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The Helicobacter pylori Methylome is Acid-Responsive due to Regulation by the Two-Component System ArsRS and the Type I DNA Methyltransferase HsdM1 (HP0463)

ABSTRACT: Helicobacter pylori colonizes the gastric epithelium and is the leading cause of gastric adenocarcinoma. There is increasing evidence that DNA methylation regulates gene expression in addition to its role in genome protection. In this study, we characterize the impact of acidity, the Two-Component System (TCS) ArsRS, Autoinducer-2, and the Type I m6A DNA methyltransferase HsdM1 (HP0463) on the Helicobacter pylori methylome. Transcription of hsdM1 increases at least 4-fold in the absence of the sensory histidine kinase ArsS, the major acid sensing protein of H. pylori. HsdM1 exists in the phase-variable operon hsdR1-hsdM1. Phase-locking hsdR1 (HP0464), the restriction endonuclease gene, has significant impacts on the transcription of hsdM1. To determine the impacts of methyltransferase transcription patterns on the methylome, we conducted methylome sequencing on samples that had undergone prolonged acid-exposure experiment. We found differentially methylated motifs between pH 7 and pH 5 growth conditions, and that deletions of arsS and hsdM1 interfere with the epigenetic acid response. Deletion of arsS leads to altered hsdM1 expression and alters the activity of multiple methyltransferases in both conditions indicating that the ArsRS TCS, in addition to its role in acid acclimation via direct effects on regulon member transcription, may also indirectly impact gene expression via regulation of the methylome. We characterized the target motif of HsdM1 (HP0463) to be (H)HTCAVN6TGY. HsdM1 may hierarchically regulate the Type II m5C DNA methyltransferase HP1121. This complex regulation of DNA methyltransferases, and thus differential methylation patterns, may have implications for the decades-long persistent infection by H. pylori.

ORGANISM(S): Helicobacter pylori

PROVIDER: GSE241991 | GEO | 2024/01/01

REPOSITORIES: GEO

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Project description:The human gastric pathogen Helicobacter pylori is extremely well adapted to the highly acidic conditions encountered in the stomach. The pronounced acid resistance of H. pylori relies mainly on the ammonia-producing enzyme urease, however, urease-independent mechanisms are likely to contribute to acid adaptation. Acid-responsive gene regulation is mediated at least in part by the ArsRS two-component system consisting of the essential OmpR-like response regulator ArsR and the non-essential cognate histidine kinase ArsS whose autophosphorylation is triggered in response to low pH. In this study by global transcriptional profiling of an ArsS-deficient H. pylori mutant grown at pH 5.0 we define the ArsR~P- dependent regulon consisting of 110 genes including the urease gene cluster, the genes encoding the aliphatic amidases AmiE and AmiF and the rocF gene encoding arginase. Transcriptome analyses were performed using a whole-genome microarray containing 1649 PCR products generated with specific primer pairs derived from the genome sequences of H. pylori 26695 (Tomb et al., 1997. The complete genome sequence of the gastric pathogen Helicobacter pylori. Nature 388:539-547) and J99 (Alm et al., 1999. Genomic-sequence comparison of two unrelated isolates of the human gastric pathogen Helicobacter pylori. Nature 397:176-180) which were spotted in duplicate. Microarrays were produced as described by Gressmann et al. (Gressmann et al., 2005. Gain and loss of multiple genes during the evolution of Helicobacter pylori. PLoS Genet 1(4):e43). To determine genes which are differentially expressed in the ArsS-deficient mutant G27/HP165::km at pH 5.0, cDNA was prepared from RNA extracted from H. pylori G27 and G27/HP165::km after exposing the bacteria for one hour to acidic pH. A total of eight RNA samples from two independent RNA preparations from strain G27 and G27/HP165::km, respectively, was used for cDNA labelling und hybridisation. Dye reversal colour swaps were performed as follows: One cDNA sample was generated using Cy3-dCTP and the other using Cy5-dCTP resulting in four labelled cDNAs per colour swap. Cy5-dCTP and Cy3-dCTP labelled cDNAs were combined and hybridized to the H. pylori microarray. The slides were scanned using ScanArray HT and analysed by using the ScanArray express software (Perkin Elmer). Spots were flagged and eliminated from analysis when the signal to background ratio was less then three or in obvious instances of high background or stray fluorescent signals. Median intensities of spots were background corrected and differences in dye bias were normalized by using the LOWESS algorithm (Yang et al., 2002. Normalization for cDNA microarray data: a robuste composite method addressing single and multiple slide systematic variation. Nucleic Acid Res. 30:e15). The signal ratios as measure of differential expression between the red and green channels were obtained from processed signal intensities. Ratios were further analysed with Microsoft Excel (Microsoft) and SAM software for statistic significance (Tusher et al., 2001. Significance analysis of microarrays applied to the ionizing radiation response. Proc. Natl. Acad. Sci. USA 98:5116-5121). To determine the significance of differential expression RNA was isolated from the H. pylori G27 wild-type grown in BHI broth (pH 5.0), and 20 Âµg of this RNA were labelled either with Cy3-dCTP or with Cy5-dCTP. The two cDNA probes generated were hybridized onto the same slide, and the data were analysed as mentioned above. Signal ratios < 0.5 and > 2.0 were analyzed further.

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The Helicobacter pylori Methylome is Acid-Responsive due to Regulation by the Two-Component System ArsRS and the Type I DNA Methyltransferase HsdM1 (HP0463)

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