發(fā)布時間：2018-06-24 19:45

  本文選題：單增李斯特桿菌 + 肺炎鏈球菌��； 參考：《吉林大學(xué)》2016年博士論文

【摘要】：單核細(xì)胞增多性李斯特桿菌(Listeria monocytogenes;LM)簡稱單增李斯特桿菌,革蘭氏陽性,機會致病,是一種重要的人獸共患菌,是李斯特桿菌病的唯一病原菌。單增李斯特桿菌能夠引起人和動物的多種疾病,其中主要包括:菌血癥、神經(jīng)系感染以及流產(chǎn)和死胎,而且,其感染致死率很高,能夠達(dá)到25%左右。近些年來,由于單增李斯特桿菌耐藥現(xiàn)象的不斷加重,以及新抗生素研發(fā)的乏力,極大地限制了對抗單增李斯特桿菌感染的臨床治療手段。革蘭氏陽性菌的很多表面蛋白都是通過分選酶的作用被錨定到細(xì)胞壁上的,其中,分選酶A(sortase A),能夠識別蛋白羧基端一段特定的保守序列:亮氨酸,脯氨酸,X,蘇氨酸,甘氨酸(LPXTG,其中X為任意氨基酸),將這段序列從蘇氨酸以及甘氨酸之間切割開,然后通過分選作用將其以共價的方式錨定到細(xì)胞壁上。很多通過分選作用被錨定到細(xì)胞壁上的蛋白,都與細(xì)菌與宿主細(xì)胞,組織之間的相互作用,以及細(xì)菌毒力之間有著密切的聯(lián)系,包括李斯特桿菌以及肺炎鏈球菌在內(nèi)的很多革蘭氏陽性菌的sortase A缺失株的致病能力都大大下降了。單增李斯特桿菌在其sortase A酶被敲除以后,位于其表面與其內(nèi)化侵染機體功能息息相關(guān)的蛋白內(nèi)化素A大量缺失。使得單增李斯特桿菌sortase A蛋白缺失株對靶器官的定植能力以及毒力都有很大程度的下降。這說明,sortase A蛋白的抑制劑是一種非常有潛力的抗感染新藥。查爾酮是一種重要的黃酮類化合物,其廣泛存在于自然界的植物中,是植物合成黃酮的前體化合物,具有廣泛的藥理學(xué)活性,包括抗真菌、抗病毒、抗HIV以及抗腫瘤活性。本研究通過蛋白結(jié)晶的方法成功解析了單增李斯特桿菌sortase A的高分辨率結(jié)構(gòu),同時利用分子對接的方式闡明了其與特異性底物多肽LPTTG相互作用的分子機制,并通過點突變的方式確定了其三個活性位點。通過體外酶活抑制實驗我們發(fā)現(xiàn),查爾酮能夠有效地抑制單增李斯特桿菌sortase A的蛋白酶活性。在細(xì)胞和細(xì)菌共培養(yǎng)體系內(nèi)加入查爾酮能夠顯著抑制其對靶細(xì)胞的內(nèi)化侵襲、降低細(xì)菌對細(xì)胞的細(xì)胞毒性作用;在單增李斯特菌小鼠感染模型中,皮下注射查爾酮能夠顯著降低LM在感染小鼠肝臟和脾臟內(nèi)的定植數(shù)量,通過眼觀以及組織病理學(xué)觀察顯示,查爾酮對治療組小鼠的肝臟和脾臟的病理性變化都有較好的保護(hù)作用,通過上述方式,最終提高了LM感染小鼠的存活率。我們進(jìn)一步應(yīng)用計算生物學(xué)方法、定點殘基突變和ITC等實驗分析了查爾酮以及LM-srt A的機制。結(jié)果表明查爾酮直接作用與LM-srt A的活性通道中,同時封閉了兩個關(guān)鍵的活性位點,Cys188以及Agr197使得LM-srt A喪失了其原有的催化活性,定點殘基突變和ITC實驗驗證了上述理論計算結(jié)果的準(zhǔn)確性。肺炎鏈球菌(Streptococcus pneumoniae)是一種重要的機會致病菌,通常定植在人類的鼻腔,中耳以及肺部,一旦機體免疫力下降,其就能夠感染機體。肺炎鏈球菌能夠引起包括肺炎、腦膜炎、中耳炎和菌血癥在內(nèi)的一系列疾病。每年,有2百萬人死于肺炎鏈球菌的感染。目前臨床上治療肺炎鏈球菌感染主要還是使用傳統(tǒng)的抗生素,然而,從1985年,能夠耐受青霉素的肺炎鏈球菌被發(fā)現(xiàn)以后,肺炎鏈球菌的耐藥問題便日趨嚴(yán)重,目前已經(jīng)分離到能夠耐受萬古霉素的肺炎鏈球菌。目前臨床上分離到的所有肺炎鏈球菌都含有分選酶A(Spn-srt A),實驗證明Spn-srt A缺失株相比野生型,其在鼻腔中無論定植時間還是定植數(shù)量都有顯著的下降。這表明Spn-srt A是預(yù)防肺炎鏈球菌感染的理想藥物作用靶點。我們希望能夠獲得一種廣譜的分選酶A抑制劑,于是我們又在體外進(jìn)行了查爾酮對Spn-srt A活性的抑制作用,結(jié)果出人意料,盡管Spn-srt A與LM-srt A在氨基酸序列上有超過50%的同源性,但是其對Spn-srt A的抑制活性卻遠(yuǎn)遠(yuǎn)低于LM-srt A。為了搞清楚上述問題,我們又解析了Spn-srt A的結(jié)構(gòu),在Spn-srt A的結(jié)構(gòu)中,我們意外的發(fā)現(xiàn)了一個目前已知的所有分選酶A結(jié)構(gòu)中都不存在Domain B,而在LM-srt A結(jié)構(gòu)中能夠與查爾酮結(jié)合的關(guān)鍵位點Cys和Arg,在Spn-srt A結(jié)構(gòu)中全部位于Domain B的loop上,這種結(jié)構(gòu)增大了Cys以及Arg的柔性,同時降低了查爾酮與Spn-srt A的結(jié)合能力。使得查爾酮抑制Spn-srt A的活性較LM-srt A大大下降了。中草藥在我國有著悠久的歷史,是我們傳統(tǒng)文化中不可或缺的瑰寶,由于其具有藥理活性廣,且毒副作用小等諸多優(yōu)點,使其成為了現(xiàn)代新藥研發(fā)的重要資源。本研究以LM-srt A為靶標(biāo),發(fā)現(xiàn)了查爾酮在體外能夠抑制LM-srt A蛋白的酶活功能;通過結(jié)構(gòu)學(xué)、計算生物學(xué)和定點殘基突變等諸多方法分析并進(jìn)一步確證了查爾酮對LM-srt A產(chǎn)生轉(zhuǎn)肽抑制活性的分子機制;同時降低LM對細(xì)胞感染的能力;小鼠LM感染模型實驗進(jìn)一步說明了查爾酮能夠抵抗LM的感染。綜合上述結(jié)果,我們的實驗結(jié)果為闡明查爾酮的抗感染機制研究提供了重要的實驗數(shù)據(jù),為以LM-srt A為靶標(biāo)的抗LM感染藥物的研發(fā)提供了重要的結(jié)構(gòu)基礎(chǔ)和先導(dǎo)化合物。同時也通過解析Spn-srt A的結(jié)構(gòu)闡明了查爾酮對Spn-srt A抑制活性急劇下降的原因。也為以Spn-srt A為靶標(biāo)的抗肺炎鏈球菌感染的藥物提夠了重要的結(jié)構(gòu)基礎(chǔ)。
[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.
【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：S852.61

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