白念珠菌多铜氧化酶基因功能及转录调控的研究

发布时间：2018-05-24 09:52

本文选题：白念珠菌 + 多铜氧化酶　；参考：《南开大学》2013年硕士论文

【摘要】：白念珠菌{Candida albicans)是念珠菌病原体中最常见的条件性致病菌,也是医院获得性真菌病的主要感染源之一。铁离子获得能力是白念珠菌在宿主中定居、存活和致病过程中的重要影响因素。多铜氧化酶(multicopper oxidase, MCO)基因是白念珠菌高亲和性铁获得系统的关键组分。白念珠菌基因组中含有5个MCO编码基因,研究这些基因的功能和表达调控关系,将有助于深入地了解白念珠菌在低铁环境中的存活机制,为今后白念珠菌的感染治疗和药物开发提供重要的理论依据。首先通过荧光定量PCR方法分析了铁、铜离子对白念珠菌MCO基因的调控作用,结果发现：CaFET3、CaFET34和CaFET99是白念珠菌应答低铁条件的主要MCO基因；低铜条件只是对部分MCO基因的表达产生轻微影响。而当铁铜同时缺乏时,所有MCO基因的表达均会明显升高,表明铁铜离子对MCO基因的表达具有重要的调控作用。为了进一步研究MCO基因在铁代谢方面的作用,构建了白念珠菌CaFET33和CaFET34基因酿酒酵母回补菌株,发现CaFET34可以有效地回补Scfet3△在低铁条件下的缺陷表型。利用PCR介导的同源重组方法进一步构建了Cafet33△/△和Cafet34△/△缺失菌株,测定了其胞内铁含量和铁吸收能力的变化。结果表明在缺铁条件下,CaFET34的缺失会明显降低细胞的铁含量和铁吸收能力,说明CaFET34在细胞铁离子动态平衡方面发挥着重要作用。对Cafet33△/△和Cafet34△/△缺失菌株在缺铁条件下其它MCO基因表达进行研究,结果表明当白念珠菌中CaFET33/FET34基因缺失或者发生功能障碍时,其它MCO基因会替代缺陷基因而发挥功能。随后我们观察了Cafet33△/△和Cafet34△/△缺失菌株在菌丝诱导条件下的菌落形态。结果发现：在固体菌丝诱导培养基中,CaFET33和CaFET34,尤其是CaFET34的缺失会显著影响白念珠菌菌落周边菌丝的生长,而外源铁的添加并不能回补此缺陷,表明CaFET33和CaFET34以非铁依赖的方式参与白念珠菌菌丝发育。小鼠系统感染实验证实了CaFET34是一种重要的毒力因子,在白念珠菌的致病过程中起着一定作用。构建CaFET34启动子与报告基因LacZ融合质粒,转化野生型、aft2△/△和rin101△/△菌株,在不同pH、不同铁离子浓度条件下测定p-半乳糖苷酶活性,表明Aft2p可能作为转录抑制子调控CaFET34基因的表达。而Rim101p可能作为转录激活子调控CaFET34基因的表达。此外还构建了fth1△/△单基因和fth1△/△fet33△/△双基因缺失菌株,测定了其在缺铁条件下的生长情况、胞内铁含量、代谢转换能力、菌丝以及菌落形态的变化,结果表明fth1△/△fet33△/△缺失菌株胞内铁含量明显降低、代谢转换受阻、菌落表面褶皱形成能力和菌丝聚集明显增强,表明CaFTH1和CaFET33基因产物可能在液泡膜上形成铁转运复合物,参与胞内铁稳态和液泡功能的维持。
[Abstract]:Candida albicans {Candida albicans is the most common opportunistic pathogen in Candida albicans, and it is also one of the main infection sources of nosocomial mycosis. Iron ion acquisition ability is an important factor in the host settlement, survival and pathogenicity of Candida albicans. The polycopper oxidase multicopper oxidase (MCO) gene is a key component in the high affinity iron extraction system of Candida albicans. The genome of Candida albicans contains five MCO coding genes. Studying the function and expression regulation of these genes will be helpful to understand the survival mechanism of Candida albicans in low iron environment. To provide an important theoretical basis for the treatment of Candida albicans infection and drug development in the future. The regulation of iron and copper ions on the MCO gene of Candida albicans was analyzed by fluorescence quantitative PCR method. The results showed that the major MCO genes of Candida albicans were CaFET34 and CaFET99 in response to low iron condition. The low copper condition had only a slight effect on the expression of some MCO genes. However, when iron and copper were deficient at the same time, the expression of all MCO genes increased significantly, indicating that iron and copper ions play an important role in regulating the expression of MCO gene. In order to further study the role of MCO gene in iron metabolism, CaFET33 and CaFET34 genes of Candida albicans Saccharomyces cerevisiae were constructed. It was found that CaFET34 could effectively complement the defective phenotypes of Scfet3 in low iron condition. Cafet33 / and Cafet34 / deficient strains were further constructed by PCR mediated homologous recombination method, and the changes of iron content and iron absorption capacity were measured. The results showed that the absence of CaFET34 could significantly reduce the iron content and iron absorption ability of the cells under iron deficiency, indicating that CaFET34 plays an important role in the dynamic equilibrium of iron ions in the cells. The expression of other MCO genes in Cafet33 / and Cafet34 / deficient strains under iron deficiency was studied. The results showed that when the CaFET33/FET34 gene in Candida albicans was absent or the function was impaired, other MCO genes would replace the defective genes and function. Then we observed the colony morphology of Cafet33 / and Cafet34 / deficient strains under hyphal induction. The results showed that the absence of CaFET33 and CaFET34, especially CaFET34, significantly affected the growth of mycelium around the colony of Candida albicans in solid hyphal induction medium, but the addition of exogenous iron could not compensate for the defect. The results showed that CaFET33 and CaFET34 participated in the development of Candida albicans hypha in a non-iron dependent manner. CaFET34 is an important virulence factor and plays a certain role in the pathogenic process of Candida albicans. The fusion plasmids of CaFET34 promoter and reporter gene LacZ were constructed and transformed into wild type CaFET34 / and rin101 / strains. The activity of pgalactosidase was determined at different pH and different iron ion concentration. It is suggested that Aft2p may act as a transcription suppressor to regulate the expression of CaFET34 gene. Rim101p may act as a transcriptional activator to regulate the expression of CaFET34 gene. In addition, fth1 / single gene and fth1 / fet33 / double gene deficient strains were constructed, and their growth, intracellular iron content, metabolic transformation ability, mycelium and colony morphology were determined. The results showed that the intracellular iron content of fth1 / fet33 / deficient strain was significantly decreased, the metabolic transition was blocked, and the fold forming ability and hyphae aggregation of colony surface were significantly enhanced. It is suggested that CaFTH1 and CaFET33 gene products may form iron transport complex on vacuolar membrane and participate in the maintenance of iron homeostasis and vacuolar function.
【学位授予单位】：南开大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R379.4;Q78

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