Global Lipidomics Reveals the Lipid Composition Heterogeneity of Extracellular Vesicles from Drug-Resistant Leishmania

<b>Background</b>: The rise of drug-resistant <i>Leishmania</i> strains presents a significant challenge in the treatment of Leishmaniasis, a neglected tropical disease. Extracellular vesicles (EVs) produced by these parasites have gained attention for their role in drug resistance and host-pathogen interactions. <b>Methods</b>: This study developed and applied a novel lipidomics workflow to explore the lipid profiles of EVs from three types of drug-resistant <i>Leishmania infatum</i> strains compared to a wild-type strain. EVs were isolated through ultracentrifugation, and their lipid content was extracted using a modified Matyash protocol. LC-MS analysis was performed, and data processing in MS-DIAL enabled lipid identification and quantification. Statistical analysis in MetaboAnalyst revealed strain-specific lipid alterations, highlighting potential links between lipid composition and drug resistance mechanisms. <b>Results</b>: Our results show distinct alterations in lipid composition associated with drug resistance. Specifically, drug-resistant strains exhibited reduced levels of phosphatidylcholine (PC) and phosphatidylglycerol (PG), particularly in the amphotericin B-resistant strain <i>Li</i>AmB1000.1. Sterol and glycerolipid species, including cholesteryl ester (CE) and triacylglycerol (TG) were also found to be diminished in <i>Li</i>AmB1000.1. These changes suggest significant lipid remodeling under drug pressure, potentially altering the biophysical properties of EV membranes and their capacity for molecule transfer. Furthermore, the lipidomic profiles of EVs from the other resistant strains, LiSb2000.1 and LiMF200.5, also displayed unique alterations, underscoring strain-specific adaptations to different drug resistance mechanisms. <b>Conclusions</b>: These significant alterations in lipid composition suggest potential lipid-based mechanisms underlying drug resistance in <i>Leishmania</i>, providing new avenues for therapeutic intervention.