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

幽门螺杆菌中两种酸调节小 RNA 的功能表征

• 批准号: 232886862
• 负责人: Professorin Dr. Cynthia Mira Sharma
• 金额: --
• 依托单位: Institut für Molekulare Infektionsbiologie (IMIB)
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2012
• 资助国家: 德国
• 起止时间: 2011-12-31 至 2016-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/232886862?language=en
• 关键词: Functional; characterization; two; acid; regulated

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.

小调节 RNA (sRNA) 是细菌应激反应或毒力过程中一类重要的转录后基因表达调节因子。特别是在肠细菌中的研究表明，sRNA 可以调节多个靶基因，并代表代谢网络中的关键调节因子。此外，RNA 伴侣 Hfq 是许多细菌中 sRNA 介导的调节（核糖调节）的关键角色。迄今为止，人们对人类主要病原体幽门螺杆菌的转录后基因调控几乎一无所知，幽门螺杆菌定植于世界一半人口的胃中，从而导致胃炎、溃疡和胃癌。大多数关于幽门螺杆菌的研究都集中在其高基因组多样性和毒力上，但对其基因调控机制知之甚少。 1.67 Mb 幽门螺杆菌基因组中的少量转录调节因子表明可能存在其他调节因子，例如 sRNA。然而，由于幽门螺杆菌与 50% 的细菌一样缺乏 Hfq，因此它甚至被认为是没有核糖调节的生物体。然而，我们最近开发的新型差异 RNA 测序方法揭示了幽门螺杆菌中意想不到的转录组复杂性。除了大量反义转录外，我们还鉴定了 60 多种 sRNA。现在我们的目标是这些 sRNA 的功能表征。在这个项目中，我们将重点关注两种丰富的 sRNA：HPnc5490 和 HPnc2420，它们被证明受到酸的调节，酸是人胃中幽门螺杆菌的重要应激条件。我们之前的实验表明 HPnc5490 RNA 抑制 TlpB（一种 pH 敏感趋化受体）。生物计算分析表明 HPnc5490 与 tlpB 5UTR 中的 G 重复区域富含 CU 的区域结合。该G重复对应于同聚重复，其是不同菌株之间长度变化的基础。在本项目中，我们将研究 HPnc5490 抑制 tlpB 的机制以及 HPnc5490 本身如何受到调节。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 HPnc2420 含有抗 Shine-Dalgarno 序列，因此可以充当反式作用反义调节因子。 HPnc2420 在 pH 7 下的加工在酸性条件下会丢失，初步数据表明酸响应性双组分系统 ArsRS 参与 HPnc2420 的调节。我们将利用生化和遗传学方法尝试确定该 sRNA 的靶标和调节因子，并研究其在酸应激反应中的作用。此外，我们将建立一个体内报告基因系统，用于验证幽门螺杆菌中sRNA-靶标相互作用。该项目的总体目标是建立幽门螺杆菌作为致病菌和无Hfq细菌的sRNA研究的新模式生物。这不仅有助于阐明幽门螺杆菌的核糖调节和毒力控制，也有助于阐明其他ε变形菌，包括弯曲杆菌等新兴病原体。