体外诱导耐药阿萨希毛孢子菌氟康唑耐药相关基因筛选

Screening of fluconazole resistance-related genes in Trichosporon asahii with induced fluconazole resistance in vitro

目的筛选阿萨希毛孢子菌氟康唑耐药相关基因,为研究该菌氟康唑耐药机制及开发新型抗真菌剂提供理论依据。方法将临床分离的氟康唑敏感阿萨希毛孢子菌通过不断倍增氟康唑药物浓度的方法进行体外诱导培养,直至药物浓度≥128μg/mL,再进行无药培养30代,测定其MIC值。分别提取敏感菌株和诱导耐药菌株的总RNA,构建RNA文库,转录组测序后进行生物信息学分析,寻找差异表达基因。相同方法诱导另外2株敏感菌株为耐药菌株,并对3对敏感与耐药菌株的基因组进行测序,比较ERG11基因序列。结果成功获得稳定的诱导耐药菌株,转录组发现耐药菌株中与药物转运相关基因、麦角固醇合成相关基因、抗氧化基因等上调表达,其中氟康唑靶酶编码基因ERG11基因表达上调了3.7倍。而基因组测序证实3株耐药菌株中ERG11基因的碱基序列未发生突变。结论阿萨希毛孢子菌耐药分子机制复杂,麦角固醇合成途径中的基因,特别是ERG11基因上调表达是其主要机制之一,同时还与外排转运基因、抗氧化基因等有关。

Objective To screen the genes associated with fluconazole resistance in Trichosporon asahii to facilitate mechanistic studies of fluconazole resistance and the development of new antifungal agents.Methods Three clinical isolates of fluconazole-sensitive Trichosporon asahii were cultured in the presence of increasing concentrations of fluconazole until the drug concentration reached 128μg/mL.After further culture for 30 generations in the absence of fluconazole,the MIC value of the induced bacteria was measured.The total RNA was extracted from both the sensitive and resistant strains of the bacteria for construction of the stranded RNA-Seq libraries and transcriptome sequencing to identify the differentially expressed genes(DEGs)in the resistant strains.The genomes of the 3 sensitive and resistant strains were sequenced for comparison of the sequence of the differentially expressed ERG11 gene.Results We successfully obtainedstable Trichosporon asahii strains with fluconazole resistance.Transcriptome sequencing data showed that the genes associated with drug transport,ergosterol synthesis and antioxidation were up-regulated in the resistant strains,and among them ERG11 gene,the target gene of fluconazole,was up-regulated by 3.7 folds.Genomic sequencing proved no mutations in the sequence of ERG11 gene in the 3 resistant strains.Conclusion The molecular mechanism for drug resistance in Trichosporon asahii is complex.The upregulation of ergosterol synthesis pathway,especially ERG11 gene,along with other genes associated with efflux transfer and oxidative stress,is one of the main mechanisms for drug resistance in Trichosporon asahii.