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

English version České info

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% β-strands, similar to pore-forming β-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.

Vyšlo v časopise: VacA Toxin/Subunit p34: Targeting of an Anion Channel to the Inner Mitochondrial Membrane. PLoS Pathog 6(4): e32767. doi:10.1371/journal.ppat.1000878
Kategorie: Research Article
prolekare.web.journal.doi_sk: https://doi.org/10.1371/journal.ppat.1000878

Souhrn

Zdroje

1. MontecuccoC

RappuoliR

2001 Living dangerously: how Helicobacter pylori survives in the human stomach. Nat Rev Mol Cell Biol 2 457 466

2. SuerbaumS

MichettiP

2002 Helicobacter pylori infection. N Engl J Med 347 1175 1186

3. KustersJG

van VlietAH

KuipersEJ

2006 Pathogenesis of Helicobacter pylori infection. Clin Microbiol Rev 19 449 490

4. PritchardDM

CrabtreeJE

2006 Helicobacter pylori and gastric cancer. Curr Opin Gastroenterol 22 620 625

5. CoverTL

BlaserMJ

2009 Helicobacter pylori in health and disease. Gastroenterology 136 1863 1873

6. BoquetP

RicciV

GalmicheA

GauthierNC

2003 Gastric cell apoptosis and H. pylori: has the main function of VacA finally been identified? Trends Microbiol 11 410 413

7. CoverTL

BlankeSR

2005 Helicobacter pylori VacA, a paradigm for toxin multifunctionality. Nat Rev Microbiol 3 320 332

8. LupettiP

HeuserJE

ManettiR

MassariP

LanzavecchiaS

1996 Oligomeric and subunit structure of Helicobacter pylori vacuolating cytotoxin. J Cell Biol 133 801 806

9. CoverTL

HansonPI

HeuserJE

1997 Acid-induced dissociation of VacA, the Helicobacter pylori vacuolating cytotoxin, reveals its pattern of assembly. J Cell Biol 138 759 769

10. AdrianM

CoverTL

DubochetJ

HeuserJE

2002 Multiple oligomeric states of the Helicobacter pylori vacuolating toxin demonstrated by cryo-electron microscopy. J Mol Biol 318 121 133

11. El-BezC

AdrianM

DubochetJ

CoverTL

2005 High resolution structural analysis of Helicobacter pylori VacA toxin oligomers by cryo-negative staining electron microscopy. J Struct Biol 151 215 228

12. IwamotoH

CzajkowskyDM

CoverTL

SzabòG

ShaoZ

1999 VacA from Helicobacter pylori: a hexameric chloride channel. FEBS Lett 450 101 104

13. CzajkowskyDM

IwamotoH

CoverTL

ShaoZ

1999 The vacuolating toxin from Helicobacter pylori forms hexameric pores in lipid bilayers at low pH. Proc Natl Acad Sci USA 96 2001 2006

14. SzabòI

BrutscheS

TombolaF

MoschioniM

SatinB

1999 Formation of anion-selective channels in the cell plasma membrane by the toxin VacA of Helicobacter pylori is required for its biological activity. EMBO J 18 5517 5527

15. McClainMS

CaoP

IwamotoH

Vinion-DubielAD

SzabòG

2001 A 12-amino-acid segment, present in type s2 but not type s1 Helicobacter pylori VacA proteins, abolishes cytotoxin activity and alters membrane channel formation. J Bacteriol 183 6499 6508

16. McClainMS

IwamotoH

CaoP

Vinion-DubielAD

LiY

SzabòG

2003 Essential role of a GXXXG motif for membrane channel formation by Helicobacter pylori vacuolating toxin. J Biol Chem 278 12101 12108

17. KimS

ChamberlainAK

BowieJU

2004 Membrane channel structure of Helicobacter pylori vacuolating toxin: role of multiple GXXXG motifs in cylindrical channels. Proc Natl Acad Sci USA 101 5988 5991

18. TorresVJ

McClainMS

CoverTL

2004 Interactions between p-33 and p-55 domains of the Helicobacter pylori Vacuolating Cytotoxin (VacA). J Biol Chem 279 2324 2331

19. GauthierNC

RicciV

LandraudL

BoquetP

2006 Helicobacter pylori VacA toxin: a tool to study novel early endosomes. Trends Microbiol 14 292 294

20. SewaldX

FischerW

HaasR

2008 Sticky socks: Helicobacter pylori VacA takes shape. Trends Microbiol 16 89 92

21. de BernardM

BurroniD

PapiniE

RapuoliR

TelfordJ

1998 Identification of the Helicobacter pylori VacA toxin domain active in the cytosol. Infect Immun 66 6014 6016

22. NguyenVQ

CaprioliRM

CoverTL

2001 Carboxy-terminal proteolytic processing of Helicobacter pylori vacuolating toxin. Infect Immun 69 543 546

23. GalmicheA

RassowJ

DoyeA

CagnolS

ContaminS

2000 The N-terminal 34 kDa fragment of Helicobacter pylori vacuolating cytotoxin targets mitochondria and induces cytochrome c release. EMBO J 19 6361 6370

24. KimuraM

GotoS

WadaA

YahiroK

NiidomeT

HatakeyamaT

1999 Vacuolating cytotoxin purified from Helicobacter pylori causes mitochondrial damage in human gastric cells. Microb Pathog 26 45 52

25. MenakerRJ

CeponisPJM

JonesNL

2004 Helicobacter pylori induces apoptosis of macrophages in association with alterations in the mitochondrial pathway. Infect Immun 72 2889 2898

26. WillhiteDC

BlankeSR

2004 Helicobacter pylori vacuolating cytotoxin enters cells, localizes to the mitochondria, and induces mitochondrial membrane permeability changes correlated to toxin channel activity. Cell Microbiol 6 143 154

27. BlankeSR

2005 Micro-managing the executioner: pathogen targeting of mitochondria. Trends Microbiol 13 64 71

28. AshktorabH

DashwoodRH

DashwoodMM

ZaidiSI

HewittSM

2008 H. pylori-induced apoptosis in human gastric cancer cells mediated via the release of apoptosis-inducing factor from mitochondria. Helicobacter 13 506 517

29. ZhangH

FangDC

LanCH

LuoYH

2007 Helicobacter pylori infection induces apoptosis in gastric cancer cells through the mitochondrial pathway. J Gastroenterol Hepatol 22 1051 1056

30. WillhiteDC

CoverTL

BlankeSR

2003 Cellular vacuolation and mitochondrial cytochrome c release are independent outcomes of Helicobacter pylori vacuolating cytotoxin activity that are each dependent on membrane channel formation. J Biol Chem 278 48204 48209

31. SeibelNM

EljouniJ

NalaskowskiMM

HampeW

2007 Nuclear localization of enhanced green fluorescent protein homomultimers. Anal Biochem 368 95 99

32. NeupertW

HerrmannJM

2007 Translocation of proteins into mitochondria. Annu Rev Biochem 76 723 749

33. StegerHF

SöllnerT

KieblerM

DietmeierKA

PfallerR

1990 Import of ADP/ATP carrier into mitochondria: two receptors act in parallel. J Cell Biol 111 2353 2363

34. Vinion-DubielAD

McClainMS

CzajkowskyDM

IwamotoH

YeD

1999 A dominant negative mutant of Helicobacter pylori vacuolating toxin (VacA) inhibits VacA-induced cell vacuolation. J Biol Chem 274 37736 37742

35. YeD

BlankeSR

2000 Mutational analysis of the Helicobacter pylori vacuolating toxin amino terminus: identification of amino acids essential for cellular vacuolation. Infect Immun 68 4354 4357

36. McClainMS

SchrawW

RicciV

BoquetP

CoverTL

2000 Acid activation of Helicobacter pylori vacuolating cytotoxin (VacA) results in toxin internalization by eukaryotic cells. Mol Microbiol 37 433 442

37. GambillBD

VoosW

KangPJ

MiaoB

LangerT

1993 A dual role for mitochondrial heat shock protein 70 in membrane translocation of preproteins. J Cell Biol 123 109 117

38. HoogenraadNJ

WardLA

RyanMT

2002 Import and assembly of proteins into mitochondria of mammalian cells. Biochim Biophys Acta 1592 97 105

39. BolenderN

SickmannA

WagnerR

MeisingerC

PfannerN

2008 Multiple pathways for sorting mitochondrial precursor proteins. EMBO Rep 9 42 49

40. ChacinskaA

KoehlerCM

MilencovicD

LithgowT

PfannerN

2009 Importing mitochondrial proteins: machineries and mechanisms. Cell 138 628 644

41. MoczkoM

EhmannB

GärtnerF

HönlingerA

SchäferE

1994 Deletion of the receptor MOM19 strongly impairs import of cleavable preproteins into Saccharomyces cerevisiae mitochondria. J Biol Chem 269 9045 9051

42. KrimmerT

RapaportD

RyanMT

MeisingerC

KassenbrockCK

2001 Biogenesis of porin of the outer mitochondrial membrane involves an import pathway via receptors and the general import pore of the TOM complex. J Cell Biol 152 289 300

43. HillK

ModelK

RyanMT

DietmeierK

MartinF

1998 Tom40 forms the hydrophilic channel of the mitochondrial import pore for preproteins. Nature 395 516 521

44. WangX-M

WattiezR

PaggliaciaC

TelfordJL

RuysschaertJ-M

2000 Membrane topology of vacA cytotoxin from H. pylori. FEBS Lett 481 96 100

45. ShanmugavadivuB

ApellH-J

MeinsT

ZethK

KleinschmidtJH

2007 Correct folding of the β-barrel of the human membrane protein VDAC requires a lipid bilayer. J Mol Biol 368 66 78

46. GarrowAG

AgnewA

WestheadDR

2005 TMB-Hunt: an amino acid composition based method to screen proteomes for beta-barrel transmembrane proteins. BMC Bioinformatics 6 56

47. RandelO

RassowJ

MotzC

2007 Separation of proteins by Blue Native Electrophoresis (BN-PAGE). Methods in Molecular Biology 390 417 427 In: Protein Targeting Protocols: Second Edition, Editor: M. van der Giezen, Humana Press inc., Totowa, NJ;

48. Kozjak-PavlovicV

RossK

RudelT

2008 Import of bacterial pathogenicity factors into mitochondria. Curr Opin Microbiol 11 1 6

49. TombolaF

CarlessoC

SzabòI

de BernardM

ReyratJM

1999 Helicobacter pylori vacuolating toxin forms anion-selective channels in planar lipid bilayers: possible implications for the mechanism of cellular vacuolation. Biophys J 76 1401 1409

50. RapaportD

2003 Finding the right organelle. Targeting signals in mitochondrial outer-membrane proteins. EMBO rep 4 948 952

51. PfannerN

WiedemannN

MeisingerC

LithgowT

2004 Assembling the mitochondrial outer membrane. Nat Struct Mol Biol 11 1044 1048

52. Kozjak-PavlovicV

Dian-LothrupEA

MeineckeM

KeppO

RossK

2009 Bacterial porin disrupts mitochondrial membrane potential and sensitizes host cells to apoptosis. PLoS Pathog 5 e1000629 doi:10.1371/journal.ppat.1000629

53. AbeY

ShodaiT

MutoT

MiharaK

ToriiH

2000 Structural basis of presequence recognition by the mitochondrial protein import receptor Tom20. Cell 100 551 560

54. BrandizziF

FrangneN

Mark-Martin S. Hawes C

NeuhausJ-M

2002 The destination for single-pass membrane proteins is influenced markedly by the length of the hydrophobic domain. Plant Cell 14 1077 1092

55. BalssJ

PapatheodorouP

MehmelM

BaumeisterD

HertelB

2008 Transmembrane domain length of viral K+ channels is a signal for mitochondria targeting. Proc Natl Acad Sci USA 105 12313 12318

56. SteenaartNAE

ShoreGC

1997 Alteration of a mitochondrial outer membrane signal anchor sequence that permits its insertion into the inner membrane. Contribution of hydrophobic residues. J Biol Chem 272 12057 12061

57. KieferD

KuhnA

1999 Hydrophobic forces drive spontaneous membrane insertion of the bacteriophage Pf3 coat protein without topological control. EMBO J 18 6299 6306

58. SchleiffE

SilviusJR

ShoreGC

1999 Direct membrane insertion of voltage-dependent anion-selective channel protein catalyzed by mitochondrial Tom20. J Cell Biol 145 973 978

59. GouauxE

1997 Channel-forming toxins: tales of transformation. Curr Opin Struct Biol 7 566 573

60. ParkerMW

FeilSC

2005 Pore-forming protein toxins: from structure to function. Prog Biophys Mol Biol 88 91 142

61. GangwerKA

MushrushDJ

StauffDL

SpillerB

McClainMS

2007 Crystal structure of the Helicobacter pylori vacuolating toxin p55 domain. Proc Natl Acad Sci USA 104 16293 16298

62. TombolaF

Del GiudiceG

PapiniE

ZorattiM

2000 Blockers of VacA provide insights into the structure of the pore. Biophys J 79 863 873

63. HilfRJ

DutzlerR

2008 X-ray structure of a procaryotic pentameric ligand-gated ion channel. Nature 452 375 379

64. JayasingheL

MilesG

BayleyH

2006 Role of the amino latch of staphylococcal α-hemolysin in pore formation: a co-operative interaction between the N terminus and position 217. J Biol Chem 281 2195 2204

65. NogueiraC

FigueiredoC

CarneiroF

GomesAT

BarreiraR

2001 Helicobacter pylori genotypes may determine gastric histopathology. Am J Pathol 158 647 654

66. GenissetC

PuharA

CaloreF

de BernardM

Dell'AntoneP

2007 The concerted action of the Helicobacter pylori cytotoxin VacA and the v-ATPase proton pump induces swelling of isolated endosomes. Cell Microbiol 9 1481 1490

67. KuckD

KolmererB

Iking-KonertC

KrammerPH

StremmelW

2001 Vacuolating cytotoxin of Helicobacter pylori induces apoptosis in the human gastric epithelial cell line AGS. Infect Immun 69 5080 5087

68. CoverTL

KrishnaUS

IsraelDE

PeekRMJr

2003 Induction of gastric epithelial cell apoptosis by Helicobacter pylori vacuolating cytotoxin. Cancer Res 63 951 957

69. YamasakiE

WadaA

KumatoriA

NakagawaI

FunaoJ

2006 Helicobacter pylori vacuolating cytotoxin induces activation of the proapoptotic proteins Bax and Bak, leading to cytochrome c release and cell death, independent of vacuolation. J Biol Chem 281 11250 11259

70. TruscottKN

KovermannP

GeisslerA

MerlinA

MeijerM

2001 A presequence- and voltage-sensitive channel of the mitochondrial preprotein translocase formed by Tim23. Nat Struct Biol 8 1074 1082

71. PapatheodorouP

DomanskaG

RassowJ

2007 Protein targeting to mitochondria of Saccharomyces cerevisiae and Neurospora crassa. Methods in Molecular Biology 390 151 166 In: Protein Targeting Protocols: Second Edition, Editor: M. van der Giezen, Humana Press inc., Totowa, NJ;

72. RassowJ

1999 Protein folding and import into organelles. Oxford Oxford University Press 43 94 In Post-Translational Processing: Practical Approach, Hames BD, Higgins SJ (Eds.).

73. MeineckeM

WagnerR

KovermannP

GuiardB

MickDU

2006 Tim50 maintains the permeability barrier of the mitochondrial inner membrane. Science 312 1523 1526

74. BeckerL

BannwarthM

MeisingerC

HillK

ModelK

2005 Preprotein translocase of the outer mitochondrial membrane: reconstituted Tom40 forms a characteristic TOM pore. J Mol Biol 353 1011 1020