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1.
PLoS Pathog ; 6(4): e1000878, 2010 Apr 29.
Article in English | MEDLINE | ID: mdl-20442789

ABSTRACT

The vacuolating toxin VacA, released by Helicobacter pylori, is an important virulence factor in the pathogenesis of gastritis and gastroduodenal ulcers. VacA contains two subunits: The p58 subunit mediates entry into target cells, and the p34 subunit mediates targeting to mitochondria and is essential for toxicity. In this study we found that targeting to mitochondria is dependent on a unique signal sequence of 32 uncharged amino acid residues at the p34 N-terminus. Mitochondrial import of p34 is mediated by the import receptor Tom20 and the import channel of the outer membrane TOM complex, leading to insertion of p34 into the mitochondrial inner membrane. p34 assembles in homo-hexamers of extraordinary high stability. CD spectra of the purified protein indicate a content of >40% beta-strands, similar to pore-forming beta-barrel proteins. p34 forms an anion channel with a conductivity of about 12 pS in 1.5 M KCl buffer. Oligomerization and channel formation are independent both of the 32 uncharged N-terminal residues and of the p58 subunit of the toxin. The conductivity is efficiently blocked by 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB), a reagent known to inhibit VacA-mediated apoptosis. We conclude that p34 essentially acts as a small pore-forming toxin, targeted to the mitochondrial inner membrane by a special hydrophobic N-terminal signal.


Subject(s)
Bacterial Proteins/metabolism , Mitochondrial Membranes/metabolism , Animals , Bacterial Proteins/chemistry , Electrophysiology , HeLa Cells , Helicobacter pylori/metabolism , Humans , Microscopy, Fluorescence , Rats
2.
PLoS Pathog ; 5(10): e1000629, 2009 Oct.
Article in English | MEDLINE | ID: mdl-19851451

ABSTRACT

The bacterial PorB porin, an ATP-binding beta-barrel protein of pathogenic Neisseria gonorrhoeae, triggers host cell apoptosis by an unknown mechanism. PorB is targeted to and imported by host cell mitochondria, causing the breakdown of the mitochondrial membrane potential (DeltaPsi(m)). Here, we show that PorB induces the condensation of the mitochondrial matrix and the loss of cristae structures, sensitizing cells to the induction of apoptosis via signaling pathways activated by BH3-only proteins. PorB is imported into mitochondria through the general translocase TOM but, unexpectedly, is not recognized by the SAM sorting machinery, usually required for the assembly of beta-barrel proteins in the mitochondrial outer membrane. PorB integrates into the mitochondrial inner membrane, leading to the breakdown of DeltaPsi(m). The PorB channel is regulated by nucleotides and an isogenic PorB mutant defective in ATP-binding failed to induce DeltaPsi(m) loss and apoptosis, demonstrating that dissipation of DeltaPsi(m) is a requirement for cell death caused by neisserial infection.


Subject(s)
Apoptosis/drug effects , Membrane Potential, Mitochondrial/drug effects , Porins/pharmacology , Bacterial Proteins/metabolism , Bacterial Proteins/pharmacology , Bacterial Proteins/physiology , Carrier Proteins/metabolism , Caspases/metabolism , Cytochromes c/metabolism , Enzyme Activation/drug effects , HeLa Cells , Host-Pathogen Interactions/physiology , Humans , Membrane Transport Proteins/metabolism , Mitochondria/drug effects , Mitochondria/metabolism , Mitochondria/ultrastructure , Mitochondrial Precursor Protein Import Complex Proteins , Models, Biological , Neisseria gonorrhoeae/chemistry , Neisseria gonorrhoeae/pathogenicity , Neisseria gonorrhoeae/physiology , Neisseriaceae Infections/metabolism , Neisseriaceae Infections/pathology , Porins/metabolism , Porins/physiology
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