Inukai Tatsuya, Watanabe Rikuto, Murakami Yoshiki, Cabral Horacio, Kuroda Masahiko, Nakamura Shigeki
Department of Microbiology, Tokyo Medical University, 6-1-1 Shinjuku, Shinjuku-ku, Tokyo, 160-8402, Japan.
Department of Molecular Pathology, Tokyo Medical University, Tokyo, Japan.
Sci Rep. 2025 May 19;15(1):17398. doi: 10.1038/s41598-025-02742-0.
Aspergillus fumigatus, which causes aspergillosis, has developed resistance to azole antifungal agents in recent years. As only three main classes of antifungal drugs are available, the development of novel therapeutic strategies is crucial. We aimed to control the expression of virulence factors by introducing microRNAs (miRNAs) into fungi as an innovative therapeutic approach. To test our hypothesis, we selected miRNA mimics targeting alb1, which is involved in the synthesis of 1,8-dihydroxynaphthalene (DHN)-melanin, a virulence factor of A. fumigatus, and transfected them into the protoplast of the fungus, resulting in a two-fold reduction in alb1 expression. Next, we created a 3×HA-tagged Alb1 protein (Alb1-HAp)-expressing strain and confirmed the regulation of translation using western blotting with an anti-HA antibody. The protein amount of Alb1-HAp was reduced by one-third after the introduction of the miRNA. Moreover, the reduction in melanin after miRNA transfection promoted the killing of fungus by hydrogen peroxide-induced oxidative stress and sensitised the fungus to neutrophil attack. Additionally, by loading miRNAs into a fungus-targeted delivery system, we demonstrated the potential of transferring miRNAs into intact fungal cells in vitro. These results indicate the potential of miRNAs to regulate target virulence factors in fungi, leading to the development of novel therapies.
引起曲霉病的烟曲霉近年来已对唑类抗真菌药物产生耐药性。由于目前仅有三类主要的抗真菌药物可用，因此开发新的治疗策略至关重要。我们旨在通过将微小RNA（miRNA）导入真菌来控制毒力因子的表达，以此作为一种创新的治疗方法。为了验证我们的假设，我们选择了靶向alb1的miRNA模拟物，alb1参与1,8-二羟基萘（DHN）-黑色素的合成，而DHN-黑色素是烟曲霉的一种毒力因子。我们将这些miRNA模拟物转染到真菌原生质体中，导致alb1表达降低了两倍。接下来，我们构建了一个表达3×HA标签的Alb1蛋白（Alb1-HAp）的菌株，并使用抗HA抗体通过蛋白质免疫印迹法确认了翻译调控情况。导入miRNA后，Alb1-HAp的蛋白量减少了三分之一。此外，miRNA转染后黑色素的减少促进了过氧化氢诱导的氧化应激对真菌的杀伤作用，并使真菌对中性粒细胞攻击更敏感。另外，通过将miRNA加载到真菌靶向递送系统中，我们证明了在体外将miRNA转移到完整真菌细胞中的潜力。这些结果表明miRNA在调节真菌中靶标毒力因子方面具有潜力，从而为新疗法的开发带来了希望。
