白念珠菌IPF7817基因的生物学功能研究
发布时间:2019-06-29 12:40
【摘要】: IPF7817是白念珠菌的功能未知基因,已有研究结果提示该基因可能参与白念珠菌的耐药和细胞内氧化还原状态的调节,但是其确切的生物学功能在本研究开展之前尚不明确。本研究利用基因敲除技术系统性地考察了IPF7817的功能:首先应用“Ura-blaster”策略将IPF7817的两个等位基因从白念珠菌的基因组中敲除,继而考察了IPF7817基因敲除对细胞内氧化还原稳态和耐药表型的影响。氧化还原稳态相关实验结果显示:IPF7817基因敲除后,细胞内的活性氧水平显著提高;线粒体功能的直接指标——线粒体膜电位升高;一些重要的氧化还原相关基因(包括GLR1、SOD2和TRR1)转录表达上调;细胞内还原型谷胱甘肽和氧化型谷胱甘肽的比值增加。上述结果提示:IPF7817在白念珠菌的氧化还原稳态调节中发挥重要作用。考察IPF7817基因对耐药表型影响的实验结果显示:IPF7817基因的缺失对白念珠菌的药物敏感性没有明显影响;对白念珠菌药物外排功能没有明显影响;但是会导致多药耐药基因MDR1的表达上调;分别用6μg/mL和24μg/mL的氟康唑诱导白念珠菌耐药性产生,MIC_(80)检测、Spot assay和Filter disk assay结果一致显示:IPF7817基因缺失菌产生氟康唑抗性的时间明显早于亲本菌,并且对其他唑类药物存在交叉耐药性;经氟康唑诱导后的基因缺失菌JH2_(F6-47)、JH2_(F24-48)相对于诱导后亲本RM1000_(F6-47)、RM1000_(F24-48),唑类药物作用的靶酶基因ERG11表达显著上调;敏感性回复实验显示:6μg/mL氟康唑诱导的耐药亲本菌可恢复对氟康唑的敏感性(MIC_(80)为0.5μg/mL),但同样条件下诱导的耐药IPF7817基因缺失菌在40天后仍保持耐药性的稳定,不发生对氟康唑敏感性的回复。此外,本研究还考察了IPF7817对白念珠菌菌丝和生物被膜形成能力的影响,IPF7817基因缺失菌和亲本菌的结果未见明显差异。综上所述,IPF7817参与了白念珠菌细胞内氧化还原稳态的调节,并影响白念珠菌诱导耐药性的产生。
[Abstract]:IPF7817 is an unknown functional gene of candida albicans. The results suggest that the gene may be involved in the regulation of drug resistance and intracellular redox status of candida albicans, but its exact biological function is not clear before the development of this study. In this study, the function of IPF7817 was systematically investigated by gene knockout technique. Firstly, two IPF7817 alleles were knockout from the genome of candida albicans by "Ura-blaster" strategy, and then the effects of IPF7817 knockout on intracellular redox homeostasis and drug resistance phenotype were investigated. The results of redox homeostasis test showed that the level of reactive oxygen species (Ros) in cells increased significantly after knockout of IPF7817 gene, the mitochondrial membrane potential, the transcriptional expression of some important redox related genes (including GLR1,SOD2 and TRR1), and the ratio of reduced Glutathion to oxidized Glutathion increased. These results suggest that IPF7817 plays an important role in the redox homeostasis regulation of candida albicans. The results of investigating the effect of IPF7817 gene on drug resistance phenotype showed that the deletion of IPF7817 gene had no significant effect on the drug sensitivity of candida albicans, but had no significant effect on the drug efflux function of candida albicans, but it would lead to the up-regulation of the expression of multi-drug resistance gene MDR1. Fluconazole (6 渭 g / mL) and 24 渭 g / mL (fluconazole) were used to induce drug resistance of candida albicans. The results of MIC_ (80) detection of, Spot assay and Filter disk assay showed that the time of fluconazole resistance of IPF7817 gene deleted bacteria was significantly earlier than that of parent bacteria, and there was cross resistance to other zole drugs. The expression of gene deletion bacteria JH2_ (F6 鈮,
本文编号:2507827
[Abstract]:IPF7817 is an unknown functional gene of candida albicans. The results suggest that the gene may be involved in the regulation of drug resistance and intracellular redox status of candida albicans, but its exact biological function is not clear before the development of this study. In this study, the function of IPF7817 was systematically investigated by gene knockout technique. Firstly, two IPF7817 alleles were knockout from the genome of candida albicans by "Ura-blaster" strategy, and then the effects of IPF7817 knockout on intracellular redox homeostasis and drug resistance phenotype were investigated. The results of redox homeostasis test showed that the level of reactive oxygen species (Ros) in cells increased significantly after knockout of IPF7817 gene, the mitochondrial membrane potential, the transcriptional expression of some important redox related genes (including GLR1,SOD2 and TRR1), and the ratio of reduced Glutathion to oxidized Glutathion increased. These results suggest that IPF7817 plays an important role in the redox homeostasis regulation of candida albicans. The results of investigating the effect of IPF7817 gene on drug resistance phenotype showed that the deletion of IPF7817 gene had no significant effect on the drug sensitivity of candida albicans, but had no significant effect on the drug efflux function of candida albicans, but it would lead to the up-regulation of the expression of multi-drug resistance gene MDR1. Fluconazole (6 渭 g / mL) and 24 渭 g / mL (fluconazole) were used to induce drug resistance of candida albicans. The results of MIC_ (80) detection of, Spot assay and Filter disk assay showed that the time of fluconazole resistance of IPF7817 gene deleted bacteria was significantly earlier than that of parent bacteria, and there was cross resistance to other zole drugs. The expression of gene deletion bacteria JH2_ (F6 鈮,
本文编号:2507827
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