Functional characterization of two acid-regulated small RNAs in Helicobacter pylori
Zusammenfassung der Projektergebnisse
To successfully survive in changing environments or to efficiently colonize a host, bacterial pathogens modulate their gene expression to adapt to these changing conditions. In this project, we aimed to provide new insights into the mechanisms of gene regulation during stress response such as acid adaptation or virulence of the gastric pathogen Helicobacter pylori with a focus on small RNA (sRNA)-mediated post-transcriptional regulation. Our work revealed novel mechanisms of sRNA-mediated regulation and new connections of sRNAs to phasevariable control. Moreover, we have identified and characterized several sRNAs (RepG, ArsZ) that directly regulate genes involved in acid-adaptation or colonization or even act as central regulators (NikS sRNA) of multiple virulence factors of H. pylori. For example, based on in-vitro and in-vivo analyses, we have demonstrated that the abundant sRNA RepG (regulator of polymeric G-repeats) sRNA represses translation of the TlpB chemotaxis receptor by base-pairing with its C/U-rich terminator loop to a variable homopolymeric G-repeat in the tlpB mRNA leader. While RepG sRNA itself is highly conserved, the length of the G-repeat in the target mRNA varies among different H. pylori isolates. Thereby, RepG can mediate both repression and activation of TlpB based on the number of Gs present in the repeat. This sRNA-mediated modulation of tlpB expression through length variation of the G-repeat represents a novel mechanism of gene expression control, as it links single sequence repeat-dependent phase variation to post-transcriptional regulation. Moreover, we observed that RepG co-regulates expression of the chemotaxis receptor TlpB and the downstream gene HP0102, a glycosyltransferase that we have revealed to be essential for mice stomach colonization due to its role in LPS O-chain biosynthesis. In addition, length variation of the G-repeat in the tlpB-hp0102 leader defines RepG-mediated control of hp0102 and, in turn, gradually modulates LPS O-chain synthesis as well as sensitivity to membrane-targeting antibiotics of H. pylori. While RepG targets a variable G-repeat, we uncovered that the sRNA NikS is itself under phase-variable control based on a length-variable T-stretch in its promoter. Moreover, we uncovered that NikS is transcriptionally repressed in response to nickel availabilities via the transcriptional regulator NikR and acts as a central regulator of H. pylori virulence phenotypes by directly repressing translation of its major virulence factors. To survive the acidic environment of the human stomach, H. pylori employs the urease enzyme, which cleaves urea into ammonia to buffer its periplasmic space. Here, we have uncovered that the H. pylori ArsZ sRNA modulates urease activity under acidic conditions by directly controlling levels of the nickel-binding protein HP1432. At low pH, HP1432 releases nickel ions, which are required as co-factors for the urease enzyme. Under neutral conditions, the continued expression of ArsZ allows the sRNA to repress translation of HP1432 on the post-transcriptional level. Upon shift to an acidic environment, the acid-responsive ArsRS twocomponent system simultaneously represses transcription of the ArsZ sRNA and activates HP1432 mRNA. Due to this regulatory circuit, the sRNA acts as a delay switch for fine-tuning the dynamics of urease induction upon exposure to an acidic environment. To the best of our knowledge, ArsZ is the first example of a trans-acting sRNA that regulates a stress-induced nickel storage protein to modulate urease maturation in a coherent feed-forward-loop network. Our functional characterization of three sRNAs demonstrates that H. pylori employs diverse mechanisms of RNA-based regulation during stress response and virulence control. Moreover, our work shows that studying sRNAs in bacteria that lack the common RNA chaperone Hfq can reveal unexpected mechanisms and intricate regulatory networks in bacterial gene expression control.
Projektbezogene Publikationen (Auswahl)
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(2014) A variable homopolymeric G-repeat defines small RNA-mediated posttranscriptional regulation of a chemotaxis receptor in Helicobacter pylori. PNAS 111(4): E501-10
Pernitzsch SR, Tirier SM, Beier D, Sharma CM
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(2014) READemption – A tool for the computational analysis of deep-sequencing based transcriptome data. Bioinformatics 30(23):3421-3
Förstner KU, Vogel J, Sharma CM
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(2015) Differential RNA-seq (dRNA-seq) for annotation of transcriptional start sites and small RNAs in Helicobacter pylori. Methods 86: 89- 101
Bischler T, Tan HS, Nieselt K, Sharma CM
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(2015) Stable isotope labeling by amino acids in cell culture based proteomics reveals differences in protein abundances between spiral and coccoid forms of the gastric pathogen Helicobacter pylori. Journal of Proteomics, 126:34-45
Müller SA, Pernitzsch SR, Haange SB, Uetz P, von Bergen M, Sharma CM, Kalkhof S
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(2016) Small RNAs in bacterial virulence and communication. Microbiology Spectrum 4(3)
Svensson SL, Sharma CM
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(2017) Identification of the RNA pyrophosphohydrolase RppH of Helicobacter pylori and global analysis of its RNA targets. Journal of Biological Chemistry 292(2): 1934-1950
Bischler T, Hsieh PK, Resch M, Liu Q, Tan HS, Foley PL, Hartleib A, Sharma CM, Belasco JG
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(2020) A repeat-associated small RNA controls the major virulence factors of Helicobacter pylori. Molecular Cell 80(2):210-226
Eisenbart Eisenbart SK#, Alzheimer M#, Pernitzsch SR, Dietrich S, Stahl S, Sharma CM
