Helicobacter pylori VacA Toxin/Subunit p34: Targeting of an Anion Channel to the Inner Mitochondrial Membrane

Domańska, Grażyna; Motz, Christian; Meinecke, Michael; Harsman, Anke; Papatheodorou, Panagiotis; Reljić, Boris; Dian-Lothrop, Elke A.; Galmiche, Antoine; Kepp, Oliver; Becker, Lars; Günnewig, Kathrin; Wagner, Richard; Rassow, Joachim

doi:10.1371/journal.ppat.1000878

Cited by 74 publications

(109 citation statements)

References 73 publications

(119 reference statements)

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“…VacA-dependent mitochondrial fragmentation was also visible within human cervical adenocarcinoma (HeLa) and human larynx carcinoma (HEp-2) cells (Fig. S2B), two nongastric cell lines that have been previously used to study VacA interactions with mitochondria (17,30). Within AZ-521 cells, mitochondrial fragmentation was detected with VacA at concentrations as low as 10 nM, but not at 1 nM (Fig.…”

Section: Resultsmentioning

confidence: 80%

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Helicobacter pylori vacuolating cytotoxin A (VacA) engages the mitochondrial fission machinery to induce host cell death

Jain

Luo

Blanke³

2011

Proc. Natl. Acad. Sci. U.S.A.

148

119

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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.cytochrome c | mitochondrial dynamics | gastric cancer | gastric ulcer | toxin

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Section: Resultsmentioning

confidence: 80%

“…We thank Dr. Marina Jendrach (Goethe University) for pEGFP-Drp1 and pEFGP-DN-Drp1 (K38A), Dr. Wei-Xing Zong (Stony Brook University) for bax +/+ and bax −/− MEFs, and Dr. Joachim Rassow (Ruhr-Universität Bochum) for the plasmid encoding p34-EGFP (30). We also thank Drs.…”

mentioning

confidence: 99%

Helicobacter pylori vacuolating cytotoxin A (VacA) engages the mitochondrial fission machinery to induce host cell death

Jain

Luo

Blanke³

2011

Proc. Natl. Acad. Sci. U.S.A.

148

119

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“…Radiolabeled proteins were synthesized in reticulocyte lysate (TNT T7 coupled reticulocyte lysate system, Promega) containing [ 35 S]Met. Rat liver mitochondria were isolated as described before (33). The mitochondria (30 g of mitochondrial protein) were incubated with 5 l of reticulocyte lysate in 50 l of 250 mM sucrose, 80 mM KCl, 20 mM potassium phosphate, 2 mM NADH, 2 mM ATP, 10 mM MOPS/KOH, pH 7.2, containing 3% (w/v) bovine serum albumin.…”

Section: In Vitro Test For Protein Interaction With Mitochondria and mentioning

confidence: 99%

A Phosphodiesterase 2A Isoform Localized to Mitochondria Regulates Respiration

Acı́n-Pérez

Russwurm

Günnewig

et al. 2011

Journal of Biological Chemistry

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119

133

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Mitochondria are central organelles in cellular energy metabolism, apoptosis, and aging processes. A signaling network regulating these functions was recently shown to include soluble adenylyl cyclase as a local source of the second messenger cAMP in the mitochondrial matrix. However, a mitochondrial cAMPdegrading phosphodiesterase (PDE) necessary for switching off this cAMP signal has not yet been identified. Here, we describe the identification and characterization of a PDE2A isoform in mitochondria from rodent liver and brain. We find that mitochondrial PDE2A is located in the matrix and that the unique N terminus of PDE2A isoform 2 specifically leads to mitochondrial localization of this isoform. Functional assays show that mitochondrial PDE2A forms a local signaling system with soluble adenylyl cyclase in the matrix, which regulates the activity of the respiratory chain. Our findings complete a cAMP signaling cascade in mitochondria and have implications for understanding the regulation of mitochondrial processes and for their pharmacological modulation.Mitochondria play central roles in cellular energy metabolism, as well as in the regulation of cell cycle progression, apoptosis, and aging processes (1, 2). Despite their importance, signaling into, from, and within mitochondria is still not well understood. Emerging signaling mechanisms in mitochondria and between the organelle and its environment include reversible protein deacetylation (3, 4), redox regulation and reactive oxygen species formation (5-7), and cyclic adenosine monophosphate (cAMP) signaling (8, 9).cAMP-dependent effects and proteins of cAMP signaling systems, such as cAMP-responsive element-binding protein (CREB), protein kinase A (PKA), and A-kinase anchoring proteins (AKAPs), 3 have been described in mitochondria (10 -12). In addition to these effector proteins, a complete cAMP signaling microdomain requires enzymes for synthesis and degradation of the second messenger. Although an intramitochondrial cAMP source has been identified recently (8), there is no known cAMP-degrading enzyme in this organelle. Cyclic AMP is formed inside mitochondria by soluble adenylyl cyclase (sAC) (8), a member of Class III of the nucleotidyl cyclase family, which also comprises the G-protein-regulated transmembrane adenylyl cyclases (13). Unique from transmembrane adenylyl cyclases, sAC is activated by bicarbonate (14), and it appears to act as a metabolic sensor (15), whose mitochondrial form(s) seems to modulate PKA-mediated regulation of respiration (8) and apoptosis (16).The opponents of the cyclic nucleotide-forming cyclases are cyclic nucleotide monophosphate (cNMP)-degrading phosphodiesterases (PDEs). Mammalian cells contain a varying subset of members of the classical PDE family, which comprises 11 PDE gene families (PDE1-11) (17, 18) and non-generic PDEs such as the protein human Prune (19,20). The isoforms of the generic PDEs comprise homologous catalytic domains, fused to varying regulatory domains, making them sensitive to a variety of signals s...

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“…Studies of VacA proteins expressed in transiently transfected HeLa cells have shown that the entire p33 domain plus the amino-terminal part of the p55 domain are required for intracellular toxin activity (39,44,45). The p33 domain has an important role in targeting VacA to mitochondria (46)(47)(48). Within the p33 domain, a predicted hydrophobic region about 30 amino acids in length is present near the amino terminus (20,49).…”

mentioning

confidence: 99%

“…Efforts to elucidate the structure of the p33 domain have been hindered by its insolubility (42), and efforts to determine the crystal structure of p88 VacA secreted by H. pylori have been hindered by its assembly into a heterogeneous assortment of oligomeric forms. Several studies have proposed structural models for regions of the p33 domain (47,52,55,56), particularly the predicted transmembrane domain that contains the GXXXG repeats (55), but at present there are no experimental data to either validate or refute the proposed models. In this study, we report crystallization and preliminary structural analysis of a nonoligomerizing VacA protein, which provides the first insight into structural properties of the p33 domain.…”

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confidence: 99%

A Nonoligomerizing Mutant Form of Helicobacter pylori VacA Allows Structural Analysis of the p33 Domain

et al. 2016

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Helicobacter pylori secretes a pore-forming VacA toxin that has structural features and activities substantially different from those of other known bacterial toxins. VacA can assemble into multiple types of water-soluble flower-shaped oligomeric structures, and most VacA activities are dependent on its capacity to oligomerize. The 88-kDa secreted VacA protein can undergo limited proteolysis to yield two domains, designated p33 and p55. The p33 domain is required for membrane channel formation and intracellular toxic activities, and the p55 domain has an important role in mediating VacA binding to cells. Previous studies showed that the p55 domain has a predominantly ␤-helical structure, but no structural data are available for the p33 domain. We report here the purification and analysis of a nonoligomerizing mutant form of VacA secreted by H. pylori. The nonoligomerizing 88-kDa mutant protein retains the capacity to enter host cells but lacks detectable toxic activity. Analysis of crystals formed by the monomeric protein reveals that the ␤-helical structure of the p55 domain extends into the C-terminal portion of p33. Fitting the p88 structural model into an electron microscopy map of hexamers formed by wild-type VacA (predicted to be structurally similar to VacA membrane channels) reveals that p55 and the ␤-helical segment of p33 localize to peripheral arms but do not occupy the central region of the hexamers. We propose that the amino-terminal portion of p33 is unstructured when VacA is in a monomeric form and that it undergoes a conformational change during oligomer assembly. Helicobacter pylori is a Gram-negative bacterium that persistently colonizes the stomach in about half of the human population worldwide (1-3). Most people tolerate the presence of this organism for long periods without any adverse consequences, but a small subset of H. pylori-infected individuals develop gastric adenocarcinoma or peptic ulcer disease.H. pylori is a relatively noninvasive organism that lives in the gastric mucus layer overlying gastric epithelial cells, sometimes adhering to the gastric epithelium. Many H. pylori-induced alterations in the gastric mucosa are attributable to the actions of secreted bacterial proteins on host cells (4-6). One such protein is the secreted vacuolating toxin, VacA (4, 7-10). VacA causes multiple alterations in gastric epithelial cells, including swelling of endosomes (vacuolation) (11, 12), permeabilization of mitochondrial membranes, and activation of mitogen-activated protein kinases (4, 7-9). In addition to its effects on gastric epithelial cells, VacA can disrupt the functions of many types of immune cells (T cells, B cells, neutrophils, eosinophils, and monocytes) (4, 7-9, 13-16). The cellular activities of VacA are attributed mainly to its ability to form anion-selective channels in host cells (17-21). VacA lacks sequence similarity to any other known bacterial toxins.H. pylori strains isolated from unrelated individuals are genetically heterogeneous, and the risk of developing gastric...

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Helicobacter pylori VacA Toxin/Subunit p34: Targeting of an Anion Channel to the Inner Mitochondrial Membrane

Cited by 74 publications

References 73 publications

Helicobacter pylori vacuolating cytotoxin A (VacA) engages the mitochondrial fission machinery to induce host cell death

Helicobacter pylori vacuolating cytotoxin A (VacA) engages the mitochondrial fission machinery to induce host cell death

A Phosphodiesterase 2A Isoform Localized to Mitochondria Regulates Respiration

A Nonoligomerizing Mutant Form of Helicobacter pylori VacA Allows Structural Analysis of the p33 Domain

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