Zhang Heng, Zhu Zhangling, Peng Mengqi, Liu Sijie, Gong Xiao, Chen Tian, Hu Qingwen, Li Linyun, Dun-Zhu Zha-Xi, Drol-Ga Lha-Zom, Sun Yi
Department of Dermatology, Jingzhou Hospital Affiliated to Yangtze University, Hubei Provincial Clinical Research Center for Diagnosis and Therapeutics of Pathogenic Fungal Infection, Jingzhou, 434100, Hubei Province, China.
Department of Ophthalmology, Jingzhou Hospital Affiliated to Yangtze University, Jingzhou, 434100, Hubei Province, China.
Mycopathologia. 2025 Sep 15;190(5):85. doi: 10.1007/s11046-025-00992-0.
Aspergillus fumigatus poses significant clinical challenges due to its increasing azole resistance. This study investigates the sdh1 gene's role in regulating azole susceptibility, mitochondrial function, and virulence.
Fungal strains were co-cultured with varying concentrations of succinate dehydrogenase inhibitors (SDHIs). Post-treatment azole minimum inhibitory concentrations (MICs) were determined using broth microdilution method, while succinate dehydrogenase subunit (SDH) expression changes were analyzed via RT-qPCR. Using A. fumigatus MFIG001 as the parental strain, sdh1 knockout mutant (Δsdh1) and complemented strain (Δsdh1::sdh1) were constructed through homologous recombination. Detect the hyphal growth rate of Δsdh1, MICs and the changes in virulence within the Galleria mellonella infection model. Mitochondrial function was evaluated by measuring SDH activity, ATP content, and reactive oxygen species (ROS) levels. Transcriptomic changes were analyzed using RNA-seq and RT-qPCR, with efflux pump activity validated through Rhodamine 6G accumulation assays.
Exposure to subinhibitory concentrations of SDHIs induced azole resistance in A. fumigatus, with 4.12% of strains exhibiting reduced susceptibility to voriconazole, itraconazole, and posaconazole. RT-qPCR analysis revealed significant downregulation of sdh1 in resistant strains, implicating its role in resistance development. Deletion of sdh1 resulted in an 8- to 16-fold increase in triazole MICs, confirming its role as a negative regulator of azole susceptibility. Phenotypically, the Δsdh1 strain exhibited impaired growth, reduced sporulation, and diminished efficacy of azole treatment in the G. mellonella infection model. Furthermore, Δsdh1 exhibited severe mitochondrial dysfunction, including reduced SDH activity, decreased ATP levels, elevated ROS, and impaired antioxidant defenses. RNA-seq analysis revealed that the deletion of sdh1 upregulated the expression of efflux pump genes (e.g., cdr1B, abcB, mdr4), while Rhodamine 6G efflux assays demonstrated significantly enhanced efflux activity.
These results identify sdh1 as a critical determinant of azole susceptibility through dual mechanisms: mitochondrial function maintenance and efflux pump regulation. The observed SDHI-induced cross-resistance suggests agricultural fungicides may drive environmental selection of azole-resistant strains. While sdh1 deletion increased drug tolerance through efflux activation, the concurrent mitochondrial damage reduced pathogenic fitness, revealing compensatory evolutionary constraints. This work highlights the need to monitor non-target effects of agricultural SDHIs on clinical antifungal resistance.
烟曲霉因其对唑类药物的耐药性不断增加而带来了重大的临床挑战。本研究调查了sdh1基因在调节唑类药物敏感性、线粒体功能和毒力方面的作用。
将真菌菌株与不同浓度的琥珀酸脱氢酶抑制剂(SDHIs)共培养。处理后,采用肉汤微量稀释法测定唑类药物的最低抑菌浓度(MICs),同时通过RT-qPCR分析琥珀酸脱氢酶亚基(SDH)的表达变化。以烟曲霉MFIG001作为亲本菌株,通过同源重组构建sdh1基因敲除突变体(Δsdh1)和互补菌株(Δsdh1::sdh1)。检测Δsdh1的菌丝生长速率、MICs以及在大蜡螟感染模型中毒力的变化。通过测量SDH活性、ATP含量和活性氧(ROS)水平来评估线粒体功能。使用RNA测序和RT-qPCR分析转录组变化,并通过罗丹明6G积累试验验证外排泵活性。
暴露于亚抑菌浓度的SDHIs可诱导烟曲霉产生唑类耐药性,4.12%的菌株对伏立康唑、伊曲康唑和泊沙康唑的敏感性降低。RT-qPCR分析显示,耐药菌株中sdh1基因显著下调,表明其在耐药性产生中的作用。删除sdh1导致三唑类药物的MICs增加8至16倍,证实其作为唑类药物敏感性负调节因子的作用。在表型上,Δsdh1菌株生长受损、孢子形成减少,并且在大蜡螟感染模型中唑类药物治疗的效果降低。此外,Δsdh1表现出严重的线粒体功能障碍,包括SDH活性降低、ATP水平下降、ROS升高以及抗氧化防御受损。RNA测序分析表明,删除sdh1会上调外排泵基因(如cdr1B、abcB、mdr4)的表达,而罗丹明6G外排试验表明外排活性显著增强。
这些结果通过线粒体功能维持和外排泵调节这两种机制确定sdh1是唑类药物敏感性的关键决定因素。观察到的SDHI诱导的交叉耐药性表明,农业杀菌剂可能推动了唑类耐药菌株的环境选择。虽然删除sdh1通过激活外排增加了药物耐受性,但同时发生的线粒体损伤降低了致病适应性,揭示了补偿性进化限制。这项工作强调了监测农业SDHIs对临床抗真菌耐药性的非靶向效应的必要性。