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 LaboratóriumČlánok vyšiel v časopise
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