查尔酮对单增李斯特杆菌以及肺炎链球菌D39分选酶A抑制活性的分子机制研究

发布时间：2018-06-24 19:45

本文选题：单增李斯特杆菌 + 肺炎链球菌　；参考：《吉林大学》2016年博士论文

【摘要】：单核细胞增多性李斯特杆菌(Listeria monocytogenes;LM)简称单增李斯特杆菌,革兰氏阳性,机会致病,是一种重要的人兽共患菌,是李斯特杆菌病的唯一病原菌。单增李斯特杆菌能够引起人和动物的多种疾病,其中主要包括:菌血症、神经系感染以及流产和死胎,而且,其感染致死率很高,能够达到25%左右。近些年来,由于单增李斯特杆菌耐药现象的不断加重,以及新抗生素研发的乏力,极大地限制了对抗单增李斯特杆菌感染的临床治疗手段。革兰氏阳性菌的很多表面蛋白都是通过分选酶的作用被锚定到细胞壁上的,其中,分选酶A(sortase A),能够识别蛋白羧基端一段特定的保守序列:亮氨酸,脯氨酸,X,苏氨酸,甘氨酸(LPXTG,其中X为任意氨基酸),将这段序列从苏氨酸以及甘氨酸之间切割开,然后通过分选作用将其以共价的方式锚定到细胞壁上。很多通过分选作用被锚定到细胞壁上的蛋白,都与细菌与宿主细胞,组织之间的相互作用,以及细菌毒力之间有着密切的联系,包括李斯特杆菌以及肺炎链球菌在内的很多革兰氏阳性菌的sortase A缺失株的致病能力都大大下降了。单增李斯特杆菌在其sortase A酶被敲除以后,位于其表面与其内化侵染机体功能息息相关的蛋白内化素A大量缺失。使得单增李斯特杆菌sortase A蛋白缺失株对靶器官的定植能力以及毒力都有很大程度的下降。这说明,sortase A蛋白的抑制剂是一种非常有潜力的抗感染新药。查尔酮是一种重要的黄酮类化合物,其广泛存在于自然界的植物中,是植物合成黄酮的前体化合物,具有广泛的药理学活性,包括抗真菌、抗病毒、抗HIV以及抗肿瘤活性。本研究通过蛋白结晶的方法成功解析了单增李斯特杆菌sortase A的高分辨率结构,同时利用分子对接的方式阐明了其与特异性底物多肽LPTTG相互作用的分子机制,并通过点突变的方式确定了其三个活性位点。通过体外酶活抑制实验我们发现,查尔酮能够有效地抑制单增李斯特杆菌sortase A的蛋白酶活性。在细胞和细菌共培养体系内加入查尔酮能够显著抑制其对靶细胞的内化侵袭、降低细菌对细胞的细胞毒性作用;在单增李斯特菌小鼠感染模型中,皮下注射查尔酮能够显著降低LM在感染小鼠肝脏和脾脏内的定植数量,通过眼观以及组织病理学观察显示,查尔酮对治疗组小鼠的肝脏和脾脏的病理性变化都有较好的保护作用,通过上述方式,最终提高了LM感染小鼠的存活率。我们进一步应用计算生物学方法、定点残基突变和ITC等实验分析了查尔酮以及LM-srt A的机制。结果表明查尔酮直接作用与LM-srt A的活性通道中,同时封闭了两个关键的活性位点,Cys188以及Agr197使得LM-srt A丧失了其原有的催化活性,定点残基突变和ITC实验验证了上述理论计算结果的准确性。肺炎链球菌(Streptococcus pneumoniae)是一种重要的机会致病菌,通常定植在人类的鼻腔,中耳以及肺部,一旦机体免疫力下降,其就能够感染机体。肺炎链球菌能够引起包括肺炎、脑膜炎、中耳炎和菌血症在内的一系列疾病。每年,有2百万人死于肺炎链球菌的感染。目前临床上治疗肺炎链球菌感染主要还是使用传统的抗生素,然而,从1985年,能够耐受青霉素的肺炎链球菌被发现以后,肺炎链球菌的耐药问题便日趋严重,目前已经分离到能够耐受万古霉素的肺炎链球菌。目前临床上分离到的所有肺炎链球菌都含有分选酶A(Spn-srt A),实验证明Spn-srt A缺失株相比野生型,其在鼻腔中无论定植时间还是定植数量都有显著的下降。这表明Spn-srt A是预防肺炎链球菌感染的理想药物作用靶点。我们希望能够获得一种广谱的分选酶A抑制剂,于是我们又在体外进行了查尔酮对Spn-srt A活性的抑制作用,结果出人意料,尽管Spn-srt A与LM-srt A在氨基酸序列上有超过50%的同源性,但是其对Spn-srt A的抑制活性却远远低于LM-srt A。为了搞清楚上述问题,我们又解析了Spn-srt A的结构,在Spn-srt A的结构中,我们意外的发现了一个目前已知的所有分选酶A结构中都不存在Domain B,而在LM-srt A结构中能够与查尔酮结合的关键位点Cys和Arg,在Spn-srt A结构中全部位于Domain B的loop上,这种结构增大了Cys以及Arg的柔性,同时降低了查尔酮与Spn-srt A的结合能力。使得查尔酮抑制Spn-srt A的活性较LM-srt A大大下降了。中草药在我国有着悠久的历史,是我们传统文化中不可或缺的瑰宝,由于其具有药理活性广,且毒副作用小等诸多优点,使其成为了现代新药研发的重要资源。本研究以LM-srt A为靶标,发现了查尔酮在体外能够抑制LM-srt A蛋白的酶活功能;通过结构学、计算生物学和定点残基突变等诸多方法分析并进一步确证了查尔酮对LM-srt A产生转肽抑制活性的分子机制;同时降低LM对细胞感染的能力;小鼠LM感染模型实验进一步说明了查尔酮能够抵抗LM的感染。综合上述结果,我们的实验结果为阐明查尔酮的抗感染机制研究提供了重要的实验数据,为以LM-srt A为靶标的抗LM感染药物的研发提供了重要的结构基础和先导化合物。同时也通过解析Spn-srt A的结构阐明了查尔酮对Spn-srt A抑制活性急剧下降的原因。也为以Spn-srt A为靶标的抗肺炎链球菌感染的药物提够了重要的结构基础。
[Abstract]:Listeria monocytogenes; LM (LM), known as the single increase of Lester bacilli, gram-positive and opportunistic, is an important zoonotic bacterium and is the only pathogen of Lester bacilli. The monocytic Bacillus monocytogenes can cause a variety of diseases of human and animal, including bacteremia and neurologic infection. As well as abortion and stillbirth, and its infection fatality rate is very high, can reach about 25%. In recent years, due to the increasing resistance to the single increase of Lester bacilli, and the lack of new antibiotic research and development, it has greatly restricted the clinical treatment methods against the infection of the single increase of Lester bacilli. The effect of the excessively selective enzyme is anchored to the cell wall, in which the separation enzyme A (sortase A) can identify a specific conservative sequence of the carboxyl terminus of the protein: leucine, proline, X, threonine, glycine (LPXTG, which X is arbitrary amino acid), cutting this sequence from threonine and glycine, and then by sorting it. The covalent approach is anchored to the cell wall. Many proteins anchored to the cell wall by sorting effect are closely related to the interaction between bacteria and host cells, tissues, and bacterial virulence, including the sortase A deletion strain of many Gram-positive bacteria, including Lester and Streptococcus pneumoniae. The ability of the disease was greatly reduced. After the sortase A enzyme was knocked out of the sortase bacillus, the protein lactoin A, which was located on its surface, was closely related to the function of the internalization of the organism, which resulted in a great decrease in the ability to colonize the target organs and the toxicity of the sortase A protein deletion strain of the Bacillus monocytogenes. The inhibitor of sortase A protein is a very potential new anti infection drug. Chalcone is an important flavone compound. It is widely used in natural plants and is a precursor compound of plant synthetic flavonoids. It has extensive pharmacological activity, including antifungal, antiviral, anti HIV and anti-tumor activity. The high resolution structure of Lester sortase A was successfully analyzed by the method of protein crystallization. At the same time, the molecular mechanism of its interaction with the specific substrate polypeptide LPTTG was elucidated by molecular docking, and three active sites were determined by point mutation. Ketone can effectively inhibit the protease activity of the sortase A sortase. The addition of chalcone in the cell and bacterial co culture system can significantly inhibit the invasion of the target cells and reduce the cytotoxicity of the bacteria to the cells. In the mouse infection model of the single increasing Lester strain mice, the hypodermic injection of chalcone can significantly reduce the L The number of colonization of M in the liver and spleen of mice showed that chalcone had a good protective effect on the pathological changes of the liver and spleen of the mice in the treatment group by ophthalmic and histopathological observation. Through the above methods, the survival rate of LM infected mice was improved. We further applied computational biology methods and fixed points. The mechanism of chalcone and LM-srt A were analyzed by the residues mutation and ITC. The results showed that the direct action of chalcone and the active channel of LM-srt A closed two key active sites. Cys188 and Agr197 made LM-srt A lost its original catalytic activity. The theoretical calculation was verified by the fixed point residue mutation and ITC experiment. The accuracy of the results. Streptococcus pneumoniae (Streptococcus pneumoniae) is an important opportunistic pathogen, usually fixed in the human nasal cavity, the middle ear and the lungs. Once the body's immunity decreases, it can infect the body. Streptococcus pneumoniae can cause a series of diseases including pneumonia, meningitis, otitis media and bacteremia. 2 million people died from Streptococcus pneumoniae infection. The main clinical treatment of Streptococcus pneumoniae infection is the use of traditional antibiotics. However, after the discovery of penicillin resistant Streptococcus pneumoniae in 1985, the problem of drug resistance of Streptococcus pneumoniae is becoming more and more serious, and it is now separated to the pneumonia that can tolerate vancomycin. Streptococcus pneumoniae. All Streptococcus pneumoniae isolated from the clinic currently contain A (Spn-srt A). Experiments have shown that the Spn-srt A deletion plant has a significant decrease in both colonization and colonization in the nasal cavity. This indicates that Spn-srt A is an ideal drug target for the prevention of Streptococcus pneumoniae infection. We hope that Spn-srt A can be used as an ideal target for the prevention of Streptococcus pneumoniae infection. In order to obtain a broad-spectrum selective enzyme A inhibitor, we also carried out the inhibitory effect of chalcone on the activity of Spn-srt A in vitro. The results were unexpected. Although Spn-srt A and LM-srt A have more than 50% homology on the amino acid sequence, the inhibitory activity to Spn-srt A is far lower than LM-srt A. to clarify the above problems. We also parsed the structure of Spn-srt A. In the structure of Spn-srt A, we accidentally discovered that there is no Domain B in all of the A structures known now, and the key loci of binding to chalcone in the A structure of LM-srt, Cys and Arg on the Spn-srt structure The flexibility of YS and Arg reduces the binding ability of chalcone and Spn-srt A. The activity of chalcone to inhibit Spn-srt A is greatly decreased than that of LM-srt A. Chinese herbal medicine has a long history in our country. It is an indispensable treasure in our traditional culture. It has many advantages, such as extensive pharmacological activity, and small side effects, and so on. For the important resources of modern drug development, this study uses LM-srt A as a target to discover the enzyme activity of chalcone in vitro that inhibits the LM-srt A protein, and further confirms the molecular mechanism of the inhibitory activity of chalcone on LM-srt A by a number of methods of structure, computational biology and site site mutagenesis. Our experimental results provide important experimental data for the study of the anti infection mechanism of chalcone, which provides important structure for the research and development of anti LM infection drugs with LM-srt A as the target of LM. The basic and pilot compounds are also explained by the analysis of the structure of Spn-srt A. The cause of the sharp decline in the inhibitory activity of chalcone to Spn-srt A is also explained. It also provides an important structural basis for the drug resistant to Streptococcus pneumoniae with Spn-srt A as the target.
【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S852.61

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