Toxoplasma gondii assembles extracellular vesicles with conserved lipid profiles 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 lipidomic analysis of TgEVs secreted by tachyzoites grown in four distinct host cell types: 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 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 enriched in TgEVs across all host conditions, suggesting active selection of neutral lipids during vesicle formation. Correlation analyses confirmed that TgEV lipidomes diverge from their cellular origin, indicating a process of active sorting rather than passive acquisition from the host membrane.Discussion These findings indicate that T. gondii produces vesicles with conserved and distinctive lipid compositions that differ from those of the host cell. This selective lipid core hints at key functions in parasite, host communication, immune modulation, nutrient acquisition, and other vesicle-cell interactions. Our work advances the molecular understanding of TgEVs and establishes a foundation for future studies into how lipid-mediated signalling contributes to the complex dynamics of T. gondii infections in different cellular environments.

• VetImaging