Theaflavin Ameliorates Streptococcus suis-Induced Infection In Vitro and In Vivo

Abstract

Streptococcus suis (S. suis) is one of the most important zoonotic pathogens that threaten the lives of pigs and humans. Even worse, the increasingly severe antimicrobial resistance in S. suis is becoming a global issue. Therefore, there is an urgent need to discover novel antibacterial alternatives for the treatment of S. suis infection. In this study, we investigated theaflavin (TF1), a benzoaphenone compound extracted from black tea, as a potential phytochemical compound against S. suis. TF1 at MIC showed significant inhibitory effects on S. suis growth, hemolytic activity, and biofilm formation, and caused damage to S. suis cells in vitro. TF1 had no cytotoxicity and decreased adherent activity of S. suis to the epithelial cell Nptr. Furthermore, TF1 not only improved the survival rate of S. suis-infected mice but also reduced the bacterial load and the production of IL-6 and TNF-α. A hemolysis test revealed the direct interaction between TF1 and Sly, while molecular docking showed TF1 had a good binding activity with the Glu198, Lys190, Asp111, and Ser374 of Sly. Moreover, virulence-related genes were downregulated in the TF1-treated group. Collectively, our findings suggested that TF1 can be used as a potential inhibitor for treating S. suis infection in view of its antibacterial and antihemolytic activity.

Keywords: Streptococcus suis; hemolytic activity; molecular docking; suilysin; theaflavin.

Figure 1

TF1 antibacterial activity. (A) Chemical structure of TF1. (B) Kinetics of the killing activity of TF1 against SC19 were monitored by OD600nm at the indicated times. (C) Kinetics of the killing activity of TF1 against SC19 were monitored by CFU counts at the indicated times.

Figure 2

Cellular shape and structure analysis of SC19. (A) TEM analysis of untreated SC19, the bar at the bottom right means 500 nm. (B) SEM analysis of untreated SC19, the bar at the bottom right means 1 μm. (C) TEM analysis of SC19 treated with TF1 at MIC, bacterial cell presented vacuolation degeneration, the bar at the bottom right means 500 nm. (D) SEM analysis of SC19 treated with TF1 at MIC, bacterial cell presented shrinkage, cell size reduction and perforation of the cell surface, the bar at the bottom right means 1 μm.

Figure 3

Hemolytic activity and biofilm formation of SC19 affected by TF1. (A,B) Hemolytic activity analysis of SC19 culture affected by TF1. Absorption was measured at 543 nm to determine Sly activity. TSB was used as a negative control. (C,D) Direct interaction of TF1 and Sly revealed by hemolytic activity analysis of SC19 culture supernatant treated by TF1. Absorption was measured at 543 nm to determine Sly activity. TSB was used as a negative control. (E,F) Biofilm formation analysis of SC19 affected by TF1. Absorption was measured at 590 nm to determine biofilm production. TSB was used as a negative control. The height of the bars indicates the mean values for the relative expression data ± SEM (ns, p > 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001).

Figure 4

TF1 inhibited the adhesion ability of SC19 and SC19-mediated cytokine production. (A) TF1 had no cytotoxic effect on the epithelial cell Nptr through LDH release measurements. (B) Adhesion ability analysis of SC19 affected by TF1. (C) TNF-αproduction in serum of SC19-infected mice affected by TF1. (D) IL-6 production in serum of SC19-infected mice affected by TF1. The height of the bars indicates the mean values for the relative expression data ± SEM (ns, p > 0.05; * p < 0.05; ** p < 0.01; *** p < 0.001).

Figure 5

TF1 reduced the pathogenicity of SC19. (A) TF1-protected mice against SC19 infection, a significant difference in survival between different groups was analyzed by log rank test. (B) Bacterial burdens in the brains of the SC19-infected mice. (C) Bacterial burdens in the lungs of the SC19-infected mice. (D) Bacterial burdens in the spleens of the SC19-infected mice. The height of the bars indicates the mean values for the relative expression data ± SEM (ns, p > 0.05; * p < 0.05; ** p < 0.01; *** p < 0.001).

Figure 6

Molecular docking and qRT-PCR results. (A) 3D structure of Sly docked with TF1. (B) Enlarged view of the pocket after molecular docking of TF1 and Sly protein, where the amino acids Glu198, Lys190, Asp111, and Ser374 react with TF1. (C) Interactions between the binding site of TF1 and the amino acid of Sly protein. The hydroxyl groups of TF1 are linked to the amino acids of the Sly protein, the five hydrogen bonds are indicated with the green dotted arrows. (D) Virulence-related genes expression analysis of TF1 treated SC19 compared to untreated SC19. The height of the bars indicated the mean values for the relative expression data ± SEM * p < 0.05; ** p < 0.01).