白念珠菌高铁还原酶基因表达调控及Aft2p转录因子的功能研究
发布时间:2018-05-18 13:38
本文选题:白念珠菌 + 高铁还原酶 ; 参考:《南开大学》2010年博士论文
【摘要】:白念珠菌是最常见的条件性致病菌,由白念珠菌引起的感染逐年上升,引起人们广泛的关注。白念珠菌细胞形态从酵母型向菌丝型的转换与其感染能力和毒力密切相关。细胞对外界pH的应答是由保守的信号传导途径RIM101途径所控制,白念珠菌中许多依赖pH表达的基因受转录因子Rim101蛋白的调控。pH条件影响着铁离子的生物可利用率,由此推测铁吸收系统受转录因子Rim101蛋白调控。铁离子的吸收是白念珠菌在宿主中定居、存活和致病过程中重要的影响因素。铁吸收系统的调控对白念珠菌生存起非常重要的作用,其中高铁还原酶是高亲和性吸收系统的第一个关键酶,高铁还原酶基因表达的开启与关闭将直接影响白念珠菌吸收铁离子的能力。白念珠菌基因中含有17个编码高铁还原酶的基因,因此研究这些基因的表达调控,通过构建基因缺失突变株分析基因的功能,发现与形态转换相关的新的转录调控因子等有助于更加深入的了解白念珠菌是如何在宿主中(低铁环境)存活的,而且本研究所鉴定与识别的靶点将为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Northern杂交的方法检测高铁还原酶基因FRE1,FRE2,FRE1O,FRE11,FRP1和FRP2在酸性(pH4)和碱性(pH8)培养条件下,以及在酸性和碱性的培养基中分别添加50μmol/L BPS,150μmol/L BIP和150μmol/L菲洛嗪(Ferrozine)铁离子螯合剂所造成低铁环境中的表达情况,结果发现FRE1O为酸性pH应答基因;FRE1、FRE2、FRE1l和FRP2为碱性应答基因;FRP1的表达较为特殊:既是碱性应答基因,又是低铁条件应答基因。 然后,利用PCR介导的同源重组的方法构建了白念珠菌高铁还原酶fre1△/△、fre2△/△、fre5△/△、fre10△/△和frp1△/△单基因缺失突变株以及fre1△/△fre2△/△、fre2△/△fre10△/△两株双基因突变株,并对上述基因缺失突变株和实验室保存的缺失突变株的细胞高铁还原酶活性进行分析,结果表明在酸性条件下(pH4),fre10△/△以及含有fre10突变的fre2△/△fre10△/△和fre5△/△fre10△/△双基因突变株都导致高铁还原酶活性大大降低,说明Fre10p是酸性条件下高铁还原酶活性的主要贡献者;碱性条件下(pH8),fre2△/△以及含有fre2突变的fre2△/△fre10△/△和fre1△/△fre2△/△双基因突变株都导致高铁还原酶活性大大降低,说明Fre2p是碱性条件下高铁还原酶活性的主要贡献者;而其它基因例如frel、fre5、fre8、fre9、fre71、frp1和frp2基因的缺失突变不影响菌株在pH4、pH4+50μmol/L BPS和pH8培养条件下高铁还原酶的活性。 通过对已获得的高铁还原酶基因缺失突变株在低铁条件下生长情况的比较发现:在YPG培养基中所有菌株都生长良好,而在YPG+150μmol/L BPS的低铁培养基中,只有fre2△/△突变株不能生长,在YPG+200μmol/L BPS的低铁培养基中,fre2△/△和frp1△/△突变株都不能生长,这说明在较低浓度的铁环境下Fre2p是吸收铁离子的主要高铁还原酶,但是在更低浓度的铁离子环境中,Fre2p和Frplp对菌株的生长同等重要,二者缺少任何一个都将导致菌株对低铁环境敏感。 构建高铁还原酶FRP1基因启动子与lacZ基因的融合结构(PFRP1-lacZ),并将PFRP1-lacZ分别转入野生型和rim101△/△菌株中,结果表明Rim101p转录因子的缺失严重降低了FRP1基因启动子的活性。在野生型菌株中,酸性条件只能较低程度上诱导FRP1基因启动子的活性;碱性条件可以明显促进FRP1基因启动子的活性,而且这种促进作用依赖于低铁环境。 通过对已经克隆得到的982bp的FRPl基因启动子序列进行分析,结果发现存在两个可能的Riml01p结合序列,分别位于-629位点和-157位点。通过电泳迁移率实验确定Rim101p能够与FRP1启动子内部的两个结合位点直接结合,再通过定点突变的方法,构建含有RimlOlp结合序列突变的FRP1基因启动子P-157mut、P-629mut和P-157,-629mut,并检测含有突变启动子菌株的β-半乳糖苷酶的活性,结果发现:-157位点的突变并没有改变启动子的活性,与含有P-lacz结构的菌株的活性相似;但是-629位点的突变造成菌株的lacZ活性比野生型降低了6倍,这表明-629位点在Rim101p调控FRP1表达的过程中起着非常重要的作用,而-157位点对于基因的表达调控并不重要。 利用同源重组的方法,构建含有FRP1:GFP融合结构的菌株,荧光显微镜下观察发现绿色荧光分布于细胞质内,并且可以清楚地发现Frp1蛋白定位于细胞的液泡中,通过软件分析表明Frplp是一个具有5次跨膜结构的膜蛋白,因此可以得知白念珠菌Frplp定位于液泡膜。 在实验过程中我们发现了白念珠菌Aft类型的转录因子Aft2p,并将AFT2基因在酿酒酵母中进行表达,结果发现CaAFT2基因在酿酒酵母PGK1强启动子的诱导下能够弥补Scaftl△菌株的生长缺陷,这说明白念珠菌Aft2p转录因子与酿酒酵母Aft类型的转录因子具有功能相似性。同时构建白念珠菌aft2△/∧缺失突变株和AFT2基因的回补突变株并进行表型分析,结果发现aft2的缺失不影响菌株在液体和固体培养基中的生长,但是基因突变改变了菌落的形态,缺失突变株在固体培养基中不能形成菌丝,同时aft2的缺失对细胞高铁还原酶活性产生较大影响,而且以小鼠为模型的系统感染实验表明白念珠菌aft2基因的缺失在较大程度上减弱了菌株的毒力。 使用Real-time PCR方法分析Aft2p转录因子对铁代谢基因表达调控时发现,Aft2p既可以促进高铁还原酶FRP1的表达,又可以抑制铁转运蛋白基因MRS4和铁离子通透酶基因FTR1的表达,因此Aft2p的调控作用是双向的,Aft2p既是转录激活因子,又是转录抑制因子。
[Abstract]:Candida albicans are the most common conditional pathogenic bacteria, and the infection caused by Candida albicans has increased year by year, causing widespread concern. The transformation of Candida albicans from yeast type to mycelium is closely related to its infection ability and virulence. The response of cells to the external pH is controlled by the RIM101 pathway of guarded signal transduction pathway. Many of the genes dependent on pH expression in Candida, regulated by the transcription factor Rim101 protein, affect the bioavailability of iron ions, thus speculates that the iron absorption system is regulated by the transcription factor Rim101 protein. The iron absorption is an important factor in the survival and disease process of Candida albicans in the host. The iron absorption line is an important factor in the disease. The regulation of the system plays a very important role in the survival of Candida albicans, in which the high iron reductase is the first key enzyme in the high affinity absorption system. The opening and closing of the gene expression of high iron reductase will directly affect the ability of Candida albicans to absorb iron ions. The gene of Candida albicans contains 17 genes encoding high iron reductase. The expression regulation of these genes and the analysis of the function of gene deletion mutants and the discovery of new transcriptional regulators associated with morphologic transformation can help to further understand how Candida albicans live in the host (low iron environment), and the target identified and identified in this study will be the infection of Candida albicans. Treatment and drug development provide an important theoretical basis.
First, the Northern hybridization method was used to detect the high iron reductase gene FRE1, FRE2, FRE1O, FRE11, FRP1 and FRP2 in acidic (pH4) and alkaline (pH8) culture conditions, as well as 50 micron mol/L BPS, 150 micron mol/L, and 150 micron Filo iron ion chelating agents in the low iron environment. The results showed that FRE1O was an acid pH response gene, FRE1, FRE2, FRE1l and FRP2 were alkaline response genes, and the expression of FRP1 was more specific: it was both alkaline response gene and low iron condition response gene.
Then, the two mutant strains of Candida albicans, high iron reductase fre1 Delta / Delta, fre2 Delta / Delta, fre5 Delta / Delta, fre10 Delta / delta and frp1 Delta / Delta, and fre1 Delta / delta fre2 Delta / Delta, fre2 Delta / delta fre10 Delta / delta two mutant strain, were constructed and preserved in the mutant and Laboratory of the gene deletion mutant and the laboratory. The activity of high iron reductase in the missing mutant cells was analyzed. The results showed that in acid conditions (pH4), fre10 Delta / delta and fre10 mutation, fre2 Delta / delta fre10 Delta / delta and fre5 Delta / delta fre10 Delta / delta double gene mutants all led to high iron reductase activity, suggesting that Fre10p was the main activity of high iron reductase under acidic conditions. To contributors, the basic conditions (pH8), fre2 Delta / delta and fre2 mutation fre2 Delta / delta fre10 Delta / delta and fre1 Delta / delta fre2 Delta / delta double gene mutants all lead to high iron reductase activity greatly reduced, indicating that Fre2p is the main contributor to the activity of high iron reductase under alkaline conditions, while other genes such as frel, fre5, fre8, fre9, etc. The deletion mutation of frp1 and frp2 genes did not affect the activity of high iron reductase in pH4, pH4+50, mol/L BPS and pH8 culture conditions.
By comparison of the growth conditions of the mutant strains of high iron reductase gene deletion in low iron conditions, it was found that all strains were well grown in the YPG medium, but only fre2 Delta / delta mutant strains could not grow in the low iron medium of YPG+150 mol/L BPS, fre2 Delta / delta and frp1 in the low iron medium of YPG+200 mu mol/L BPS. The delta / delta mutant strain can not grow, which indicates that Fre2p is the main high iron reductase in the lower concentration of iron environment, but in the lower concentration of the iron ion environment, Fre2p and Frplp are equally important for the growth of the strain. The lack of any one of the two will cause the strain to be sensitive to the low iron environment.
The fusion structure (PFRP1-lacZ) of the promoter of the high iron reductase FRP1 gene and the lacZ gene was constructed, and PFRP1-lacZ was transferred into the wild type and rim101 delta strain respectively. The results showed that the deletion of Rim101p transcriptional factors seriously reduced the activity of the FRP1 gene promoter. In the wild type, the acidic condition could only induce the FRP1 gene in a lower degree. The activity of FRP1 promoter can be obviously promoted by alkaline condition, and this promotion effect depends on the low iron environment.
By analyzing the FRPl gene promoter sequence of the cloned 982bp, it was found that there were two possible Riml01p binding sequences at the -629 and -157 sites. By the electrophoresis mobility test, Rim101p could be directly combined with the two binding sites within the FRP1 promoter, and then through the fixed-point mutation method, Constructing FRP1 gene promoter, P-157mut, P-629mut and P-157, -629mut, and detecting the activity of beta galactosidase containing mutant promoter strains. The results showed that the mutation of the -157 site did not change the activity of the promoter, similar to the activity of the strain containing the P-lacz structure, but the mutation of the -629 loci was found. The lacZ activity of the strain is 6 times lower than that of the wild type, which indicates that the -629 site plays a very important role in the process of Rim101p regulation of FRP1 expression, and the regulation of -157 loci is not important for the regulation of gene expression.
By means of homologous recombination, a strain containing FRP1:GFP fusion structure was constructed. Under the fluorescence microscope, the green fluorescence was found in the cytoplasm, and it was found that the Frp1 protein was located in the vacuole of the cell. The software analysis showed that Frplp was a membrane protein with 5 transmembrane structures. The Frplp was located in the vacuolar membrane.
During the experiment, we found the transcription factor Aft2p of the Aft type of Candida albicans and the expression of the AFT2 gene in Saccharomyces cerevisiae. The results showed that the CaAFT2 gene could make up the growth defects of Scaftl delta strain under the induction of the strong promoter of Saccharomyces cerevisiae, which indicates that the Aft2p transcription factor of Candida albicans and the Aft type of Saccharomyces cerevisiae. The transcriptional factor had functional similarity. Meanwhile, the mutant strain of Candida albicans aft2 Delta / a deletion mutant and AFT2 gene was constructed and the phenotypic analysis was carried out. The results showed that the deletion of aft2 did not affect the growth of the strain in the liquid and solid medium, but the mutation of the gene changed the morphology of the colony, and the missing mutant was not in the solid medium. Mycelium can form mycelium, and the loss of aft2 has a great effect on the activity of cell high iron reductase, and the system infection test table in mice shows that the deletion of the aft2 gene of Candida albicans weakens the virulence of the strain to a large extent.
The Real-time PCR method was used to analyze the regulation of Aft2p transcription factors to iron metabolism gene expression. Aft2p could promote the expression of high iron reductase FRP1 and inhibit the expression of the iron transporter gene MRS4 and the iron ion permeable enzyme gene FTR1. Therefore, the regulation of Aft2p is bidirectional, Aft2p is both a transcription activator and a transcription factor. Inhibitory factor.
【学位授予单位】:南开大学
【学位级别】:博士
【学位授予年份】:2010
【分类号】:R379
【参考文献】
相关期刊论文 前1条
1 陈曦,陈江野;白色念珠菌形态转换的调控[J];生命的化学;2000年05期
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