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

• Applicant: Professorin Dr. Cynthia Mira Sharma
• Subject Area: Metabolism, Biochemistry and Genetics of Microorganisms
• Term: from 2012 to 2017
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 232886862

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.

• DFG Programme: Research Grants