Anti- Toxoplasma gondii Effects of Lipopeptide Derivatives of Lycosin-I

Abstract

Toxoplasmosis, caused by Toxoplasma gondii (T. gondii), is a serious zoonotic parasitic disease. We previously found that Lycosin-I exhibited anti-T. gondii activity, but its serum stability was not good enough. In this study, we aimed to improve the stability and activity of Lycosin-I through fatty acid chain modification, so as to find a better anti-T. gondii drug candidate. The α/ε-amino residues of different lysine residues of Lycosin-I were covalently coupled with lauric acid to obtain eight lipopeptides, namely L-C₁₂, L-C₁₂-1, L-C₁₂-2, L-C₁₂-3, L-C₁₂-4, L-C₁₂-5, L-C₁₂-6, and L-C₁₂-7. Among these eight lipopeptides, L-C₁₂ showed the best activity against T. gondii in vitro in a trypan blue assay. We then conjugated a shorter length fatty chain, aminocaproic acid, at the same modification site of L-C₁₂, namely L-an. The anti-T. gondii effects of Lycosin-I, L-C₁₂ and L-an were evaluated via an invasion assay, proliferation assay and plaque assay in vitro. A mouse model acutely infected with T. gondii tachyzoites was established to evaluate their efficacy in vivo. The serum stability of L-C₁₂ and L-an was improved, and they showed comparable or even better activity than Lycosin-I did in inhibiting the invasion and proliferation of tachyzoites. L-an effectively prolonged the survival time of mice acutely infected with T. gondii. These results suggest that appropriate fatty acid chain modification can improve serum stability and enhance anti-T. gondii effect of Lycosin-I. The lipopeptide derivatives of Lycosin-I have potential as a novel anti-T. gondii drug candidate.

Keywords: Lycosin-I; Toxoplasma gondii; fatty acid chain modification; lipopeptide.

Figure 1

The effect of lipopeptides against T. gondii was evaluated using the trypan blue assay. (A) The modification site of the lipopeptides. Label X₁ indicates that lauric acid was coupled to the α-amino or ε-amino group of Lys. Label X₂ indicates that aminocaproic acid was coupled to the α-amino group of Lys. (B) Visualization of the secondary structure of Lycosin-I; blue indicates the lysine. (C) Tachyzoites treated with the control group (PBS) and different concentrations of lipopeptides. Tachyzoite viability was observed under a light microscope, and five fields were randomly selected to calculate tachyzoite mortality (ns > 0.05, * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001 in comparison with PBS). (D) Figure of normalized concentration–response curves (mortality as a percentage of PBS control) constructed from the bar graphs in C.

Figure 2

(A–C) Mortality of lipopeptides treated with serum on tachyzoites. There was a significant difference for Lycosin-I but not for L-C₁₂ and L-an (* p < 0.05, ns > 0.05). (D) A CCK-8 assay was used to evaluate the toxicity of lipopeptides on HFFs (* p < 0.05, ns > 0.05).

Figure 3

Effect of lipopeptides on invasion and proliferation of tachyzoites into host cells. (A) Statistical analysis of the invasion rate of tachyzoites. Tachyzoites were pretreated with DMEM (negative control), SDZ (a clinical drug for toxoplasmosis, 10 μM), Lycosin-I (5 μM and 10 μM), L-C₁₂ (5 μM and 10 μM), and L-an (5 μM and 10 μM) before exposure to HFFs. (B) The statistics of the proliferation rate of tachyzoites (the proliferation rate of the tachyzoites in the negative group was determined as 100%). HFFs infected with tachyzoites were treated with DMEM (negative control), SDZ (a clinical drug for toxoplasmosis, 10 μM), Lycosin-I (5 μM and 10 μM), L-C₁₂ (5 μM and 10 μM), and L-an (5 μM and 10 μM) for 24 h. (C) The proportion of PVs with different numbers of tachyzoites in 100 PVs. (D) Photograph of a representative well from each group of plaque assays (scale bars = 1 cm). HFFs not infected with tachyzoites were treated with DMEM (blank control). HFFs infected with tachyzoites were treated with DMEM (negative control, DMEM), SDZ (a clinical drug for toxoplasmosis, 10 μM), Lycosin-I(10 μM), L-C₁₂ (10 μM), and L-an (10 μM) for 7 days. (E) Enlargement of the red rectangle selected in D (scale bars = 5 mm). (F,G) Number and area of plaques calculated using Adobe Photoshop version 2020 (* p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001 compared to DMEM; ^# p < 0.05, ^## p < 0.01 and ^### p < 0.001 compared to Lycosin-I).

Figure 4

Anti-T. gondii of lipopeptides in vivo. Mice acutely infected with T. gondii were treated with PBS (negative control), SDZ (4 mg/kg, positive control), Lycosin-I (4 mg/kg), L-C₁₂ (4 mg/kg) and L-an (4 mg/kg). (A) The survival time of mice was recorded for 15 days (n = 8 for each group; *** p < 0.001 and **** p < 0.0001 compared to PBS). (B–G) The number of tachyzoites in the peritoneal fluid of mice was directly counted using a blood cell counting plate, while the expression of SAG1 for tachyzoites was detected via qRT-PCR in the heart, liver, spleen, lung and brain of mice. Analysis was performed using the comparative threshold cycle method (2^−∆∆Ct) (* p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001 compared to PBS).

Figure 5

Expression of inflammatory factors in the spleen of mice. Mice acutely infected with T. gondii were treated with PBS (negative control), SDZ (4 mg/kg, positive control), Lycosin-I (4 mg/kg), L-C₁₂ (4 mg/kg) and L-an (4 mg/kg). After 5 days of treatment, the expression level of IFN-γ (A), TNF-α (B), IL-4 (C) and IL-10 (D) in the spleen was determined via qRT-PCR. Analysis was performed using the comparative threshold cycle method (2^−∆∆Ct) (* p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001 compared to PBS; ^# p < 0.05, ^## p < 0.01, ^### p < 0.001 and ^#### p < 0.0001 compared to Lycosin-I).