Effective Antibacterial and Antihemolysin Activities of Ellipticine Hydrochloride against Streptococcus suis in a Mouse Model

Abstract

Streptococcal toxic shock-like syndrome (STSLS) caused by the epidemic strain of Streptococcus suis leads to severe inflammation and high mortality. The life and health of humans and animals are also threatened by the increasingly severe antimicrobial resistance in Streptococcus suis There is an urgent need to discover novel strategies for the treatment of S. suis infection. Suilysin (SLY) is considered to be an important virulence factor in the pathogenesis of S. suis In this study, ellipticine hydrochloride (EH) was reported as a compound that antagonizes the hemolytic activity of SLY. In vitro, EH was found to effectively inhibit SLY-mediated hemolytic activity. Furthermore, EH had a strong affinity for SLY, thereby directly binding to SLY to interfere with the hemolytic activity. Meanwhile, it was worth noting that EH was also found to have a significant antibacterial activity. In vivo, compared with traditional ampicillin, EH not only significantly improved the survival rate of mice infected with S. suis 2 strain Sc19 but also relieved lung pathological damage. Furthermore, EH effectively decreased the levels of inflammatory cytokines (interleukin-6 [IL-6], tumor necrosis factor alpha [TNF-α]) and blood biochemistry enzymes (alanine transaminase [ALT], aspartate transaminase [AST], creatine kinase [CK]) in Sc19-infected mice. Additionally, EH markedly reduced the bacterial load of tissues in Sc19-infected mice. In conclusion, our findings suggest that EH can be a potential compound for treating S. suis infection in view of its antibacterial and antihemolysin activity.IMPORTANCE In recent years, the inappropriate use of antibiotics has unnecessarily caused the continuous emergence of resistant bacteria. The antimicrobial resistance of Streptococcus suis has also become an increasingly serious problem. Targeting virulence can reduce the selective pressure of bacteria on antibiotics, thereby alleviating the development of bacterial resistance to a certain extent. Meanwhile, the excessive inflammatory response caused by S. suis infection is considered the primary cause of acute death. Here, we found that ellipticine hydrochloride (EH) exhibited effective antibacterial and antihemolysin activities against S. suisin vitroIn vivo, compared with ampicillin, EH had a significant protective effect on S. suis serotype 2 strain Sc19-infected mice. Our results indicated that EH, with dual antibacterial and antivirulence effects, will contribute to treating S. suis infections and alleviating the antimicrobial resistance of S. suis to a certain extent. More importantly, EH may develop into a promising drug for the prevention of acute death caused by excessive inflammation.

Keywords: Streptococcus suis; antibacterial; antihemolysin; ellipticine hydrochloride; suilysin.

FIG 1

Survival of S. suis was determined after treatment with EH at the concentration of 4× MIC.

FIG 2

Antihemolysin activity of EH against S. suis. (A) Chemical structure of EH. (B) The hemolytic effect of Sc19 culture supernatant on defibrated sheep erythrocytes was determined by measuring the optical density at 543 nm. Positive control, 2.5% Triton X-100. Negative control, culture medium alone. (C) The hemolytic effect of Sc19 Δsly culture supernatant on defibrated sheep erythrocytes was determined as described above. (D) EH inhibited the hemolytic activity of Sc19 culture supernatant. The 125-μl culture’s supernatant was incubated with different concentrations of EH at 37°C for 30 min and then incubated with 875 μl PBS containing 2% defibrated sheep red blood at 37°C for 30 min. (E) EH reduced the hemolytic activity of the purified SLY protein. In the hemolytic assay, the hemolytic activity of the purified SLY protein was determined after coincubation with the different concentrations of EH. (F) Effect of EH at final concentrations of 1, 2, 4, 8, 16, 32, 64, and 128 μg/ml on defibrated sheep erythrocytes. EH at different concentrations was incubated with cells for 1 h at 37°C. Positive control, 2.5% Triton X-100. The two-tailed unpaired t test was used for statistical analysis. The data shown are representative of three independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

FIG 3

Interaction between EH and SLY. (A) EH was bound into the pose1 of SLY. (B) EH is indicated with green sticks; the two hydrogen bonds are indicated with the black dotted line. (C) Interaction between RED-NHS second-generation dye-labeled purified Sly with EH. Data are presented as the mean ± standard error of the mean (s.e.m.) from three independent experiments. (D) Interaction between RED-NHS second-generation dye-labeled purified L110A/S84A-Sly with EH. Data are presented as the mean ± s.e.m. from three independent experiments. (E) The interaction of SLY protein and EH was analyzed using the ITC assay. EH (0.2 mmol/liter) was dripped into the purified SLY protein (0.02 mmol/liter) in PBS buffer (pH 7.4) at 25°C. The data were analyzed to obtain the equilibrium dissociation constant (K_d,  1.235 × 10⁻⁷ mol/liter), stoichiometry (n = 2.079), and changes of enthalpy (ΔH, −142.8 kJ/mol) and entropy (ΔS, –362.2J/mol/K). (F) An LSPR assay explored the kinetic parameters of the binding reaction between SLY and EH. The equilibrium dissociation constants (K_d) of SLY and EH were found to be 1.86 × 10⁻⁶ M using Trace Drawer software.

FIG 4

Survival rate and tissue pathological changes of Sc19-infected mice. The dose and interval of each treatment were 5 mg/kg and 12 h, respectively. (A) Survival rate of mice per day. Compared with ampicillin, EH has a higher protection rate for Sc19-infected mice (log-rank and chi-square tests, n = 10). (B) Pathological changes of lung and brain tissue after EH and ampicillin treatment. EH alleviated tissue damage of infected mice.

FIG 5

Levels of inflammatory cytokines and blood biochemistry enzymes in Sc19-infected mice. The dose and interval of each treatment were 5 mg/kg and 12 h, respectively. (A) Expression levels of IL-6 and TNF-α in infected mice. Compared with ampicillin, EH reduced the production of inflammatory cytokines. (B) Levels of ALT, AST, and CK in the blood of infected mice. Compared with ampicillin, EH decreased the levels of blood biochemistry enzymes of Sc19-infected mice. The two-tailed unpaired t test was applied for statistical analysis. The data shown are representative of three independent experiments. *, P < 0.05; ***, P < 0.001.

FIG 6

Tissue bacterial load in Sc19-infected mice. The dose and interval of each treatment were 5 mg/kg and 12 h, respectively. Both EH and ampicillin can reduce the bacterial titers in different tissues in mice infected with Sc19. The bacterial counts of Sc19 in the lung, spleen, kidney, and liver are shown. The two-tailed unpaired t test was applied for statistical analysis. The data shown are representative of three independent experiments. **, P < 0.01; ***, P < 0.001.