#PAGE_PARAMS# #ADS_HEAD_SCRIPTS# #MICRODATA#

Evidence That Intracellular Stages of Utilize Amino Sugars as a Major Carbon Source


Intracellular parasites, such as Leishmania spp, must acquire suitable carbon sources from the host cell in order to replicate. Here we present evidence that intracellular amastigote stages of Leishmania exploit amino sugars in the phagolysosome of mammalian macrophages as a source of carbon and energy. L. major parasites are capable of using N-acetylglucosamine and glucosamine as primarily carbon sources and contain key enzymes required for conversion of these sugars to fructose-6-phosphate. The last step in this pathway is catalyzed by glucosamine-6-phosphate deaminase (GND), which was targeted to glycosomes via a canonical C-terminal targeting signal when expressed as a GFP fusion protein. Mutant parasites lacking GND were unable to grow in medium containing amino sugars as sole carbohydrate source and rapidly lost viability, concomitant with the hyper-accumulation of hexosamine-phosphates. Expression of native GND, but not a cytosolic form of GND, in Δgnd parasites restored hexosamine-dependent growth, indicating that toxicity is due to depletion of glycosomal pools of ATP. Non-lethal increases in hexosamine phosphate levels in both Δgnd and wild type parasites was associated with a defect in promastigote metacyclogenesis, suggesting that hexosamine phosphate levels may influence parasite differentiation. Promastigote and amastigote stages of the Δgnd mutant were unable to replicate within macrophages and were either completely cleared or exhibited reduced lesion development in highly susceptible Balb/c mice. Our results suggest that hexosamines are a major class of sugars in the macrophage phagolysosome and that catabolism of scavenged amino sugars is required to sustain essential metabolic pathways and prevent hexosamine toxicity.


Vyšlo v časopise: Evidence That Intracellular Stages of Utilize Amino Sugars as a Major Carbon Source. PLoS Pathog 6(12): e32767. doi:10.1371/journal.ppat.1001245
Kategorie: Research Article
prolekare.web.journal.doi_sk: https://doi.org/10.1371/journal.ppat.1001245

Souhrn

Intracellular parasites, such as Leishmania spp, must acquire suitable carbon sources from the host cell in order to replicate. Here we present evidence that intracellular amastigote stages of Leishmania exploit amino sugars in the phagolysosome of mammalian macrophages as a source of carbon and energy. L. major parasites are capable of using N-acetylglucosamine and glucosamine as primarily carbon sources and contain key enzymes required for conversion of these sugars to fructose-6-phosphate. The last step in this pathway is catalyzed by glucosamine-6-phosphate deaminase (GND), which was targeted to glycosomes via a canonical C-terminal targeting signal when expressed as a GFP fusion protein. Mutant parasites lacking GND were unable to grow in medium containing amino sugars as sole carbohydrate source and rapidly lost viability, concomitant with the hyper-accumulation of hexosamine-phosphates. Expression of native GND, but not a cytosolic form of GND, in Δgnd parasites restored hexosamine-dependent growth, indicating that toxicity is due to depletion of glycosomal pools of ATP. Non-lethal increases in hexosamine phosphate levels in both Δgnd and wild type parasites was associated with a defect in promastigote metacyclogenesis, suggesting that hexosamine phosphate levels may influence parasite differentiation. Promastigote and amastigote stages of the Δgnd mutant were unable to replicate within macrophages and were either completely cleared or exhibited reduced lesion development in highly susceptible Balb/c mice. Our results suggest that hexosamines are a major class of sugars in the macrophage phagolysosome and that catabolism of scavenged amino sugars is required to sustain essential metabolic pathways and prevent hexosamine toxicity.


Zdroje

1. IsbergRR

O'ConnorTJ

HeidtmanM

2009

The Legionella pneumophila replication vacuole: making a cosy niche inside host cells.

Nat Rev Microbiol

7

13

24

2. SwansonMS

Fernandez-MoreiraE

2002

A microbial strategy to multiply in macrophages: the pregnant pause.

Traffic

3

170

177

3. SchaibleUE

KaufmannSH

2005

A nutritive view on the host-pathogen interplay.

Trends Microbiol

13

373

380

4. AppelbergR

2006

Macrophage nutriprive antimicrobial mechanisms.

J Leukoc Biol

79

1117

1128

5. NadererT

McConvilleMJ

2008

The Leishmania-macrophage interaction: a metabolic perspective.

Cell Microbiol

10

301

308

6. DaviesCR

KayeP

CroftSL

SundarS

2003

Leishmaniasis: new approaches to disease control.

BMJ

326

377

382

7. PetersNC

EgenJG

SecundinoN

DebrabantA

KimblinN

2008

In vivo imaging reveals an essential role for neutrophils in leishmaniasis transmitted by sand flies.

Science

321

970

974

8. BelkaidY

MendezS

LiraR

KadambiN

MilonG

2000

A natural model of Leishmania major infection reveals a prolonged “silent” phase of parasite amplification in the skin before the onset of lesion formation and immunity.

J Immunol

165

969

977

9. PetersN

SacksD

2006

Immune privilege in sites of chronic infection: Leishmania and regulatory T cells.

Immunol Rev

213

159

179

10. BurchmoreRJ

Rodriguez-ContrerasD

McBrideK

MerkelP

BarrettMP

2003

Genetic characterization of glucose transporter function in Leishmania mexicana.

Proc Natl Acad Sci U S A

100

3901

3906

11. NadererT

EllisMA

SerneeMF

De SouzaDP

CurtisJ

2006

Virulence of Leishmania major in macrophages and mice requires the gluconeogenic enzyme fructose-1,6-bisphosphatase.

Proc Natl Acad Sci U S A

103

5502

5507

12. WinchesterB

2005

Lysosomal metabolism of glycoproteins.

Glycobiology

15

1R

15R

13. NadererT

WeeE

McConvilleMJ

2008

Role of hexosamine biosynthesis in Leishmania growth and virulence.

Mol Microbiol

69

858

869

14. KumarMJ

JamaluddinMS

NatarajanK

KaurD

DattaA

2000

The inducible N-acetylglucosamine catabolic pathway gene cluster in Candida albicans: discrete N-acetylglucosamine-inducible factors interact at the promoter of NAG1.

Proc Natl Acad Sci U S A

97

14218

14223

15. MeibomKL

LiXB

NielsenAT

WuCY

RosemanS

2004

The Vibrio cholerae chitin utilization program.

Proc Natl Acad Sci U S A

101

2524

2529

16. AlvarezFJ

KonopkaJB

2007

Identification of an N-acetylglucosamine transporter that mediates hyphal induction in Candida albicans.

Mol Biol Cell

18

965

975

17. SinghP

GhoshS

DattaA

2001

Attenuation of virulence and changes in morphology in Candida albicans by disruption of the N-acetylglucosamine catabolic pathway.

Infect Immun

69

7898

7903

18. da SilvaR

SacksDL

1987

Metacyclogenesis is a major determinant of Leishmania promastigote virulence and attenuation.

Infect Immun

55

2802

2806

19. LorenzMC

FinkGR

2002

Life and death in a macrophage: role of the glyoxylate cycle in virulence.

Eukaryot Cell

1

657

662

20. BurchmoreRJ

Rodriguez-ContrerasD

McBrideK

MerkelP

BarrettMP

2003

Genetic characterization of glucose transporter function in Leishmania mexicana.

Proc Natl Acad Sci USA

100

3901

3906

21. Alvarez-AnorveLI

CalcagnoML

PlumbridgeJ

2005

Why does Escherichia coli grow more slowly on glucosamine than on N-acetylglucosamine? Effects of enzyme levels and allosteric activation of GlcN6P deaminase (NagB) on growth rates.

J Bacteriol

187

2974

2982

22. NatarajanK

DattaA

1993

Molecular cloning and analysis of the NAG1 cDNA coding for glucosamine-6-phosphate deaminase from Candida albicans.

J Biol Chem

268

9206

9214

23. HaanstraJR

van TuijlA

KesslerP

ReijndersW

MichelsPA

2008

Compartmentation prevents a lethal turbo-explosion of glycolysis in trypanosomes.

Proc Natl Acad Sci U S A

105

17718

17723

24. FuruyaT

KesslerP

JardimA

SchnauferA

CrudderC

2002

Glucose is toxic to glycosome-deficient trypanosomes.

Proc Natl Acad Sci U S A

99

14177

14182

25. AlbertMA

HaanstraJR

HannaertV

Van RoyJ

OpperdoesFR

2005

Experimental and in silico analyses of glycolytic flux control in bloodstream form Trypanosoma brucei.

J Biol Chem

280

28306

28315

26. Rodriguez-ContrerasD

FengX

KeeneyKM

BouwerHG

LandfearSM

2007

Phenotypic characterization of a glucose transporter null mutant in Leishmania mexicana.

Mol Biochem Parasitol

153

9

18

27. MaugeriDA

CazzuloJJ

BurchmoreRJ

BarrettMP

OgbunudePO

2003

Pentose phosphate metabolism in Leishmania mexicana.

Mol Biochem Parasitol

130

117

125

28. IlgT

2002

Generation of myo-inositol-auxotrophic Leishmania mexicana mutants by targeted replacement of the myo-inositol-1-phosphate synthase gene.

Mol Biochem Parasitol

120

151

156

29. RaltonJE

NadererT

PirainoHL

BashtannykTA

CallaghanJM

2003

Evidence that intracellular β1-2 mannan is a virulence factor in Leishmania parasites.

J Biol Chem

278

40757

40763

30. SerneeMF

RaltonJE

DinevZ

KhairallahGN

O'HairRA

2006

Leishmania β-1,2-mannan is assembled on a mannose-cyclic phosphate primer.

Proc Natl Acad Sci U S A

103

9458

9463

31. DennisJW

NabiIR

DemetriouM

2009

Metabolism, cell surface organization, and disease.

Cell

139

1229

1241

32. ZeidanQ

HartGW

2010

The intersections between O-GlcNAcylation and phosphorylation: implications for multiple signaling pathways.

J Cell Sci

123

13

22

33. BanerjeeS

RobbinsPW

SamuelsonJ

2009

Molecular characterization of nucleocytosolic O-GlcNAc transferases of Giardia lamblia and Cryptosporidium parvum.

Glycobiology

19

331

336

34. CunninghamML

TitusRG

TurcoSJ

BeverleySM

2001

Regulation of differentiation to the infective stage of the protozoan parasite Leishmania major by tetrahydrobiopterin.

Science

292

285

287

35. JiangD

LiangJ

NoblePW

2007

Hyaluronan in tissue injury and repair.

Annu Rev Cell Dev Biol

23

435

461

36. RussellDG

XuS

ChakrabortyP

1992

Intracellular trafficking and the parasitophorous vacuole of Leishmania mexicana-infected macrophages.

J Cell Sci

103

1193

1210

37. JoshiMB

RogersME

ShakarianAM

YamageM

Al-HarthiSA

2005

Molecular characterization, expression, and in vivo analysis of LmexCht1: the chitinase of the human pathogen, Leishmania mexicana.

J Biol Chem

280

3847

3861

38. RogersME

HajmovaM

JoshiMB

SadlovaJ

DwyerDM

2008

Leishmania chitinase facilitates colonization of sand fly vectors and enhances transmission to mice.

Cell Microbiol

10

1363

1372

39. AndradeLO

AndrewsNW

2005

The Trypanosoma cruzi-host-cell interplay: location, invasion, retention.

Nat Rev Microbiol

3

819

823

40. EbikemeCE

PeacockL

CoustouV

RiviereL

BringaudF

2008

N-acetyl D-glucosamine stimulates growth in procyclic forms of Trypanosoma brucei by inducing a metabolic shift.

Parasitology

135

585

594

41. AzemaL

ClaustreS

AlricI

BlonskiC

WillsonM

2004

Interaction of substituted hexose analogues with the Trypanosoma brucei hexose transporter.

Biochem Pharmacol

67

459

467

42. De SouzaDP

SaundersEC

McConvilleMJ

LikicVA

2006

Progressive peak clustering in GC-MS Metabolomic experiments applied to Leishmania parasites.

Bioinformatics

22

1391

1396

43. RaltonJE

McConvilleMJ

1998

Delineation of three pathways of glycosylphosphatidylinositol biosynthesis in Leishmania mexicana - precursors from different pathways are assembled on distinct pools of phosphatidylinositol and undergo fatty acid remodeling.

J Biol Chem

273

4245

4257

44. MitchellGF

HandmanE

1983

Leishmania tropica major in mice: vaccination against cutaneous leishmaniasis in mice of high genetic susceptibility.

Aust J Exp Biol Med Sci

61

11

25

45. OlivaG

FontesMR

GarrattRC

AltamiranoMM

CalcagnoML

1995

Structure and catalytic mechanism of glucosamine 6-phosphate deaminase from Escherichia coli at 2.1 A resolution.

Structure

3

1323

1332

Štítky
Hygiena a epidemiológia Infekčné lekárstvo Laboratórium

Článok vyšiel v časopise

PLOS Pathogens


2010 Číslo 12
Najčítanejšie tento týždeň
Najčítanejšie v tomto čísle
Kurzy

Zvýšte si kvalifikáciu online z pohodlia domova

Aktuální možnosti diagnostiky a léčby litiáz
nový kurz
Autori: MUDr. Tomáš Ürge, PhD.

Všetky kurzy
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

#ADS_BOTTOM_SCRIPTS#