cytochrome-c-t and Helicobacter-Infections

Helicobacter pylori (H. pylori) infection is associated with chronic gastritis, peptic ulcer and gastric cancer. Apoptosis induced by microbial infections is implicated in the pathogenesis of H. pylori infection. Here we show that human gastric epithelial cells sensitized to H. pylori confer susceptibility to TRAIL-mediated apoptosis via modulation of death receptor signaling. Human gastric epithelial cells are intrinsically resistant to TRAIL-mediated apoptosis. The induction of TRAIL sensitivity by H. pylori is dependent on the activation of caspase-8 and its downstream pathway. H. pylori induces caspase-8 activation via enhanced assembly of the TRAIL death-inducing signaling complex (DISC) through downregulation of cellular FLICE-inhibitory protein (FLIP). Overexpression of FLIP abolished the H. pylori-induced TRAIL sensitivity in human gastric epithelial cells. Our study thus demonstrates that H. pylori induces sensitivity to TRAIL apoptosis by regulation of FLIP and assembly of DISC, which initiates caspase activation, resulting in the breakdown of resistance to apoptosis, and provides insight into the pathogenesis of gastric damage in Helicobacter infection. Modulation of host apoptosis signaling by bacterial interaction adds a new dimension to the pathogenesis of Helicobacter.

Topics: Apoptosis; BH3 Interacting Domain Death Agonist Protein; CASP8 and FADD-Like Apoptosis Regulating Protein; Caspase 8; Cell Line, Tumor; Cytochromes c; Death Domain Receptor Signaling Adaptor Proteins; Dose-Response Relationship, Drug; Enzyme Activation; Epithelial Cells; Gastric Mucosa; Helicobacter Infections; Helicobacter pylori; Humans; Mitochondria; Recombinant Proteins; Signal Transduction; Stomach; Time Factors; TNF-Related Apoptosis-Inducing Ligand

A number of pathogenic bacteria target mitochondria to modulate the host's apoptotic machinery. Studies here revealed that infection with the human gastric pathogen Helicobacter pylori disrupts the morphological dynamics of mitochondria as a mechanism to induce host cell death. The vacuolating cytotoxin A (VacA) is both essential and sufficient for inducing mitochondrial network fragmentation through the mitochondrial recruitment and activation of dynamin-related protein 1 (Drp1), which is a critical regulator of mitochondrial fission within cells. Inhibition of Drp1-induced mitochondrial fission within VacA-intoxicated cells inhibited the activation of the proapoptotic Bcl-2-associated X (Bax) protein, permeabilization of the mitochondrial outer membrane, and cell death. Our data reveal a heretofore unrecognized strategy by which a pathogenic microbe engages the host's apoptotic machinery.

Topics: Animals; Apoptosis; Bacterial Proteins; bcl-2-Associated X Protein; Cell Line; Cell Line, Tumor; Cells, Cultured; Cytochromes c; Dynamins; Fibroblasts; Flow Cytometry; Green Fluorescent Proteins; GTP Phosphohydrolases; HeLa Cells; Helicobacter Infections; Helicobacter pylori; Host-Pathogen Interactions; Humans; Membrane Potential, Mitochondrial; Mice; Mice, Knockout; Microscopy, Fluorescence; Microtubule-Associated Proteins; Mitochondria; Mitochondrial Proteins; Mutation; Stomach Neoplasms

Helicobacter pylori is a gastric bacterial pathogen that evades host immune responses in vivo and is associated with the development of gastritis, peptic ulcer disease, and gastric cancers. Induction of macrophage apoptosis is a method employed by multiple pathogens to escape host immune responses. Therefore, we hypothesized that H. pylori induces apoptosis of infected macrophages. RAW 264.7 cells were infected with H. pylori strain 60190, and apoptosis was assessed. Transmission electron microscopy and fluorescence microscopy showed that infected macrophages displayed morphological features characteristic of apoptosis. Quantification by acridine orange-ethidium bromide fluorescent-dye staining showed that apoptosis was dose and time dependent, and apoptosis was further confirmed by increased binding of annexin V-fluorescein isothiocyanate (FITC) to externalized phosphatidylserine of infected but not of control macrophages. Macrophages infected with isogenic mutants of H. pylori strain 60190 deficient in either cagA or vacA induced significantly less apoptosis than the parental strain, as assessed by increased binding of annexin V-FITC. Western blot analysis of whole-cell protein lysates revealed that infection with strain 60190 induced a time-dependent increase in cleavage of procaspase 8 and disappearance of full-length Bid compared with uninfected cells. Furthermore, pharmacological inhibition of caspase 8 caused a decrease in levels of apoptosis. Finally, infection caused a time-dependent increase in mitochondrial-membrane permeability and release of cytochrome c into the cytosol. These results suggest that H. pylori induces apoptosis of macrophages in association with alterations in the mitochondrial pathway. Elimination of this key immunomodulatory cell may represent a mechanism employed by the bacterium to evade host immune responses.

Topics: Animals; Antigens, Bacterial; Apoptosis; Bacterial Proteins; BH3 Interacting Domain Death Agonist Protein; Carrier Proteins; Caspase 8; Caspases; Cell Line; Cytochromes c; Genes, Bacterial; Helicobacter Infections; Helicobacter pylori; Macrophages; Mice; Microscopy, Electron; Mitochondria; Mutation; Phosphatidylserines

Glutamine synthetase is a key enzyme necessary for ammonia detoxification in the brain, but excessive activation of this enzyme can be cytotoxic to neural cells as a consequence of excessive consumption of ATP and glutamate. The stomach also expresses high levels of glutamine synthetase and this study aimed to investigate a possible pathophysiological role of glutamine synthetase in ammonia-induced gastric mucosal injury.. Normal rat gastric mucosal epithelial (RGM-1) cells were treated with ammonia, and a specific glutamine synthetase inhibitor (methionine sulfoximine) was used to assess the action of glutamine synthetase.. Treatment with ammonia induced apoptotic cell death. Increased expression of p21 and Bax, decreased expression of Bcl-2, cytochrome C release from the mitochondria into the cytosol and subsequent activation of caspase-9 and -3 were identified in the cells treated with ammonia, although there was no apparent change in p53 expression. On the other hand, pretreatment with various concentrations of methionine sulfoximine reduced the glutamine synthetase activity in ammonia-treated RGM-1 cells, and prevented the induction of apoptosis and the reduction in intracellular ATP levels in a dose-dependent manner.. Our results suggested that the energy exhaustion which resulted from an overload of ammonia to glutamine synthetase may have initiated the apoptotic signaling in gastric mucosal cells.

Topics: Adenosine Triphosphate; Ammonia; Animals; Apoptosis; bcl-2-Associated X Protein; Cell Culture Techniques; Cytochromes c; Energy Metabolism; Gene Expression Profiling; Glutamate-Ammonia Ligase; Helicobacter Infections; Helicobacter pylori; Intestinal Mucosa; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Rats; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction; Tumor Suppressor Protein p53

Helicobacter pylori infects the human stomach by escaping the host immune response. One mechanism of bacterial survival and mucosal damage is induction of macrophage apoptosis, which we have reported to be dependent on polyamine synthesis by arginase and ornithine decarboxylase. During metabolic back-conversion, polyamines are oxidized and release H(2)O(2), which can cause apoptosis by mitochondrial membrane depolarization. We hypothesized that this mechanism is induced by H. pylori in macrophages. Polyamine oxidation can occur by acetylation of spermine or spermidine by spermidine/spermine N(1)-acetyltransferase prior to back-conversion by acetylpolyamine oxidase, but recently direct conversion of spermine to spermidine by the human polyamine oxidase h1, also called spermine oxidase, has been demonstrated. H. pylori induced expression and activity of the mouse homologue of this enzyme (polyamine oxidase 1 (PAO1)) by 6 h in parallel with ornithine decarboxylase, consistent with the onset of apoptosis, while spermidine/spermine N(1)-acetyltransferase activity was delayed until 18 h when late stage apoptosis had already peaked. Inhibition of PAO1 by MDL 72527 or by PAO1 small interfering RNA significantly attenuated H. pylori-induced apoptosis. Inhibition of PAO1 also significantly reduced H(2)O(2) generation, mitochondrial membrane depolarization, cytochrome c release, and caspase-3 activation. Overexpression of PAO1 by transient transfection induced macrophage apoptosis. The importance of H(2)O(2) was confirmed by inhibition of apoptosis with catalase. These studies demonstrate a new mechanism for pathogen-induced oxidative stress in macrophages in which activation of PAO1 leads to H(2)O(2) release and apoptosis by a mitochondrial-dependent cell death pathway, contributing to deficiencies in host defense in diseases such as H. pylori infection.

Topics: Acetyltransferases; Animals; Apoptosis; Caspase 3; Caspases; Cell Line; Cytochromes c; Enzyme Induction; Gene Expression Regulation, Enzymologic; Helicobacter Infections; Helicobacter pylori; Hydrogen Peroxide; In Situ Nick-End Labeling; Macrophages; Membrane Potentials; Mice; Mitochondria; Ornithine Decarboxylase; Oxidoreductases Acting on CH-NH Group Donors; Polyamine Oxidase