Lipid Flippase Mediated Membrane Asymmetry Governs Extracellular Vesicles Biogenesis and Host Interactions in Cryptococcus neoformans

Authors

Pawar, S., Zhnag, Y., Varsanayi, C., Gadiyar, V., Avina, S., Birge, R., Xue, C.

Abstract

Cryptococcus neoformans is the leading cause of fungal meningitis in immunocompromised patients. Alveolar macrophages are the first line of defense against Cryptococcus infection. Our previous study showed that deletion of Cdc50, the regulatory subunit of P4-ATPase (lipid flippase) complex, results in increased phagocytosis and macrophage killing, and avirulence in animal models. However, how fungal flippase dysfunction modulates Cryptococcus-macrophage interaction remains unknown. Here we identify Cdc50 as a central determinant of membrane lipid homeostasis, extracellular vesicle (EV) biogenesis and macrophage responses in C. neoformans. Our whole cell lipidomic analysis revealed that loss of Cdc50 disrupted membrane lipid homeostasis leading to phospholipid enrichment in cdc50{Delta} mutant, and a reduction in fatty acid production accompanied by pronounced ultrastructural defects in membrane architecture. Loss of Cdc50 also induced a hyper-vesiculating phenotype, with cdc50{Delta}producing significantly more extracellular vesicles (EVs) than wild type H99 cells. Lipidomic profiling of cdc50{Delta} EVs revealed enrichment of phospholipids, including phosphatidylserine (PS), indicating active lipid sorting during vesicle biogenesis. Functional analysis showed that EVs from the wildtype H99 suppress phagocytosis whereas cdc50{Delta} EVs enhance phagocytosis, indicating a differential macrophage priming. Despite increased PS externalization in cdc50{Delta} cells and EVs, macrophage recognition and uptake occur independent of PS-mediated efferocytosis pathways, including PS receptor MertK. Following macrophage uptake, cdc50{Delta} were intrinsically vulnerable to macrophage killing due to rapid phagosome acidification. Together, we demonstrate that Cdc50 dependent lipid homeostasis regulates EV production, lipid composition, membrane architecture and drives the intracellular fate of C. neoformans.

Preprint server: bioRxiv
The authors list and abstract were imported from bioRxiv on 14 Jun 2026.

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