Bacterial strains and plasmids utilized for cloning and mutant construction are detailed in Table 1. The SS2 virulent wild-type (WT) strain of HA9801, isolated from diseased pigs which mainly manifested septicemia among SS2 outbreak in Jiangsu, China, was kindly offered by Professor Weihuan Fang at Zhejiang University. The S. suis strains were cultured in Brain heart infusion (BHI; Oxoid, UK) at 37°C. Escherichia coli DH5α cells were cultured in Luria–Bertani medium at 37°C. The various antibiotics (Solarbio, Beijing, China) used for cloning and the selection of mutants were incorporated into the culture media at specific concentrations: 50 μg/mL of ampicillin for E. coli, 100 μg/mL and 50 μg/mL of spectinomycin for SS2 and E. coli, respectively.

The cbpD deletion mutant (ΔcbpD) based on wild-type strain HA9801 was constructed as previously described (34). In brief, the upstream and downstream flanking fragments of the cbpD gene were amplified by PCR based on the template of the HA9801 genome and the primers listed in Table 2. The flanking fragments were then fused by overlapping PCR. The product was subjected to endonuclease enzymatic digestion, ligated with the thermo-sensitive vector of pSET4s, and then transformed into the DH5α competent cells to generate the knockout plasmid of pSET4s::cbpD after PCR identification and DNA sequencing. The recombinant pSET4s::cbpD shuttle vector was introduced into HA9801 competent cells via electroporation. The resulting ΔcbpD mutant was obtained through a series of screening steps including repeated SS2 passage, targeted gene double exchange, and positive mutant colony isolation, and confirmed by PCR verification and DNA re-sequencing. To construct the allelic cbpD complementation strain of cΔcbpD, the previously reported impdh promoter was fused with the cbpD-ORF and subsequently integrated into pSET2 to yield the complemental plasmid pSET2::cbpD. The recombinant plasmid was electroporated into the ΔcbpD competent cells and the resulting cΔcbpD isolate was screened by spectinomycin resistance and PCR confirmation.

Growth capability was examined by the protocol instructed previously (35). A single isolated colony of the WT, ΔcbpD, and cΔcbpD strains was inoculated into BHI broth for a 12 h incubation at 37°C with 150 rpm shaking. The cultures were centrifuged to form a precipitate and then adjusted to an OD_600nm of 0.4 in BHI. Subsequently, 250 μL of the bacterial suspension each was transferred into 25 mL of BHI medium. The cultures were used for spectrophotometric measurement at OD_600nm every 2 h after 37°C incubation at 150 rpm. Triplicate assays with three wells each were conducted and determined for this experiment.

The SS2-WT, ΔcbpD, and cΔcbpD strains were Gram-stained on the glass slides following the staining guide and observed by microscopy under oil immersion. The numbers of cocci among these strains in 50 chains each under light microscopy were counted and average bacterial cells per chain were examined. Stress tolerance phenotypes of SS2 strains upon thermo-induced treatment at 42°C, acetic acid treatment at pH5.5 for 37°C or oxidative treatment with 20 mM H₂O₂ at 37°C for 1 h, were conducted and examined as previously described, respectively (36).

The adhesion to and invasion of host cells by SS2 was assessed in accordance with a previous study by using the human laryngeal epithelial HEp-2 cells and the murine brain microvascular endothelial bEND3.0 cells, respectively (36). Briefly, bacterial cultures were added to the cells in a 24-well culture plate at a multiplicity of infection (MOI) of 50, with three wells for each strain. The plates were incubated for 1 h at 37°C, 5% CO₂. To remove non-adherent bacteria, the wells were washed four times with 1 mL of 10 mM PBS. For the adherence assay, the sterile ddH₂O was added to lyse infected cells and then plated to BHI agar by 10-fold dilution in PBS. After 24 h incubation at 37°C, the bacteria colonies were enumerated. The adhesion rate was defined as (viable CFU_Adh / inoculated CFU_Total) × 100%. The adhesive inhibition assay was conducted the same as the aforementioned procedure (37), with the exception of the 10% inactivated rabbit polyclonal targeted or pre-immune negative sera treatment of the WT and ΔcbpD for 1 h prior to infection.

For the invasion assay, DMEM consisting of gentamicin (300 μg/mL) was introduced into the bacterial infection cells following adhesion assay to eradicate extracellular dissociated SS2 for 1 h at 37°C. The cells underwent subsequently triple washes with PBS and were further lysed by using sterile distilled water. The intracellular evasive bacteria were determined by BHI plate counting. The invasion rate was defined as (viable intracellular CFU_Inv / inoculated CFU_Total) × 100%.

Murine macrophage cell line RAW264.7 was used for the phagocytosis assay according to a previously reported protocol (34, 38). Cells were subcultured and inoculated into 24-well plates at 37°C, 5% CO₂. The cells were cultivated for 12 h until the cell confluence reached above 90%, washed with PBS, and incubated in DMEM containing 10% inactivated fetal bovine serum. The suspensions of both the WT and ΔcbpD strains were pre-adjusted to an OD_600nm of 0.4 and then added to the cells at an MOI of 50. After 1 h incubation, the cells were subjected to PBS washing and then treated with gentamicin (300 μg/mL) for another 1 h. Subsequently, the mixture was washed three times by PBS, lysed with sterile distilled water, and plated on BHI agar to count the viable SS2 colonies. The phagocytosis rate was determined by calculating the percentage of surviving CFU in the BHI medium compared to the CFU in the original inoculum. This assay was performed three times for each strain, with triplicate wells tested on each occasion.

Survival assay of SS2 within mice whole blood was executed following the established protocol (35). Cultures in the mid-exponential growth phase were standardized to an optical density of 0.4 at 600 nm. A 50 μL aliquot of either WT or ΔcbpD, was added to 450 μL of fresh whole blood collected from clinically healthy mice and then incubated at 37°C for 1 h. The viable bacterial counts were determined via BHI agar plate enumeration. Each assay was conducted in triplicate and replicated three times.

In each designated group, 12 female BALB/c mice, aged 4 weeks, were randomly chosen and subjected to intraperitoneal injection with 1 mL of WT, ΔcbpD, and cΔcbpD at approximately 5 × 10⁸ CFU/mL, respectively. An equal volume of PBS inoculation served as a negative control. The clinical manifestations and mortality of mice were monitored every 12 h and continuously recorded for 1 week. Furthermore, the heart, liver, spleen, lung, kidney, and brain organs from infected mice showing clinical symptoms were aseptically collected, homogenized, and subsequently subjected to BHI plate counting for bacterial load determination in another SS2 infection experiment. Meanwhile, 4% paraformaldehyde was used to fix tissue samples and then paraffin-embedded sections of the lesion tissues were prepared, stained by Hematoxylin and Eosin, and the histopathological changes of the tissues were examined under a microscope as described previously (37). Animal infection experiments conformed to the animal ethics guidelines from the Medical Ethics Committee of Yichun University in Yichun City, Jiangxi Province, China (2022018).

Bacterial cultures, upon reaching the mid-logarithmic growth phase, were harvested. The cells were then pelleted through centrifugation at 5,000 rpm for 10 min. Total RNA extraction from the bacterial pellets was carried out following the manufacturer’s protocol by the RNA Extraction Kit (TransGen, China) (39). Subsequently, synthesis and amplification of cDNA were performed via a reverse transcription kit (Vazyme, China). To quantify the transcriptional levels of the target genes listed in Table 2, relative qPCR was conducted using the ChamQ Universal SYBR qPCR Master Mix (Vazyme, China) with Bio-Rad CFX96 thermocycler. The 16S rRNA gene was determined as an internal reference to normalize the gene expression data.

The effect of CbpD deficiency on the virulence-related factors expression levels was analyzed by Western blotting. Overnight cultures of WT and ΔcbpD strains were centrifuged at 5,000 rpm for 10 min to collect the bacteria suspension and supernatants. Total protein samples from bacteria suspension, except for two secreted proteins Sly and TGase, which were directly determined from the harvested bacteria supernatant, were utilized for the analysis of protein expression changes. The total SS2 cellular proteins were extracted by cell wall cleavage buffer including 3 mg/mL of lysozyme in 37°C for 1.5 h and homogenizing the mixture following the previously established protocol, and the protein concentration was quantified by the Bradford method (40). SDS-PAGE was performed, followed by protein transfer onto PVDF membranes, blocking, and incubation with rabbit polyclonal antibodies (prepared in-house) at 4°C for 12 h. Then, the PVDF membranes underwent incubation of HRP-conjugated goat anti-rabbit monoclonal IgG, washed five times with TBST containing 0.5% skimmed milk powder, and visualized by chemiluminescent imaging system after a chemiluminescence reaction kit (GLPBIO, United States). Protein changes in grayscale were accomplished via ImageJ software (ImageJ 1.5, NIH, United States).

Each experimental assay was performed in triplicate and repeated on at least three distinct occasions, and the resulting data were presented as mean ± standard deviations (SDs). Statistical significance was determined using a paired, two-tailed Student’s t-test, facilitated by the GraphPad Prism 5.0 software. For animal infection experiments, the log-rank test was used to assess the differences in survival rate. The p-value threshold of 0.05 was established and considered as the criterion for statistical significance.

The construction of the cbpD mutant and complementation strains was performed using homologous recombination with the shuttle vectors pSET4s for the mutant and pSET2 for the complementation strain. The specific gene knockout plasmid of recombinant pSET4s::cbpD was generated as shown in Figure 1A. After electroporation, repeated subculture, and screening, the suspected ΔcbpD mutant and allelic cΔcbpD colonies were identified by PCR using the outer-, inner-, and S. suis-specific 16S rRNA gene primers, and subjected to verification by DNA sequencing. The cbpD mutant with its allelic complementary strain of cΔcbpD was successfully obtained, as evidenced by PCR identification (Figure 1B).

Figure 2A demonstrated that the growth patterns between the WT and ΔcbpD strains were predominantly congruent, and the ablation of cbpD does not impinge upon the growth competence of SS2. Morphological observations disclosed that in contrast to the WT strain, the ΔcbpD possessed enhanced chain-forming capability, with an average of 3–5 bacterial cells per chain, and manifested a propensity to aggregate (Figures 2B,C). The stress tolerance analysis discovered that the deletion of the cbpD gene would significantly decrease the survival rate of ΔcbpD and render this strain more susceptible to cultures at 42°C, pH5.5, or 20 mM H₂O₂ stress conditions (Figure 2D). The abovementioned deviations could be restored in phenotype after cbpD replenishment in cΔcbpD strain.

To examine the potential role of CbpD in SS2 infection, in vitro cell infection models previously developed were used to investigate the differences in adhesion, invasion, resistance to phagocytosis, and survival capability among WT, ΔcbpD, and cΔcbpD strains. The findings revealed a prominent increase in the adhesion rate of the cbpD-deficient strain to HEp-2 epithelial cells (p < 0.05, Figure 3A), while the colonies of ΔcbpD invading endothelial bEND3.0 cells were notably decreased (p < 0.05, Figure 3B). These cbpD-deficient SS2 were prone to be phagocytosed by macrophage cells of RAW264.7 and showed an 80% reduction in survival when incubated in ex vivo mouse whole blood (p < 0.01, Figures 3C,D). The introduction of the cbpD gene could partially restore these phenotypic changes. These data implied that the CbpD factor was involved in SS2 infection by mediating the immune invasion and phagocytosis process.

An in vivo mouse infection model was used to evaluate the impact of cbpD gene deletion on SS2 virulence at a lethal dose. The findings indicated that 50% of the mice in the WT infection group died within 24 h, and all died within 72 h. The clinical symptoms in the WT-infected mice were noticeable, with common manifestations such as distinguished disheveled hair, tearing and blindness, mental signs, and movement disorders. Nevertheless, a significant decrease in the virulence of the cbpD deletion strain was shown as none of the mice died 3 days after infection, resulting in a survival rate of 41.67% as well as strikingly alleviated clinical symptoms (Figure 4A). The results of bacterial load in mouse organs affirmed that the susceptible strain of the ΔcbpD was easier to clear since the prominently reduced colonization colonies of the ΔcbpD were shown in mice organs in Figure 4B. In accordance with the results of bacterial burden in organs, the milder and relieved pathological damages, such as myolysis and disruption of cardiac muscle fibers in the heart, hepatocellular necrosis, and inflammatory cell infiltration with hemorrhage in the liver, disintegration of the splenic nodules along with bleeding in the spleen, congestion, and rupture of the alveoli accompanied by hemorrhage in the lung, glomerular enlargement, and infiltration with inflammatory cells in the kidney, and purulent meningitis, neuronal cell swelling and degeneration in the brain in the WT infection group, were induced by the CbpD deficiency SS2 infection based on the microscopic histopathological observation (Figure 4C). The results illustrated that cbpD deletion attenuated the virulence of S. suis 2.

To dissect the impact of the cbpD mutation on bacterial virulence factors expression, seven previously reported virulence-associated factors including the Sly, TGase, SodA, adhesion factor of chaperone DnaJ, Stp, membrane-associated protein of enolase, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), were examined by transcriptional qPCR and Western blotting analysis. The qPCR results indicated that the deletion of the cbpD gene induces the upregulated expression of dnaJ and the conspicuously downregulated expression of three genes of tgase, enolase, and stp (Figure 5A). Further protein level detection by Western blotting proved the corresponding differential alterations of these targeted factors in transcriptional levels (Figures 5B,C).

The adhesive inhibition assay was executed to investigate whether the elevated expression of the adhesion factor of DnaJ in ΔcbpD strain prompted the augmented bacterial adherence to HEp-2 cells. The surprising results in Figure 5D revealed that pre-blocked treatment of the ΔcbpD mutant with monoclonal DnaJ antisera significantly attenuated its adhesion to HEp-2 cells when compared to the negative pre-immune antisera treatment group (p < 0.01).