Label-Free Quantitative Proteomic Analysis Reveals the Effects of Biogenic Silver Nanoparticles on Fusarium keratoplasticum and Their Therapeutic Potential in Galleria mellonella Larvae

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 MIC50 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 (105 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.