Extracellular vesicle-mediated protein secretion in filamentous fungi

Fungi use intracellular and extracellular mechanisms, like organelle maintenance, endomembrane metabolism, and secretion, to adapt and survive in changing environments. Intracellular vesicles transport materials between membranes to support cellular homeostasis and growth, but fungal extracellular vesicles (EVs) have gained recent attention for their specific role in the extracellular environment. This project studies EVs in fungal protein secretion using the model organism, Aspergillus nidulans, focusing on EVs as carriers of extracellular enzymes. It is hypothesized that enzymes are packaged into EVs which are released from the cell by an unknown mechanism. To investigate this, A. nidulans’ EVs were isolated through ultrafiltration and quantified via flow cytometry at various growth points. High vesicle counts were observed between 48 and 72 hours. At 72 hours of growth, EVs were analyzed for quantity and xylanase activity by comparing two strains: A1149 (reference strain) and PFI-XLN7, a xylanase overproducing strain. Xylanase activity was detected in EVs from both strains when grown in xylose or xylan, with PFI-XLN7 showing significantly higher EV counts and xylanase activity. To track xylanase secretion, the A. nidulans reference strain was transformed to insert mCherry into the xylanase (xlnC) locus, creating strain XynMC11. When grown in xylose, mCherry was detected in isolated EVs, but not when grown in glucose, indicating regulated EV-mediated xylanase secretion. Fluorescence microscopy of XynMC11 in xylose showed vesicle-like structures containing mCherry outside the cells after 72 hours of growth. mCherry was also found within biofilms, indicating that EVs deposit cargo into biofilms. Furthermore, a strain lacking the native xlnC signal peptide (SP) produced fewer EVs, showing the role of SPs and the endoplasmic reticulum in EV cargo loading. Overexpressing mCherry in place of XlnC (strain XlnR7MC7) in A. nidulans led to growth and biofilm defects, but increased EV production compared to XynmC11, though mCherry-containing EVs was about 26% of the total for both strains, showing regulated EV production. Overall, this work highlights a regulated pathway of enzyme secretion via EVs in A. nidulans, providing evidence that EVs originate from the ER and contribute to biofilm composition.