Center for Global Health, School of Public Health, Nanjing Medical University, Nanjing, China.
Key Laboratory of Vector Biology and Pathogen Control of Zhejiang Province, Huzhou Universitygrid.411440.4, Huzhou Central Hospital, Huzhou, China.
Antimicrob Agents Chemother. 2022 Jun 21;66(6):e0015122. doi: 10.1128/aac.00151-22. Epub 2022 Jun 1.
In Aspergillus fumigatus, the most prevalent resistance to azoles results from mutational modifications of the azole target protein Cyp51A, but there are non- mutants resistant to azoles, and the mechanisms underlying the resistance of these strains remain to be explored. Here, we identified a novel cytochrome oxidase, (W56*), nonsense mutation in the laboratory and found that it caused reduced colony growth and resistance to multiantifungal agents. Meanwhile, we revealed that cold storage is responsible for increased tolerance of conidia to itraconazole (ITC) stress, which further advances azole-resistant mutations (cryopreservation→ITC tolerance→azole resistance). The deletion or mutation of results explicitly in resistance to antifungal-targeting enzymes, including triazoles, polyenes, and allylamines, required for ergosterol synthesis, or resistance to fungal ergosterol. A high-performance liquid chromatography (HPLC) assay showed that the knockout strain decreased intracellular itraconazole concentration. In addition, the lack of Cox7c resulted in the accumulation of intracellular heme B. We validated that an endogenous increase in, or the exogenous addition of, heme B was capable of eliciting azole resistance, which was in good accordance with the phenotypic resistance analysis of mutants. Furthermore, RNA sequencing verified the elevated transcriptional expression levels of multidrug transport genes. Additionally, lower itraconazole-induced reactive oxygen species generation in mycelia of a -deletion strain suggested that this reduction may, in part, contribute to drug resistance. These findings increase our understanding of how A. fumigatus's direct responses to azoles promote fungal survival in the environment and address genetic mutations that arise from patients or environments.
在烟曲霉中，最常见的唑类药物耐药性是由于唑类药物靶蛋白 Cyp51A 的突变修饰引起的，但也存在非突变体对唑类药物耐药的情况，这些菌株耐药的机制仍有待探索。在这里，我们在实验室中鉴定了一种新型细胞色素氧化酶 （W56*），并发现它导致菌落生长减少和对多抗真菌剂的耐药性。同时，我们揭示了冷储存是导致分生孢子对伊曲康唑（ITC）应激增加耐受性的原因，这进一步促进了唑类耐药突变（冷冻保存→ITC 耐受性→唑类耐药性）。缺失或突变 明确导致对抗真菌靶向酶（包括三唑类、多烯类和烯丙胺类）的耐药性，这些酶是合成麦角固醇所必需的，或者导致真菌麦角固醇耐药性。高效液相色谱（HPLC）分析表明， 敲除株减少了细胞内伊曲康唑的浓度。此外，Cox7c 的缺失导致细胞内血红素 B 的积累。我们验证了内源增加或外源添加血红素 B 能够引发唑类耐药性，这与 突变体的表型耐药性分析结果相符。此外，RNA 测序证实了多药转运基因的转录表达水平升高。此外，在 -缺失菌株的菌丝中，较低的伊曲康唑诱导活性氧生成表明，这种减少可能部分导致耐药性。这些发现增加了我们对烟曲霉对唑类药物的直接反应如何促进真菌在环境中的生存以及解决来自患者或环境的遗传突变的理解。
