【作者】 张旭；

【导师】 孙宝林；

【作者基本信息】 中国科学技术大学 ， 微生物学， 2015， 博士

【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。更多还原

【Abstract】 Staphylococcus aureus is a gram-positive and opportunistic human pathogen, which is widely distributed in the environment. The bacterium has become a major clinical pathogen and causes millions of infectious cases in hospital every year. S. aureus can cause a variety of infections ranging from localized skin infections to life-threatening systemic infections. The infection ability of S. aureus mainly depends on the production of many virulence factors, in which a-toxin is a notorious one. All these virulence factors are under control of different regulatory pathways, including two-component systems, transcriptional regulator proteins, and non-coding RNAs. Recent studies have pointed to the importance of small-noncoding RNAs (sRNAs) in bacterial virulence control.Here, we report the identification and functional analyses of a novel sRNA, ArtR, which is overlapped with luxS gene on the opposite strand. The full length of ArtR is345nt, as determined by RACE experiment. Our data showed that the AgrA protein can bind to the artR promoter and repress artR transcription, suggesting that, besides RNAIII, ArtR is the second sRNA regulated by AgrA. Therefore, we designated it as ArtR (AgrA-repressed and toxin regulating sRNA). Furthermore, ArtR is unique in S. aureus and involved in virulence regulation by activating a-toxin expression. ArtR can directly bind to the5’untranslated region of the sarT mRNA, thus promoting the degradation of sarT mRNA by RNase III and arresting the translation of SarT. This suggests that the activation of ArtR on the a-toxin is through SarT. This study reveals another kind of staphylococcal regulatory small RNA that plays a role in virulence control. It also indicates the diversity of small RNA-target mRNA interactions and how these multiple interactions can mediate virulence regulation in S. aureus.RNA regulation may involves proteins such as RNase, RNA chaperones and those proteins targeted by sRNA. Since it is difficult to identify RNA-binding proteins (RBPs), very little was known about the RBPs in S. aureus, especially those associated with sRNAs. Here we adopted a tRNA scaffold streptavidin aptamer based pull-down assay to identify RBPs in S. aureus. The tethered RNA was successfully captured by the streptavidin magnetic beads, and proteins binding to RNAIII were isolated and analyzed by mass spectrometry. We have identified81proteins, and expressed heterologously9of them in Escherichia coli. The binding ability of the recombinant proteins with RNAIII was further analyzed by electrophoresis mobility shift assay. RNase III has an RNA-binding motif, and exhibited RNA degradation activity. CshA, RNase J2, and Era also have RNA-binding motifs and showed binding ability with RNAIII. Hu and WalR have DNA-binding domain, and also showed RNAIII binding ability. Surprisingly, Pyk and FtsZ, without RNA/DNA-binding domains, also had binding activity with RNAIII. PfkA did not bind to RNAIII even at high concentration. PNPase and Enolase were suggested to be the components of RNA degradosome, but they were not found in our pull-down assay and they did not bind to RNAIII as expected. This study suggests that some proteins can bind to RNAIII in S. aureus, and may be involved in RNAIII functions. And tRSA based pull-down assay is an effective method to search for RBPs in bacteria, and this method should facilitate the identification and functional study of RBPs in diverse bacterial species.更多还原