Study on the Effect of Phillyrin on Streptococcus suis In Vivo and In Vitro

Abstract

As a zoonotic pathogen, S. suis serotype 2 (SS2) can cause severe diseases in both pigs and humans, and develop resistance to antibiotics. Plant natural compounds are regarded as promising alternatives to conventional antibiotics. Phillyrin is the major bioactive components of Chinese herbal medicine Forsythia suspensa. In this study, we explored the activity and action mechanism of phillyrin against SS2. The results showed that phillyrin could disrupt membrane integrity, destroy intracellular structures, and increase the exosmosis of DNA. Results of PCR revealed that phillyrin affected bacterial-virulence-related genes' expression levels. Meanwhile, phillyrin significantly decreased the adhesion activity, inhibited lactate dehydrogenase (LDH) secretion, and reduced biofilm formation of SS2 in Newborn pig trachea epithelial (NPTr) cells. Furthermore, phillyrin protected tight junction protein of NPTr cells from SS2. We reported that phillyrin (0.1 mg/kg) treatment after bacterial challenge significantly improved the survival rate, ameliorated pulmonary inflammation, and inhibited the accumulation of multiple cytokines (IL-1, IL-6, IL-8, and TNF-α). Molecular docking showed that phillyrin had a good binding activity with the Ala88 and Asp111 of suilysin (SLY), one of the most important virulence factors of SS2. Collectively, phillyrin possesses antibacterial and anti-inflammatory activities, and is a promising candidate for preventing SS2 infection.

Keywords: S. suis; action mechanism; biofilm; cell adhesion; phillyrin.

Figure 1

Antibacterial activity of phillyrin against SC19 in vitro. (A) Chemical structure of phillyrin. (B) Analysis of phillyrin by Mass Spectrum. (C) The growth curve of SC19 affected by phillyrin was determined by OD600 nm at the indicated times. (D) The growth curve of SC19 affected by phillyrin was determined by CFU counts at the indicated times. (ns, p > 0.05; ** p < 0.01; *** p < 0.001; **** p< 0.0001).

Figure 2

Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) of SS2 after treatment with phillyrin. (A) TEM observation of phillyrin-untreated SC19; the bar at the bottom right means 1 µm. (B) TEM observation of phillyrin-treated SC19; the bar at the bottom right means 1 µm. (C) SEM observation of phillyrin-untreated SC19; the bar at the bottom right means 300 nm. (D) SEM observation of phillyrin-treated SC19; the bar at the bottom right means 300 nm. Bacterial cell presented shrinkage, cell size reduction, and perforation of the cell surface. Control cells without treatment appeared with normal shape.

Figure 3

Phillyrin signifcantly improves the DNA exosmosis of SS2. SC19 was cultured to the logarithmic stage, blended to 107 CFU/mL and treated with phillyrin at 64 µg/mL and 128 µg/mL respectively.SC19 without phillyrin were used as a negative control. Supernatants were collected after co-incubated for 0, 1, 2, 4, 6 and 8 h, and the DNA content was determinated with a microspectrophotometer.

Figure 4

Phillyrin inhibits the secretion of hemolysin and biofilm information of SS2. (A,B) Hemolytic activity analysis of SC19 affected by PHI. SC19 was cultured to the logarithmic stage, treated with different concentrations (1/4 MIC, 1/2 MIC, MIC, 1/4 MBC and 1/2 MBC) of phillyrin. After incubation with defibrillated sheep blood, the hemolysin was collected and detected by a spectrophotometer. (C,D) Biofilm formation analysis of SC19 affected by phillyrin. The biofilms of the SC19 with phillyrin at MIC and MBC (512 µg/mL) were stained by the crystal violet method and OD600 was determined after it dissolved. ns, p>0.05; * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001 compared to the respective control.

Figure 5

Effect of phillyrin on NPTr cells. (A) Analysis of cytotoxicity of phillyrin by double fluorescence staining of NPTr cells. (B) Cell viability was determined by a CCK8 assay. (C) Analysis of the cytotoxic activity of phillyrin by standard LDH release assay. ns, p > 0.05. (D) Results were expressed as the percentage of LDH release compared to the non-phillyrin cells. Error bars represent the standard deviation of three independent experiments performed in triplicate. **** p < 0.0001.

Figure 6

Adhesion ability analysis of SS2 affected by phillyrin. NPTr cells were treated by SC19 with phillyrin, and then the number of adherent bacteria was measured. The experiment was performed in triplicate and repeated at least three times. * p < 0.05; ** p < 0.01.

Figure 7

Phillyrin reduces damage of cell tight junction protein by SS2. (A) Expression of cell tight junction protein ZO-1 was detected by Western blot. (B) Western blot analysis of phillyrin treatment on the tight junction protein ZO-1 of NPTr cells infected by SC19 with or without 64 µg/mL phillyrin. The height of the bars indicates the mean values for the relative expression data ± SEM (**** p < 0.0001). Original images of (A) can be found in supplementary materials.

Figure 8

Phillyrin suppresses genes expression of SS2.The real-time PCR results of virulence factor genes (mrp, epf, sly, stk), cell adhesion-related genes (ccpA, fbps), and cell division-related genes (gor) of SC19. ** p < 0.01; *** p < 0.001 compared to the respective control.

Figure 9

Phillyrin reduced the virulence of SS2 in mice. (A) Virulence assay of SC19, SC19+phillyrin, and normal saline by comparing the survival of posttreatment mice. Bacterial load in the blood (B) and lung (C) tissues of the SC19-infected mice. Secretion of IL-1ß (D), IL-6 (E), IL-8 (F), and TNF-α (G) in the serum of mice infected with SC19. 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; **** p < 0.0001).

Figure 10

Molecular docking of phillyrin and SLY. (A,B) Three-dimensional structure of SLY docked with phillyrin, revealing the amino acids Ala88 and Asp111 react with phillyrin. (C) Predictive interaction between phillyrin and SLY protein. Two key bonds indicated with the dotted arrows were established.