ABSTRACT: The increasing problem of antifungal drug resistance is a serious concern, making it necessary to find new treatment options. In this study, we evaluated the antifungal activity and molecular effects of biogenic silver nanoparticles (AgNPs) synthesized using the culture filtrate of Epicoccum nigrum against Fusarium keratoplasticum, a fungal genera with high resistance to antifungal drugs. The AgNPs showed strong antifungal activity, with a MIC50 of 1.79 μg/mL, leading to 92.85% inhibition of fungal growth at the highest concentration tested (5.92 μg/mL). To understand how AgNPs affect F. keratoplasticum, we performed label-free quantitative proteomic mass spectrometric (LFQ-MS) analysis. We identified 853 proteins, 52 of which were significantly altered in abundance after AgNPs treatment. Principal component analysis (PCA) and hierarchical clustering showed a clear difference between the proteomes of control and AgNP-treated samples. Several proteins related to oxidative stress response, mitochondrial function, and riboflavin biosynthesis were affected. Proteins involved in riboflavin biosynthesis and electron transport decreased in abundance, suggesting that AgNPs disrupt fungal metabolism and energy production. On the other hand, proteins related to oxidative stress response and heat shock increased in abundance, showing that the fungus is under strong stress when exposed to AgNPs. To check the toxicity and antifungal activity of AgNPs in vivo, we used the Galleria mellonella larval model. At a dose of 2.58 mg/kg, AgNPs were not highly toxic, with 90% of the larvae surviving after seven days. Hemocyte density increased after AgNPs treatment, showing that there was some immune activation, but it returned to normal levels after 48 hours. Also, proteomic analysis of G. mellonella hemolymph after AgNPs exposure showed only small changes in protein abundance, mostly in immune response and metabolism, confirming that AgNPs do not cause serious damage to the host. The antifungal effect of AgNPs was also tested in G. mellonella larvae infected with F. keratoplasticum. Infection with 10⁵ conidia/mL caused a 90% mortality rate by day seven in untreated larvae. However, AgNP-treated larvae had a fivefold increase in survival, with 50% surviving by day seven, proving that AgNPs can help fight fungal infections in vivo. These results confirm that biogenic AgNPs are effective antifungal agents that act through oxidative stress, metabolic disruption, and mitochondrial damage in F. keratoplasticum. The combination of proteomic analysis and in vivo experiments provides strong evidence that AgNPs are effective and safe in insect model. Future studies should focus on testing long-term toxicity, and exploring their use in medicine and agriculture to help combat the increasing problem of antifungal resistance.
Glaucia Caruso 
