Catechin gallate triggers metabolomic and lipidomic alteration in Toxoplasma gondii.

Abstract

Background: Toxoplasma gondii is a zoonotic parasite, the causative agent of toxoplasmosis, which has global importance owing to its significant socioeconomic, public health, and veterinary burdens. Toxoplasmosis is currently treated with a combination of pyrimethamine and sulfadiazine. These drugs have treatment failures and toxicity and are ineffective against the bradyzoite stage. Hence, there is a need for new inhibitors against T. gondii. Catechin gallate (CG) is a known antioxidant with demonstrated antiparasitic properties. However, little is known about its anti-Toxoplasma gondii activity and mechanism of action.

Methods: Here, we assess the effect of CG on human telomerase reverse transcriptase immortalized foreskin fibroblast (hTERT) cells, cytotoxicity, and inhibitory activity of the RH-RFP (type I) strain of T. gondii tachyzoite. Inhibitory and cytotoxicity activities were measured by a fluorescent plate reader, and the data were analyzed using Graph Pad Prism software. In addition, to predict the possible mechanism of CG action, hTERT cells were cultured in a T25 flask and infected with RH-RFP parasites, followed by CG administration and incubation for 48 h. Parasites were quenched under ice, and the parasites were purified from host cells and extracted with chloroform-methanol. The extracts containing the lipids and metabolites were analyzed using liquid chromatography-mass spectrometry (LC-MS).

Results: To address this research question, we tested the in vitro inhibitory activity of CG against parasite growth at 48 h and 72 h. The half-maximal inhibitory concentration (IC₅₀) values against tachyzoite growth were calculated to be 10.07 (8.31-12.20) µM and 7.057 (5.98-8.32) µM for 48 h and 72 h, respectively. We identified 5-formyl-tetrahydromethanopterin; 5-(6-hydroxy-6-methyloctyl)-2,5-dihydrofuran-2-one; trans-3-indoleacrylic acid; 5,5-dimethyl-2-{[(2-phenylacetyl)amino]methyl}-1,3-thiazolane-4-carboxylic acid; 5'-S-Ethyl-5'-thioadenosine; L-Norleucine; and norepinephrine sulfate as the most produced during the CG treatment. For the lipidomics analysis, we identified the production of several sphingolipid species, including ceramides, dihydroceramide, and sphingosine, which are associated with apoptosis and autophagy. The limited number of sphingomyelin and sphingosine-1-phosphate identified, which are known to promote proliferation, suggests that CG may be affecting T. gondii parasites' proliferation. In addition, oxidized fatty acids (3-hydroxypropyl stearate and (R)-3-hydroxy myristic acid) were observed in both treatments with low production, which confers oxidative stress induction on parasites.

Conclusions: The study showed that CG had inhibitory activity against T. gondii growth and caused metabolite and lipid alterations in T. gondii. This requires future studies on the enzymes associated with the biosynthesis of these metabolite/lipid pathways that are altered in these in vitro studies.