NIH4215: A mutation-prone thiamine auxotrophic clinical Aspergillus fumigatus isolate.
NIH4215: A mutation-prone thiamine auxotrophic clinical Aspergillus fumigatus isolate.
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NIH4215：一种容易发生突变的硫胺素营养营养性临床曲霉曲霉分离株。
DOI：
10.3389/ffunb.2022.908343
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发表时间：
2022
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烟曲霉菌是威胁生命的侵袭性曲霉病的主要原因。尽管有各种抗真菌药物可用，但治疗仍然具有挑战性，需要进一步研究。因此，来源于一例致命的人类肺曲霉菌病的临床烟曲霉菌分离株NIH4215经常被用于药物疗效研究。出乎意料的是，我们最初试图为NIH4215菌株产生生物发光报告，用于体内药物疗效研究，但失败了，因为NIH4215不能在定义的最低限度的培养基上生长。随后的分析发现，菌株NIH4215有一个以前未被描述的硫胺营养缺陷症，并将烟曲霉Af293的硫胺生物合成基因转化为nmt1基因，证实了该基因是导致硫胺营养缺陷症的原因。对有缺陷的nmt1基因进行测序发现，在一个基本的铁结合基序中，半胱氨酸密码子丢失。随后，野生型nmt1基因在NIH4215中通过同时删除akuB基因座成功地产生了一株生物发光报告菌株。由此产生的生物发光ΔAKUB菌株显示出高频率的同源整合，这一点被PYRG和NIAD缺失突变体的产生所证实。当在梅隆格列氏菌感染模型中进行测试时，硫胺素营养不良和akuB基因缺失对毒力都没有显著影响。然而，除了硫胺素营养缺陷区外，菌株NIH4215及其衍生物的菌落边缘经常出现形态改变和白化突变的扇区，并成功地分离到稳定的白化突变株。通过对自发产生的耐氟酸突变株的筛选，证实了NIH4215基因突变率的提高。在耐氟玫瑰花酸的NIH4215菌株中发现了pyrG和pyre基因的独立突变，突变频率比临床烟曲霉菌CBS144.89菌株的突变频率高至少一个数量级。综上所述，尽管NIH4215菌株在动物模型中具有毒力，但它是一种硫胺素营养缺陷体，容易积累突变。我们的结果表明，硫胺素的生物合成对于宿主感染是必不可少的，而像NIH4215这样的易突变菌株可能会促进在环境中越来越多的观察到的唑类耐药性菌株的进化。
Aspergillus fumigatus is the main cause of life-threatening invasive aspergillosis. Despite the availability of various antifungals, therapy remains challenging and requires further studies. Accordingly, the clinical A. fumigatus isolate NIH4215 deriving from a fatal case of human pulmonary aspergillosis has frequently been used in drug efficacy studies. Unexpectedly, our initial attempts to generate a bioluminescent reporter of strain NIH4215 for in vivo drug efficacy studies failed, as NIH4215 was unable to grow on defined minimal medium. Subsequent analyses discovered a previously undescribed thiamine auxotrophy of strain NIH4215 and transformation with thiamine biosynthesis genes from A. fumigatus strain Af293 identified the nmt1 gene as cause of the thiamine auxotrophy. Sequencing of the defective nmt1 gene revealed the loss of a cysteine codon within an essential iron-binding motif. Subsequently, the wild-type nmt1 gene was successfully used to generate a bioluminescent reporter strain in NIH4215 by simultaneously deleting the akuB locus. The resulting bioluminescent ΔakuB strains showed a high frequency of homologous integration as confirmed by generation of pyrG and niaD deletion mutants. When tested in a Galleria mellonella infection model, neither thiamine auxotrophy nor the deletion of the akuB locus had a significant effect on virulence. However, besides thiamine auxotrophy, sectors with altered morphology and albino mutants frequently arose on colony edges of strain NIH4215 and its derivatives, and stable albino mutants were successfully isolated. A proposed increased mutation rate of NIH4215 was confirmed by screening for spontaneous occurrence of fluoorotic acid resistant mutants. Independent mutations in the pyrG and pyrE gene were identified in the fluoroorotic acid resistant NIH4215 isolates and the frequency of mutation was by at least one order of magnitude higher than that observed for the clinical A. fumigatus isolate CBS144.89. In summary, despite its virulence in animal models, strain NIH4215 is a thiamine auxotroph and prone to accumulate mutations. Our results suggest that thiamine biosynthesis is dispensable for host infection and mutation-prone strains such as NIH4215 could potentially facilitate the evolution of azole resistant strains as increasingly observed in the environment.