本文選題：致病酵母YJM789 + PDR5��； 參考：《浙江大學(xué)》2012年博士論文

【摘要】：真菌感染在臨床上能導(dǎo)致高致死率,在艾滋病病人和接受器官移植或者進(jìn)行化療的免疫缺陷群體中更為明顯�？拐婢幬锏拈L期使用可能導(dǎo)致藥物耐受和治療失敗。以致病微生物為對(duì)象研究藥物抗性產(chǎn)生的機(jī)制對(duì)于解決日益嚴(yán)重的耐藥問題非常重要。 致病釀酒酵母菌株YJM789具有許多與致病性相關(guān)的表型。在本研究中,我們使用通過全基因組鋪瓦芯片進(jìn)行基因分型的四分體分離子庫對(duì)導(dǎo)致釀酒酵母BY4741(實(shí)驗(yàn)室菌株)與YJM789菌株氟康唑抗性差異的基因進(jìn)行遺傳定位,結(jié)果顯示YJM789中PDR5基因序列的巨大變異是導(dǎo)致其對(duì)氟康唑高度敏感的關(guān)鍵因素。 PDR5基因編碼一個(gè)外排泵,它通過外排大量結(jié)構(gòu)差異很大的毒性化合物賦予細(xì)胞多藥物抗性。我們使用GFP分別標(biāo)記了BY4741與YJM789中的PDR5,熒光顯微鏡觀察表明YJM789中Pdr5p-GFP融合蛋白定位于細(xì)胞膜,且過表達(dá)YJM789PDR5無法回補(bǔ)BY4741pdr5缺失株的抗藥表型。這些結(jié)果說明在YJM789中由Pdr5p所介導(dǎo)的唑類藥物抗性下降不是由于Pdr5p錯(cuò)誤定位或表達(dá)下調(diào)而是由Pdr5p功能受損所引起。 我們?cè)谒幬锃傊桨寮耙后w培養(yǎng)液中比較了YJM789與BY4741對(duì)唑類及多烯類抗真菌藥物的抗性,結(jié)果表明雖然YJM789中PDR5突變導(dǎo)致其對(duì)氟康唑外排能力嚴(yán)重受損,但同樣的突變卻賦予細(xì)胞在多烯抗生素下生長的優(yōu)勢(shì)。 為研究序列變異對(duì)YJM789Pdr5p功能的影響,我們通過片段替換的方法將劃分的BY4741PDR5片段替換為YJM789PDR5的相應(yīng)片段,構(gòu)建了一序列PDR5嵌合表達(dá)載體。通過使用氟康唑與放線菌酮抗性實(shí)驗(yàn)評(píng)估各Pdr5p嵌合體的外排功能,同時(shí)研究其表達(dá)、亞細(xì)胞定位、ATPase活性以及羅丹明6G外排效率等特性,結(jié)果顯示位于預(yù)測(cè)的連接第十與第十一跨膜螺旋胞內(nèi)環(huán)內(nèi)1352位點(diǎn)由丙氨酸到甲硫氨酸的突變可導(dǎo)致Pdr5p藥物外排能力嚴(yán)重下降,且其影響程度與突變殘基的大小相關(guān)。我們的研究為解釋ABC轉(zhuǎn)運(yùn)蛋白的功能機(jī)制、研制更為有效的抗真菌藥物提供理論基礎(chǔ)。
[Abstract]:Fungal infections can lead to high mortality rates, especially among AIDS patients and immune deficiency groups receiving organ transplantation or chemotherapy. Long-term use of antifungal drugs may lead to drug tolerance and treatment failure. It is very important to study the mechanism of drug resistance in pathogenic microorganisms in order to solve the problem of drug resistance. The pathogenic Saccharomyces cerevisiae YJM789 has many phenotypes related to pathogenicity. In this study, we used a tetrad ion library that was genotyped by whole-genome tile chip to localize the genes that led to the difference in fluconazole resistance between Saccharomyces cerevisiae BY4741 (laboratory strain) and YJM789 strain. The results showed that the great variation of PDR5 gene sequence in YJM789 was the key factor leading to its high sensitivity to fluconazole. The PDR5 gene encodes an efflux pump that endows cells with multidrug resistance through a large number of structurally different toxic compounds. GFP was used to label PDR5 in BY4741 and YJM789 respectively. Fluorescence microscopy showed that the Pdr5p-GFP fusion protein in YJM789 was located on cell membrane, and the overexpression of YJM789PDR5 could not compensate for the drug-resistant phenotype of BY4741pdr5 deficient strain. These results suggest that the decrease in YJM789 mediated by Pdr5p is not due to the mislocalization or down-regulation of Pdr5p expression, but to the impaired function of Pdr5p. We compared the resistance of YJM789 and BY4741 to antifungal agents of azoles and polyenes in drug Agar plate and liquid culture medium. The results showed that although the PDR5 mutation in YJM789 resulted in serious damage to fluconazole efflux ability. But the same mutation gives cells the advantage of growing under polyene antibiotics. In order to study the effect of sequence mutation on the function of YJM789Pdr5p, we constructed a sequence PDR5 chimeric expression vector by replacing the divided BY4741PDR5 fragment with the corresponding fragment of YJM789PDR5. The efflux function of each Pdr5p chimera was evaluated by fluconazole and actinomycin resistance test. The expression of fluconazole, the activity of subcellular locator ATPase and the effeciency of Rhodamine 6G efflux were also studied. The results showed that the mutation of 1352 site from alanine to methionine in the predicted tenth and eleventh transmembrane helical cytosol resulted in a serious decrease in the drug efflux capacity of Pdr5p, and the degree of influence was related to the size of the mutant residues. Our study provides a theoretical basis for explaining the functional mechanism of ABC transporter and developing more effective antifungal drugs.
【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2012
【分類號(hào)】：R341

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