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.

抗真菌药物耐药性问题日益严峻，亟需开发新型治疗替代方案。本研究评估了以黑团孢霉（Epicoccum nigrum）培养滤液合成的生物源银纳米颗粒（biogenic silver nanoparticles, AgNPs）对抗高耐药性真菌物种角状镰孢（Fusarium keratoplasticum）的抗真菌活性及其分子作用机制。该生物源AgNPs展现出优异的抗真菌活性：半数抑菌浓度（MIC50）为1.79 μg/mL，在5.92 μg/mL浓度下可实现92.85%的菌丝生长抑制率。无标记定量蛋白质组学分析（label-free quantitative proteomic analysis, LFQ-MS）结果显示，经AgNP处理后，共有52种蛋白质的表达丰度发生显著改变，这些蛋白涉及氧化应激应答、线粒体功能及核黄素生物合成通路。核黄素生物合成与电子传递相关蛋白的表达下调，提示真菌代谢与能量代谢受到干扰；而氧化应激应答相关蛋白及热休克蛋白（heat shock proteins）表达上调，则表明真菌处于应激状态。为在体内评估AgNPs的毒性与抗真菌效力，本研究以2.58 mg/kg的剂量暴露大蜡螟（Galleria mellonella）幼虫，结果显示处理后7天幼虫存活率达90%。幼虫血淋巴细胞密度出现一过性升高，未观察到长期免疫功能损伤。对幼虫血淋巴的蛋白质组学分析显示，仅有少量蛋白质表达丰度发生变化，且主要与免疫应答及代谢过程相关。在真菌感染实验中，接种角状镰孢（105个分生孢子/mL）的幼虫死亡率达90%，但经AgNP处理后幼虫存活率提升了5倍（第7天时存活率为50%）。上述研究结果证实，生物源AgNPs可通过诱导角状镰孢产生氧化应激、干扰代谢过程及损伤线粒体发挥抗真菌作用。蛋白质组学与体内实验数据的联合分析证实了该制剂的抗真菌有效性与生物安全性。未来可进一步开展长期毒性研究，并探索其在医药与农业领域对抗真菌耐药性的潜在应用价值。