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Effect of Dhps deletion on macrophage activation by Helicobacter pylori

ABSTRACT: Bone marrow-derived macrophages from Dhps fl/fl mice and mice with specific deletion of Dhps in myeloid cells were infected with Helicobacter pylori PMSS1 for 24 hours. Cells were washed and lysed. Proteins were analyzed by isobaric tags for relative and absolute quantitation (iTRAQ).

INSTRUMENT(S): Q Exactive Plus, Q Exactive HF

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Bone Marrow-derived Macrophage, Macrophage

DISEASE(S): Bacterial Gastritis

SUBMITTER: Kristie Lindsey Rose

LAB HEAD: Kristie Lindsey Rose

PROVIDER: PXD018187 | Pride | 2021-09-09

REPOSITORIES: Pride

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Publications

Hypusination Orchestrates the Antimicrobial Response of Macrophages.

Gobert Alain P AP Finley Jordan L JL Latour Yvonne L YL Asim Mohammad M Smith Thaddeus M TM Verriere Thomas G TG Barry Daniel P DP Allaman Margaret M MM Delagado Alberto G AG Rose Kristie L KL Calcutt M Wade MW Schey Kevin L KL Sierra Johanna C JC Piazuelo M Blanca MB Mirmira Raghavendra G RG Wilson Keith T KT

Cell reports 20201201 11

Innate responses of myeloid cells defend against pathogenic bacteria via inducible effectors. Deoxyhypusine synthase (DHPS) catalyzes the transfer of the N-moiety of spermidine to the lysine-50 residue of eukaryotic translation initiation factor 5A (EIF5A) to form the amino acid hypusine. Hypusinated EIF5A (EIF5A<sup>Hyp</sup>) transports specific mRNAs to ribosomes for translation. We show that DHPS is induced in macrophages by two gastrointestinal pathogens, Helicobacter pylori and Citrobacter ...[more]

PMID: 33326776

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Macrophage Responses to Helicobacter pylori

Project description:The purpose of this study was to examine macrophage proteomic changes induced by Helicobacter pylori. Macrophages utilized were the RAW 264.7 murine cell line. Macrophages were treated with H. pylori for 24 hours. The experimental design was a 4-plex isobaric tags for relative and absolute quantification (iTRAQ). In addition to uninfected control and H. pylori infected, the additional two conditions included an inhibitor of deoxyhypusine synthase (N1-guanyl-1,7-diamine-heptane, 1-(7-ammonioheptyl)guanidinium sulfate; GC7) an enzyme involved in the hypusination translation pathway, and the inhibitor plus H. pylori.

2020-05-26 | PXD010082 | Pride

ChIP-seq of Helicobacter pylori N6 wild-type and HP1021 deletion mutant under microaerophilic and aerobic conditions (optimal growth and oxidative stress conditions, respectively).

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.

2023-09-23 | E-MTAB-13026 | biostudies-arrayexpress

RNA-seq of Helicobacter pylori N6 wild-type and HP1021 deletion mutant under microaerophilic and aerobic conditions (optimal growth and oxidative stress conditions, respectively)

Project description:We performed transcriptome (RNA-seq) analyses for Helicobacter pylori N6 wild-type (WT) and HP1021 deletion mutant (ΔHP1021::aphA-3) under oxidative stress and optimal microaerobic growth conditions. The expression of 411 genes was affected by oxidative stress in stressed wild-type cells (WTS) compared to non-stressed cells (WT). Interestingly, ΔHP1021 did not respond to oxidative stress. A comparison of genes expressed in the ΔHP1021 and WT strains under optimal growth conditions revealed 191 differently expressed genes. Moreover, transcriptional changes and overall final protein levels correlated across multiple genes. The data were validated through RT-qPCR and phenotype experiments for selected processes.

2023-09-23 | E-MTAB-13025 | biostudies-arrayexpress

Helicobacter pylori pathogenic mechanism

Project description:Helicobacter pylori infection reprograms host gene expression and influences various cellular processes, which have been investigated by cDNA microarray in vitro culture cells and in vivo patients of the chronic abdominal complaint. In this study，the effects of H. pylori infection on host gene expression in the gastric antral mucosa of patients with chronic gastritis were examined. The gastric antral mucosa was obtained from a total of 6 untreated patients undergoing gastroscopic and pathologic confirmation of chronic superficial gastritis. Three patients infected by H. pylori and 3 patients uninfected were used to cDNA microarray experiment.

2012-04-29 | E-GEOD-30042 | biostudies-arrayexpress

Expression data from Helicobacter pylori isolates infecting a gastric epithelial progenitor cell line

Project description:Helicobacter pylori clinical isolates can establish themselves in gastric epithelial stem cells and this interaction may have implications for gastric tumorigenesis. Mouse gastric epithelial progenitor cells (mGEPs) were infected for 24hrs with Helicobacter pylori clinical isolates Kx1 and Kx2. Kx1 and Kx2 were also grown in cell media in the absence of cells. Kx1 was isolated from a patient with chronic atrophic gastritis (ChAG) and Kx2 from the same patient 4 years later, when he progressed to gastric adenocarcinoma. Keywords: RNA Expression Array H. pylori strains Kx1 or Kx2 that had been grown to log phase were used to infect mGEP cells.. After 24h, media and non-attached bacteria were washed off and the cells harvested by trypsinization. RNA was prepared from bacteria infecting mGEP cells or bacterial cultures of Kx1 and Kx2 grown in cell media for 24h in the absence of mGEP cells.

2010-05-26 | E-GEOD-10261 | biostudies-arrayexpress

Helicobacter pylori outer membrane vesicle proteome

Project description:Gram negative bacteria release outer membrane vesicles (OMVs) as part of their natural growth. These OMVs take part in a biological functions including bacterial communication, exchange of genetic material and bacterial pathogenesis. However, the relationship between bacterial growth stage and OMV protein composition has not been explored before nor how their proteome is different to their parent bacterium. In this study, we examined the proteome of Helicobacter pylori and its OMVs from early log, late log or stationary phase of growth and found that globally, the protein content of OMVs over time is vastly different to one another as well as to their parent bacterium. OMVs purified from early log phase of growth contained the greatest number of unique proteins and a significant upregulation of virulence and pathogenic proteins. However, when compared back to their parent bacterium, OMVs from later stages of growth contained more unique proteins than those from earlier stages of growth. We show for the first time the regulation of OMV protein composition by H. pylori that is dependent on bacterial growth stage. Our results have expanded our understanding of the fundamental production of OMVs and the selective packaging of cargo in OMVs that result in specific functions.

2021-03-23 | PXD024918 | Pride

Delineation of the pH-responsive regulon controlled by the Helicobacter pylori ArsRS two2 component system

Project description:Helicobacter pylori encounters a wide range of pH within the human stomach. In a comparison of H. pylori cultured in vitro under neutral or acidic conditions, about 15% of genes are26 differentially expressed, and corresponding changes are detectable for many of the encoded proteins. The ArsRS two-component system (TCS), comprised of the sensor kinase ArsS and its cognate response regulator ArsR, has an important role in mediating pH-responsive changes in H. pylori gene expression. In this study, we sought to delineate the pH-responsive ArsRS regulon and further define the role of ArsR in pH-responsive gene expression. We compared H. pylori strains containing an intact ArsRS system with an arsS null mutant or strains containing site32 specific mutations of a conserved aspartate residue (D52) in ArsR, which is phosphorylated in response to signals relayed by the cognate sensor kinase ArsS. We identified 178 genes that were pH-responsive in strains containing an intact ArsRS system but not in ∆arsS or arsR mutants. These constituents of the pH-responsive ArsRS regulon include genes involved in acid acclimatization (ureAB, amidases), oxidative stress responses (katA, sodB), transcriptional regulation related to iron or nickel homeostasis (fur, nikR), and genes encoding outer membrane proteins [including sabA, alpA, alpB, hopD (labA), and horA]. When comparing H. pylori strains containing an intact ArsRS TCS with arsRS mutants, each cultured at neutral pH, relatively few genes are differentially expressed. Collectively, these data suggest that ArsRS41 mediated gene regulation has an important role in H. pylori adaptation to changing pH conditions.

2022-02-16 | PXD023679 | Pride

Isolation method and characterization of outer membranes vesicles of Helicobacter pylori grown in a chemically defined medium

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.

2021-05-31 | PXD025393 | Pride

RNase R is associated in a functional complex with the RhpA DEAD-box RNA helicase in Helicobacter pylori

Project description:Ribonucleases are central players in post-transcriptional regulation, a major level of gene expression regulation in all cells. Here, we characterized the 3’-5’ exoribonuclease RNase R from the bacterial pathogen Helicobacter pylori. The "prototypical" Escherichia coli RNase R displays both exoribonuclease and helicase activities, but whether this latter RNA unwinding function is a general feature of bacterial RNase R had not been addressed. We observed that H. pylori HpRNase R protein does not carry the domains responsible for helicase activity and accordingly the purified protein is unable to degrade in vitro RNA molecules with secondary structures. The lack of RNase R helicase domains is widespread among the Campylobacterota, that include Helicobacter and Campylobacter genera, and this loss occurred gradually during their evolution. An in vivo interaction between HpRNase R and RhpA, the sole DEAD-box RNA helicase of H. pylori was discovered. Purified RhpA facilitates the degradation of double stranded RNA by HpRNase R, showing that this complex is functional. HpRNase R has a minor role in 5S rRNA maturation and few targets in H. pylori, all included in the RhpA regulon. We concluded that during evolution, HpRNase R has co-opted the RhpA helicase to compensate for its lack of helicase activity.

2021-04-08 | PXD023837 | Pride

Profiling of the Helicobacter pylori redox switch HP1021 regulon using a multi-omics approach

Project description:The gastric human pathogen Helicobacter pylori has developed mechanisms to combat stress factors, including reactive oxygen species (ROS). Here, we present a comprehensive study on the redox switch protein HP1021 regulon combining transcriptomic, proteomic and DNA-protein interactions analyses. Our results indicated that HP1021 modulates H. pylori's response to oxidative stress. HP1021 controlled the transcription of 497 genes, including 407 genes related to response to oxidative stress. 79 proteins were differently expressed in the HP1021 deletion mutant. HP1021 regulated typical ROS response pathways (katA, rocF) and less canonical ones, particularly DNA uptake and central carbohydrate metabolism. We identified HP1021 as the first molecular regulator of competence in H. pylori, as HP1021-dependent repression of the comB DNA uptake genes was relieved under oxidative conditions, increasing natural competence. Furthermore, HP1021 controlled glucose consumption by directly regulating the gluP transporter and had an important impact on maintaining the energetic balance in the cell.

2023-09-20 | PXD041978 | Pride

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