Insight into the role of Streptococcus suis zinc metalloprotease C from the new serotype causing meningitis in piglets

Abstract

Streptococcus suis (S. suis) is an important gram-positive pathogen and an emerging zoonotic pathogen that causes meningitis in swine and humans. Although several virulence factors have been characterized in S. suis, the underlying mechanisms of pathogenesis are not fully understood. In this study, we identified Zinc metalloproteinase C (ZmpC) probably as a critical virulence factor widely distributed in S. suis strains. ZmpC was identified as a critical facilitator in the development of bacterial meningitis, as evidenced by the detection of increased expression of TNF-α, IL-8, and matrix metalloprotease 9 (MMP-9). Subcellular localization analysis further revealed that ZmpC was localized to the cell wall surface and gelatin zymography analysis showed that ZmpC could cleave human MMP-9. Mice challenge demonstrated that ZmpC provided protection against S. suis CZ130302 (serotype Chz) and ZY05719 (serotype 2) infection. In conclusion, these results reveal that ZmpC plays an important role in promoting CZ130302 to cause mouse meningitis and may be a potential candidate for a S. suis CZ130302 vaccine.

Keywords: Streptococcus suis; Meningitis; Protective antigen; Serotype Chz; Virulence; Zinc metalloprotease.

Fig. 1

Distribution and phylogenetic analysis of Zmp in S. suis. (A) The NCBI database was searched for Zmp of S. suis using conserved HEXXH…E motifs. The number of Zmps in each S. suis strain was analyzed using GraphPad Prism 8. Chz type strains are highlighted in red. (B) Phylogenetic analysis was constructed by Neighbor-Joining Tree using MEGA-X software. IgA1 proteases (H020_RS0105710), ZmpC (H020_RS0100375), ZmpD (H020_RS0103275) and ZmpB (C4N11_03120) in S. pneumoniae were used as references and are highlighted with blue boxes. Zmps in the Chz type CZ130302 strain are highlighted with red boxes

Fig. 2

ZmpC contributes to the virulence of CZ130302. (A) Zmps in CZ130302 and Δzmp were detected by PCR using primer pairs. The original gel is presented in Supplementary material 4. (B) Effect of the deletion mutants on the growth of S. suis CZ130302. The results are indicated as the means ± SEM of the results from 3 independent experiments (P>0.05). (C) Survival curves of 5-week-old BALB/c mice infected with wild-type or mutant strains at 5 × 10⁷ CFU/mouse. The control group received only 1×PBS. Ten mice from each group were monitored over a 7-day period. Log-rank (Mantel-Cox) test to determine differences in survival between groups: ** P < 0.01. Infected mice were euthanized 12 h after infection to determine the bacteria burden in the brain (D), liver (E), and spleen (F). Unpaired two-tailed Student’s t-test: * P < 0.05; ** P < 0.01; **** P < 0.0001

Fig. 3

ZmpC in CZ130302 contributes to the development of meningitis. (A) Transcriptional level of TNF-α (A), IL-8 (B), and MMP-9 (C) encoding gene in the brains of the mice infected with the indicated strains. Data are represented as mean ± SEM of three independent repeats. Unpaired two-tailed Student’s t-test: * P < 0.05; *** P < 0.001

Fig. 4

ZmpC in CZ130302 cleaves human MMP-9. (A) The three-dimensional structures of ZmpC in S. pneumoniae TIGR4 (left) and S. suis CZ130302 (right) were predicted using the online site SWISS-MODEL. Conserved HEXXH…E motifs in α-helix are shown in red boxes. (B) Schematic representation of the structure of ZmpC in S. suis CZ130302. ZmpC in CZ130302 contains complete M26 N-terminal and C-terminal domains. (C) The purified recombinant protein ZmpC-M26 was incubated with human MMP-9 for gelatin zymography analysis. The first lane indicates that the sample has only 2 µg human MMP-9, the second to sixth lanes indicate that 2 µg human MMP-9 was incubated with different concentrations of ZmpC-M26 (900 ng to 1300 ng), and the eighth lane indicates that the sample has only ZmpC-M26. The red arrow indicates the detection of a bright band of approximately 52 kDa. The results showed that ZmpC cleaves human MMP-9. (D) The purified recombinant protein ZmpE-M26 (900 ng to 1300 ng) was incubated with human MMP-9 for gelatin zymography analysis. No other bright bands were detected in the incubation group, indicating that ZmpE cannot cleave human MMP-9. The original gel is presented in Supplementary material 4

Fig. 5

ZmpC is a protective antigen that localizes to the cell surface. (A) Western blot analysis identified the localization of ZmpC in CZ130302. The cell wall proteins in CZ130302 and ΔzmpC were extracted and Western blot was performed. A band consistent with the size of ZmpC was detected only in CZ130302 lane. The original blot is presented in Supplementary material 4. (B) Immunofluorescence assays were used to identify ZmpC anchored to the cell wall with anti-ZmpC-M26 and negative serum, respectively. It should be noted that intracellular proteins are not recognized by extracellular antisera. White bars represent 10 μm. To explore the protective potential of ZmpC, mice immunized with recombinant protein ZmpC-M26 were challenged with Chz type CZ130302 strain (C) and serotype 2 ZY05719 strain (D), respectively. ZmpC-M26 can provide effective protection against CZ130302 and ZY05719 infection