Salt regulated gene expression in Helicobacter pylori
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ABSTRACT: In this study, we tested the hypothesis that high salt concentrations might alter gene expression in H. pylori. Analysis here provides into the role that salt may play in H. pylori pathogenesis. . H. pylori strain 26695 (SC#7) was grown for 15 h in BB-FBS medium containing 0.5% sodium chloride (i.e. BB-FBS-0.5%) The bacterial cells were then grown for 10 h, in fresh brucella broth at either 0.25% or 1.5% salt, harvested by centrifugation, and frozen at -80oC. RNA isolated was reverse transcribed and used to probe H. pylori Panorama arrays
Project description:Helicobacter pylori (H. pylori) is a ε-proteobacterium that colonizes the stomach of about half of the world's population. Persistent infections have been associated with several gastric diseases. Mainly rod- or spiral shaped but also coccoid H. pylori forms have been isolated from mucus layer biopsies of patients. It is still being debated whether the coccoid form can be transformed back into the spiral form or whether this morphology is a result of bacterial cell death or persistence. We established stable isotope labeling by amino acids in cell culture (SILAC) for quantitative proteomics of H. pylori and applied it to investigate differences between the spiral and the coccoid morphology. We detected 72% and were able to relatively quantify 47% of the H. pylori proteome. Proteins involved in cell division and transcriptional and translational processes showed a lower abundance in coccoid cells. Additionally, proteins related to host colonization, including CagA, the arginase RocF, and the TNF-α inducing protein were down-regulated. The fact that outer membrane proteins were observed at higher abundances might represent a mechanism for immune evasion but also preserves adherence to host cells. The established protocol for relative protein quantification of H. pylori samples offers new possibilities for research on H. pylori.
Project description:In this study transcriptional start sites (TSS) for H. pylori 26695 were determined To detect the complement of transcripts expressed from H. pylori, we collected three independent biological replicates (B1 – B3) from 26695 wild type strain grown to mid-exponential (OD 600 ~0.6) phase under microaerophilic conditions at 37°C in BHI medium. For all three samples, total RNA was extracted and subjected to differential RNA-seq (dRNA-seq) library preparation for primary transcriptome analysis as described previously (Sharma et al., 2010). Specifically, prior to cDNA library construction half of each RNA sample was treated with 5’ terminator exonuclease (+TEX samples), which degrades RNAs containing a 5’-monophosphate (5’-P) and, thus, enriches for primary transcripts containing 5’-triphosphates (5’-PPP). The other half of each sample was left untreated (-TEX samples) and thus contains both primary transcripts (5’-PPP) and processed RNAs (5’-P).
Project description:Transcriptional profiling of Helicobacter pylori comparing 26695 wild-type strain and a HP0244-deficient mutant 26695/âHP0244::km treated at three different pH conditions (pH 7.4, pH 4.5 without urea, or pH 2.5 with 30 mM urea) for 30 min to define the HP0244 acid-responsive regulon Keywords: Genetic modification and stress response Wild type vs HP0244-deficient mutant at three different pH conditions (pH7.4, pH4.5 without urea, and pH2.5 with 30 mM urea). Three biological replicates for each pH condition: 3 wild type and 3 mutant, independently grown, pH treated, and harvested.
Project description:We performed DNA-protein interaction (ChIP-seq) analyses for Helicobacter pylori N6 wild-type (WT) and HP1021 deletion mutant (ΔHP1021::aphA-3) under oxidative stress (21% O2) and optimal microaerobic growth (5% O2) conditions. We detected 100 binding sites of HP1021 on the H. pylori N6 chromosome, most of which are promoter-located, likely affecting gene transcription. 84 of 100 identified HP1021 binding sites were located near promoter regions. EMSA and ChIP-qPCR confirmed the binding of HP1021 to the promoter region of a few genes.
Project description:The present study was aimed at analyzing (i) the biological cost of RNA polymerase (rpoB) mutations conferring rifampin resistance on H.pylori, (ii) the relationship between the cost of rpoB mutations and the chromosomal mutaion, (iii) the relationship between the cost of rpoB mutations and the transcription profile of sensitive and resistantrif strains of H.pylori (iv) and rpoB mutations in view of the possible fitness burden associated with resistance to another antibiotics. H.pylori reference strain 26695 was routinely maintained on Columbia agar plates and H. pylori-selective antibiotic mix Dent. Liquid culture was grown in BHI broth. Both plates and broth cultures were incubated at 37C under atmosphere enriched with 5% CO2 for 2-3 days . Mutant strains were selected by culturing H. pylori 26695 on selective plates containing rifampicin. In 5 days resistant colonies were picked up and passed under rifampicin pressure. RNA isolated was reverse transcribed and used to probe H. pylori home-made arrays
Project description:The CrdR-ChIP profiling is comparing H. pylori gDNA without crdR interaction as control and CrdR interact with H. pylori gDNA (WT vs crdR). The goal was determine the crdR binding site on H. pylori genome, it provide possible crdR-regulated genes. 2 samples: H. pylori 26695 is control, and CrdR is experiment.
Project description:Ribonucleases (RNases) are central actors in post-transcriptional regulation, a major level of regulation of gene expression in all cells. This control plays an important role in the bacterial pathogen Helicobacter pylori, although only the function of RNase J was characterized so far. Here, we studied the RNase R enzyme from H. pylori, a 3’-5’ exoribonuclease whose ortholog in Escherichia coli was reported to display, in addition, helicase activity and to be able to hydrolyze RNA substrates with double stranded structures. We observed that HpRNase R protein does not carry the domains responsible for helicase activity in E. coli and accordingly that the purified protein is unable to degrade in vitro RNA molecules with secondary structures. The RNase R helicase domain loss is widespread among the Campylobacterota and occurred gradually during their evolution. Furthermore, an in vivo interaction between HpRNase R and RhpA, the sole DEAD-box RNA helicase of H. pylori, was discovered. Phylogenomics suggests that this interaction might occur in other bacteria of the phylum Campylobacterota. Purified RhpA facilitates the degradation of double stranded RNA substrates by HpRNase R, showing that this complex is functional. HpRNase R has a minor role of in 5S rRNA maturation and, as shown by RNA-Seq, few targets in H. pylori all of them being included in the RhpA regulon. In conclusion, we describe a new type of RNase R that lacks some of the features that were considered as hallmarks of RNase R proteins, but that has co-opted another RNA helicase, which we hypothesize helps it accomplish some of its functions in vivo.
Project description:Analysis of Helicobacter pylori strain 26695 after 20 minutes of 0.25μg/ml Clarithromycin. Results provide insight into the mechanisms employed by the bacterium that help it adapt to Clarithromycin stress In the study presented here, we compared the gene expression profile between H.pylori without CLA treatment and H.pylori treated with 0.25μg/ml Clarithromycin.
Project description:Helicobacter pylori, a pathogenic member of phylum Campylobacterota (formerly Epsilonproteobacteria), is recognized as the leading cause of several human gastric pathologies, including acute and chronic gastritis, peptic ulcer disease, gastric adenocarcinoma, and gastric mucosa-associated lymphoid tissue (MALT) lymphoma. In the last two decades, the alarming increase of the antibiotic resistance levels to first-line and even “rescue” antibiotics, especially clarithromycin, metronidazole and levofloxacin, has led to a marked decrease of the eradication rates of traditional therapies. In previous works, we have validated the essential protein HsrA as an effective therapeutic target for H. pylori infection. HsrA is an OmpR/PhoB-type orphan response regulator, unique and highly conserved in members of phylum Campylobacterota, which appears involved in a variety of crucial physiological processes. In the present work, we carried out a transcriptomic analysis in order to discern the global effects of lethal concentrations of a bactericidal HsrA inhibitor on the H. pylori physiology. Treatment with the bactericidal HsrA inhibitor significantly changed the transcript levels of 367 open reading frames (ORF), of which 212 genes appeared upregulated and 155 genes resulted in downregulation, as compared with control samples. Thus, in vivo HsrA inhibition influenced, directly or indirectly, the expression of 23% of ORFs encoded by the H. pylori 26696 genome. Among the 268 differentially expressed genes (DEGs) with defined functions, two functional categories were highly enriched with downregulated genes involved in essential physiological processes: (1) ribosome biogenesis, and (2) electron transfer and oxidative phosphorylation.
Project description:Outer membrane vesicles (OMVs) are small vesicles constitutively shed by all Gram-negative bacterium, which have been proposed to play a role in Helicobacter pylori persistence and pathogenesis. The methods currently available for the isolation of H. pylori OMVs are diverse and time-consuming, raising the need for a protocol standardization, which was the main aim of this study. Here, we showed that the chemically defined F12 medium, supplemented with cholesterol, nutritionally supports bacterial growth and maintains H. pylori viability for at least 72 h. Additionally, we developed an abridged protocol for isolation of OMVs from these bacterial cultures, which comprises a low-speed centrifugation, supernatant filtration through a 0.45 µm pore, and two ultracentrifugations for OMVs’ recovery and washing. Using this approach, a good yield of highly pure bona fide OMVs was recovered from cultures of different H. pylori strains and in different periods of bacterial growth. Analysis of the proteome of OMVs isolated from H. pylori F12-cholesterol cultures validated the efficacy of our isolation protocol in attaining a higher sample purity with low representation of flagella proteins. The comparison with the proteomes of H. pylori OMVs available in the literature demonstrated that OMVs carry a selective protein cargo, suggesting their importance for H. pylori survival and pathogenesis. In conclusion, this work proposes a time- and cost-efficient protocol for the isolation of H. pylori OMVs from a chemically defined culture medium that is suitable for implementation in research and in the biopharmaceutical field.