Proteomic and Ultrastructural Analyses Reveal Stress Granule Clearance in Aspergillus Flavus Mycelia Triggered by Antifungal Metabolites of Bacillus Amyloliquefaciens

This study aimed at evaluating the antifungal potential of two metabolite fractions extracted from the antagonistic Bacillus amyloliquefaciens (IS5) culture filtrate using ethyl acetate (EAF) and ammonium sulfate precipitate (ASF) on ultrastructure and protein profile of Aspergillus flavus (A115). The results indicated that EAF exhibited more antifungal potential on A. flavus than ASF. Likewise, TEM examination of EAF-treated mycelium revealed disintegrated cell membranes and vast cytoplasmic disorganization. Whereas ASF-treated mycelium showed a dismantled outermost cell wall layer and granular cytoplasmic foci, seemingly stress granules (SGs), accompanied by extensive vacuolation indicating an autophagy-like mechanism. Non-targeting shotgun proteomic analysis was adopted using NanoLC-ESI-MS/MS. Mycelium treated with EAF showed increased abundance of ROS-related proteins such as glutathione S-transferase, superoxide dismutase and elevated fold change (FC) of redoxin and thioredoxin domain-containing protein in respect to control. On the other side, despite ASF treatment exerting a common down-regulation for the central carbon and energy metabolism, ATP synthesis and transport machinery have not followed this trend except for a few cases such as ATP-dependent RNA helicase eIF4A that was down-regulated, implying the inhibition of translation process. Then significant abundance was observed for ribosomal proteins (RPs), polyadenylate-binding protein, and GTP-binding protein, which are well documented as stress granules (SGs) constituents. Taken together, while EAF treatment-indicated a typical oxidative stress in A. flavus mycelium, ASF treatment induced translation inhibition resulted in SGs constitution, then autophagy was triggered to clear SGs. Further studies are so far needed to uncover the exact constituents of both extracted fractions (EAF and ASF).