Helicobacter pylori cell translocating kinase (CtkA/JHP0940) is pro-apoptotic in mouse macrophages and acts as auto-phosphorylating tyrosine kinase

Tenguria, Shivendra; Ansari, Suhail A.; Khan, Nooruddin; Ranjan, Amit; Devi, Savita; Tegtmeyer, Nicole; Lind, Judith; Backert, Steffen; Ahmed, Niyaz

doi:10.1016/j.ijmm.2014.07.017

Cited by 25 publications

(32 citation statements)

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“…Many effector proteins of H. pylori are known to be involved in the activation of NF-κB and AP-1 (9–11,16,17) which further regulate the production of IL-8 and TNF-α (37). In congruence with this, a significant difference in NF-κB activation in AGS cells at mRNA level was observed when compared to WT and tie A KO H. pylori (Figure 5).…”

Section: Resultsmentioning

confidence: 99%

“…The spectrum of disease manifestations caused by H. pylori is attributable to various secretory effector proteins (9–11,28,41). Despite triggering vigorous innate and adaptive immune responses, these secreted effector proteins also facilitate survival of the pathogen in the gastric mucosa (9,16).…”

Section: Discussionmentioning

confidence: 99%

“…The reaction mixture was incubated at 30°C for 15 min as described elsewhere (9). DNA-PK substrate and DNA-PK kinase were replaced by TieA in the test reaction for autophosphorylation.…”

Section: Methodsmentioning

confidence: 99%

“…Several strain specific genes such as ice A , ctk A , dup A , cag A , vac A , jhp0947 and others have been reported for their association with gastric diseases and inflammatory responses via secretion of TNF-α, IL-1β, IL-6 and IL-8 (8–13). Furthermore, vac A and ctk A induce host cell apoptosis, a balancing effect on epithelial cells turnover (9,13). Although about 20% of PZ genes are H. pylori specific, they lack any significant homologues available in the public databases.…”

Section: Introductionmentioning

confidence: 99%

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Multipronged regulatory functions of a novel endonuclease (TieA) fromHelicobacter pylori

et al. 2016

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Helicobacter pylori portrays a classical paradigm of persistent bacterial infections. A well balanced homeostasis of bacterial effector functions and host responses is purported to be the key in achieving long term colonization in specific hosts. H. pylori nucleases have been shown to assist in natural transformation, but their role in virulence and colonization remains elusive. Therefore, it is imperative to understand the involvement of these nucleases in the pathogenesis of H. pylori. Here, we report the multifaceted role of a TNFR-1 interacting endonuclease A (TieA) from H. pylori. tieA expression is differentially regulated in response to environmental stress and post adherence to gastric epithelial cells. Studies with isogenic knockouts of tieA revealed it to be a secretory protein which translocates into the host gastric epithelial cells independent of a type IV secretion system, gets phosphorylated by DNA-PK kinase and auto-phosphorylates as serine kinase. Furthermore, TieA binds to and cleaves DNA in a non-specific manner and promotes Fas mediated apoptosis in AGS cells. Additionally, TieA induced pro-inflammatory cytokine secretion via activation of transcription factor AP-1 and signaled through MAP kinase pathway. Collectively, TieA with its multipronged and moonlighting functions could facilitate H. pylori in maintaining a balance of bacterial adaptation, and elimination by the host responses.

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

“…The reaction mixture was incubated at 30°C for 15 min as described elsewhere (9). DNA-PK substrate and DNA-PK kinase were replaced by TieA in the test reaction for autophosphorylation.…”

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Multipronged regulatory functions of a novel endonuclease (TieA) fromHelicobacter pylori

et al. 2016

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“…Briefly, 10 10 cells were lysed in 1 mL of kinase buffer as described previously and 30 μL of the H. pylori lysate were mixed with two units of human c-Abl tyrosine kinase in the presence of 1 μmol/L of ATP (NEB, Frankfurt/Germany) [57]. After incubation for 30 min at 30 °C, the reactions were stopped by heating the samples at 95 °C for 5 min [48].…”

Section: Methodsmentioning

confidence: 99%

Systematic analysis of phosphotyrosine antibodies recognizing single phosphorylated EPIYA-motifs in CagA of East Asian-type Helicobacter pylori strains

et al. 2016

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BackgroundHighly virulent strains of the gastric pathogen Helicobacter pylori encode a type IV secretion system (T4SS) that delivers the effector protein CagA into gastric epithelial cells. Translocated CagA undergoes tyrosine phosphorylation by members of the oncogenic c-Src and c-Abl host kinases at EPIYA-sequence motifs A, B and D in East Asian-type strains. These phosphorylated EPIYA-motifs serve as recognition sites for various SH2-domains containing human proteins, mediating interactions of CagA with host signaling factors to manipulate signal transduction pathways. Recognition of phospho-CagA is mainly based on the use of commercial pan-phosphotyrosine antibodies that were originally designed to detect phosphotyrosines in mammalian proteins. Specific anti-phospho-EPIYA antibodies for each of the three sites in CagA are not forthcoming.ResultsThis study was designed to systematically analyze the detection preferences of each phosphorylated East Asian CagA EPIYA-motif by pan-phosphotyrosine antibodies and to determine a minimal recognition sequence. We synthesized phospho- and non-phosphopeptides derived from each predominant EPIYA-site, and determined the recognition patterns by seven different pan-phosphotyrosine antibodies using Western blotting, and also investigated representative East Asian H. pylori isolates during infection. The results indicate that a total of only 9–11 amino acids containing the phosphorylated East Asian EPIYA-types are required and sufficient to detect the phosphopeptides with high specificity. However, the sequence recognition by the different antibodies was found to bear high variability. From the seven antibodies used, only four recognized all three phosphorylated EPIYA-motifs A, B and D similarly well. Two of the phosphotyrosine antibodies preferentially bound primarily to the phosphorylated motif A and D, while the seventh antibody failed to react with any of the phosphorylated EPIYA-motifs. Control experiments confirmed that none of the antibodies reacted with non-phospho-CagA peptides and in accordance were able to recognize phosphotyrosine proteins in human cells.ConclusionsThe results of this study disclose the various binding preferences of commercial anti-phosphotyrosine antibodies for phospho-EPIYA-motifs, and are valuable in the application for further characterization of CagA phosphorylation events during infection with H. pylori and risk prediction for gastric disease development.

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Inflammasome Activation by Helicobacter pylori and Its Implications for Persistence and Immunity

Pachathundikandi

Müller

Backert

2016

Current Topics in Microbiology and Immunology

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Infection with the Gram-negative pathogen Helicobacter pylori is the most prevalent chronic bacterial infection affecting about 50 % of the human world population and is the main risk factor for gastric cancer development. The pro-inflammatory cytokine IL-1β plays a crucial role in the development of gastric tumors, and polymorphisms in the IL-1 gene cluster resulting in increased IL-1β production have been associated with increased risk for gastric cancer. Recently, Helicobacter pylori was postulated to activate the inflammasome in human and mouse immune cells, and the molecular mechanisms and the bacterial virulence factors activating the inflammasome were elucidated in cell culture as well as animal models. It appears that H. pylori-induced IL-1β secretion is mediated by activation of toll-like receptor 2 (TLR-2), Nod-like receptor family member NLRP3 and caspase-1. The cag pathogenicity island-encoded type IV secretion system, lipopolysaccharide, vacuolating cytotoxin, and urease B subunit appear to play a role in inflammasome activation. In addition, recent results indicate that the TLR-2 → NLRP3 → caspase-1 → IL-18 axis is critical to H. pylori-specific immune regulation conferring protection against allergen-induced asthma and inflammatory bowel disease in murine models. The present chapter will review the proposed mechanisms of NLRP3 inflammasome activation during H. pylori infection and discuss the recent progress in this important research field.

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Helicobacter pylori cell translocating kinase (CtkA/JHP0940) is pro-apoptotic in mouse macrophages and acts as auto-phosphorylating tyrosine kinase

Cited by 25 publications

References 57 publications

Multipronged regulatory functions of a novel endonuclease (TieA) fromHelicobacter pylori

Multipronged regulatory functions of a novel endonuclease (TieA) fromHelicobacter pylori

Systematic analysis of phosphotyrosine antibodies recognizing single phosphorylated EPIYA-motifs in CagA of East Asian-type Helicobacter pylori strains

Inflammasome Activation by Helicobacter pylori and Its Implications for Persistence and Immunity

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