Abstract
Antifungal drug resistance is a growing concern, necessitating new therapeutic alternatives. This study evaluated the antifungal activity and molecular effects of biogenic silver nanoparticles (AgNPs) synthesized using the culture filtrate of Epicoccum nigrum against Fusarium keratoplasticum, a highly resistant fungal species. AgNPs exhibited strong antifungal activity, with a MIC(50) of 1.79 μg/mL and 92.85% growth inhibition at 5.92 μg/mL. Label-free quantitative proteomic analysis (LFQ-MS) revealed 52 proteins with significantly altered abundance after AgNP treatment, affecting the oxidative stress response, mitochondrial function, and riboflavin biosynthesis. Decreased levels of proteins involved in riboflavin biosynthesis and electron transport suggest metabolic and energy disruption, while increased levels of oxidative stress response and heat shock proteins indicate fungal stress. To assess toxicity and antifungal efficacy in vivo, Galleria mellonella larvae were exposed to AgNPs at 2.58 mg/kg, showing a 90% survival rate after 7 days. Hemocyte density increased temporarily with no long-term immune disruption. Proteomic analysis of hemolymph revealed minor protein abundance changes, mostly related to the immune response and metabolism. In fungal infection assays, larvae infected with F. keratoplasticum (10(5) conidia/mL) had a 90% mortality rate, but AgNP treatment increased survival 5-fold (50% by day seven). These findings confirm that biogenic AgNPs act through the induction of oxidative stress, metabolic disruption, and mitochondrial damage in F. keratoplasticum. The combination of proteomic and in vivo data supports their efficacy and safety. Further studies should explore long-term toxicity and potential applications in medicine and agriculture to combat antifungal resistance.