本文選題：白念珠菌 + 高鐵還原酶��； 參考：《南開大學(xué)》2010年博士論文

【摘要】：白念珠菌是最常見的條件性致病菌,由白念珠菌引起的感染逐年上升,引起人們廣泛的關(guān)注。白念珠菌細(xì)胞形態(tài)從酵母型向菌絲型的轉(zhuǎn)換與其感染能力和毒力密切相關(guān)。細(xì)胞對(duì)外界pH的應(yīng)答是由保守的信號(hào)傳導(dǎo)途徑RIM101途徑所控制,白念珠菌中許多依賴pH表達(dá)的基因受轉(zhuǎn)錄因子Rim101蛋白的調(diào)控。pH條件影響著鐵離子的生物可利用率,由此推測(cè)鐵吸收系統(tǒng)受轉(zhuǎn)錄因子Rim101蛋白調(diào)控。鐵離子的吸收是白念珠菌在宿主中定居、存活和致病過程中重要的影響因素。鐵吸收系統(tǒng)的調(diào)控對(duì)白念珠菌生存起非常重要的作用,其中高鐵還原酶是高親和性吸收系統(tǒng)的第一個(gè)關(guān)鍵酶,高鐵還原酶基因表達(dá)的開啟與關(guān)閉將直接影響白念珠菌吸收鐵離子的能力。白念珠菌基因中含有17個(gè)編碼高鐵還原酶的基因,因此研究這些基因的表達(dá)調(diào)控,通過構(gòu)建基因缺失突變株分析基因的功能,發(fā)現(xiàn)與形態(tài)轉(zhuǎn)換相關(guān)的新的轉(zhuǎn)錄調(diào)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(低鐵環(huán)境)存活的,而且本研究所鑒定與識(shí)別的靶點(diǎn)將為白念珠菌的感染治療和藥物開發(fā)提供重要的理論依據(jù)。 首先,通過Northern雜交的方法檢測(cè)高鐵還原酶基因FRE1,FRE2,FRE1O,FRE11,FRP1和FRP2在酸性(pH4)和堿性(pH8)培養(yǎng)條件下,以及在酸性和堿性的培養(yǎng)基中分別添加50μmol/L BPS,150μmol/L BIP和150μmol/L菲洛嗪(Ferrozine)鐵離子螯合劑所造成低鐵環(huán)境中的表達(dá)情況,結(jié)果發(fā)現(xiàn)FRE1O為酸性pH應(yīng)答基因；FRE1、FRE2、FRE1l和FRP2為堿性應(yīng)答基因；FRP1的表達(dá)較為特殊：既是堿性應(yīng)答基因,又是低鐵條件應(yīng)答基因。 然后,利用PCR介導(dǎo)的同源重組的方法構(gòu)建了白念珠菌高鐵還原酶fre1△/△、fre2△/△、fre5△/△、fre10△/△和frp1△/△單基因缺失突變株以及fre1△/△fre2△/△、fre2△/△fre10△/△兩株雙基因突變株,并對(duì)上述基因缺失突變株和實(shí)驗(yàn)室保存的缺失突變株的細(xì)胞高鐵還原酶活性進(jìn)行分析,結(jié)果表明在酸性條件下(pH4),fre10△/△以及含有fre10突變的fre2△/△fre10△/△和fre5△/△fre10△/△雙基因突變株都導(dǎo)致高鐵還原酶活性大大降低,說明Fre10p是酸性條件下高鐵還原酶活性的主要貢獻(xiàn)者；堿性條件下(pH8),fre2△/△以及含有fre2突變的fre2△/△fre10△/△和fre1△/△fre2△/△雙基因突變株都導(dǎo)致高鐵還原酶活性大大降低,說明Fre2p是堿性條件下高鐵還原酶活性的主要貢獻(xiàn)者；而其它基因例如frel、fre5、fre8、fre9、fre71、frp1和frp2基因的缺失突變不影響菌株在pH4、pH4+50μmol/L BPS和pH8培養(yǎng)條件下高鐵還原酶的活性。 通過對(duì)已獲得的高鐵還原酶基因缺失突變株在低鐵條件下生長(zhǎng)情況的比較發(fā)現(xiàn)：在YPG培養(yǎng)基中所有菌株都生長(zhǎng)良好,而在YPG+150μmol/L BPS的低鐵培養(yǎng)基中,只有fre2△/△突變株不能生長(zhǎng),在YPG+200μmol/L BPS的低鐵培養(yǎng)基中,fre2△/△和frp1△/△突變株都不能生長(zhǎng),這說明在較低濃度的鐵環(huán)境下Fre2p是吸收鐵離子的主要高鐵還原酶,但是在更低濃度的鐵離子環(huán)境中,Fre2p和Frplp對(duì)菌株的生長(zhǎng)同等重要,二者缺少任何一個(gè)都將導(dǎo)致菌株對(duì)低鐵環(huán)境敏感。 構(gòu)建高鐵還原酶FRP1基因啟動(dòng)子與lacZ基因的融合結(jié)構(gòu)(PFRP1-lacZ),并將PFRP1-lacZ分別轉(zhuǎn)入野生型和rim101△/△菌株中,結(jié)果表明Rim101p轉(zhuǎn)錄因子的缺失嚴(yán)重降低了FRP1基因啟動(dòng)子的活性。在野生型菌株中,酸性條件只能較低程度上誘導(dǎo)FRP1基因啟動(dòng)子的活性；堿性條件可以明顯促進(jìn)FRP1基因啟動(dòng)子的活性,而且這種促進(jìn)作用依賴于低鐵環(huán)境。 通過對(duì)已經(jīng)克隆得到的982bp的FRPl基因啟動(dòng)子序列進(jìn)行分析,結(jié)果發(fā)現(xiàn)存在兩個(gè)可能的Riml01p結(jié)合序列,分別位于-629位點(diǎn)和-157位點(diǎn)。通過電泳遷移率實(shí)驗(yàn)確定Rim101p能夠與FRP1啟動(dòng)子內(nèi)部的兩個(gè)結(jié)合位點(diǎn)直接結(jié)合,再通過定點(diǎn)突變的方法,構(gòu)建含有RimlOlp結(jié)合序列突變的FRP1基因啟動(dòng)子P-157mut、P-629mut和P-157,-629mut,并檢測(cè)含有突變啟動(dòng)子菌株的β-半乳糖苷酶的活性,結(jié)果發(fā)現(xiàn)：-157位點(diǎn)的突變并沒有改變啟動(dòng)子的活性,與含有P-lacz結(jié)構(gòu)的菌株的活性相似；但是-629位點(diǎn)的突變?cè)斐删甑膌acZ活性比野生型降低了6倍,這表明-629位點(diǎn)在Rim101p調(diào)控FRP1表達(dá)的過程中起著非常重要的作用,而-157位點(diǎn)對(duì)于基因的表達(dá)調(diào)控并不重要。 利用同源重組的方法,構(gòu)建含有FRP1：GFP融合結(jié)構(gòu)的菌株,熒光顯微鏡下觀察發(fā)現(xiàn)綠色熒光分布于細(xì)胞質(zhì)內(nèi),并且可以清楚地發(fā)現(xiàn)Frp1蛋白定位于細(xì)胞的液泡中,通過軟件分析表明Frplp是一個(gè)具有5次跨膜結(jié)構(gòu)的膜蛋白,因此可以得知白念珠菌Frplp定位于液泡膜。 在實(shí)驗(yàn)過程中我們發(fā)現(xiàn)了白念珠菌Aft類型的轉(zhuǎn)錄因子Aft2p,并將AFT2基因在釀酒酵母中進(jìn)行表達(dá),結(jié)果發(fā)現(xiàn)CaAFT2基因在釀酒酵母PGK1強(qiáng)啟動(dòng)子的誘導(dǎo)下能夠彌補(bǔ)Scaftl△菌株的生長(zhǎng)缺陷,這說明白念珠菌Aft2p轉(zhuǎn)錄因子與釀酒酵母Aft類型的轉(zhuǎn)錄因子具有功能相似性。同時(shí)構(gòu)建白念珠菌aft2△/∧缺失突變株和AFT2基因的回補(bǔ)突變株并進(jìn)行表型分析,結(jié)果發(fā)現(xiàn)aft2的缺失不影響菌株在液體和固體培養(yǎng)基中的生長(zhǎng),但是基因突變改變了菌落的形態(tài),缺失突變株在固體培養(yǎng)基中不能形成菌絲,同時(shí)aft2的缺失對(duì)細(xì)胞高鐵還原酶活性產(chǎn)生較大影響,而且以小鼠為模型的系統(tǒng)感染實(shí)驗(yàn)表明白念珠菌aft2基因的缺失在較大程度上減弱了菌株的毒力。 使用Real-time PCR方法分析Aft2p轉(zhuǎn)錄因子對(duì)鐵代謝基因表達(dá)調(diào)控時(shí)發(fā)現(xiàn),Aft2p既可以促進(jìn)高鐵還原酶FRP1的表達(dá),又可以抑制鐵轉(zhuǎn)運(yùn)蛋白基因MRS4和鐵離子通透酶基因FTR1的表達(dá),因此Aft2p的調(diào)控作用是雙向的,Aft2p既是轉(zhuǎn)錄激活因子,又是轉(zhuǎn)錄抑制因子。
[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.
【學(xué)位授予單位】：南開大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2010
【分類號(hào)】：R379

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 陳曦,陳江野;白色念珠菌形態(tài)轉(zhuǎn)換的調(diào)控[J];生命的化學(xué);2000年05期

，

本文編號(hào)：1906036