新生隐球菌外分泌蛋白致病机制及新型药物抗真菌作用研究

发布时间：2018-12-11 02:27

【摘要】：研究目的新生隐球菌是临床最常见的侵袭性病原真菌之一,可附着于活体植入材料表面形成生物膜结构,从而显著增强其对抗真菌药物的耐药性。本研究第一部分筛选鉴定了新生隐球菌生物膜态的3个外分泌差异蛋白,并初步论证其对隐球菌生物膜形成及毒力的功能影响。第二部分,我们将隐球菌金属蛋白酶Cn Mpr1在酿酒酵母细胞中表达后发现,和野生株比,Cn MPR1表达株ScCn MPR1获得了穿越体外血脑屏障模型的能力,说明Cn Mpr1可以独立于透明质酸-CD44等其他途径单独在隐球菌穿越血脑屏障中其重要作用。在本部分研究中,我们拟利用蛋白质组学技术系统分析脑微血管内皮细胞潜在的与Cn Mpr1相互作用的受体蛋白。在第三部分的研究中,我们合成了不同直径的银包钯纳米片以检测它们对各种主要侵袭性致病真菌的抑制作用。研究方法第一部分利用同源重组原理和基因枪技术构建各差异蛋白的基因缺失株,结合生物膜表型检测及细胞、动物实验探索其对隐球菌生物膜形成与毒力的影响。第二部分我们利用免疫磁珠法提取h CMEC/D3细胞表面蛋白,通过与ScCn MPR1(野生株作对照)共同孵育后提取蛋白进行MALDI-TOF-MS质谱检测与分析。第三部分我们首先利用微量稀释法测试不同直径的银包钯纳米片对各种深部致病真菌的抗真菌活性。随后我们就银包钯纳米片对新生隐球菌的抑菌作用及其作用机制进行探究。结果第一部分中,体外生物膜检测发现,较之标准株,vid△表现出显著的生物膜形成能力的下降,同时其在巨噬细胞内的生存率及对小鼠吸入感染模型的毒力显著减弱。第二部分蛋白质组学研究中,我们共发现79个脑微血管内皮细胞蛋白与ScCn MPR1的结合能力增强。第三部分我们成功合成了不同直径的银包钯纳米片,发现其对主要深部致病真菌特别是新生隐球菌具有明显的抑制作用。银包钯纳米片的抗真菌作用明显优于氟康唑,且接近两性霉素B,而其溶血作用远小于两性霉素B。银包钯纳米片和两性霉素B具有一定的联合杀菌作用。新生隐球菌和银包钯纳米片相互作用后,细胞膜的完整性遭到破坏,通透性明显增加。结论一、我们的研究首次发现外分泌蛋白Vid对新生隐球菌的生物膜形成以及致病感染具有重要的调节功能。二、与ScCn MPR1相互作用的宿主蛋白质中,将近三分之一的蛋白质与细胞骨架和膜重排相关;分别有16%的蛋白质与代谢过程以及蛋白质合成有关;以及其他一些与细胞运输、免疫、蛋白质折叠和细胞信号转导相关的蛋白质。我们推测这些蛋白在隐球菌穿越血脑屏障过程以及与Mpr1的相互作用中起着重要作用。三、我们的研究首次发现银包钯纳米片具有抗新生隐球菌作用,其作用机制可能与破坏细胞膜通透性,打乱细胞内蛋白合成与能量代谢有关。
[Abstract]:Objective Cryptococcus neoformans (Cryptococcus neoformans) is one of the most common invasive fungi in clinic. In the first part of this study, three exocrine differentially secreted proteins of Cryptococcus neoformans biofilm state were identified, and their effects on biofilm formation and virulence of Cryptococcus neoformans were preliminarily demonstrated. In the second part, we expressed Cryptococcus metalloproteinase Cn Mpr1 in Saccharomyces cerevisiae cells and found that compared with the wild strain, ScCn MPR1 obtained the ability to cross the blood-brain barrier model in vitro. The results suggest that Cn Mpr1 can play an important role in Cryptococcus Blood-brain Barrier independently of other pathways such as hyaluronic acid-CD44. In this part of the study, we intend to use proteomics techniques to analyze the potential receptor proteins interacting with Cn Mpr1 in brain microvascular endothelial cells. In the third part, we synthesized silver coated palladium nanoparticles with different diameters to detect their inhibitory effects on various major invasive fungi. Methods in the first part, the homologous recombination principle and gene gun technique were used to construct the gene deletion strains of different proteins. Combined with biofilm phenotypic detection and cell, animal experiments were conducted to explore its effect on the biofilm formation and virulence of Cryptococcus. In the second part, the surface proteins of h CMEC/D3 cells were extracted by immunomagnetic bead method. The extracted proteins were detected and analyzed by MALDI-TOF-MS mass spectrometry after incubation with ScCn MPR1 (wild strain as control). In the third part, the antifungal activity of silver coated palladium nanoparticles with different diameters against various deep pathogenic fungi was tested by microdilution method. Then we studied the bacteriostatic effect and mechanism of silver-coated palladium nanoparticles on Cryptococcus neoformans. Results in the first part, in vitro biofilm assay showed that vid showed a significant decrease in biofilm formation, and its survival rate in macrophages and its toxicity to mouse model of inhalation infection were significantly decreased compared with the standard strain. In the second part of proteomics, we found that 79 brain microvascular endothelial cell proteins had enhanced binding ability to ScCn MPR1. In the third part, we successfully synthesized silver coated palladium nanoparticles with different diameters, and found that they had obvious inhibitory effects on the main deep pathogenic fungi, especially Cryptococcus neoformans (Cryptococcus neoformans). The antifungal effect of silver coated palladium nanoparticles was better than fluconazole and was close to amphotericin B, but its hemolytic effect was much less than amphotericin B. Silver-coated palladium nanoparticles and amphotericin B have a combined bactericidal effect. After interaction between Cryptococcus neoformans and silver coated palladium nanoparticles, the integrity of cell membrane was destroyed and the permeability increased significantly. Conclusion: for the first time, we found that exocrine protein Vid plays an important role in regulating biofilm formation and pathogenic infection of Cryptococcus neoformans. Secondly, nearly 1/3 of the host proteins interacting with ScCn MPR1 were related to cytoskeleton and membrane rearrangement, 16% of proteins were related to metabolic process and protein synthesis, respectively. And other proteins related to cell transport, immunity, protein folding, and cell signal transduction. We speculate that these proteins play an important role in Cryptococcus Blood-Brain Barrier and interaction with Mpr1. Third, we first found that silver coated palladium nanoparticles have anti-Cryptococcus neoformans action mechanism may be related to the destruction of cell membrane permeability, disrupting intracellular protein synthesis and energy metabolism.
【学位授予单位】：第二军医大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R379.5

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