Lipidomic analysis of Toxoplasma gondii extracellular vesicles across host cell types

Introduction. Toxoplasma gondii is an obligate intracellular parasite with an exceptional capacity to colonize a broad range of host species and cell types. Successful infection depends on its ability to manipulate host metabolism, including lipid pathways that are essential for membrane biogenesis, signalling, and immune modulation. Extracellular vesicles (EVs) are increasingly recognized as critical mediators of parasite–host interactions, but while their protein and nucleic acid cargo has been studied, the lipid composition of T. gondii EVs (TgEVs) remains poorly defined.
Methods. In this study, we performed a comprehensive lipidomic analysis of TgEVs secreted by tachyzoites grown in four distinct host cell environments: fibroblasts, Vero cells, myoblasts, and porcine intestinal epithelial cells (IPEC). Cells and TgEVs were isolated from five biological replicates per condition and analysed by liquid chromatography coupled to high-resolution tandem mass spectrometry. Comparative lipid profiling of TgEVs and their corresponding host cells was performed after total ion current normalization, followed by principal component analysis to capture global compositional patterns and pairwise differential abundance testing to identify significantly enriched or depleted lipid species.
Results: We identified 194 lipid species across 15 classes. Despite pronounced metabolic differences among host cell types, TgEVs displayed a highly conserved and distinctive lipid profile. Glycerophospholipids such as phosphatidylcholine (PC) and phosphatidylethanolamine (PE) were the most abundant components, while sphingolipids, including sphingomyelin and ceramides, were consistently present and likely contribute to vesicle biogenesis and cargo organization. Notably, triacylglycerols (TG) were significantly enriched in TgEVs across all host conditions, suggesting active selection of neutral lipids during vesicle formation. Correlation analyses (r = 0.21–0.34) confirmed that TgEV lipidomes diverge from their cellular origin, indicating a process of active, selective sorting rather than passive acquisition from the host membrane.
Discussion: These findings demonstrate that T. gondii actively remodels host-derived lipids into vesicles with conserved and functionally specialized compositions. This selective lipid core likely underpins key aspects of parasite–host communication, including immune modulation, nutrient acquisition, and vesicle–cell interactions. Our work fundamentally advances the molecular understanding of TgEVs and establish a foundation for future studies into how lipid-mediated signalling contributes to the complex dynamics of T. gondii infection.