金黄色葡萄球菌ArtR的功能研究及RNAIII结合蛋白的鉴定

发布时间：2018-05-18 14:23

本文选题：金黄色葡萄球菌 + 非编码RNA　；参考：《中国科学技术大学》2015年博士论文

【摘要】：金黄色葡萄球菌是典型的革兰氏阳性条件致病菌,在环境中分布广泛。近年来,金黄色葡萄球菌已成为主要的临床感染菌,医院内每年都会有数以百万的金黄色葡萄球菌感染发生。金黄色葡萄球菌感染能够引发的症状有食物中毒、溃疡、心内膜炎、骨髓炎、肺炎等,甚至引起败血性休克而导致死亡。金黄色葡萄球菌之所以能引起如此多的病症,是因为它能分泌多种致病因子,其中a溶血素是非常重要的毒素之一。在感染过程中,这些毒性因子受到了广泛而精细的调控,从而帮助细菌响应和适应各种复杂的环境。这些调控因子包括二元信号系统、转录调控蛋白和非编码RNA等。我们在前期研究LuxS/AI-2信号系统时,发现了一个与luxS反向重复的sRNA。Northern blot和RACE分析证实,这个sRNA全长为345nt,并将其命名为ArtR。序列分析和RT-PCR等实验表明,ArtR是金黄色葡萄球菌中所特有的一种sRNA,它的表达受到Agr群体感应系统的抑制。但是这种抑制效应不是通过Agr群体感应系统常用的RNAIII来实现,而是直接通过AgrA蛋白与ArtR的启动子相互作用来实现。基因敲除实验发现,ArtR的缺失影响了金黄色葡萄球菌α溶血素的水平。而凝胶阻滞分析实验证明,ArtR并不能直接结合α溶血素的mRNA。于是我们寻找了a溶血素相关的转录因子,发现ArtR突变株中α溶血素的抑制因子SarT的水平有所升高。通过凝胶阻滞分析和RNA酶解等实验我们证实ArtR通过与sarT5'UTR上的一段区域与相互作用,促进了RNase Ⅲ对sarT mRNA的降解。这项研究鉴定了金黄色葡萄球菌属中一个新的毒性相关的sRNA,并且阐明它是通过与转录因子mRNA相互作用来实现其调控过程的。 sRNA调控不仅局限于sRNA与靶RNA的相互作用,可能有很多蛋白质参与其中。但是由于技术的限制,对金黄色葡萄球菌中sRNA相关的蛋白质知之甚少。我们通过前期的研究工作成功建立了利用tRNA融合RNA适配体(简称tRSA)进行sRNA蛋白质pull-down的实验体系,选取了金黄色葡萄球菌中重要的sRNA——RNAIII,对其结合蛋白进行钓取。实验发现tRSA标记的RNA能够较好被链霉亲和素磁珠捕获,用这个体系成功钓取了81个能够结合RNAⅢ的候选蛋白质。我们进一步通过蛋白质表达、凝胶阻滞分析验证了这些候选蛋白与RNAⅢ的结合能力。选取的9个蛋白质中,RNase Ⅲ对RNAⅢ具有降解活性,7个蛋白(CshA、RNase J2、Era、Hu、WalR、PyK、FtsZ)可以结合RNAⅢ,PflcA没有检测出结合能力。同时选取了两个pull-down未鉴定出的,但属于RNA降解体的蛋白,发现它们并不能够直接结合RNAⅢ。该实验表明金黄色葡萄球菌中可能有很多蛋白质结合RNAⅢ,并有可能参与它的调控过程；tRSA的体系能够很好的运用于金黄色葡萄球菌sRNA结合蛋白的pull-down实验,并且成功鉴定出部分RNAⅢ的结合蛋白质。深入研究这些蛋白质对于sRNA调控以及金黄色葡萄球菌致病性调控的作用,能够深化我们对细菌sRNA调控机制和复杂网络的认识。
[Abstract]:Staphylococcus aureus is a typical gram positive condition pathogenic bacteria and is widely distributed in the environment. In recent years, Staphylococcus aureus has become the main clinical infection bacteria. In hospitals, millions of Staphylococcus aureus infection occurs every year in hospitals. The symptoms of Staphylococcus aureus can be caused by food poisoning and ulcers. Endocarditis, osteomyelitis, pneumonia, and even septic shock cause death. Staphylococcus aureus can cause so many diseases because it can secrete a variety of pathogenic factors, in which a hemolysin is one of the most important toxins. In the process of infection, these toxic factors are widely and finely regulated. It helps bacteria respond to and adapt to various complex environments. These regulatory factors include two element signal system, transcriptional regulatory protein and non coding RNA.
In our previous study of the LuxS/AI-2 signal system, we found a sRNA.Northern blot and RACE analysis that repeats the reverse of luxS. This sRNA full length is 345nt, and it is named ArtR. sequence analysis and RT-PCR and other experiments show that ArtR is a kind of sRNA in Staphylococcus aureus, and its expression is subjected to the Agr population induction system. But this inhibitory effect was not realized by the common RNAIII of the Agr quorum sensing system, but was realized directly through the interaction of the AgrA protein with the promoter of ArtR. The gene knockout experiment found that the deletion of ArtR affected the level of the alpha hemolysin of Staphylococcus aureus. And the gel block analysis proved that ArtR was not. Directly combining mRNA. with alpha hemolysin we searched for the transcription factors associated with a hemolysin, and found that the level of the inhibitory factor SarT of alpha hemolysin in the ArtR mutant was elevated. Through gel block analysis and RNA enzymolysis experiments, we confirmed that ArtR has promoted RNase III to sarT mR through the interaction with a segment of sarT5'UTR. The degradation of NA. This study identified a new toxic related sRNA in the genus Staphylococcus and clarified that it was regulated by interaction with the transcription factor mRNA.
SRNA regulation is not only limited to the interaction between sRNA and target RNA, and there may be a lot of proteins involved. But because of the limitations of technology, there is little knowledge about sRNA related proteins in Staphylococcus aureus. We successfully established a tRNA fusion RNA aptamer (tRSA) for sRNA protein pull-down through previous research work. The experimental system selected the important sRNA - RNAIII in Staphylococcus aureus to catch the binding protein. The experiment found that the tRSA labeled RNA could be better captured by the streptomycin magnetic beads, and 81 candidate proteins that could be combined with RNA III were caught with this system. We further through protein expression, gel block The binding ability of these candidate proteins to RNA III was verified. Of the 9 proteins selected, RNase III had biodegradation activity to RNA III, and 7 proteins (CshA, RNase J2, Era, Hu, WalR, PyK, FtsZ) could be combined with RNA III and did not detect the binding ability. At the same time, two proteins were not identified, but the proteins belonging to the descending disaggregation were found. They are not able to combine RNA III directly. This experiment shows that there may be a lot of proteins associated with RNA III in Staphylococcus aureus and may be involved in its regulatory process. The system of tRSA can be well used in the pull-down experiment of Staphylococcus aureus sRNA binding protein and successfully identified some of the binding proteins of RNA III. A thorough study of the effects of these proteins on sRNA regulation and the pathogenicity of Staphylococcus aureus can deepen our understanding of the regulatory mechanism of bacterial sRNA and the complex network.
【学位授予单位】：中国科学技术大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R378.11

【共引文献】