Metabolic Changes in Glucose Transporter-deficient Leishmania mexicana and Parasite Virulence

Abstract

Leishmania mexicana are parasitic protozoa that express a variety of glycoconjugates that play important roles in their biology as well as the storage carbohydrate β-mannan, which is an essential virulence factor for survival of intracellular amastigote forms in the mammalian host. Glucose transporter null mutants, which are viable as insect form promastigotes but not as amastigotes, do not take up glucose and other hexoses but are still able to synthesize these glycoconjugates and β-mannan, although at reduced levels. Synthesis of these carbohydrate-containing macromolecules could be accounted for by incorporation of non-carbohydrate precursors into carbohydrates by gluconeogenesis. However, the significantly reduced level of the virulence factor β-mannan in the glucose transporter null mutants compared with wild-type parasites may contribute to the non-viability of these null mutants in the disease-causing amastigote stage of the life cycle. Leishmania mexicana are parasitic protozoa that express a variety of glycoconjugates that play important roles in their biology as well as the storage carbohydrate β-mannan, which is an essential virulence factor for survival of intracellular amastigote forms in the mammalian host. Glucose transporter null mutants, which are viable as insect form promastigotes but not as amastigotes, do not take up glucose and other hexoses but are still able to synthesize these glycoconjugates and β-mannan, although at reduced levels. Synthesis of these carbohydrate-containing macromolecules could be accounted for by incorporation of non-carbohydrate precursors into carbohydrates by gluconeogenesis. However, the significantly reduced level of the virulence factor β-mannan in the glucose transporter null mutants compared with wild-type parasites may contribute to the non-viability of these null mutants in the disease-causing amastigote stage of the life cycle. Leishmaniae are pathogenic protozoa of the order Kinetoplastida that cause important diseases in humans and other vertebrates (1.Herwaldt B.L. Lancet. 1999; 354: 1191-1199Abstract Full Text Full Text PDF PubMed Scopus (1356) Google Scholar). There are two principal stages in the parasite life cycle, (i) extracellular flagellated promastigotes that live within the alimentary tract of the sand fly vector and (ii) non-flagellated amastigotes that reside within the acidified phagolysosomal vesicles of vertebrate host macrophages. Both life cycle stages of Leishmania mexicana can be cultivated in vitro (2.Bates P.A. Parasitology. 1994; 108: 1-9Crossref PubMed Scopus (126) Google Scholar) facilitating biochemical and genetic studies. Uptake and metabolism of glucose in Leishmania species has been of considerable interest, as this sugar provides a major source of carbon and energy to these parasites (3.Cazzulo J.J. FASEB J. 1992; 6: 3153-3161Crossref PubMed Scopus (174) Google Scholar). In L. mexicana, glucose catabolism is 10–20-fold higher in the promastigotes compared with amastigotes (4.Hart D.T. Coombs G.H. Exp. Parasitol. 1982; 54: 397-409Crossref PubMed Scopus (131) Google Scholar). Furthermore, amastigotes transport glucose at a much lower rate than promastigotes and derive metabolic energy primarily from fatty acid oxidation (4.Hart D.T. Coombs G.H. Exp. Parasitol. 1982; 54: 397-409Crossref PubMed Scopus (131) Google Scholar, 5.Burchmore R.J. Hart D.T. Mol. Biochem. Parasitol. 1995; 74: 77-86Crossref PubMed Scopus (44) Google Scholar). The higher rates of glucose uptake and utilization by promastigotes in relation to amastigotes is consistent with the high concentrations of sugars that promastigotes experience from the plant sap ingested by the sand fly vector (6.Schlein Y. Parasitol. Today. 1986; 2: 175-177Abstract Full Text PDF PubMed Scopus (49) Google Scholar) compared with the apparently sugar-limited environment that amastigotes experience (7.Burchmore R.J. Barrett M.P. Int. J. Parasitol. 2001; 31: 1311-1320Crossref PubMed Scopus (137) Google Scholar) in the macrophage phagolysosome. The L. mexicana genome encompasses a cluster of three glucose transporter genes, LmGT1, LmGT2, and LmGT3, which encode closely related isoforms (8.Burchmore R.J. Landfear S.M. J. Biol. Chem. 1998; 273: 29118-29126Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). A L. mexicana glucose transporter “knock-out” line (Δlmgt) (9.Burchmore R.J. Rodriguez-Contreras D. McBride K. Merkel P. Barrett M.P. Modi G. Sacks D. Landfear S.M. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 3901-3906Crossref PubMed Scopus (115) Google Scholar), in which the entire cluster of glucose transporter genes was eliminated by targeted gene replacement, exhibited no detectable glucose transport activity. The Δlmgt promastigotes were able to grow, although at a reduced rate compared with that of wild-type cells. In contrast, Δlmgt cells exhibited dramatically reduced viability inside macrophages compared with wild-type parasites and were unable to grow as axenic amastigotes, demonstrating that glucose transporters are essential for amastigote viability. Furthermore, axenically cultured wild-type amastigotes required glucose for viability (9.Burchmore R.J. Rodriguez-Contreras D. McBride K. Merkel P. Barrett M.P. Modi G. Sacks D. Landfear S.M. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 3901-3906Crossref PubMed Scopus (115) Google Scholar). The observation of the essential nature of glucose transporters in amastigotes originally posed a conundrum. Unexpectedly, the glucose carriers were essential in the life cycle stage in which they were least used for energy production. This suggested that glucose and the other hexoses (fructose, mannose, and galactose) 2D. Rodriquez-Contreras, X. Feng, K. Keeney, and S. M. Landfear, submitted for publication.2D. Rodriquez-Contreras, X. Feng, K. Keeney, and S. M. Landfear, submitted for publication. that these permeases transport may be building blocks for essential macromolecules in the mammalian stage of the parasite life cycle. Leishmania parasites synthesize a large number of abundant glycoconjugates, many of which are expressed on the parasite membrane or are secreted, that require hexoses for their biosynthesis. These include a major plasma membrane glycoconjugate lipophosphoglycan (LPG) 3The abbreviations used are: LPG, lipophosphoglycan; mPPG, membrane proteophosphoglycan; gp63, glycoprotein 63 (the major surface protease of 63 kDa); sAP, secreted acid phosphatase; GIPL, glycosylinositolphospholipid; iFBS, heat-inactivated fetal bovine serum; MES, 2-(N-morpholino)ethanesulfonic acid; mAb, monoclonal antibody; HPTLC, high performance thin layer chromatography; PEPCK, phosphoenolpyruvate carboxylase; PPDK, phosphate dikinase.3The abbreviations used are: LPG, lipophosphoglycan; mPPG, membrane proteophosphoglycan; gp63, glycoprotein 63 (the major surface protease of 63 kDa); sAP, secreted acid phosphatase; GIPL, glycosylinositolphospholipid; iFBS, heat-inactivated fetal bovine serum; MES, 2-(N-morpholino)ethanesulfonic acid; mAb, monoclonal antibody; HPTLC, high performance thin layer chromatography; PEPCK, phosphoenolpyruvate carboxylase; PPDK, phosphate dikinase. (10.Beverley S.M. Turco S.J. Trends Microbiol. 1998; 6: 35-40Abstract Full Text PDF PubMed Scopus (144) Google Scholar), a high molecular weight proteophosphoglycan (PPG) (11.Ilg T. Stierhof Y.D. Craik D. Simpson R. Handman E. Bacic A. J. Biol. Chem. 1996; 271: 21583-21596Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar), a family of small neutral glycosylinositolphospholipids (GIPLs) (12.Ralton J.E. McConville M.J. J. Biol. Chem. 1998; 273: 4245-4257Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar), the major cell surface glycoprotein, a metalloproteinase designated gp63 (13.Yao C. Donelson J.E. Wilson M.E. Mol. Biochem. Parasitol. 2003; 132: 1-16Crossref PubMed Scopus (212) Google Scholar), and a secreted glycoprotein enzyme designated secreted acid phosphatase (sAP) (14.Ilg T. Stierhof Y.D. Etges R. Adrian M. Harbecke D. Overath P. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 8774-8778Crossref PubMed Scopus (49) Google Scholar). Several such glycoconjugates have been shown to play important functions in the biology of the parasite. Leishmania parasites also elaborate an intracellular storage carbohydrate, β1–2 mannan, that is an important virulence factor required for viability of the amastigote stage of the life cycle, possibly because it protects against nutrient deprivation and other stresses encountered in the macrophage (15.Ralton J.E. Naderer T. Piraino H.L. Bashtannyk T.A. Callaghan J.M. McConville M.J. J. Biol. Chem. 2003; 278: 40757-40763Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). Because hexoses are necessary for biosynthesis of these carbohydrate-containing macromolecules, it is possible that genetic ablation of the glucose (hexose) transporters would also prevent their biosynthesis, thus explaining the lethal phenotype of the Δlmgt mutation in amastigotes. Indeed, evidence for an essential role of glycoconjugates and β-mannan emerged from previous studies on the gene encoding the enzyme GDP-mannose pyrophosphorylase (15.Ralton J.E. Naderer T. Piraino H.L. Bashtannyk T.A. Callaghan J.M. McConville M.J. J. Biol. Chem. 2003; 278: 40757-40763Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar, 16.Garami A. Mehlert A. Ilg T. Mol. Biol. 2001; PubMed Scopus Google Scholar). have the of the Δlmgt null mutants to synthesize these carbohydrate-containing have that Δlmgt parasites are still able to synthesize glycoconjugates and β-mannan to However, the reduced level of the essential virulence factor β-mannan in Δlmgt parasites may contribute to the of the glucose transporter null mutants to as amastigotes, the of in promastigotes and amastigotes. and of L. mexicana wild-type and null cell were cultured at in with heat-inactivated fetal bovine The and null mutants were by and Ilg amastigotes of wild-type parasites were by promastigotes at in J. Parasitol. PubMed Scopus Google Scholar), with and amastigote were by in of LPG, mPPG, gp63, and LPG, parasites were in and with for at T. J. PubMed Scopus Google Scholar) and with for at to carbohydrate that are also by the T. G. Etges R. D. Overath P. J. Sci. 1991; PubMed Google Scholar). In a was from cells as of mPPG, cell were as J.E. McConville M.J. Biochem. J. PubMed Scopus Google Scholar). gp63, membrane were by as M.E. J. Google Scholar). The and gp63 that is from the cell were as C. A. Donelson J.E. Wilson M.E. Mol. Biochem. Parasitol. PubMed Scopus Google Scholar) by the promastigotes in by in the cells were by and the were and with a the of was the of in gp63 and of gp63 and T. Stierhof Y.D. M. McConville M.J. Overath P. Parasitology. 1994; 108: PubMed Scopus Google Scholar, A. Mol. Biochem. Parasitol. 1994; PubMed Scopus Google Scholar), or from wild-type and the null cell were with in a phosphate as suggested by the The of in the was as a in the molecular by of LPG, mPPG, gp63, and by of cell or of cells were by to or and by of and mPPG, the which with the T. Harbecke D. M. Overath P. J. Biochem. PubMed Scopus Google Scholar) was used as the as this is glycoconjugates (11.Ilg T. Stierhof Y.D. Craik D. Simpson R. Handman E. Bacic A. J. Biol. Chem. 1996; 271: 21583-21596Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar, T. Harbecke D. M. Overath P. J. Biochem. PubMed Scopus Google Scholar), and is in the The of gp63 and in Δlmgt was by of the or cell gp63 and a against Leishmania gp63 M.E. J. Google Scholar) by which with L. mexicana gp63, and the of against T. Harbecke D. M. Overath P. J. Biochem. PubMed Scopus Google Scholar) by and to were as by the were the and of LPG, and and by and β-mannan were by of promastigotes with as (15.Ralton J.E. Naderer T. Piraino H.L. Bashtannyk T.A. Callaghan J.M. McConville M.J. J. Biol. Chem. 2003; 278: 40757-40763Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar) with The cell was by with for of for was by for and with The were and by in such as were in the in for The lower β-mannan was with and by was from by as in M.J. Bacic A. Handman E. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). The carbohydrate were on by and by with and to for cell from the number of cells were to the and as (12.Ralton J.E. McConville M.J. J. Biol. Chem. 1998; 273: 4245-4257Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar, J.E. Naderer T. Piraino H.L. Bashtannyk T.A. Callaghan J.M. McConville M.J. J. Biol. Chem. 2003; 278: 40757-40763Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar, T. Etges R. Overath P. McConville M.J. J. J. J. Biol. Chem. 1992; Full Text PDF PubMed Google Scholar). was also on as of the in wild-type and Δlmgt cell β-mannan was the acid which the carbohydrate in the from the number of wild-type and Δlmgt cells and of The Scholar). The sugar of was a for and glycoconjugates the by the a was as A. Turco S.J. S.M. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). were in and was at least three wild-type and Δlmgt were a and was of and and Δlmgt promastigotes were with sugars by the cells with and in with of wild-type and Δlmgt promastigotes and wild-type axenic amastigotes were in the with and of the acid and acid for the cells were in and used to and β-mannan and as The was also from and used as the for the metabolic β-mannan were on and with by was to and by of the in by and In was to acid at for S.J. P.A. PubMed Scopus Google Scholar), to the carbohydrate of the was the In this the in the of to the and was from the carbohydrate of LPG, which not to the The in the carbohydrate of was on a and promastigotes were and for as Landfear S.M. Mol. Biochem. Parasitol. PubMed Scopus Google Scholar). The was used at a of was used as a at a of the were with the were in by in and by a were Leishmania major genome L. G. M. E. E. R. A. P. C. A. S.M. G. K. G. M. E. L. C. A. J. A. G. A. M. C. A. A. D. C. D. Y. S. A. M. L. A. T. M. D. K. S. S. S. K. S. M. C. E. R. M. J. J. L. S. D. M. J. K. A. S. D. R. S. C. G. R. T. J. S. J.M. PubMed Scopus Google Scholar) was with the acid of the and phosphoenolpyruvate or or and the at and the phosphate or the acid of the enzyme was of Glucose on the of Δlmgt cells are unable to transport hexoses (9.Burchmore R.J. Rodriguez-Contreras D. McBride K. Merkel P. Barrett M.P. Modi G. Sacks D. Landfear S.M. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 3901-3906Crossref PubMed Scopus (115) Google Scholar), the of the Δlmgt to as amastigotes may be to the of of glycoconjugates by the of LPG, mPPG, gp63, and in this also the of these glycoconjugates in the L. mexicana wild-type the that not synthesize T. J. PubMed Scopus Google Scholar), and the null which not glycoconjugates or β-mannan A. Ilg T. J. 2001; PubMed Scopus Google Scholar). and were in Δlmgt as by of cells or cell with the were from wild-type and Δlmgt promastigotes were by Δlmgt promastigotes synthesize detectable of the and and the was no was in the and mutants In contrast, and were in the but not in the The of the gp63 and in Δlmgt were by of the gp63, not or cell secreted gp63 and A in the molecular weight of and gp63 was in wild-type and Δlmgt with that the Δlmgt null can These were by studies the demonstrating the of glycoconjugates on the surface of Δlmgt cells of glycoconjugates in the surface of L. mexicana wild-type and the null cell and promastigotes were with the as a and the as a A or of cell line is the level of glycoconjugates the wild-type and Δlmgt a the parasite cell as A. Turco S.J. S.M. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). that the by the is reduced in Δlmgt promastigotes compared with wild-type Δlmgt promastigotes are in than wild-type the in was not to because the was to for the the two that glycoconjugates to be by Δlmgt cells in the of although at reduced levels. of by Δlmgt also Δlmgt promastigotes are able to synthesize β-mannan, because to β-mannan (15.Ralton J.E. Naderer T. Piraino H.L. Bashtannyk T.A. Callaghan J.M. McConville M.J. J. Biol. Chem. 2003; 278: 40757-40763Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar) could the glucose transporter null not as amastigotes. The that β-mannan was by Δlmgt promastigotes but apparently at lower than in wild-type this compared the of β-mannan the number of wild-type and Δlmgt cell These that β-mannan was abundant in Δlmgt than in wild-type promastigotes were cells in the of not wild-type cells glucose sugar of β-mannan compared with Δlmgt cells in contrast, of β-mannan not Δlmgt cells in the or of Δlmgt of for that the null was not sugar for biosynthesis of glycoconjugates and β-mannan, wild-type and Δlmgt promastigotes with and the incorporation of into β-mannan and The although was into β-mannan and not in wild-type no such incorporation could be in Δlmgt were with and not the of Δlmgt promastigotes to sugar the of sugars in the glycoconjugates by Δlmgt parasites be by by the plasma or other that in level uptake in the of glycoconjugates in Δlmgt parasites derive hexoses from a than from the for in Δlmgt that the Δlmgt parasites could sugars for of carbohydrate-containing would be to synthesize by the and are to glucose L. and Scholar). functions in Δlmgt promastigotes to precursors for glycoconjugate biosynthesis by wild-type and Δlmgt promastigotes with precursors in the of a that from in wild-type promastigotes is a also wild-type cells in be that from was into β-mannan by Δlmgt promastigotes in the of In and as wild-type parasites not significantly from this into the β-mannan However, of glucose from the of wild-type promastigotes incorporation of from into β-mannan were from Δlmgt and wild-type promastigotes was In the from this be into the and the carbohydrate of the incorporation of from into the carbohydrate of from Δlmgt but not from wild-type by the carbohydrate incorporation into the of wild-type and Δlmgt parasites was that wild-type cells were were and and not that wild-type promastigotes can of glucose and Δlmgt which are unable to take up to sugars necessary for glycoconjugate and β-mannan biosynthesis, in the of extracellular in also in amastigotes. Because the Δlmgt null not grow as amastigotes (9.Burchmore R.J. Rodriguez-Contreras D. McBride K. Merkel P. Barrett M.P. Modi G. Sacks D. Landfear S.M. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 3901-3906Crossref PubMed Scopus (115) Google Scholar), the of wild-type axenic amastigotes (2.Bates P.A. Parasitology. 1994; 108: 1-9Crossref PubMed Scopus (126) Google Scholar) for glucose to by the cells with the in the or in the of incorporation into the of amastigotes was or higher in cells sugar not that amastigotes were for the used in these that amastigotes, to were able to from the into β-mannan also compared the incorporation of precursors into the β-mannan in promastigotes and amastigotes in the or of sugar in the and the to Both life cycle stages of precursors of sugar sugar that amastigotes and promastigotes have to gluconeogenesis. However, promastigotes and amastigotes is that the exhibited incorporation of into β-mannan in the of glucose in the that is by glucose in amastigotes, compared with the This is possible because glucose transport R.J. Hart D.T. Mol. Biochem. Parasitol. 1995; 74: 77-86Crossref PubMed Scopus (44) Google Scholar) and the (4.Hart D.T. Coombs G.H. Exp. Parasitol. 1982; 54: 397-409Crossref PubMed Scopus (131) Google Scholar, G.H. Hart D.T. Mol. Biochem. Parasitol. 1982; PubMed Scopus Google Scholar) are in amastigotes, thus the of that would of into β-mannan in or Glucose or in a of Δlmgt of and carbohydrate-containing to be for the survival and of Leishmania parasites in the sand fly vector and the mammalian host (15.Ralton J.E. Naderer T. Piraino H.L. Bashtannyk T.A. Callaghan J.M. McConville M.J. J. Biol. Chem. 2003; 278: 40757-40763Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar, 16.Garami A. Mehlert A. Ilg T. Mol. Biol. 2001; PubMed Scopus Google Scholar, D. S. 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The L. mexicana parasites from into β-mannan and However, it is not as a that can of carbohydrates in these The for in Leishmania species is shown in This is by the of genes encoding the of and the of the enzyme in L. mexicana or related Furthermore, this the of and to as precursors for of for Glucose in essential role for glucose transporters in amastigotes is by the observation that Δlmgt promastigotes macrophages as as wild-type parasites and into the Δlmgt amastigotes are unable to inside the macrophage and the of the wild-type amastigotes (9.Burchmore R.J. Rodriguez-Contreras D. McBride K. Merkel P. Barrett M.P. Modi G. Sacks D. Landfear S.M. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 3901-3906Crossref PubMed Scopus (115) Google Scholar). Furthermore, amastigotes require because wild-type axenic amastigotes they are to glucose (9.Burchmore R.J. Rodriguez-Contreras D. McBride K. Merkel P. Barrett M.P. Modi G. Sacks D. Landfear S.M. Proc. Natl. Acad. Sci. U. S. 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PubMed Scopus Google Scholar) has that a null in the gene encoding the enzyme in L. major is also unable to as amastigotes, and in this has a phenotype to that of the glucose transporter null these the of carbohydrates in amastigotes and that the intracellular stage of the life cycle glucose transporters and for viability. and Wilson for and cell Turco for and McConville for to this also and for for in and and for the