). Peritoneal exudate cells were harvested 4 days later and identified by microscopic analysis and non-specific esterase staining (33). When >90% of the exudate cells were identified as macrophages, the cells were plated at a density of 10⁶ cells per well in 12-well plates.

RAW 264.7 cells were exposed to 10 µg⋅ml⁻¹ HP1330 protein, lipopolysaccharide (LPS, 100 ng⋅ml⁻¹, Sigma), and LPS inhibitor polymyxin B (Poly.B, 10 µg⋅ml⁻¹, Sigma) for 6 h, as described (34).

After RAW 264.7 cells were stimulated, the expressions of TNF-α, MCP-1, and IL-1β were measured by qRT-PCR as reported previously (35). Briefly, the total RNA of cells was extracted using the TRIzol^® reagent (Invitrogen, Paisley, UK). Then, complementary DNA was synthesized from 4 µg of the total RNA by using AMV reverse transcriptase (Takara, Japan), as previously described (36). qRT-PCR was performed using ViiA™ 7 Software (Applied Biosystems) with SYBR green PCR Kit (Roche). All of the primers used in qRT-PCR were listed in Table 2. The relative amounts of target gene expression were normalized with GAPDH housekeeping gene, using 2^−ΔΔCt method (37).

“The concentrations of TNF-α, MCP-1, and IL-1β in the cell culture supernatants or serums were determined using commercially available ELISA kits (BioLegend), following the manufacturer’s instructions” (18).

The Δhp1330 mutant strain was constructed as previously described (29). The left (714 bp) and right (688 bp) DNA fragments of hp1330 were prepared from the SC19 genome using PCR with the primers hp1330L-1/2 and hp1330R-1/2, respectively. The products were inserted into the pSET4s vector to generate plasmid p4Δhp1330. Next, the recombinant vector was electrotransformed into SC19 competent cells. The mutant strain was screened based on spectinomycin resistance and thermosensitive suicide of the pSET4s vector. The suspected mutant was verified using three pairs of primers: P1/P2 (to detect the pSET4s vector), P3/P4 (to detect gdh), and P5/P6 (to detect hp1330).

The complemented strain of hp1330 was obtained according to a previous procedure (27). A DNA fragment covering the hp1330 ORF region and its promoter region was prepared by PCR using the primers chp1330-1 and chp1330-2. This fragment was then cloned into pSET2 to generate plasmid p2CΔhp1330. To obtain the complemented strain CΔhp1330, the recombinant plasmid was electrotransformed into Δhp1330.

In vitro, the WT (SC19), Δhp1330, and CΔhp1330 strains were used to infect RAW 264.7 cells at a dose of 5 × 10⁶ colony-forming units (CFUs). After 6 h, culture supernatants and RAW 264.7 cells were collected for ELISA and qRT-PCR analysis, respectively.

This study was carried out in accordance with the recommendations of the Guide for the Care and Use of Laboratory Animals Monitoring Committee of Hubei Province, China, and the protocol was approved by the Committee on the Ethics of Animal Experiments at the College of Veterinary Medicine, Huazhong Agricultural University. For virulence studies, 6-week-old female C57BL/6 mice (10 mice/group) were challenged i.p. with 6 × 10⁸ CFUs of the SC19, Δhp1330, or CΔhp1330 strain. The infected mice were monitored for clinical signs and survival times for 7 days. In addition, another batch of 60 6-week-old female C57BL/6 mice was randomly assigned to three groups with 20 mice/group and challenged i.p. with a non-lethal dose (2 × 10⁸ CFUs per mouse) of the SC19, Δhp1330, or CΔhp1330 strain. At 3, 6, 9, and 12 h postinfection, an equal number of mice in each group were sacrificed to collect blood, which was used for bacteria counts and ELISAs to measure TNF-α, MCP-1, and IL-1β production (26, 38).

It is reported that “TLR2 is the major immune receptor involved in S. suis recognition” (39, 40). To investigate which receptor was specifically responsible for HP1330-mediated cytokine upregulation, we first detected TLR2 changes after HP1330 stimulation by qRT-PCR, with TLR4 as a control. Second, antibody blocking assays were performed as previously described (41). After pretreatment with 8 µg of an anti-TLR2 (BioLegend) or anti-TLR4 (BioLegend) antibody for 30 min, RAW264.7 cells were incubated with 10 µg⋅ml⁻¹ HP1330 for 6 h. The concentrations of TNF-α, MCP-1, and IL-1β in the culture supernatants were determined by ELISA. On the basis of these experiments, we identified the recognition receptor of HP1330. Finally, TLR2−/− and TLR4−/− macrophages were isolated from TLR2−/− and TLR4−/− mice to verify the above results.

For cell-signaling analysis, RAW 264.7 cells were incubated with the following specific inhibitors 30 min prior to the addition of HP1330, including SB203580 (for p38 MAPK, 10 µM; Cayman Chemical), SP600125 (for JNK, 10 µM; Cayman Chemical), pyrrolidine dithiocarbamate (PDTC; for NF-κB, 20 µM; Sigma), LY294002 (for PI3K, 20 µM; Cayman Chemical), and U0126 (for ERK1/2, 10 µM; Cayman Chemical) (42). After HP1330 stimulation for 6 h, culture supernatants were collected for ELISAs to measure TNF-α, MCP-1, and IL-1β production. According to the conditions used for cytokine activation, we screened for the signal-transduction molecule induced by HP1330.

To verify the above analysis results, the phosphorylation of HP1330-induced signal-transduction molecules was confirmed by western blotting (43). Briefly, after stimulation with 10 µg⋅ml⁻¹ HP1330 for 6 h, RAW 264.7 cells were washed once with cold PBS and incubated on ice for 15 min using radioimmunoprecipitation assay lysis buffer with phosphatase inhibitors (Roche). The supernatants were collected, and their protein concentrations were quantified by Bradford protein assay. Then 40 µg proteins were resolved on a 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel, followed by electrotransfer to a 0.22-µm nitrocellulose membrane. Activation of ERK1/2 was assessed using a specific antibody against phosphorylated ERK1/2 (Cell Signaling Technology). In addition, β-actin was detected by an anti-β-actin antibody (Wuhan PMK Biotechnology Co., Ltd.), as an internal control. Protein bands were visualized by incubation with a horseradish peroxidase-conjugated secondary antibody and then detected using the ECL System (Amersham Life Science, Arlington Heights, IL, USA).

Statistical analyses were performed by an unpaired Student’s t-test. All assays were repeated at least three times, and a P value < 0.05 was considered significant. In the figures, * and ** represent P values < 0.05 and <0.01, respectively.

The purity of the recombinant protein HP1330 was analyzed by SDS-PAGE (Figure 1A) and western blotting (Figure 1B), revealing that HP1330 was successfully purified. After endotoxin removal, the protein concentration and endotoxin level of HP1330 were approximately 1.3 mg⋅ml⁻¹ and 0.01 endotoxin unit⋅ml⁻¹, respectively. Next, we investigated the pro-inflammatory activity of HP1330 in RAW 264.7 cells. As shown in Figure 2, HP1330 significantly induced TNF-α, MCP-1, and IL-1β expression, and the cytokine response induced by HP1330 was not reduced by Poly.B, an inhibitor of negatively charged molecules like LPS that is normally used to exclude the effects of contaminating endotoxins (44, 45). These results showed that HP1330 has robust pro-inflammatory activity in RAW 264.7 cells, and that residual bacterial endotoxins were not responsible for the effect.

To determine whether heat treatment influences the pro-inflammatory activity of HP1330, we next pretreated HP1330 using previously reported conditions [100°C for 10 min (42)] before adding it to RAW264.7 cells. Heat-treated HP1330 failed to induce TNF-α, MCP-1, and IL-1β production in RAW264.7 cells after a 6-h stimulation (Figure 3). These results indicated that the pro-inflammatory activity of HP1330 in RAW 264.7 cells is heat-sensitive.

To confirm the deletion of hp1330, PCR analysis was performed with three pairs of primers (P1/P2, P3/P4, and P5/P6). hp1330 could be detected in the SC19 strain, but not in Δhp1330. As a control, gdh could be detected in both SC19 and Δhp1330. In addition, the pSET4s vector could not be detected in Δhp1330 (Figure 4A). These results indicated that the hp1330 gene was deleted from the bacterial chromosome. To further examine the influence of the hp1330 deletion, the growth curves and Gram staining of the SC19, Δhp1330, and CΔhp1330 strains were determined by culturing to logarithmic growth phase in TSB. As shown in Figure 4B, no significant difference was found among the growth curves of the three strains, while the chain of Δhp1330 strain was obvious longer than SC19 or CΔhp1330 (Figure 4C). To avoid the influence of chain length differences on the accuracy of bacterial counting, we then examined the morphologies of the SC19, Δhp1330, and CΔhp1330 strains cultured on TSA via Gram staining. The result showed that no marked difference occurred among these three strains (Figure 4D). Thus, in subsequent cellular and animal experiments, we cultured SC19, Δhp1330, and CΔhp1330 on TSA. In addition, transmission electron microscopy was used to detect the capsules of SC19, Δhp1330, and CΔhp1330 strains, respectively. There was no obvious difference in capsular thickness and morphology among these three strains (Figure 4E). This indicated that HP1330 does not regulate the capsule of S. suis.

After RAW 264.7 cells were incubated with the SC19, Δhp1330, or CΔhp1330 strain, the levels of TNF-α, MCP-1, and IL-1β were measured by ELISA and qRT-PCR. Deleting the hp1330 gene significantly reduced the pro-inflammatory ability of SS2 (Figure 5). These results indicated that HP1330 plays an important role in SS2-induced, pro-inflammatory responses in vitro.

To examine the role of HP1330 in SS2 infection, C57BL/6 mice were used as an experimental infection model. Firstly, for virulence testing, mice were inoculated with the SC19, Δhp1330, or CΔhp1330 strain at a dose of 6 × 10⁸ CFUs, which was a lethal dose of SC19 for C57BL/6 mice. The group of mice infected with SC19 developed obvious clinical signs of SS2 infection, including a rough hair coat, weight loss, depression, shivering, and suppuration of the eyes during the first day postinfection. Only 20% of the mice survived to 7 days postinfection. However, mice in the Δhp1330 group showed an overall survival rate of 90%, with no obvious symptoms (Figure 6). These results indicated that deleting the hp1330 gene significantly decreased the virulence of SS2 to mice (P < 0.01). Second, to further analyze why the Δhp1330 deletion resulted in attenuated virulence, mice were challenged i.p. with a non-lethal dose of the SC19, Δhp1330, or CΔhp1330 strain, and then cytokine concentrations and bacterial loads in the blood were determined. As a result, the blood of mice infected with Δhp1330 contained lower cytokine concentrations at 6 and 12 h postinfection, and lower bacterial loads at 6 h postinfection (Figures 7A,C), whereas the chain length of the SC19, Δhp1330, and CΔhp1330 strains showed no obvious differences (Figure 7B). Overall, our data suggested that HP1330 may contribute to SS2 virulence by inducing high-level pro-inflammatory responses and influencing in vivo bacterial loads.

The qRT-PCR assay results showed that TLR2 could be obviously upregulated in RAW264.7 cells by HP1330 stimulation, but TLR4 not (Figure 8A). This implied that TLR2 may be the inflammatory recognition receptor of HP1330. An antibody blocking assay was performed to test this possibility. Compared with the positive control, the anti-TLR2 antibody significantly reduced the expression levels of TNF-α, MCP-1, and IL-1β (Figure 8B). In addition, TLR2−/− macrophages were isolated from TLR2−/− mice and then used to evaluate the pro-inflammatory activity of HP1330. The results showed that HP1330 could stimulate obvious pro-inflammatory responses in WT macrophages, but not in TLR2−/− macrophages (Figure 8C). All of the above experiments demonstrated that HP1330-induced cytokine secretion depends on TLR2.

To further elucidate the mechanisms through which HP1330-induced pro-inflammatory responses, we investigated HP1330-dependent signal-transduction pathways in RAW264.7 cells. Inhibitors of p38 MAPK, JNK, NF-κB, PI3K, and ERK1/2 were used to analyze which signaling pathway was responsible for HP1330-induced pro-inflammatory responses. As shown in Figure 9A, the ERK 1/2 MAPK inhibitor (U0126) significantly suppressed cytokine production induced by HP1330, whereas the other four inhibitors (SB203580, SP600125, PDTC, and LY294002) did not. This indicated that HP1330-induced pro-inflammatory responses likely depended on ERK 1/2 MAPK phosphorylation. To test this hypothesis, the phosphorylation of ERK 1/2 MAPK was measured by western blot analysis in HP1330-stimulated RAW264.7 cells. Compared with that in the control group, ERK 1/2 MAPK phosphorylation was significantly enhanced in the HP1330-stimulated group, detecting actin as a loading control (Figure 9B). These results indicated that HP1330 activated pro-inflammatory responses in an ERK1/2 phosphorylation-dependent manner.