Functional characterization of two acid-regulated small RNAs in Helicobacter pylori

小分子调控RNA（Small regulatory RNAs，sRNA）是一类重要的转录后基因表达调控因子，在细菌的胁迫反应或毒力过程中起重要作用。特别是在肠杆菌中的研究表明，sRNA可以调控多个靶基因，并代表代谢网络中的关键调控因子。此外，RNA伴侣Hfq是许多细菌中sRNA介导的调节（核糖核酸调节）的关键参与者。到目前为止，人们对人类主要病原体幽门螺杆菌（Helicobacter pylori）的转录后基因调控几乎一无所知，幽门螺杆菌在世界一半人口的胃中定植，从而导致胃炎、溃疡和胃癌。对H. pylori的研究主要集中在基因组多样性和毒力方面，但对其基因调控机制的研究却很少。在1.67Mb的H. pylori基因组表明可能存在额外的调节因子，如sRNA。然而，自从H.幽门螺杆菌与50%的细菌一样缺乏Hfq，甚至被认为是一种没有核糖核酸调控的生物。幽门。除了大量的反义转录外，我们还鉴定了60多种sRNA。现在，我们的目标是这些sRNA的功能特性。在本项目中，我们将重点关注两个丰富的sRNA，HPnc 5490和HPnc 2420，这两个sRNA被证明是受酸调节的，酸是H. pylori在人类的胃里我们以前的实验已经表明，HPnc 5490 RNA抑制TlpB，pH敏感趋化性受体。生物计算分析表明，HPnc 5490与富含CU的区域结合到tlpB 5 UTR中的G重复序列。该G-重复对应于均聚重复，其是不同菌株之间长度变化的基础。在这个项目中，我们将研究HPnc 5490抑制tlpB的机制以及HPnc 5490本身是如何调节的。此外，我们将研究G-重复序列在tlpB表达和调节中的作用。第二个sRNA，HPnc 2420，具有抗Shine-Dalgarno序列，因此，可以作为反式作用的反义调节剂。HPnc 2420在pH 7下的处理在酸性条件下丢失，初步数据表明酸响应性双组分系统ArsRS参与HPnc 2420的调节。利用生物化学和遗传学的方法，我们将试图确定这种sRNA的目标和监管机构，并调查其在酸胁迫反应中的作用。此外，我们将建立一个体内报告基因系统，用于验证H.幽门。本项目的总体目标是建立H. pylori作为一种新的模式生物，用于病原菌和不含Hfq的细菌的sRNA研究。这不仅有助于阐明核糖体调控和H. pylori，但也存在于其他Epsilonproteobacteria中，包括新出现的病原体如弯曲杆菌。

Small regulatory RNAs (sRNAs) are an important class of post-transcriptional gene expression regulators during bacterial stress response or virulence. Especially work in enterobacteria showed that sRNAs can regulate multiple target genes and represent key regulators in metabolic networks. Moreover, the RNA chaperone Hfq is a key player in sRNA-mediated regulation (riboregulation) in many bacteria. So far, almost nothing is known about post-transcriptional gene regulation in the major human pathogen, Helicobacter pylori, which colonizes the stomachs of half of the world's population and thereby causes gastritis, ulcer, and gastric cancer. Most studies on H. pylori focused on its high genomic diversity and virulence, but only little is known about its gene regulation mechanisms. The small number of transcriptional regulators in the 1.67 Mb H. pylori genome indicates that there might be additional regulators, such as sRNAs. However, since H. pylori lacks Hfq, like 50 % of all bacteria, it was even considered as an organism without riboregulation.However, our recently developed novel differential RNA-sequencing approach revealed an unexpected transcriptome complexity in H. pylori. Besides a massive antisense transcription we have identified more than 60 sRNAs. Now we aim at the functional characterization of these sRNAs. In this project, we will focus on two abundant, sRNAs, HPnc5490 and HPnc2420, which turned out to be regulated by acid, an important stress condition for H. pylori in the human stomach. Our previous experiments have shown that HPnc5490 RNA represses TlpB, a pH-sensing chemotaxis receptor. Biocomputational analysis indicated that HPnc5490 binds with a CU-rich region to a G-repeat in the tlpB 5UTR. This G-repeat corresponds to a homopolymeric repeat which underlies length variation among different strains. In this project we will investigate the mechanism of tlpB repression by HPnc5490 and how HPnc5490 itself is regulated. Moreover, we will investigate the role of the G-repeat in tlpB expression and regulation. The second sRNA, HPnc2420, harbors an anti-Shine-Dalgarno sequence and, thus, could act as a trans-acting antisense regulator. Processing of HPnc2420 at pH 7, is lost under acidic conditions and preliminary data indicate an involvement of the acid-responsive two-component system ArsRS in regulation of HPnc2420. Using biochemical and genetic methods we will try to identify the targets and regulators of this sRNA and to investigate its role during the acid stress response. Moreover, we will establish an in vivo reporter gene system for the validation of sRNA-target interactions in H. pylori. The overall goal of this project is to establish H. pylori as a new model organism for sRNA research in pathogenic bacteria and bacteria without Hfq. This will not only help shed light on riboregulation and control of virulence in H. pylori, but also in other Epsilonproteobacteria, including emerging pathogens like Campylobacter.