EspA Acts as a Critical Mediator of ESX1-Dependent Virulence in by Affecting Bacterial Cell Wall Integrity

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Mycobacterium tuberculosis (Mtb) requires the ESX1 specialized protein secretion system for virulence, for triggering cytosolic immune surveillance pathways, and for priming an optimal CD8+ T cell response. This suggests that ESX1 might act primarily by destabilizing the phagosomal membrane that surrounds the bacterium. However, identifying the primary function of the ESX1 system has been difficult because deletion of any substrate inhibits the secretion of all known substrates, thereby abolishing all ESX1 activity. Here we demonstrate that the ESX1 substrate EspA forms a disulfide bonded homodimer after secretion. By disrupting EspA disulfide bond formation, we have dissociated virulence from other known ESX1-mediated activities. Inhibition of EspA disulfide bond formation does not inhibit ESX1 secretion, ESX1-dependent stimulation of the cytosolic pattern receptors in the infected macrophage or the ability of Mtb to prime an adaptive immune response to ESX1 substrates. However, blocking EspA disulfide bond formation severely attenuates the ability of Mtb to survive and cause disease in mice. Strikingly, we show that inhibition of EspA disulfide bond formation also significantly compromises the stability of the mycobacterial cell wall, as does deletion of the ESX1 locus or individual components of the ESX1 system. Thus, we demonstrate that EspA is a major determinant of ESX1-mediated virulence independent of its function in ESX1 secretion. We propose that ESX1 and EspA play central roles in the virulence of Mtb in vivo because they alter the integrity of the mycobacterial cell wall.

Vyšlo v časopise: EspA Acts as a Critical Mediator of ESX1-Dependent Virulence in by Affecting Bacterial Cell Wall Integrity. PLoS Pathog 6(6): e32767. doi:10.1371/journal.ppat.1000957
Kategorie: Research Article
prolekare.web.journal.doi_sk: https://doi.org/10.1371/journal.ppat.1000957

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Zdroje

1. LewisKN

LiaoR

GuinnKM

HickeyMJ

SmithS

2003 Deletion of RD1 from Mycobacterium tuberculosis Mimics Bacille Calmette-Guerin Attenuation. J Infect Dis 187 117 123

2. HsuT

Hingley-WilsonSM

ChenB

ChenM

DaiAZ

2003 The primary mechanism of attenuation of bacillus Calmette-Guerin is a loss of secreted lytic function required for invasion of lung interstitial tissue. Proc Natl Acad Sci U S A 100 12420 12425

3. PymAS

BrodinP

BroschR

HuerreM

ColeST

2002 Loss of RD1 contributed to the attenuation of the live tuberculosis vaccines Mycobacterium bovis BCG and Mycobacterium microti. Mol Microbiol 46 709 717

4. PymAS

BrodinP

MajlessiL

BroschR

DemangelC

2003 Recombinant BCG exporting ESAT-6 confers enhanced protection against tuberculosis. Nat Med 9 533 539

5. StanleySA

RaghavanS

HwangWW

CoxJS

2003 Acute infection and macrophage subversion by Mycobacterium tuberculosis require a specialized secretion system. Proc Natl Acad Sci U S A 100 13001 13006

6. GuinnKM

HickeyMJ

MathurSK

ZakelKL

GrotzkeJE

2004 Individual RD1-region genes are required for export of ESAT-6/CFP-10 and for virulence of Mycobacterium tuberculosis. Mol Microbiol 51 359 370

7. StanleySA

JohndrowJE

ManzanilloP

CoxJS

2007 The Type I IFN response to infection with Mycobacterium tuberculosis requires ESX-1-mediated secretion and contributes to pathogenesis. J Immunol 178 3143 3152

8. LeberJH

CrimminsGT

RaghavanS

Meyer-MorseNP

CoxJS

2008 Distinct TLR- and NLR-mediated transcriptional responses to an intracellular pathogen. PLoS Pathog 4 e6 doi:10.1371/journal.ppat.0040006

9. KooIC

WangC

RaghavanS

MorisakiJH

CoxJS

2008 ESX-1-dependent cytolysis in lysosome secretion and inflammasome activation during mycobacterial infection. Cell Microbiol 10 1866 1878

10. GaoLY

GuoS

McLaughlinB

MorisakiH

EngelJN

2004 A mycobacterial virulence gene cluster extending RD1 is required for cytolysis, bacterial spreading and ESAT-6 secretion. Mol Microbiol 53 1677 1693

11. van der WelN

HavaD

HoubenD

FluitsmaD

van ZonM

2007 M. tuberculosis and M. leprae translocate from the phagolysosome into the cytosol in myeloid cells. Cell 129 1287 1298

12. BrodinP

RosenkrandsI

AndersenP

ColeST

BroschR

2004 ESAT-6 proteins: protective antigens and virulence factors? Trends Microbiol 12 500 508

13. PandeyAK

YangY

JiangZ

FortuneSM

CoulombeF

2009 NOD2, RIP2 and IRF5 play a critical role in the type I interferon response to Mycobacterium tuberculosis. PLoS Pathog 5 e1000500 doi:10.1371/journal.ppat.1000500

14. StammLM

MorisakiJH

GaoLY

JengRL

McDonaldKL

2003 Mycobacterium marinum escapes from phagosomes and is propelled by actin-based motility. J Exp Med 198 1361 1368

15. de JongeMI

Pehau-ArnaudetG

FretzMM

RomainF

BottaiD

2007 ESAT-6 from Mycobacterium tuberculosis dissociates from its putative chaperone CFP-10 under acidic conditions and exhibits membrane-lysing activity. J Bacteriol 189 6028 34

16. SmithJ

ManoranjanJ

PanM

BohsaliA

XuJ

Evidence for pore formation in host cell membranes by ESX-1-secreted ESAT-6 and its role in Mycobacterium marinum escape from vacuole. Infect Immun 76 5478 5487

17. CorosA

CallahanB

BattaglioliE

DerbyshireKM

2008 The specialized secretory apparatus ESX-1 is essential for DNA transfer in Mycobacterium smegmatis. Mol Microbiol 69 794 808

18. PallenMJ

2002 The ESAT-6/WXG100 superfamily – and a new Gram-positive secretion system? Trends Microbiol 10 209 212

19. FlintJL

KowalskiJC

KarnatiPK

DerbyshireKM

2004 The RD1 virulence locus of Mycobacterium tuberculosis regulates DNA transfer in Mycobacterium smegmatis. Proc Natl Acad Sci U S A 101 12598 12603

20. São-JoséC

BaptistaC

SantosMA

2004 Bacillus subtilis operon encoding a membrane receptor for bacteriophage SPP1. J Bacteriol 186 8337 8346

21. CalmetteA

1927 La Vacinne Preventive Contre la Tuberculose Paris Masson et cie. 250

22. SteenkenW

1938 Spontaneous lysis of tubercle bacilli on artificial culture media. Am Rev Tuberc 38 777 790

23. FriguiW

BottaiD

MajlessiL

MonotM

JosselinE

2008 Control of M. tuberculosis ESAT-6 secretion and specific T cell recognition by PhoP. PLoS Pathog 4 e33 doi:10.1371/journal.ppat.0040033

24. ChampionPA

StanleySA

ChampionMM

BrownEJ

CoxJS

2006 C-terminal signal sequence promotes virulence factor secretion in Mycobacterium tuberculosis. Science 313 1632 1636

25. RenshawPS

LightbodyKL

VeverkaV

MuskettFW

KellyG

2005 Structure and function of the complex formed by the tuberculosis virulence factors CFP-10 and ESAT-6. EMBO J 24 2491 2498

26. RaghavanS

ManzanilloP

ChanK

DoveyC

CoxJS

2008 Secreted transcription factor controls Mycobacterium tuberculosis virulence. Nature 454 717 721

27. FortuneSM

JaegerA

SarracinoDA

ChaseMR

SassettiCM

2005 Mutually dependent secretion of proteins required for mycobacterial virulence. Proc Natl Acad Sci U S A 102 10676 10681

28. XuJ

LaineO

MasciocchiM

ManoranjanJ

SmithJ

2007 A unique Mycobacterium ESX-1 protein co-secretes with CFP-10/ESAT-6 and is necessary for inhibiting phagosome maturation. Mol Microbiol 66 787 800

29. McLaughlinB

ChonJS

MacGurnJA

CarlssonF

ChengTL

2007 A mycobacterium ESX-1-secreted virulence factor with unique requirements for export. PLoS Pathog 3 e105 doi:10.1371/journal.ppat.0030105

30. HoffmannC

LeisA

NiederweisM

PlitzkoJM

EngelhardtH

2008 Disclosure of the mycobacterial outer membrane: cryo-electron tomography and vitreous sections reveal the lipid bilayer structure. Proc Natl Acad Sci U S A 105 3963 3967

31. OjhaA

AnandM

BhattA

KremerL

JacobsWRJr

2005 GroEL1: a dedicated chaperone involved in mycolic acid biosynthesis during biofilm formation in mycobacteria. Cell 123 861 873

32. KadokuraH

KatzenF

BeckwithJ

2003 Protein disulfide bond formation in prokaryotes. Annu Rev Biochem 72 111 135

33. EhrtS

GuoXV

HickeyCM

RyouM

MonteleoneM

2005 Controlling gene expression in mycobacteria with anhydrotetracycline and Tet repressor. Nucleic Acids Res 33 e21

34. LiuH

SadygovRG

YatesJR3rd

2004 A model for random sampling and estimation of relative protein abundance in shotgun proteomics. Anal Chem 76 4193 4201

35. OldWM

Meyer-ArendtK

Aveline-WolfL

PierceKG

MendozaA

2005 Comparison of label-free methods for quantifying human proteins by shotgun proteomics. Mol Cell Proteomics 4 1487 1502

36. MacGurnJA

RaghavanS

StanleySA

CoxJS

2005 A non-RD1 gene cluster is required for Snm secretion in Mycobacterium tuberculosis. Mol Microbiol 57 1653 1663

37. DiGiuseppe ChampionPA

ChampionMM

ManzanilloP

CoxJS

2009 ESX-1 secreted virulence factors are recognized by multiple cytosolic AAA ATPases in pathogenic mycobacteria. Mol Microbio 73 950 962

38. KamathAB

WoodworthJ

XiongX

TaylorC

WengY

2004 Cytolytic CD8+ T cells recognizing CFP10 are recruited to the lung after Mycobacterium tuberculosis infection. J Exp Med 200 1479 1489

39. LewinsohnDM

ZhuL

MadisonVJ

DillonDC

FlingSP

2001 Classically restricted human CD8+ T lymphocytes derived from Mycobacterium tuberculosis-infected cells: definition of antigenic specificity. J Immunol 166 439 446

40. WoodworthJS

FortuneSM

BeharSM

2008 Bacterial protein secretion is required for priming of CD8+ T cells specific for the Mycobacterium tuberculosis antigen CFP10. Infect Immun 76 4199 4205

41. PangX

VuP

ByrdTF

GhannyS

SoteropoulosP

2007 Evidence for complex interactions of stress-associated regulons in an mprAB deletion mutant of Mycobacterium tuberculosis. Microbiology 153(Pt 4) 1229 1242

42. HeH

ZahrtTC

2005 Identification and characterization of a regulatory sequence recognized by Mycobacterium tuberculosis persistence regulator MprA. J Bacteriol 187 202 212

43. FisherMA

PlikaytisBB

ShinnickTM

2002 Microarray analysis of the Mycobacterium tuberculosis transcriptional response to the acidic conditions found in phagosomes. J Bacteriol 184 4025 4032

44. PathakSK

BasuS

BasuKK

BanerjeeA

PathakS

2007 Direct extracellular interaction between the early secreted antigen ESAT-6 of Mycobacterium tuberculosis and TLR2 inhibits TLR signaling in macrophages. Nat Immunol 8 610 618

45. AbdallahAM

Gey van PittiusNC

ChampionPA

CoxJ

LuirinkJ

2007 Type VII secretion–mycobacteria show the way. Nat Rev Microbiol 5 883 891

46. SaniM

HoubenEN

GeurtsenJ

PiersonJ

de PunderK

2010 Direct visualization by cryo-EM of the mycobacterial capsular layer: a labile structure containing ESX-1-secreted proteins. PLoS Pathog 6 e1000794 doi:10.1371/journal.ppat.1000794

47. MostowyS

CletoC

ShermanDR

BehrMA

2004 The Mycobacterium tuberculosis complex transcriptome of attenuation. Tuberculosis (Edinb) 84 197 204

48. VandalOH

RobertsJA

OdairaT

SchnappingerD

NathanCF

2009 Acid-susceptible mutants of Mycobacterium tuberculosis share hypersusceptibility to cell wall and oxidative stress and to the host environment. J Bacteriol 191 625 631

49. CosmaCL

KleinK

KimR

BeeryD

RamakrishnanL

2006 Mycobacterium marinum Erp is a virulence determinant required for cell wall integrity and intracellular survival. Infect Immun 74 3125 3133

50. BanaeiN

KincaidEZ

LinSY

DesmondE

JacobsWRJr

2009 Lipoprotein processing is essential for resistance of Mycobacterium tuberculosis to malachite green. Antimicrob Agents Chemother 53 3799 3802

51. SiegristMS

UnnikrishnanM

McConnellMJ

BorowskyM

ChengTY

2009 Mycobacterial Esx-3 is required for mycobactin-mediated iron acquisition. Proc Natl Acad Sci U S A 106 18792 18797

52. EliasJE

GygiSP

2007 Target-decoy search strategy for increased confidence in large-scale protein identifications by mass spectrometry. Nat Methods 4 207 214

53. NesvizhskiiAI

KellerA

KolkerE

AebersoldR

2003 A statistical model for identifying proteins by tandem mass spectrometry. Anal Chem 75 4646 4658

54. ZhangB

VerBerkmoesNC

LangstonMA

UberbacherE

HettichRL

2006 Detecting differential and correlated protein expression in label-free shotgun proteomics. J Proteome Res 5 2909 2918

55. RengarajanJ

BloomBR

RubinEJ

2005 Genome-wide requirements for Mycobacterium tuberculosis adaptation and survival in macrophages. Proc Natl Acad Sci U S A 102 8327 8332

56. FortuneSM

SolacheA

JaegerA

HillPJ

BelisleJT

2004 Mycobacterium tuberculosis inhibits macrophage responses to IFN-gamma through myeloid differentiation factor 88-dependent and -independent mechanisms. J Immunol 172 6272 6280