The biological role of MutT in the pathogenesis of the zoonotic pathogen Streptococcus suis serotype 2

Abstract

Streptococcus suis (S. suis) is an important rising pathogen that causes serious diseases in humans and pigs. Although some putative virulence factors of S. suis have been identified, its pathogenic mechanisms are largely unclear. Here, we identified a putative virulence-associated factor MutT, which is unique to S. suis serotype 2 (SS2) virulent strains. To investigate the biological roles of MutT in the SS2 virulent strain ZY05719, the mutT knockout mutant (ΔmutT) was generated and used to explore the phenotypic and virulent variations between the parental and ΔmutT strains. We found that the mutT mutation significantly inhibited cell growth ability, shortened the chain length, and displayed a high susceptibility to H₂O₂-induced oxidative stress. Moreover, this study revealed that MutT induced the adhesion and invasion of SS2 to host cells. Deletion of mutT increased microbial clearance in host tissues of the infected mice. Sequence alignment results suggested that mutT was encoded in a strain-specific manner, in which the detection was strongly linked to bacterial pathogenicity. In both zebrafish and mice infection models, the virulence of ΔmutT was largely reduced compared with that of ZY05719. Overall, this study provides compelling evidence that MutT is indispensable for the virulence of SS2 and highlights the biological role of MutT in bacteria pathogenesis during infection.

Keywords: MutT; Streptococcus suis; pathogenesis; virulence.

Figure 1.

Confirmation and characterization of the mutT mutant strain. (a) The mutT mutation was confirmed through combined PCR. (b) Growth curves of the wild-type and mutant strains using OD₆₀₀ measurements under vigorous shaking. (c) Growth curves of the wild-type and mutant strains using OD₆₀₀ measurements under static conditions. Each experiment was performed in triplicate. *P < 0.05; **P < 0.01; ***P < 0.001. Gram staining of ZY05719 and ΔmutT grown in THB (d) or cultured on THA (e) were evaluated by light microscopy (× 1000). (f) Morphology characteristics of ZY05719 and ΔmutT were examined by TEM (× 50,000)

Figure 2.

The role of MutT in oxidative stress tolerance of SS2. ZY05719 and ΔmutT were cultured in THB to an OD₆₀₀ of 0.6 and collected by centrifugation. Then, the bacteria were subjected to stress challenges of H₂O₂ (a), high temperature (b), and acidic conditions (c). Bacterial survival was determined by plating diluted samples onto THB agar before and after stress challenges. Each experiment was performed in triplicate. *P < 0.05; **P < 0.01; ***P < 0.001

Figure 3.

The involvement of MutT in adhesion and invasion of SS2 to host cells. (a) Adhesion and invasion of ZY05719 and ΔmutT to HBMEC cells. (b) Adhesion and invasion of ZY05719 and ΔmutT to HEp-2 cells. Each experiment was performed in triplicate. *P < 0.05; **P < 0.01; ***P < 0.001

Figure 4.

Colonization of the ΔmutT and wild-type strains in various tissues of mice. Mice were intraperitoneally injected with a dose of 5 × 10⁷ CFU of ZY05719 or ΔmutT, respectively. At 24 h post-infection, the bacteria numbers recovered in the brain (a), blood (b), spleen (c), and liver (d) samples. Results were shown as the mean ± SEM from four infected mice. *P < 0.05; **P < 0.01; ***P < 0.001

Figure 5.

Effect of mutT mutation on the virulence of SS2. (a) Survival curve of ZY05719 and ∆mutT in a mouse infection model. Infected mice were monitored for seven days post-infection. (b) Histopathology of brain tissues of mice infected with ZY05719, ΔmutT, or PBS. BALB/c mice were challenged with 2 × 10⁷ CFU of ZY05719 or ΔmutT, respectively. At 72 h after challenge, the infected mice of each group were sacrificed for histological analysis. Brain tissue sections from ZY05719 infection group displayed meningeal thickening and neutrophilic infiltration (green arrow). No obvious pathological changes were found in ∆mutT group or PBS control group