High Content Phenotypic Cell-Based Visual Screen Identifies Acyltrehalose-Containing Glycolipids Involved in Phagosome Remodeling


The ability of the tubercle bacillus to arrest phagosome maturation is considered one major mechanism that allows its survival within host macrophages. To identify mycobacterial genes involved in this process, we developed a high throughput phenotypic cell-based assay enabling individual sub-cellular analysis of over 11,000 Mycobacterium tuberculosis mutants. This very stringent assay makes use of fluorescent staining for intracellular acidic compartments, and automated confocal microscopy to quantitatively determine the intracellular localization of M. tuberculosis. We characterised the ten mutants that traffic most frequently into acidified compartments early after phagocytosis, suggesting that they had lost their ability to arrest phagosomal maturation. Molecular analysis of these mutants revealed mainly disruptions in genes involved in cell envelope biogenesis (fadD28), the ESX-1 secretion system (espL/Rv3880), molybdopterin biosynthesis (moaC1 and moaD1), as well as in genes from a novel locus, Rv1503c-Rv1506c. Most interestingly, the mutants in Rv1503c and Rv1506c were perturbed in the biosynthesis of acyltrehalose-containing glycolipids. Our results suggest that such glycolipids indeed play a critical role in the early intracellular fate of the tubercle bacillus. The unbiased approach developed here can be easily adapted for functional genomics study of intracellular pathogens, together with focused discovery of new anti-microbials.


Vyšlo v časopise: High Content Phenotypic Cell-Based Visual Screen Identifies Acyltrehalose-Containing Glycolipids Involved in Phagosome Remodeling. PLoS Pathog 6(9): e32767. doi:10.1371/journal.ppat.1001100
Kategorie: Research Article
prolekare.web.journal.doi_sk: https://doi.org/10.1371/journal.ppat.1001100

Souhrn

The ability of the tubercle bacillus to arrest phagosome maturation is considered one major mechanism that allows its survival within host macrophages. To identify mycobacterial genes involved in this process, we developed a high throughput phenotypic cell-based assay enabling individual sub-cellular analysis of over 11,000 Mycobacterium tuberculosis mutants. This very stringent assay makes use of fluorescent staining for intracellular acidic compartments, and automated confocal microscopy to quantitatively determine the intracellular localization of M. tuberculosis. We characterised the ten mutants that traffic most frequently into acidified compartments early after phagocytosis, suggesting that they had lost their ability to arrest phagosomal maturation. Molecular analysis of these mutants revealed mainly disruptions in genes involved in cell envelope biogenesis (fadD28), the ESX-1 secretion system (espL/Rv3880), molybdopterin biosynthesis (moaC1 and moaD1), as well as in genes from a novel locus, Rv1503c-Rv1506c. Most interestingly, the mutants in Rv1503c and Rv1506c were perturbed in the biosynthesis of acyltrehalose-containing glycolipids. Our results suggest that such glycolipids indeed play a critical role in the early intracellular fate of the tubercle bacillus. The unbiased approach developed here can be easily adapted for functional genomics study of intracellular pathogens, together with focused discovery of new anti-microbials.


Zdroje

1. RussellDG

2001 Mycobacterium tuberculosis: here today, and here tomorrow. Nat Rev Mol Cell Biol 2 569 577

2. VergneI

ChuaJ

SinghSB

DereticV

2004 Cell biology of mycobacterium tuberculosis phagosome. Annu Rev Cell Dev Biol 20 367 394

3. RussellDG

2003 Phagosomes, fatty acids and tuberculosis. Nat Cell Biol 5 776 778

4. HestvikAL

HmamaZ

Av-GayY

2005 Mycobacterial manipulation of the host cell. FEMS Microbiol Rev 29 1041 1050

5. NguyenL

PietersJ

2005 The Trojan horse: survival tactics of pathogenic mycobacteria in macrophages. Trends Cell Biol 15 269 276

6. VergneI

FrattiRA

HillPJ

ChuaJ

BelisleJ

2004 Mycobacterium tuberculosis phagosome maturation arrest: mycobacterial phosphatidylinositol analog phosphatidylinositol mannoside stimulates early endosomal fusion. Mol Biol Cell 15 751 760

7. VandalOH

PieriniLM

SchnappingerD

NathanCF

EhrtS

2008 A membrane protein preserves intrabacterial pH in intraphagosomal Mycobacterium tuberculosis. Nat Med 14 849 854

8. WalburgerA

KoulA

FerrariG

NguyenL

Prescianotto-BaschongC

2004 Protein kinase G from pathogenic mycobacteria promotes survival within macrophages. Science 304 1800 1804

9. MacGurnJA

CoxJS

2007 A genetic screen for Mycobacterium tuberculosis mutants defective for phagosome maturation arrest identifies components of the ESX-1 secretion system. Infect Immun 75 2668 2678

10. PetheK

SwensonDL

AlonsoS

AndersonJ

WangC

2004 Isolation of Mycobacterium tuberculosis mutants defective in the arrest of phagosome maturation. Proc Natl Acad Sci U S A 101 13642 13647

11. StewartGR

PatelJ

RobertsonBD

RaeA

YoungDB

2005 Mycobacterial mutants with defective control of phagosomal acidification. PLoS Pathog 1 269 278

12. BurguiereA

HitchenPG

DoverLG

KremerL

RidellM

2005 LosA, a key glycosyltransferase involved in the biosynthesis of a novel family of glycosylated acyltrehalose lipooligosaccharides from Mycobacterium marinum. J Biol Chem 280 42124 42133

13. RenH

DoverLG

IslamST

AlexanderDC

ChenJM

2007 Identification of the lipooligosaccharide biosynthetic gene cluster from Mycobacterium marinum. Mol Microbiol 63 1345 1359

14. CamineroJA

PenaMJ

Campos-HerreroMI

RodriguezJC

GarciaI

2001 Epidemiological evidence of the spread of a Mycobacterium tuberculosis strain of the Beijing genotype on Gran Canaria Island. Am J Respir Crit Care Med 164 1165 1170

15. Gonzalo AsensioJ

MaiaC

FerrerNL

BariloneN

LavalF

2006 The virulence-associated two-component PhoP-PhoR system controls the biosynthesis of polyketide-derived lipids in Mycobacterium tuberculosis. J Biol Chem 281 1313 1316

16. CamachoLR

EnsergueixD

PerezE

GicquelB

GuilhotC

1999 Identification of a virulence gene cluster of Mycobacterium tuberculosis by signature-tagged transposon mutagenesis. Mol Microbiol 34 257 267

17. ColeST

BroschR

ParkhillJ

GarnierT

ChurcherC

1998 Deciphering the biology of Mycobacterium tuberculosis from the complete genome sequence. Nature 393 537 544

18. BangeFC

CollinsFM

JacobsWRJr

1999 Survival of mice infected with Mycobacterium smegmatis containing large DNA fragments from Mycobacterium tuberculosis. Tuber Lung Dis 79 171 180

19. DaffeM

McNeilM

BrennanPJ

1991 Novel type-specific lipooligosaccharides from Mycobacterium tuberculosis. Biochemistry 30 378 388

20. van SoolingenD

HoogenboezemT

de HaasPE

HermansPW

KoedamMA

1997 A novel pathogenic taxon of the Mycobacterium tuberculosis complex, Canetti: characterization of an exceptional isolate from Africa. Int J Syst Bacteriol 47 1236 1245

21. GutierrezMC

BrisseS

BroschR

FabreM

OmaisB

2005 Ancient origin and gene mosaicism of the progenitor of Mycobacterium tuberculosis. PLoS Pathog 1 e5

22. VergneI

ChuaJ

DereticV

2003 Tuberculosis toxin blocking phagosome maturation inhibits a novel Ca2+/calmodulin-PI3K hVPS34 cascade. J Exp Med 198 653 659

23. SavinaA

VargasP

GuermonprezP

LennonAM

AmigorenaS

Measuring pH, ROS production, maturation, and degradation in dendritic cell phagosomes using cytofluorometry-based assays. Methods Mol Biol 595 383 402

24. RobackP

BeardJ

BaumannD

GilleC

HenryK

2007 A predicted operon map for Mycobacterium tuberculosis. Nucleic Acids Res 35 5085 5095

25. 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

26. CoxJS

ChenB

McNeilM

JacobsWRJr

1999 Complex lipid determines tissue-specific replication of Mycobacterium tuberculosis in mice. Nature 402 79 83

27. Rosas-MagallanesV

Stadthagen-GomezG

RauzierJ

BarreiroLB

TailleuxL

2007 Signature-tagged transposon mutagenesis identifies novel Mycobacterium tuberculosis genes involved in the parasitism of human macrophages. Infect Immun 75 504 507

28. Astarie-DequekerC

Le GuyaderL

MalagaW

SeaphanhFK

ChalutC

2009 Phthiocerol dimycocerosates of M. tuberculosis participate in macrophage invasion by inducing changes in the organization of plasma membrane lipids. PLoS Pathog 5 e1000289

29. VoskuilMI

SchnappingerD

RutherfordR

LiuY

Schoolnik GK 2004 Regulation of the Mycobacterium tuberculosis PE/PPE genes. Tuberculosis (Edinb) 84 256 262

30. BitterW

HoubenEN

BottaiD

BrodinP

BrownEJ

2009 Systematic genetic nomenclature for type VII secretion systems. PLoS Pathog 5 e1000507

31. McLaughlinB

ChonJS

MacGurnJA

CarlssonF

ChengTL

2007 A mycobacterium ESX-1-secreted virulence factor with unique requirements for export. PLoS Pathog 3 e105

32. BrodinP

MajlessiL

MarsollierL

de JongeMI

BottaiD

2006 Dissection of ESAT-6 system 1 of Mycobacterium tuberculosis and impact on immunogenicity and virulence. Infect Immun 74 88 98

33. 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

34. 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

35. 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

36. PeirsP

LefevreP

BoarbiS

WangXM

DenisO

2005 Mycobacterium tuberculosis with disruption in genes encoding the phosphate binding proteins PstS1 and PstS2 is deficient in phosphate uptake and demonstrates reduced in vivo virulence. Infect Immun 73 1898 1902

37. 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

38. 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

39. BecqJ

GutierrezMC

Rosas-MagallanesV

RauzierJ

GicquelB

2007 Contribution of horizontally acquired genomic islands to the evolution of the tubercle bacilli. Mol Biol Evol 24 1861 1871

40. HatziosSK

SchelleMW

HolsclawCM

BehrensCR

BotyanszkiZ

2009 PapA3 is an acyltransferase required for polyacyltrehalose biosynthesis in Mycobacterium tuberculosis. J Biol Chem 284 12745 12751

41. DubeyVS

SirakovaTD

KolattukudyPE

2002 Disruption of msl3 abolishes the synthesis of mycolipanoic and mycolipenic acids required for polyacyltrehalose synthesis in Mycobacterium tuberculosis H37Rv and causes cell aggregation. Mol Microbiol 45 1451 1459

42. SirakovaTD

ThirumalaAK

DubeyVS

SprecherH

KolattukudyPE

2001 The Mycobacterium tuberculosis pks2 gene encodes the synthase for the hepta- and octamethyl-branched fatty acids required for sulfolipid synthesis. J Biol Chem 276 16833 16839

43. SchelleMW

BertozziCR

2006 Sulfate metabolism in mycobacteria. Chembiochem 7 1516 1524

44. KumarP

SchelleMW

JainM

LinFL

PetzoldCJ

2007 PapA1 and PapA2 are acyltransferases essential for the biosynthesis of the Mycobacterium tuberculosis virulence factor sulfolipid-1. Proc Natl Acad Sci U S A 104 11221 11226

45. WaltersSB

DubnauE

KolesnikovaI

LavalF

DaffeM

2006 The Mycobacterium tuberculosis PhoPR two-component system regulates genes essential for virulence and complex lipid biosynthesis. Mol Microbiol 60 312 330

46. GorenMB

D'Arcy HartP

YoungMR

ArmstrongJA

1976 Prevention of phagosome-lysosome fusion in cultured macrophages by sulfatides of Mycobacterium tuberculosis. Proc Natl Acad Sci U S A 73 2510 2514

47. SchaibleUE

Sturgill-KoszyckiS

SchlesingerPH

RussellDG

1998 Cytokine activation leads to acidification and increases maturation of Mycobacterium avium-containing phagosomes in murine macrophages. J Immunol 160 1290 1296

48. ViaLE

FrattiRA

McFaloneM

Pagan-RamosE

DereticD

1998 Effects of cytokines on mycobacterial phagosome maturation. J Cell Sci 111 Pt7 897 905

49. PerezE

SamperS

BordasY

GuilhotC

GicquelB

2001 An essential role for phoP in Mycobacterium tuberculosis virulence. Mol Microbiol 41 179 187

50. ChristopheT

JacksonM

JeonHK

FenisteinD

Contreras-DominguezM

2009 High content screening identifies decaprenyl-phosphoribose 2′ epimerase as a target for intracellular antimycobacterial inhibitors. PLoS Pathog 5 e1000645

51. BeletskiiA

CooperM

SriramanP

ChiriacC

ZhaoL

2005 High-throughput phagocytosis assay utilizing a pH-sensitive fluorescent dye. Biotechniques 39 894 897

52. FenisteinD

LenseigneB

ChristopheT

BrodinP

GenovesioA

2008 A fast fully automated cell segmentation algorithm for high throughput and high content screening. Cytometry 73 958 964

53. Prod'homG

LagierB

PelicicV

HanceAJ

GicquelB

1998 A reliable amplification technique for the characterization of genomic DNA sequences flanking insertion sequences. FEMS Microbiol Lett 158 75 81

54. KordulakovaJ

GilleronM

MikusovaK

PuzoG

BrennanPJ

2002 Definition of the first mannosylation step in phosphatidylinositol mannoside synthesis. PimA is essential for growth of mycobacteria. J Biol Chem 277 31335 31344

55. NigouJ

VercelloneA

PuzoG

2000 New structural insights into the molecular deciphering of mycobacterial lipoglycan binding to C-type lectins: lipoarabinomannan glycoform characterization and quantification by capillary electrophoresis at the subnanomole level. J Mol Biol 299 1353 1362

56. GilleronM

RonetC

MempelM

MonsarratB

GachelinG

2001 Acylation state of the phosphatidylinositol mannosides from Mycobacterium bovis bacillus Calmette Guerin and ability to induce granuloma and recruit natural killer T cells. J Biol Chem 276 34896 34904

Štítky
Hygiena a epidemiológia Infekčné lekárstvo Laboratórium

Článok vyšiel v časopise

PLOS Pathogens


2010 Číslo 9
Najčítanejšie tento týždeň
Najčítanejšie v tomto čísle
Prihlásenie
Zabudnuté heslo

Zadajte e-mailovú adresu, s ktorou ste vytvárali účet. Budú Vám na ňu zasielané informácie k nastaveniu nového hesla.

Prihlásenie

Nemáte účet?  Registrujte sa