Molecular markers of adaptive responses in Toxoplasma gondii to Pyrimethamine treatment

In this study, we employed bio-orthogonal non-canonical amino acid tagging (BONCAT) combined with iTRAQ-based quantitative proteomics to investigate the effects of pyrimethamine (PM) on newly synthesised proteins (NSPs) in Toxoplasma gondii. Parasites were treated with varying concentrations of PM, followed by mass spectrometric analysis to identify and quantify NSPs. We observed a concentration-dependent response, where several NSPs were downregulated at low PM doses but upregulated at higher concentrations. Notably, ribosomal proteins, invasion-related proteins (e.g., ROP26), and stress response proteins (e.g., serine/threonine-protein phosphatase 2B) showed significant changes in expression. The upregulation of calcineurin-related proteins suggests an activation of calcium-dependent stress response pathways, possibly as a compensatory mechanism to counteract the drug’s inhibitory effects. Our findings reveal the dynamic regulatory mechanisms of T. gondii under PM treatment, highlighting the parasite’s adaptive strategies to survive drug-induced stress. This study underscores the utility of BONCAT-iTRAQ proteomics in uncovering the molecular responses of pathogens to therapeutic interventions, providing insights that could inform the development of more effective anti-parasitic strategies.