白念珠菌新基因MRF1的耐药相关功能研究
发布时间：2018-10-10 15:29
【摘要】： 白念珠菌疾病易于感染、难以防治的特点使其在临床治疗中表现为强烈的致病性和高耐药性。前期研究显示白念珠菌耐药性形成过程中可能伴随着线粒体氧化呼吸功能的改变,并发现与线粒体呼吸相关的基因MRF1在耐药株中表达上调。为了确证白念珠菌耐药形成的“呼吸体抑制”假说,进一步阐明白念珠菌的耐药形成机制。本课题采用Ura-Blaster基因敲除策略靶向敲除和pCaEXP质粒异位表达线粒体呼吸功能相关的基因MRF1,构建了MRF1基因缺失菌和高表达菌。将构建好的菌株通过比浊法测定生长曲线；通过spot assay和微量液基实验来考察MRF1缺失菌对唑类药物的敏感性及过氧化氢的刺激耐受性是否改变；通过荧光染料检测MRF1基因缺失菌的线粒体膜电位、ATP产生和内源性活性氧水平(ROS),通过实时定量PCR考察了MRF1基因高表达后,其他已知耐药相关基因表达量的变化；又考察了MRF1缺失菌生物被膜、菌丝的形成能力、细胞膜甾醇成份和外排能力的改变情况,较全面的研究了MRF1基因的相关功能。结论：成功构建白念珠菌MRF1基因缺失菌和基因高表达菌；MRF1基因缺失后：菌株线粒体膜电位、ATP含量略降低,菌株生长速度延缓、对氧化刺激敏感性略增加、ROS产生略增多。酿酒酵母中MRF1编码的Ybr026p为2-烯酰硫酯还原酶,它催化线粒体脂肪酸合成通路Ⅱ的最后一步反应,产生作为线粒体膜成份之一的脂肪酸及合成硫辛酸的前体。由于白念MRF1p与Ybr026p同源,因此我们推测上述结果是因为MRF1缺失后不能正常合成2-烯酰硫酯还原酶,造成线粒体膜一定程度上的破损,细胞色素内容物降低,进而减弱呼吸作用而导致的。但我们通过测定基因缺失菌的药物MIC80值和spot assay实验,发现其对唑类药物的耐药性并没有显著性的改变,也不影响细胞膜的甾醇含量和外排能力,这一现象在Real-time结果中也得以证实。由于线粒体膜的功能维持涉及了大量的编码因子,推测MRF1基因非其决定性因素,所以其缺失不能对白念线粒体呼吸产生明显的抑制作用。此外,本研究发现白念珠菌MRF1基因能明显抑制细胞菌丝和被膜的形成,激光共聚焦实验也验证了这一现象,具体机制有待进一步探讨。
[Abstract]:Candida albicans is susceptible to infection and difficult to prevent and cure, which makes it strong pathogenicity and high drug resistance in clinical treatment. Previous studies have shown that the formation of resistance of Candida albicans may be accompanied by changes in mitochondrial oxidative respiratory function, and the expression of mitochondrial respiratory related gene MRF1 is up-regulated in drug-resistant strains. In order to confirm the hypothesis of "respiratory inhibition" caused by Candida albicans resistance, the mechanism of drug resistance of Candida albicans was further elucidated. In this study, MRF1 gene deletion bacteria and high expression bacteria were constructed using Ura-Blaster gene knockout strategy targeting knockout and pCaEXP plasmid ectopic expression of mitochondrial respiratory function related gene MRF1,. The growth curve was determined by turbidimetric method, the sensitivity of MRF1 deficient bacteria to azolides and the tolerance of hydrogen peroxide stimulation were investigated by spot assay and micro liquid-based test. The mitochondrial membrane potential, ATP production and endogenous reactive oxygen species (Ros) of MRF1 gene deletion bacteria were detected by fluorescence dye. The changes of other known drug-resistance related genes were examined by real-time quantitative PCR. The changes of biofilm, mycelium formation ability, cell membrane sterol composition and efflux ability of MRF1 deficient bacteria were also investigated. The related functions of MRF1 gene were studied comprehensively. Conclusion: Candida albicans MRF1 gene deletion and high gene expression bacteria were successfully constructed, and after MRF1 gene deletion, mitochondrial membrane potential, ATP content and growth rate of the strain were slightly decreased, the sensitivity to oxidative stimulation was slightly increased, and ROS production was slightly increased. The Ybr026p encoded by MRF1 in Saccharomyces cerevisiae is 2-allythioate reductase, which catalyzes the last step reaction of mitochondrial fatty acid synthesis pathway 鈪，
本文编号：2262300
[Abstract]:Candida albicans is susceptible to infection and difficult to prevent and cure, which makes it strong pathogenicity and high drug resistance in clinical treatment. Previous studies have shown that the formation of resistance of Candida albicans may be accompanied by changes in mitochondrial oxidative respiratory function, and the expression of mitochondrial respiratory related gene MRF1 is up-regulated in drug-resistant strains. In order to confirm the hypothesis of "respiratory inhibition" caused by Candida albicans resistance, the mechanism of drug resistance of Candida albicans was further elucidated. In this study, MRF1 gene deletion bacteria and high expression bacteria were constructed using Ura-Blaster gene knockout strategy targeting knockout and pCaEXP plasmid ectopic expression of mitochondrial respiratory function related gene MRF1,. The growth curve was determined by turbidimetric method, the sensitivity of MRF1 deficient bacteria to azolides and the tolerance of hydrogen peroxide stimulation were investigated by spot assay and micro liquid-based test. The mitochondrial membrane potential, ATP production and endogenous reactive oxygen species (Ros) of MRF1 gene deletion bacteria were detected by fluorescence dye. The changes of other known drug-resistance related genes were examined by real-time quantitative PCR. The changes of biofilm, mycelium formation ability, cell membrane sterol composition and efflux ability of MRF1 deficient bacteria were also investigated. The related functions of MRF1 gene were studied comprehensively. Conclusion: Candida albicans MRF1 gene deletion and high gene expression bacteria were successfully constructed, and after MRF1 gene deletion, mitochondrial membrane potential, ATP content and growth rate of the strain were slightly decreased, the sensitivity to oxidative stimulation was slightly increased, and ROS production was slightly increased. The Ybr026p encoded by MRF1 in Saccharomyces cerevisiae is 2-allythioate reductase, which catalyzes the last step reaction of mitochondrial fatty acid synthesis pathway 鈪，
本文编号：2262300
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