PMT4在新生隱球菌生物膜形成及其耐藥中的作用
發(fā)布時(shí)間：2019-05-17 22:39
【摘要】： 選題背景: 在隱球菌病的研究中亟待解決的問題是對(duì)隱球菌的致病機(jī)制和耐藥機(jī)制還缺乏足夠的了解。近年來生物膜(biofilm)對(duì)真菌致病力的作用日益為人們所重視。 研究發(fā)現(xiàn)真菌生物膜對(duì)抗真菌藥物的敏感性比游離狀態(tài)下低30-2000倍。那么,生物膜形成的相關(guān)機(jī)制是什么?有哪些基因影響了生物膜的形成?真菌生物膜的研究還處于起步階段,還有大量未解之謎。 本課題選取了有代表性的A血清型新生隱球菌H99 MATα型菌株和PMT4基因作為研究對(duì)象。Pmt4蛋白對(duì)于真菌分泌蛋白和部分膜蛋白的修飾極其重要,缺乏pmt酶活性可導(dǎo)致菌株生長(zhǎng)率下降、細(xì)胞壁不穩(wěn)定甚至死亡。已發(fā)表的研究顯示,PMT基因?qū)Π啄钪榫锬さ男纬珊蜕锬そ閷?dǎo)的耐藥至關(guān)重要。鑒于新生隱球菌和白念珠菌的諸多相似性,我們可以推測(cè),PMT基因很可能在新生隱球菌的生物膜形成及其介導(dǎo)的耐藥上起重要作用。由于這一基因編碼的蛋白存在于真菌內(nèi),植物細(xì)胞則無,它與哺乳動(dòng)物細(xì)胞內(nèi)的類似酶在性質(zhì)和催化作用上有著很大的不同。因此對(duì)其的進(jìn)一步研究也有可能為研發(fā)新的抗真菌藥物提供靶點(diǎn)。 另外,在隱球菌生物膜的研究中缺乏實(shí)用的體內(nèi)模型,因此本課題除采用已有的體外模型外,還構(gòu)建了一種新的基于血管內(nèi)插管的動(dòng)物模型。 研究目的: 1.構(gòu)建新生隱球菌H99的PMT4基因缺陷株。 2.構(gòu)建新的新生隱球菌生物膜的動(dòng)物模型。 3.觀察新生隱球菌H99野生株和PMT4缺陷株在生物膜形成方面的差異。 4.觀察新生隱球菌H99野生株和PMT4缺陷株所形成的生物膜在抗真菌藥物敏感性方面的差異。 研究方法: 1.構(gòu)建新生隱球菌H99的PMT4基因缺陷株。 使用PCR介導(dǎo)的長(zhǎng)側(cè)翼同源重組法來進(jìn)行PMT4基因的敲除工作,用一個(gè)G418抗性基因替換掉H99菌株基因組中的PMT4基因。 初步篩選采用含G418的YPD平板進(jìn)行,所得陽性轉(zhuǎn)化子分別進(jìn)行PCR驗(yàn)證。 PCR驗(yàn)證通過的陽性轉(zhuǎn)化子經(jīng)基因測(cè)序驗(yàn)證。 2.構(gòu)建新的新生隱球菌生物膜的動(dòng)物模型。 采用基礎(chǔ)培養(yǎng)基、聚苯乙烯96孔板內(nèi)孵育的方法建立體外模型。 采用兔頸外靜脈插管并在導(dǎo)管內(nèi)置聚苯乙烯薄膜的方法建立動(dòng)物模型。 3.觀察新生隱球菌H99野生株和PMT4缺陷株在生物膜形成方面的差異。 采用倒置相差顯微鏡和共聚集激光掃描顯微鏡觀察生物膜的大體形態(tài)和結(jié)構(gòu)。 采用比較集落形成單位(CFU)數(shù)量的方法比較H99野生株和PMT4缺陷株生物膜中有增殖活性的細(xì)胞比例。 4.觀察新生隱球菌H99野生株和PMT4缺陷株所形成的生物膜在抗真菌藥物敏感性方面的差異。 用美國(guó)CLSI的M27A2微量稀釋法對(duì)H99野生株和PMT4缺陷株游離細(xì)胞以及它們的生物膜細(xì)胞進(jìn)行藥敏實(shí)驗(yàn)。 用四甲基偶氮唑鹽(MTT)法測(cè)定H99野生株及其PMT4缺陷株生物膜代謝活性,并以此反應(yīng)這兩種菌株在體內(nèi)外模型中形成的生物膜對(duì)抗真菌藥物的敏感性。根據(jù)體外模型的實(shí)驗(yàn)結(jié)果決定動(dòng)物模型中采用的藥物濃度。 結(jié)果: 1.構(gòu)建新生隱球菌H99的PMT4基因缺陷株。 G418平板培養(yǎng)、對(duì)最終獲得的12號(hào)轉(zhuǎn)化子的PCR驗(yàn)證以及對(duì)其基因組相關(guān)基因的測(cè)序分析均證明12號(hào)轉(zhuǎn)化子的PMT4基因已被成功敲除。 2.構(gòu)建新的新生隱球菌生物膜的動(dòng)物模型。 實(shí)驗(yàn)證實(shí)H99野生株及其PMT4缺陷株均可在基礎(chǔ)培養(yǎng)基加96孔板的體外模型和新構(gòu)建的血管內(nèi)插管加導(dǎo)管內(nèi)置聚苯乙烯膜的動(dòng)物模型中形成生物膜。證實(shí)本課題采用的體外模型和新的動(dòng)物模型可用于生物膜的研究。 3.新生隱球菌H99野生株和PMT4缺陷株在生物膜形成方面的差異。 新生隱球菌H99野生株及其PMT4缺陷株在動(dòng)物模型中形成的生物膜與體外模型中所形成的生物膜在形成過程和大體形態(tài)方面非常相似,也是在24小時(shí)后開始成熟。但后者同時(shí)期生物膜的細(xì)胞數(shù)量和生物膜厚度比前者小。 用倒置顯微鏡和共聚集激光掃描顯微鏡均觀察到PMT4缺陷株所形成的生物膜在接種24小時(shí)后出現(xiàn)大量類似假菌絲樣的結(jié)構(gòu)。 在同等數(shù)量的接種細(xì)胞中,PMT4缺陷株生物膜細(xì)胞的集落形成單位數(shù)明顯少于H99野生株生物膜細(xì)胞。 4.新生隱球菌H99野生株和PMT4缺陷株所形成的生物膜在抗真菌藥物敏感性方面的差異。 M27A2微量稀釋法藥敏實(shí)驗(yàn)發(fā)現(xiàn)FCZ、AmB、5-FC、ITR四種抗真菌藥物對(duì)H99野生株及其PMT4缺陷株游離細(xì)胞的MIC值(μg/ml)分別是4、1、4、≤0.125和1、0.25、0.5、≤0.125;對(duì)生物膜細(xì)胞的MIC值(μg/ml)分別是4、2、8、≤0.125和2、0.125、4、≤0.125。 體外模型生物膜細(xì)胞的MTT檢測(cè)發(fā)現(xiàn),這四種藥物使?jié)岫认陆?0%所需的最低藥物濃度(μg/ml)(這一指標(biāo)類似上述MIC值)在H99野生株生物膜和PMT4缺陷株生物膜中分別是≥64、32、≥64、16和2、0.5、4、1。 動(dòng)物模型生物膜細(xì)胞的MTT檢測(cè)發(fā)現(xiàn),在64μg/ml FCZ作用下,與各自的空白對(duì)照相比,PMT4缺陷株生物膜細(xì)胞的代謝活性抑制率為96.7%,H99野生株生物膜細(xì)胞為8.7%。 結(jié)論: 通過PCR驗(yàn)證和基因測(cè)序證實(shí)12號(hào)轉(zhuǎn)化子中的PMT4基因確實(shí)被敲除了,這一PMT4缺陷株,可用于對(duì)PMT4基因進(jìn)一步的研究。首次利用兔中心靜脈插管的方式成功構(gòu)建了隱球菌血管內(nèi)插管的動(dòng)物模型,可作為研究平臺(tái)用于模擬臨床最為常見的植入物表面生物膜形成的情況,對(duì)于隱球菌生物膜的研究有著重要意義。 首次采用導(dǎo)管內(nèi)置入聚苯乙烯薄膜的方法構(gòu)建動(dòng)物模型,導(dǎo)管內(nèi)覆的聚苯乙烯薄膜可取出,避免了導(dǎo)管內(nèi)生物膜體內(nèi)模型只能在共聚集激光掃描顯微鏡下利用Z軸成像的方式進(jìn)行觀察的弊端,使得導(dǎo)管內(nèi)生物膜也可直接在普通光鏡下觀察。 實(shí)驗(yàn)結(jié)果顯示無論是H99野生株還是PMT4缺陷株所形成的生物膜均在24小時(shí)后進(jìn)入成熟期。接種48小時(shí)后進(jìn)入相對(duì)的穩(wěn)定期。PMT4缺陷株各時(shí)期生物膜的細(xì)胞量均少于H99野生株。 另外發(fā)現(xiàn)成熟的PMT4缺陷株生物膜中具有增殖活性的細(xì)胞的比例小于同時(shí)期的野生株生物膜。這意味著PMT4缺陷株形成的生物膜向外釋放菌細(xì)胞、擴(kuò)大感染范圍的能力弱于野生株。 在生物膜結(jié)構(gòu)方面,相較于H99野生株生物膜,PMT4缺陷株生物膜的一個(gè)顯著特點(diǎn)是進(jìn)入成熟期后產(chǎn)生具有大量的假菌絲樣結(jié)構(gòu)。這可能與PMT4變異株的細(xì)胞壁缺陷有關(guān),但為何進(jìn)入成熟期才出現(xiàn)尚不清楚。 藥敏實(shí)驗(yàn)顯示,PMT4缺陷株游離細(xì)胞對(duì)抗真菌藥物的敏感性比野生株稍高,這可能也與其細(xì)胞壁的缺陷有關(guān)。對(duì)隱球菌生物膜細(xì)胞進(jìn)行的M27A2藥敏顯示其對(duì)藥物的敏感性與游離細(xì)胞差異不大,可能是由于生物膜細(xì)胞在從生物膜中分離出來在營(yíng)養(yǎng)豐富的培養(yǎng)基中長(zhǎng)時(shí)間培養(yǎng)又逐漸恢復(fù)了游離細(xì)胞生物學(xué)特性的緣故。由此可見,將生物膜細(xì)胞分離出來成為懸浮細(xì)胞后再進(jìn)行常規(guī)M27A2藥敏實(shí)驗(yàn)并不能反映生物膜本身對(duì)抗真菌藥物的真實(shí)敏感性。 體內(nèi)外模型生物膜細(xì)胞的MTT檢測(cè)均表明,H99野生株生物膜細(xì)胞比PMT4缺陷株生物膜細(xì)胞對(duì)抗真菌藥物的抵抗能力強(qiáng)。在體外模型中, PMT4缺陷株生物膜細(xì)胞對(duì)不同的抗真菌藥物的敏感性比野生株高16-64倍。在體內(nèi)模型中,同等藥物濃度下,PMT4缺陷株生物膜細(xì)胞的代謝活性幾乎被完全抑制,而野生株生物膜細(xì)胞卻不受影響。 這些結(jié)果提示PMT4基因?qū)τ谛律[球菌生物膜的耐藥特性是至關(guān)重要的,PMT4基因的缺失可導(dǎo)致新生隱球菌生物膜耐藥性幾乎完全喪失。由于真菌的PMT4基因與哺乳動(dòng)物的類似基因差異很大,因此PMT4基因及其編碼的酶有可能成為新的抗真菌藥物的作用靶點(diǎn)。
[Abstract]:Background of the subject: The problem to be solved in the study of cryptococcosis is that the pathogenesis of cryptococcus and the mechanism of drug resistance are not enough In recent years, the effect of biofilms on the pathogenicity of fungi is becoming more and more important. The study found that the sensitivity of the fungal biofilm to the fungal drug was lower than in the free state. 0-2000 times. So, the biofilm is formed What is the relevant mechanism? What gene effects? Biofilm formation? The study of fungal biofilm is still in the early stage, and also A representative A-type Cryptococcus neoformans H99 MAT-1 strain and P were selected in this subject. The MT4 gene is the subject of the study. The Pmt4 protein is extremely important for the modification of the fungal secretory protein and the partial membrane protein, and the absence of the pmt enzyme activity can lead to a decrease in the growth rate of the strain. Cell wall instability and even death. Published studies have shown that the PMT gene is formed and produced by Candida albicans biofilm The drug resistance mediated by the membrane is of great importance. In view of the similarity of the neoformans and the Candida albicans, we can speculate that the PMT gene is likely to be formed in the biofilms of the neoformans. The protein encoded by this gene is present in the fungus, and the plant cell is not present, and it is similar to the enzyme in the mammalian cell in nature and in a hurry. There is a great difference in the process of research, so it is possible to research and develop the further research. In addition, a practical in vivo model is lacking in the research of cryptococcus biofilms. new-based An animal model of an intravascular cannula. The purpose of this study is to: 1. Construction of PMT4 Gene of Cryptococcus neoformans H99 2. Construction of a new animal model of the new Cryptococcus neoformans biofilm. The differences in biofilm formation of H99 wild and PMT4 strains of Cryptococcus neoformans.4. The observation of Cryptococcus neoformans H99 wild The biofilm formed by the strain and the PMT4 deficient strain is in the form of an antifungal drug, 1. The method of the study:1. Construction of a PMT4 gene-deficient strain of Cryptococcus neoformans H99. The long-flank homologous recombination mediated by PCR was used. The knock-out of the PT4 gene was carried out by the group method, and the H99 strain group was replaced with a G418-resistant gene. The PMT4 gene in the group. The primary screening was used. YPD plate with G418 for positive conversion and the PCR verification is carried out respectively, and the positive conversion sub-channels passed by the PCR are subjected to PCR verification, And 2. establishing a new animal model of the new Cryptococcus neoformans biofilms. An in vitro model was established using a basic medium, a method of incubation in a polystyrene 96-well plate. An animal model was established using a rabbit's external jugular vein and a method of built-in polystyrene film in the catheter. 3. The difference of H99 wild strain and PMT4 deficient strain on the formation of biofilm was observed. The inversion phase-difference microscope was used. And the general morphology and structure of the biofilms were observed by means of a confocal laser scanning microscope. Methods: The cell ratio of the proliferation activity of H99 wild strain and PMT4 deficient strain was compared with that of H99 wild strain and PMT4 deficient strain. And the free cells of the H99 wild strain and the PMT4 defective strain are determined by the M27A2 microdilution method of the American CLSI. Their biological membrane cells were tested for drug sensitivity. Salt (M Determination of H99 wild strain and its PMT4 deficient strain by TT method The membrane metabolic activity and the susceptibility of the two strains to the biofilms formed in the in vitro model against the fungal drug were determined, and the drug concentration used in the animal model was determined according to the experimental results of the in vitro model. Results:1. Construction of Cryptococcus neoformans The PMT4 gene-deficient strain of H99. G418 plate culture, the PCR validation of the finally obtained transformant No.12 and the sequencing analysis of its genome-related genes demonstrated the transformation of No.12 The PMT4 gene of the son has been knocked out successfully. An animal model of the bacterial biofilm, and the strain of H99 wild strain and its PMT4 were confirmed by the experiment. The biological membrane can be formed in an in vitro model of a basic culture medium and a 96-well plate and an animal model of a newly constructed intravascular cannula and a catheter built-in polystyrene membrane, .3. Cryptococcus neoformans H99 wild strain and PMT The difference of 4 defective strains in the formation of biofilm. The biofilm and in vitro model of H99 wild strain of Cryptococcus neoformans and its PMT4 deficient strain in animal model The biofilm formed is very similar in the formation process and the general morphology, and is mature after 24 hours, but the latter is in the same period of biofilms. The number of cells and the thickness of the biofilm were smaller than the former. The PMT and the confocal laser scanning microscope were used to observe the PMT. In the same number of cells, the number of colony-forming units of the biofilm cells of the PMT4-deficient strain was significantly less than that of the H99 wild-type biofilm cells.4. The difference of the biofilms formed by the H99 wild strain and the PMT4 deficient strain of the new Cryptococcus neoformans in the anti-fungal drug sensitivity was found. The MIC values (. mu.g/ ml) of the four antifungal agents of AmB,5-FC and ITR for the free cells of the H99 wild strain and the PMT4 defective strain were 4,1,4,1 0.125 and 1, 0.25, 0.5, and 0.125 respectively; the MIC values (. mu.g/ ml) to the biofilm cells were 4,2, 8, 0.125, and 2, 0.125,4, and 0.125.125. The MTT assay of the in vitro model of the biofilm cells found that the four drugs reduced the turbidity by 80% of the minimum drug concentration (. mu.g/ ml) (this indicator is similar to the above-mentioned MI) C鍊，
本文編號(hào)：2479457
[Abstract]:Background of the subject: The problem to be solved in the study of cryptococcosis is that the pathogenesis of cryptococcus and the mechanism of drug resistance are not enough In recent years, the effect of biofilms on the pathogenicity of fungi is becoming more and more important. The study found that the sensitivity of the fungal biofilm to the fungal drug was lower than in the free state. 0-2000 times. So, the biofilm is formed What is the relevant mechanism? What gene effects? Biofilm formation? The study of fungal biofilm is still in the early stage, and also A representative A-type Cryptococcus neoformans H99 MAT-1 strain and P were selected in this subject. The MT4 gene is the subject of the study. The Pmt4 protein is extremely important for the modification of the fungal secretory protein and the partial membrane protein, and the absence of the pmt enzyme activity can lead to a decrease in the growth rate of the strain. Cell wall instability and even death. Published studies have shown that the PMT gene is formed and produced by Candida albicans biofilm The drug resistance mediated by the membrane is of great importance. In view of the similarity of the neoformans and the Candida albicans, we can speculate that the PMT gene is likely to be formed in the biofilms of the neoformans. The protein encoded by this gene is present in the fungus, and the plant cell is not present, and it is similar to the enzyme in the mammalian cell in nature and in a hurry. There is a great difference in the process of research, so it is possible to research and develop the further research. In addition, a practical in vivo model is lacking in the research of cryptococcus biofilms. new-based An animal model of an intravascular cannula. The purpose of this study is to: 1. Construction of PMT4 Gene of Cryptococcus neoformans H99 2. Construction of a new animal model of the new Cryptococcus neoformans biofilm. The differences in biofilm formation of H99 wild and PMT4 strains of Cryptococcus neoformans.4. The observation of Cryptococcus neoformans H99 wild The biofilm formed by the strain and the PMT4 deficient strain is in the form of an antifungal drug, 1. The method of the study:1. Construction of a PMT4 gene-deficient strain of Cryptococcus neoformans H99. The long-flank homologous recombination mediated by PCR was used. The knock-out of the PT4 gene was carried out by the group method, and the H99 strain group was replaced with a G418-resistant gene. The PMT4 gene in the group. The primary screening was used. YPD plate with G418 for positive conversion and the PCR verification is carried out respectively, and the positive conversion sub-channels passed by the PCR are subjected to PCR verification, And 2. establishing a new animal model of the new Cryptococcus neoformans biofilms. An in vitro model was established using a basic medium, a method of incubation in a polystyrene 96-well plate. An animal model was established using a rabbit's external jugular vein and a method of built-in polystyrene film in the catheter. 3. The difference of H99 wild strain and PMT4 deficient strain on the formation of biofilm was observed. The inversion phase-difference microscope was used. And the general morphology and structure of the biofilms were observed by means of a confocal laser scanning microscope. Methods: The cell ratio of the proliferation activity of H99 wild strain and PMT4 deficient strain was compared with that of H99 wild strain and PMT4 deficient strain. And the free cells of the H99 wild strain and the PMT4 defective strain are determined by the M27A2 microdilution method of the American CLSI. Their biological membrane cells were tested for drug sensitivity. Salt (M Determination of H99 wild strain and its PMT4 deficient strain by TT method The membrane metabolic activity and the susceptibility of the two strains to the biofilms formed in the in vitro model against the fungal drug were determined, and the drug concentration used in the animal model was determined according to the experimental results of the in vitro model. Results:1. Construction of Cryptococcus neoformans The PMT4 gene-deficient strain of H99. G418 plate culture, the PCR validation of the finally obtained transformant No.12 and the sequencing analysis of its genome-related genes demonstrated the transformation of No.12 The PMT4 gene of the son has been knocked out successfully. An animal model of the bacterial biofilm, and the strain of H99 wild strain and its PMT4 were confirmed by the experiment. The biological membrane can be formed in an in vitro model of a basic culture medium and a 96-well plate and an animal model of a newly constructed intravascular cannula and a catheter built-in polystyrene membrane, .3. Cryptococcus neoformans H99 wild strain and PMT The difference of 4 defective strains in the formation of biofilm. The biofilm and in vitro model of H99 wild strain of Cryptococcus neoformans and its PMT4 deficient strain in animal model The biofilm formed is very similar in the formation process and the general morphology, and is mature after 24 hours, but the latter is in the same period of biofilms. The number of cells and the thickness of the biofilm were smaller than the former. The PMT and the confocal laser scanning microscope were used to observe the PMT. In the same number of cells, the number of colony-forming units of the biofilm cells of the PMT4-deficient strain was significantly less than that of the H99 wild-type biofilm cells.4. The difference of the biofilms formed by the H99 wild strain and the PMT4 deficient strain of the new Cryptococcus neoformans in the anti-fungal drug sensitivity was found. The MIC values (. mu.g/ ml) of the four antifungal agents of AmB,5-FC and ITR for the free cells of the H99 wild strain and the PMT4 defective strain were 4,1,4,1 0.125 and 1, 0.25, 0.5, and 0.125 respectively; the MIC values (. mu.g/ ml) to the biofilm cells were 4,2, 8, 0.125, and 2, 0.125,4, and 0.125.125. The MTT assay of the in vitro model of the biofilm cells found that the four drugs reduced the turbidity by 80% of the minimum drug concentration (. mu.g/ ml) (this indicator is similar to the above-mentioned MI) C鍊，
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