PMT4在新生隐球菌生物膜形成及其耐药中的作用
发布时间：2019-05-17 22:39
【摘要】： 选题背景: 在隐球菌病的研究中亟待解决的问题是对隐球菌的致病机制和耐药机制还缺乏足够的了解。近年来生物膜(biofilm)对真菌致病力的作用日益为人们所重视。 研究发现真菌生物膜对抗真菌药物的敏感性比游离状态下低30-2000倍。那么,生物膜形成的相关机制是什么?有哪些基因影响了生物膜的形成?真菌生物膜的研究还处于起步阶段,还有大量未解之谜。 本课题选取了有代表性的A血清型新生隐球菌H99 MATα型菌株和PMT4基因作为研究对象。Pmt4蛋白对于真菌分泌蛋白和部分膜蛋白的修饰极其重要,缺乏pmt酶活性可导致菌株生长率下降、细胞壁不稳定甚至死亡。已发表的研究显示,PMT基因对白念珠菌生物膜的形成和生物膜介导的耐药至关重要。鉴于新生隐球菌和白念珠菌的诸多相似性,我们可以推测,PMT基因很可能在新生隐球菌的生物膜形成及其介导的耐药上起重要作用。由于这一基因编码的蛋白存在于真菌内,植物细胞则无,它与哺乳动物细胞内的类似酶在性质和催化作用上有着很大的不同。因此对其的进一步研究也有可能为研发新的抗真菌药物提供靶点。 另外,在隐球菌生物膜的研究中缺乏实用的体内模型,因此本课题除采用已有的体外模型外,还构建了一种新的基于血管内插管的动物模型。 研究目的: 1.构建新生隐球菌H99的PMT4基因缺陷株。 2.构建新的新生隐球菌生物膜的动物模型。 3.观察新生隐球菌H99野生株和PMT4缺陷株在生物膜形成方面的差异。 4.观察新生隐球菌H99野生株和PMT4缺陷株所形成的生物膜在抗真菌药物敏感性方面的差异。 研究方法: 1.构建新生隐球菌H99的PMT4基因缺陷株。 使用PCR介导的长侧翼同源重组法来进行PMT4基因的敲除工作,用一个G418抗性基因替换掉H99菌株基因组中的PMT4基因。 初步筛选采用含G418的YPD平板进行,所得阳性转化子分别进行PCR验证。 PCR验证通过的阳性转化子经基因测序验证。 2.构建新的新生隐球菌生物膜的动物模型。 采用基础培养基、聚苯乙烯96孔板内孵育的方法建立体外模型。 采用兔颈外静脉插管并在导管内置聚苯乙烯薄膜的方法建立动物模型。 3.观察新生隐球菌H99野生株和PMT4缺陷株在生物膜形成方面的差异。 采用倒置相差显微镜和共聚集激光扫描显微镜观察生物膜的大体形态和结构。 采用比较集落形成单位(CFU)数量的方法比较H99野生株和PMT4缺陷株生物膜中有增殖活性的细胞比例。 4.观察新生隐球菌H99野生株和PMT4缺陷株所形成的生物膜在抗真菌药物敏感性方面的差异。 用美国CLSI的M27A2微量稀释法对H99野生株和PMT4缺陷株游离细胞以及它们的生物膜细胞进行药敏实验。 用四甲基偶氮唑盐(MTT)法测定H99野生株及其PMT4缺陷株生物膜代谢活性,并以此反应这两种菌株在体内外模型中形成的生物膜对抗真菌药物的敏感性。根据体外模型的实验结果决定动物模型中采用的药物浓度。 结果: 1.构建新生隐球菌H99的PMT4基因缺陷株。 G418平板培养、对最终获得的12号转化子的PCR验证以及对其基因组相关基因的测序分析均证明12号转化子的PMT4基因已被成功敲除。 2.构建新的新生隐球菌生物膜的动物模型。 实验证实H99野生株及其PMT4缺陷株均可在基础培养基加96孔板的体外模型和新构建的血管内插管加导管内置聚苯乙烯膜的动物模型中形成生物膜。证实本课题采用的体外模型和新的动物模型可用于生物膜的研究。 3.新生隐球菌H99野生株和PMT4缺陷株在生物膜形成方面的差异。 新生隐球菌H99野生株及其PMT4缺陷株在动物模型中形成的生物膜与体外模型中所形成的生物膜在形成过程和大体形态方面非常相似,也是在24小时后开始成熟。但后者同时期生物膜的细胞数量和生物膜厚度比前者小。 用倒置显微镜和共聚集激光扫描显微镜均观察到PMT4缺陷株所形成的生物膜在接种24小时后出现大量类似假菌丝样的结构。 在同等数量的接种细胞中,PMT4缺陷株生物膜细胞的集落形成单位数明显少于H99野生株生物膜细胞。 4.新生隐球菌H99野生株和PMT4缺陷株所形成的生物膜在抗真菌药物敏感性方面的差异。 M27A2微量稀释法药敏实验发现FCZ、AmB、5-FC、ITR四种抗真菌药物对H99野生株及其PMT4缺陷株游离细胞的MIC值(μg/ml)分别是4、1、4、≤0.125和1、0.25、0.5、≤0.125;对生物膜细胞的MIC值(μg/ml)分别是4、2、8、≤0.125和2、0.125、4、≤0.125。 体外模型生物膜细胞的MTT检测发现,这四种药物使浊度下降80%所需的最低药物浓度(μg/ml)(这一指标类似上述MIC值)在H99野生株生物膜和PMT4缺陷株生物膜中分别是≥64、32、≥64、16和2、0.5、4、1。 动物模型生物膜细胞的MTT检测发现,在64μg/ml FCZ作用下,与各自的空白对照相比,PMT4缺陷株生物膜细胞的代谢活性抑制率为96.7%,H99野生株生物膜细胞为8.7%。 结论: 通过PCR验证和基因测序证实12号转化子中的PMT4基因确实被敲除了,这一PMT4缺陷株,可用于对PMT4基因进一步的研究。首次利用兔中心静脉插管的方式成功构建了隐球菌血管内插管的动物模型,可作为研究平台用于模拟临床最为常见的植入物表面生物膜形成的情况,对于隐球菌生物膜的研究有着重要意义。 首次采用导管内置入聚苯乙烯薄膜的方法构建动物模型,导管内覆的聚苯乙烯薄膜可取出,避免了导管内生物膜体内模型只能在共聚集激光扫描显微镜下利用Z轴成像的方式进行观察的弊端,使得导管内生物膜也可直接在普通光镜下观察。 实验结果显示无论是H99野生株还是PMT4缺陷株所形成的生物膜均在24小时后进入成熟期。接种48小时后进入相对的稳定期。PMT4缺陷株各时期生物膜的细胞量均少于H99野生株。 另外发现成熟的PMT4缺陷株生物膜中具有增殖活性的细胞的比例小于同时期的野生株生物膜。这意味着PMT4缺陷株形成的生物膜向外释放菌细胞、扩大感染范围的能力弱于野生株。 在生物膜结构方面,相较于H99野生株生物膜,PMT4缺陷株生物膜的一个显著特点是进入成熟期后产生具有大量的假菌丝样结构。这可能与PMT4变异株的细胞壁缺陷有关,但为何进入成熟期才出现尚不清楚。 药敏实验显示,PMT4缺陷株游离细胞对抗真菌药物的敏感性比野生株稍高,这可能也与其细胞壁的缺陷有关。对隐球菌生物膜细胞进行的M27A2药敏显示其对药物的敏感性与游离细胞差异不大,可能是由于生物膜细胞在从生物膜中分离出来在营养丰富的培养基中长时间培养又逐渐恢复了游离细胞生物学特性的缘故。由此可见,将生物膜细胞分离出来成为悬浮细胞后再进行常规M27A2药敏实验并不能反映生物膜本身对抗真菌药物的真实敏感性。 体内外模型生物膜细胞的MTT检测均表明,H99野生株生物膜细胞比PMT4缺陷株生物膜细胞对抗真菌药物的抵抗能力强。在体外模型中, PMT4缺陷株生物膜细胞对不同的抗真菌药物的敏感性比野生株高16-64倍。在体内模型中,同等药物浓度下,PMT4缺陷株生物膜细胞的代谢活性几乎被完全抑制,而野生株生物膜细胞却不受影响。 这些结果提示PMT4基因对于新生隐球菌生物膜的耐药特性是至关重要的,PMT4基因的缺失可导致新生隐球菌生物膜耐药性几乎完全丧失。由于真菌的PMT4基因与哺乳动物的类似基因差异很大,因此PMT4基因及其编码的酶有可能成为新的抗真菌药物的作用靶点。
[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，
本文编号：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，
本文编号：2479457
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