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.