The non-conserved region of MRP is involved in the virulence of Streptococcus suis serotype 2

Abstract

Muramidase-released protein (MRP) of Streptococcus suis serotype 2 (SS2) is an important epidemic virulence marker with an unclear role in bacterial infection. To investigate the biologic functions of MRP, 3 mutants named Δmrp, Δmrp domain 1 (Δmrp-d1), and Δmrp domain 2 (Δmrp-d2) were constructed to assess the phenotypic changes between the parental strain and the mutant strains. The results indicated that MRP domain 1 (MRP-D1, the non-conserved region of MRP from a virulent strain, a.a. 242-596) played a critical role in adherence of SS2 to host cells, compared with MRP domain 1* (MRP-D1*, the non-conserved region of MRP from a low virulent strain, a.a. 239-598) or MRP domain 2 (MRP-D2, the conserved region of MRP, a.a. 848-1222). We found that MRP-D1 but not MRP-D2, could bind specifically to fibronectin (FN), factor H (FH), fibrinogen (FG), and immunoglobulin G (IgG). Additionally, we confirmed that mrp-d1 mutation significantly inhibited bacteremia and brain invasion in a mouse infection model. The mrp-d1 mutation also attenuated the intracellular survival of SS2 in RAW246.7 macrophages, shortened the growth ability in pig blood and decreased the virulence of SS2 in BALB/c mice. Furthermore, antiserum against MRP-D1 was found to dramatically impede SS2 survival in pig blood. Finally, immunization with recombinant MRP-D1 efficiently enhanced murine viability after SS2 challenge, indicating its potential use in vaccination strategies. Collectively, these results indicated that MRP-D1 is involved in SS2 virulence and eloquently demonstrate the function of MRP in pathogenesis of infection.

Keywords: Streptococcus suis serotype 2; infection; muramidase-released protein; vaccine; virulence.

Figure 1.

Multiple sequence alignments of SS2 MRP. ((A)and B) Multiple sequence alignments of the 12 SS2 MRP proteins at the amino acid level. All the sequence analyses were performed by DNAMAN software. MRP has a variable region that differs between highly virulent and low virulent strains and the C-terminus of MRP protein is extremely conserved, whereas the N-terminus differs considerably.

Figure 2.

Construction of Δmrp, Δmrp-d1, and Δmrp-d2 strains and growth curves of the WT and 3 mutant strains. (A) Schematic representation of the 3 knockout mutants and 3 recombinant proteins used in this study. The constructs were designed mainly according to the multiple sequence alignments of SS2 MRP. S: signal sequence; LPxTG: cell wall anchoring motif; Black arrows denote the truncated forms of MRP. (B) PCR confirmation of the knockout mutant strains. Three different primer sets, including internal (E/F), flanking (A/D), and external (X/Y) regions used in the PCR analysis are indicated above the lanes. Genomic DNAs from the WT and mutant strains were used as templates. (C) Western blotting confirmation of the mutant strains. The released MRP in the extracellular proteins of WT and mutant strains were probed with anti-MRP-D1 antibodies or anti-MRP-D2 antibodies. Extracellular protein samples of culture supernatant were prepared as described previously. Western blotting analysis confirmed that the expression of MRP was detected in the WT, Δmrp-d1, and Δmrp-d2 strains but was not detected in the Δmrp strain. (D) Growth characteristics of WT and mutant strains using OD₆₀₀ values.

Figure 3.

Evaluating the effect of recombinant MRP-D1, MRP-D1*, MRP-D2, and HP07325 on the adherence ability of SS2 to human cells. (A) Adherence of recombinant MRP-D1, MRP-D1*, MRP-D2, and HP07325 to HEp-2 cells was confirmed by indirect immunofluorescence assay. The HEp-2 cells without the primary antibody were used as blank control. (B) Adherence of recombinant MRP-D1, MRP-D1*, MRP-D2, and HP07325 to bEnd.3 cells was confirmed by indirect immunofluorescence assay. The bEnd.3 cells without the primary antibody were used as a blank control. (C) Recombinant proteins pulled down by HEp-2 cells. (D) Western blotting analysis of recombinant proteins with an anti–His tagged monoclonal antibody. (E) Effect of Δmrp, Δmrp-d1, and Δmrp-d2 strains on the adherence ability of SS2 to HEp-2 cells. The adherence rate of the WT strain was significantly higher than that of the 3 mutant strains. Data were expressed as the mean ± SEM of 5 independent experiments performed in triplicate. Significant differences are indicated (***p < 0.001; **p < 0.01; *p < 0.05).

Figure 4.

Assessment of the binding region of MRP to host components (FN, FH, FG, and IgG) by Far-western blotting. Recombinant MRP-D1, MRP-D1*, and MRP-D2 were separated by SDS-PAGE and stained with Coomassie blue (A) or transferred to PVDF membranes and then incubated with FN (B), FH (C), FG (D), or IgG (E). Bound host components were detected using primary antibodies or HRP-conjugated secondary antibodies, respectively.

Figure 5.

Effect of WT and mutant strains on phagocytosis and killing of SS2 by RAW246.7 macrophages. (A) Phagocytosis analysis of different SS2 strains by macrophages. The uptake of bacteria by macrophages was monitored for 1 h. At the 0 h time point, antibiotics were added to kill bacteria extracellular to macrophages. Intracellular bacteria in RAW246.7 macrophages were determined by quantitative plating at the 1 h time point. (B) Intracellular survival analysis of SS2 strains at different time points. The numbers of intracellular bacteria were recovered from cell lysates at different time points. The initial number of viable intracellular bacteria at 1 h was designated as 100%. The survival rate of the Δmrp and Δmrp-d1 strains was significantly lower than that of the Δmrp-d2 and WT strains. Data were expressed as the mean ± SEM of 3 independent experiments performed in triplicate. Significant differences are indicated (***p < 0.001; **p < 0.01; *p < 0.05).

Figure 6.

Effect of WT and mutant strains on the virulence of SS2. ((A)and B) The elimination of SS2 in mice blood and brain tissue in infection of BALB/c mice. The WT and mutant strains were separately inoculated into mice at 2 × 10⁷ CFU/mouse. The asterisk suggested that the bacterial counts of the Δmrp-d1 strain in blood and brain tissue were significantly lower than other strains at 24 h and 48 h time points. Data are expressed as the mean ± SEM of 5 infected mice per time point. (C) MRP-D1 promotes the survival of SS2 in pig blood at the measured time points (1 h and 2 h). The survival rate of the Δmrp and Δmrp-d1 strains was significantly attenuated compared with the WT and Δmrp-d2 strains. Significant differences are indicated (***p < 0.001; **p < 0.01; *p < 0.05). (D) Comparative analysis of bacterial virulence of the WT strain with the mutant strains using a mouse model. The WT and mutant strains were separately inoculated into mice at 2 × 10⁸ CFU/mouse. Mice challenged with the Δmrp-d1 strain had a higher survival rate than those challenged with the Δmrp-d2 strain or WT strain, indicating that deletion of mrp-d1 dramatically attenuated the virulence of SS2.

Figure 7.

MRP-D1 confers partial protection against SS2 infection. (A) Serum antibody responses induced by recombinant MRP-D1 and MRP-D2 in mice. The antibody titers against recombinant MRP-D1 and MRP-D2 from the immunized group were significantly higher than the control groups. (B) Evaluating the protection effect of recombinant MRP-D1 and MRP-D2 in mice. Survival curves of mice immunized with recombinant MRP-D1 were significantly higher than that of mice immunized with recombinant MRP-D2. PBS emulsified with adjuvant served as a negative control and mice were immunized with PBS alone as a blank control. (C) Assessment of the bactericidal activity of anti-MRP-D1/MRP-D2 antibodies in pig blood. Pre-incubation with anti-MRP-D1 sera significantly decreased the survival of SS2 in whole blood, confirming the result obtained in the mice protection assay. Significant differences are indicated (***p < 0.001; **p < 0.01; *p < 0.05).