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
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Hygiena a epidemiológia Infekčné lekárstvo LaboratóriumČlánok vyšiel v časopise
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