Characterization of a ricin-resistant mutant of Leishmania donovani that expresses lipophosphoglycan.

The abundant cell-surface lipophosphoglycan (LPG) of Leishmania parasites plays a central role throughout the eukaryote's life cycle. A number of LPG-defective mutants and their complementing genes have been isolated and have proven invaluable in assessing the importance of LPG and related glycoconjugates in parasite virulence. While ricin agglutination selection protocols frequently result in lpg- mutants, one  Leishmania donovani variant we isolated, named JABBA, was found to be lpg+. Procyclic (logarithmic) JABBA expresses significant amounts of a large-sized LPG, larger than observed from procyclic wild type but similar in size to LPG from wild type from metacyclic (stationary) phase. Structural analysis of the LPG from logarithmically grown JABBA by capillary electrophoresis protocols revealed that it averaged 30 repeat units composed of the unsubstituted Gal(β1,4)Man(α1)-PO4 typical of wild-type L. donovani. Analysis of JABBA LPG caps indicated that 20% is branched trisaccharide Gal(β1,4)[Glc(β1,2)]Man and tetrasaccharide Gal(β1,4)[Glc(β1,2)Man(α1,2)]Man instead of the usual Gal(β1,4)Man and Man(α1,2)Man terminating caps. Consistent with these structural observations, analyses of the relevant glycosyltransferases in JABBA microsomes involved in LPG biosynthesis showed a 2-fold increase in elongating mannosylphosphoryltransferase activity and up-regulation of a β-glucosyltransferase activity. Furthermore, the caps of JABBA LPG are cryptic in presentation as shown by the loss of binding by the lectins, ricin, peanut agglutinin and concanavalin A and reduced accessibility of the terminal galactose residues to oxidation by galactose oxidase. These results indicate that LPG from JABBA is intriguingly similar to the larger LPG in wild-type parasites that arises following the differentiation of the non-infectious procyclic promastigotes to infectious, metacyclic forms.

[1]  M. Lehrman,et al.  Letter to the Glycoforum: Improved protocols for preparing lipid-linked and related saccharides for Fluorophore-Assisted Carbohydrate Electrophoresis (FACE). , 2013, Glycobiology.

[2]  K. Chang,et al.  The role of surface glycoconjugates in Leishmania midgut attachment examined by competitive binding assays and experimental development in sand flies , 2013, Parasitology.

[3]  R. Soares,et al.  Two biochemically distinct lipophosphoglycans from Leishmania braziliensis and Leishmania infantum trigger different innate immune responses in murine macrophages , 2013, Parasites & Vectors.

[4]  R. Soares,et al.  Glycoconjugates in New World species of Leishmania: polymorphisms in lipophosphoglycan and glycoinositolphospholipids and interaction with hosts. , 2012, Biochimica et biophysica acta.

[5]  Antony Bacic,et al.  Determining the polysaccharide composition of plant cell walls , 2012, Nature Protocols.

[6]  P. Kaye,et al.  Leishmaniasis: complexity at the host–pathogen interface , 2011, Nature Reviews Microbiology.

[7]  P. Pimenta,et al.  Leishmania infantum: Lipophosphoglycan intraspecific variation and interaction with vertebrate and invertebrate hosts. , 2011, International journal for parasitology.

[8]  D. Dobson,et al.  Leishmania major Survival in Selective Phlebotomus papatasi Sand Fly Vector Requires a Specific SCG-Encoded Lipophosphoglycan Galactosylation Pattern , 2010, PLoS pathogens.

[9]  S. Turco,et al.  Quantitation of Leishmania lipophosphoglycan repeat units by capillary electrophoresis. , 2006, Biochimica et biophysica acta.

[10]  Clare L. Bennett,et al.  A critical role for lipophosphoglycan in proinflammatory responses of dendritic cells to Leishmania mexicana , 2005, European journal of immunology.

[11]  A. Warburg,et al.  Leishmania tropica: intraspecific polymorphisms in lipophosphoglycan correlate with transmission by different Phlebotomus species. , 2004, Experimental parasitology.

[12]  S. Beverley,et al.  Leishmania LPG3 encodes a GRP94 homolog required for phosphoglycan synthesis implicated in parasite virulence but not viability , 2002, The EMBO journal.

[13]  I. C. Almeida,et al.  Leishmania chagasi: lipophosphoglycan characterization and binding to the midgut of the sand fly vector Lutzomyia longipalpis. , 2002, Molecular and biochemical parasitology.

[14]  S. Beverley,et al.  The Leishmania GDP-mannose transporter is an autonomous, multi-specific, hexameric complex of LPG2 subunits. , 2000, Biochemistry.

[15]  S. Beverley,et al.  The role of phosphoglycans in Leishmania-sand fly interactions. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[16]  S. Turco,et al.  Characterization of the glucosyltransferases that assemble the side chains of the Indian Leishmania donovani lipophosphoglycan. , 1999, Archives of biochemistry and biophysics.

[17]  A. Descoteaux,et al.  Glycoconjugates in Leishmania infectivity. , 1999, Biochimica et biophysica acta.

[18]  G. Modi,et al.  Intra-species and stage-specific polymorphisms in lipophosphoglycan structure control Leishmania donovani-sand fly interactions. , 1999, Biochemistry.

[19]  E. Saraiva,et al.  Lectin-binding properties of different Leishmania species , 1999, Parasitology Research.

[20]  S. Beverley,et al.  Leishmania donovani has distinct mannosylphosphoryltransferases for the initiation and elongation phases of lipophosphoglycan repeating unit biosynthesis. , 1998, Molecular and biochemical parasitology.

[21]  M. Shahabuddin,et al.  A novel role for the peritrophic matrix in protecting Leishmania from the hydrolytic activities of the sand fly midgut , 1997, Parasitology.

[22]  M. McConville,et al.  Developmentally regulated changes in the cell surface architecture of Leishmania parasites. , 1997, Behring Institute Mitteilungen.

[23]  S. Beverley,et al.  Golgi GDP-mannose Uptake Requires Leishmania LPG2 , 1997, The Journal of Biological Chemistry.

[24]  J. S. Brimacombe,et al.  Regulation of the expression of nitric oxide synthase and leishmanicidal activity by glycoconjugates of Leishmania lipophosphoglycan in murine macrophages. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[25]  S. Beverley,et al.  Identification of genes mediating lipophosphoglycan biosynthesis by functional complementation of Leishmania donovani mutants. , 1995, Annals of tropical medicine and parasitology.

[26]  S. Beverley,et al.  A specialized pathway affecting virulence glycoconjugates of Leishmania. , 1995, Science.

[27]  M. McConville,et al.  Stage-specific binding of Leishmania donovani to the sand fly vector midgut is regulated by conformational changes in the abundant surface lipophosphoglycan , 1995, The Journal of experimental medicine.

[28]  S. Turco,et al.  Defective galactofuranose addition in lipophosphoglycan biosynthesis in a mutant of Leishmania donovani. , 1993, The Journal of biological chemistry.

[29]  L. Garraway,et al.  Isolation of virulence genes directing surface glycosyl-phosphatidylinositol synthesis by functional complementation of Leishmania. , 1993, Proceedings of the National Academy of Sciences of the United States of America.

[30]  M. Ferguson,et al.  The glycoinositol phospholipids of Leishmania mexicana promastigotes. Evidence for the presence of three distinct pathways of glycolipid biosynthesis. , 1993, The Journal of biological chemistry.

[31]  M. Quinten,et al.  Expression of lipophosphoglycan, high-molecular weight phosphoglycan and glycoprotein 63 in promastigotes and amastigotes of Leishmania mexicana. , 1993, Molecular and biochemical parasitology.

[32]  S. Beverley,et al.  Plasticity in chromosome number and testing of essential genes in Leishmania by targeting. , 1993, Proceedings of the National Academy of Sciences of the United States of America.

[33]  M. Ferguson,et al.  Developmental modification of lipophosphoglycan during the differentiation of Leishmania major promastigotes to an infectious stage. , 1992, The EMBO journal.

[34]  S. Turco,et al.  Biosynthesis of lipophosphoglycan from Leishmania donovani: characterization of mannosylphosphate transfer in vitro. , 1992, Archives of biochemistry and biophysics.

[35]  M. Ferguson,et al.  Structure of Leishmania mexicana lipophosphoglycan. , 1992, The Journal of biological chemistry.

[36]  M. Ferguson,et al.  Refined structure of the lipophosphoglycan of Leishmania donovani. , 1992, The Journal of biological chemistry.

[37]  M. Ferguson,et al.  Purification and characterization of an extracellular phosphoglycan from Leishmania donovani. , 1992, The Journal of biological chemistry.

[38]  S. Homans,et al.  Solution structure of the lipophosphoglycan of Leishmania donovani. , 1992, Biochemistry.

[39]  J. Blackwell,et al.  Developmental changes in the glycosylated phosphatidylinositols of Leishmania donovani. Characterization of the promastigote and amastigote glycolipids. , 1991, The Journal of biological chemistry.

[40]  S. Turco,et al.  Cell-free biosynthesis of lipophosphoglycan from Leishmania donovani. Characterization of microsomal galactosyltransferase and mannosyltransferase activities. , 1991, The Journal of biological chemistry.

[41]  R. Tesh,et al.  A test for genetic exchange in mixed infections of Leishmania major in the sand fly Phlebotomus papatasi. , 1991, The Journal of protozoology.

[42]  M. Ferguson,et al.  Structure of the lipophosphoglycan from Leishmania major. , 1990, The Journal of biological chemistry.

[43]  S. Beverley,et al.  Stable transfection of the human parasite Leishmania major delineates a 30-kilobase region sufficient for extrachromosomal replication and expression , 1990, Molecular and cellular biology.

[44]  T. Pearson,et al.  The immunochemical structure and surface arrangement of Leishmania donovani lipophosphoglycan determined using monoclonal antibodies. , 1989, Molecular and biochemical parasitology.

[45]  A. M. Wu,et al.  Immunochemical studies on the N-acetyllactosamine beta-(1----6)-linked trisaccharide specificity of Ricinus communis agglutinin. , 1988, Carbohydrate research.

[46]  B. Ullman,et al.  Methotrexate-resistant Leishmania donovani genetically deficient in the folate-methotrexate transporter. , 1988, The Journal of biological chemistry.

[47]  S. Turco,et al.  A ricin agglutinin-resistant clone of Leishmania donovani deficient in lipophosphoglycan. , 1988, Molecular and biochemical parasitology.

[48]  A. Bacic,et al.  Lipophosphoglycan of Leishmania major that vaccinates against cutaneous leishmaniasis contains an alkylglycerophosphoinositol lipid anchor. , 1987, Proceedings of the National Academy of Sciences of the United States of America.

[49]  R. Dwek,et al.  Structure of the major carbohydrate fragment of the Leishmania donovani lipophosphoglycan. , 1987, Biochemistry.

[50]  P. Orlandi,et al.  Structure of the lipid moiety of the Leishmania donovani lipophosphoglycan. , 1987, The Journal of biological chemistry.

[51]  G. Cross,et al.  Glycosyl-sn-1,2-dimyristylphosphatidylinositol is covalently linked to Trypanosoma brucei variant surface glycoprotein. , 1985, The Journal of biological chemistry.

[52]  M. Lehrman,et al.  Alternative and sources of reagents and supplies of fluorophore-assisted carbohydrate electrophoresis(FACE). , 2003, Glycobiology.

[53]  S. Kamhawi,et al.  Molecular aspects of parasite-vector and vector-host interactions in leishmaniasis. , 2001, Annual review of microbiology.

[54]  F. Liew,et al.  Immune effector mechanism in parasitic infections. , 1999, Immunology letters.

[55]  S. Beverley,et al.  Lipophosphoglycan (LPG) and the identification of virulence genes in the protozoan parasite Leishmania. , 1998, Trends in microbiology.

[56]  S. Turco,et al.  Biosynthesis of Leishmania lipophosphoglycan: solubilization and partial characterization of the initiating mannosylphosphoryltransferase. , 1997, Glycobiology.

[57]  R. Townsend,et al.  High-pH anion-exchange chromatography of glycoprotein-derived carbohydrates. , 1994, Methods in enzymology.

[58]  A. Descoteaux,et al.  The lipophosphoglycan of Leishmania parasites. , 1992, Annual review of microbiology.

[59]  D. Iovannisci,et al.  Single cell cloning of Leishmania parasites in purine-defined medium: isolation of drug-resistant variants. , 1984, Advances in experimental medicine and biology.