Sterculic Acid and Its Analogues Are Potent Inhibitors of Toxoplasma gondii

Abstract

Toxoplasmosis is a serious disease caused by Toxoplasma gondii, one of the most widespread parasites in the world. Lipid metabolism is important in the intracellular stage of T. gondii. Stearoyl-CoA desaturase (SCD), a key enzyme for the synthesis of unsaturated fatty acid is predicted to exist in T. gondii. Sterculic acid has been shown to specifically inhibit SCD activity. Here, we examined whether sterculic acid and its methyl ester analogues exhibit anti-T. gondii effects in vitro. T. gondii-infected Vero cells were disintegrated at 36 hr because of the propagation and egress of intracellular tachyzoites. All test compounds inhibited tachyzoite propagation and egress, reducing the number of ruptured Vero cells by the parasites. Sterculic acid and the methyl esters also inhibited replication of intracellular tachyzoites in HFF cells. Among the test compounds, sterculic acid showed the most potent activity against T. gondii, with an EC50 value of 36.2 μM, compared with EC50 values of 248-428 μM for the methyl esters. Our study demonstrated that sterculic acid and its analogues are effective in inhibition of T. gondii growth in vitro, suggesting that these compounds or analogues targeting SCD could be effective agents for the treatment of toxoplasmosis.

Keywords: Toxoplasma gondii; anti-Toxoplasma gondii effect; stearoyl-CoA desaturase; sterculic acid.

Fig. 1.

Molecular structures of test compounds. Sterculic acid was coded as INT16. The methyl esters INT13, INT14, and INT15 are different in the length of carbon chain. INT21 has the same length of carbon chain with sterculic acid but contains a methoxy moiety.

Fig. 2.

Sterculic acid and methyl esters inhibited the egress of tachyzoites from Vero cells. Representative pictures were presented to show that most of host cells remained intact after incubation with INT13 (A), INT14 (B), INT15 (C), INT16 (D), or INT21 (E) for 36 hr, with a small number of released tachyzoites, compared with numerous tachyzoites and a smaller number of intact host cells in the negative control group (F). Scale bar=100 μM. Extracellular tachyzoites are marked in red circles. (G) The number of released tachyzoites in the culture incubated with the test compounds was decreased relative to the DMSO control. Extracellular tachyzoites were counted in 6 random fields under the microscope per group. Data are presented as mean±SD. ^**P<0.01.

Fig. 3.

Sterculic acid and all methyl ester analogues inhibit tachyzoite proliferation. Freshly released parasites were used to infect HFF cell monolayers. (A) Invaded parasites were allowed to replicate for 18 hr in the culture medium containing the test compounds, and the number of vacuoles containing 1, 2, 4, or 8 parasites was counted under a fluorescence microscope after anti-T. gondii staining. (B) Representative fluorescent microscopic images were presented to show that many vacuoles contained 8 tachyzoites in DMSO control groups, (C) while 4 or 2 tachyzoites were often observed in a vacuole in INT21-treated groups after culture for 18 hr. The magnified images of the areas marked with white boxes in (B) and (C) were presented at the bottom in order. Data are presented as mean±SD from 3 independent experiments each in triplicate. ^**P<0.01.

Fig. 4.

Relative cell viability was calculated from the MTS assay. The cell viability of 0.1% DMSO was regarded as 100%. (A) Sterculic acid was cytotoxic to Vero cells at high concentrations while the methyl esters displayed no cytotoxicity. (B) All test compounds inhibited T. gondii growth as shown by increased viability of Vero cells infected with T. gondii.