The cell wall integrity pathway kinase Mpk1 regulates extracellular vesicle production and apoptotic-like vesicle release in Cryptococcus neoformans

Extracellular vesicles (EVs) are important mediators of fungal physiology and host interactions, yet the mechanisms governing their production remain poorly understood. In Cryptococcus neoformans , EV production is tightly regulated by environmental conditions, suggesting the existence of dedicated signaling pathways. Here, we screened a kinase deletion library to identify regulators of EV production and found that the cell wall integrity (CWI) pathway acts as a major negative regulator of EV production. Deletion of the CWI kinases MPK1 , MKK2 , or BCK1 increased EV release. Unexpectedly, the size of EVs produced by CWI mutants differed markedly from wild type. Accordingly, cryo-electron microscopy revealed the accumulation of larger vesicles with altered size distributions in the mpk1 Δ mutant. These EVs lacked the characteristic mannoprotein-based fibrillar surface decoration observed on most wild-type vesicles, exhibited reduced association with capsular polysaccharide, and contained substantially less protein. Quantitative proteomic analysis demonstrated extensive remodeling of the EV proteome, with depletion of glycosylphosphatidylinositol (GPI)-anchored proteins, and enrichment of ribosomal proteins and histones. Finally, mpk1 Δ EVs displayed several hallmarks of mammalian apoptotic EVs, including phosphatidylserine exposure, and were produced under conditions associated with reduced cell viability. Together, these findings identify the CWI pathway as a central regulator of fungal EV production and provide evidence that C. neoformans produces distinct EV populations through genetically regulated pathways. Our results provide evidence for an apoptotic-like EV population in fungi and reveal an unexpected link between cell wall integrity signaling, cell death, and EV diversity. IMPORTANCE Fungal extracellular vesicles (EVs) contribute to virulence, antifungal resistance, and host-pathogen interactions, yet the mechanisms governing their production remain poorly understood. Here, we identify the cell wall integrity signaling pathway as a major regulator of EV production in the human fungal pathogen Cryptococcus neoformans . We further show that disruption of one of the major kinases of this pathway induces the production of a distinct population of EVs with structural and molecular features resembling mammalian apoptotic EVs. These findings demonstrate that fungi produce multiple EV populations through genetically regulated pathways rather than a single constitutive mechanism. By establishing the existence of apoptotic-like EVs in fungi, our work provides new insight into fungal EV production and lays the foundation for investigating how different EV populations contribute to fungal physiology, pathogenesis, and antifungal resistance.