Acta Parasitologica

, Volume 63, Issue 1, pp 75–86 | Cite as

Characterization of phosphate transporter(s) and understanding their role in Leishmania donovani parasite

  • K. J. Sindhu
  • Amit Kumar Kureel
  • Sheetal Saini
  • Smita Kumari
  • Pankaj Verma
  • Ambak Kumar RaiEmail author


Inorganic phosphate (Pi) is shown to be involved in excretion of methylglyoxal (MG) in the promastigote form of Leishmania donovani parasite. Absence of Pi leads to its accumulation inside the parasite. Accumulation of MG is toxic to the parasite and utilizes glyoxylase as well as excretory pathways for its detoxification. In addition, Pi is also reported to regulate activities of ectoenzymes and energy metabolism (glucose to pyruvate) etc. Thus, it is known to cumulatively affect the growth of Leishmania parasite. Hence the transporters, which allow the movement of Pi across the membrane, can prove to be a crucial drug target. Therefore, we characterized two phosphate transporters in Leishmania (i) H+ dependent myo-inositol transporter (LdPHO84), and (ii) Na+ dependent transporter (LdPHO89), based on similar studies done previously on other lower organisms and trypanosomatids. We tried to understand the secondary structure of these two proteins and confirm modulation in their expression with the change in Pi concentration outside. Moreover, their modes of action were also measured in the presence of specific inhibitors (LiF, CCCP). Further analysis on the physiological role of these transporters in various stages of the parasite life cycle needs to be entrenched.


Leishmania inorganic phosphate Pi transporters methylglyoxal drug resistance 


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. Arnold K., Bordoli L., Kopp J., Schwede T. 2006. The SWISS-MODEL Workspace: A web-based environment for protein structure homology modelling. Bioinformatics, 22, 195–201. DOI: 10.1093/bioinformatics/bti770CrossRefGoogle Scholar
  2. Benkert P., Tosatto S.C., Schomburg D. 2008. QMEAN: A comprehensive scoring function for model quality assessment. Proteins, 71, 261–277. DOI: 10.1002/prot.21715CrossRefGoogle Scholar
  3. Bernsel A., Viklund H., Hennerdal A., Elofsson A. 2009. TOPCONS: consensus prediction of membrane protein topology. Nucleic Acids Research, 37, W465–468. DOI: 10.1093/nar/gkp363CrossRefGoogle Scholar
  4. Boutet E., Lieberherr D., Tognolli M., Schneider M., Bansal P., Bridge A.J. et al. 2016. UniProtKB/Swiss-Prot, the Manually Annotated Section of the UniProt KnowledgeBase: How to Use the Entry View. Methods in Molecular Biology, 1374, 23–54. DOI: 10.1007/978-1-4939-3167-5_2CrossRefGoogle Scholar
  5. Burns D.J., Beever R.E. 1977. Kinetic characterization of the two phosphate uptake systems in the fungus Neurospora crassa. Journal of Bacteriology, 132, 511–519PubMedPubMedCentralGoogle Scholar
  6. Callens M., Kuntz D.A., Opperdoes F.R. 1991. Characterization of pyruvate kinase of Trypanosoma brucei and its role in the regulation of carbohydrate metabolism. Molecular and Biochemical Parasitology, 47, 19–29CrossRefGoogle Scholar
  7. Coombs G.H., Craft J.A., Hart D.T. 1982. A comparative study of Leishmania mexicana amastigotes and promastigotes. Enzyme activities and subcellular locations. Molecular and Biochemical Parasitology, 5, 199–211PubMedGoogle Scholar
  8. Desjeux P. 2001. The increase in risk factors for leishmaniasis worldwide. Transactions of the Royal Society of Tropical Medicine and Hygiene, 95, 239–243CrossRefGoogle Scholar
  9. Desjeux P. 2004. Leishmaniasis: current situation and new perspectives. Comparative Immunology, Microbiology & Infectious Diseases, 27, 305–318. DOI: 10.1016/j.cimid.2004.03.004Google Scholar
  10. Dick C.F., Dos-Santos A.L., Fonseca-de-Souza A.L., Rocha-Ferreira J., Meyer-Fernandes J.R. 2010. Trypanosoma rangeli: differential expression of ecto-phosphatase activities in response to inorganic phosphate starvation. Experimental Parasitology, 124, 386–393. DOI: 10.1016/j.exppara.2009.12.006CrossRefGoogle Scholar
  11. Dick C.F., Dos-Santos A.L., Majerowicz D., Gondim K.C., Caruso-Neves C., Silva I.V., et al. 2012. Na+-dependent and Na+-independent mechanisms for inorganic phosphate uptake in Trypanosoma rangeli. Biochimica et Biophysica Acta, 1820, 1001–1008. DOI: 10.1016/j.bbagen.2012.02.019CrossRefGoogle Scholar
  12. Dick C.F., Dos-Santos A.L., Meyer-Fernandes J.R. 2014. Inorganic phosphate uptake in unicellular eukaryotes. Biochimica et Biophysica Acta, 1840, 2123–2127. DOI: 10.1016/j.bbagen.2014.03.014CrossRefGoogle Scholar
  13. Docampo R., Ulrich P., Moreno S. N. 2010. Evolution of acidocalcisomes and their role in polyphosphate storage and osmoregulation in eukaryotic microbes. Philosophical Transactions of the Royal Society Biological Sciences, 365, 775–784. DOI: 10.1098/rstb.2009.0179CrossRefGoogle Scholar
  14. Drew M.E., Langford C.K., Klamo E.M., Russell D.G., Kavanaugh M.P., Landfear S.M. 1995. Functional Expression of a myo-Inositol/H1 Symporter from Leishmania donovani. Molecular and Cellular Biology, 15, 5508–5515CrossRefGoogle Scholar
  15. Dujardin J.C. 2006. Risk factors in the spread of leishmaniases: towards integrated monitoring? Trends in Parasitology, 22, 4–6. DOI: 10.1016/ Scholar
  16. Dunker A.K., Lawson J.D., Brown C.J., Williams R.M., Romero P., Oh J.S., et al. 2001. Intrinsically disordered protein. Journal of Molecular Graphics and Modelling, 19, 26–59. DOI:10.1016/S1093-3263(00)00138-8CrossRefGoogle Scholar
  17. Dunker A.K., Silman I., Uversky V.N., Sussman J.L. 2008. Function and structure of inherently disordered proteins. Current Opinion in Structural Biology, 18, 756–764. DOI: 10.1016/ Scholar
  18. Dyson H.J., Wright P.E. 2005. Intrinsically unstructured proteins and their functions. Nature Reviews Molecular Cell Biology, 6, 197–208. DOI: 10.1038/nrm1589CrossRefGoogle Scholar
  19. Ernest I., Callens M., Opperdoes F.R., Michels P.A. 1994. Pyruvate kinase of Leishmania mexicana mexicana. Cloning and analysis of the gene, overexpression in Escherichia coli and characterization of the enzyme. Molecular and Biochemical Parasitology, 64, 43–54CrossRefGoogle Scholar
  20. Felsenstein J. 1985. Confidence limits on phylogenies: An approach using the bootstrap. Evolution, 39(4), 783–791. DOI: 10.1111/j.1558-5646.1985.tb00420.xCrossRefGoogle Scholar
  21. Fiser A., Sali A. 2003. ModLoop: automated modeling of loops in protein structures. Bioinformatics, 19, 2500–2501CrossRefGoogle Scholar
  22. Fiser A., Do R.K., Sali A. 2000. Modeling of loops in protein structures. Protein Science, 9, 1753–1773. DOI: 10.1110/ps.9.9.1753CrossRefGoogle Scholar
  23. Guex N., Peitsch M.C., Schwede T. 2009. Automated comparative protein structure modeling with SWISS-MODEL and Swiss-PdbViewer: A historical perspective. Electrophoresis, 30, 162–173. DOI: 10.1002/elps.200900140CrossRefGoogle Scholar
  24. Kumar S., Stecher G., Tamura K. 2015. MEGA7: Molecular Evolutionary Genetics Analysis Version 7.0 for Bigger Datasets. Molecular Biology and Evolution, 33, 1870–1874. DOI: 10.1093/molbev/msw054CrossRefGoogle Scholar
  25. Lamarche M.G., Wanner B.L., Crepin S., Harel J. 2008. The phosphate regulon and bacterial virulence: a regulatory network connecting phosphate homeostasis and pathogenesis. FEMS Microbiology Reviews, 32, 461–473. DOI: 10.1111/j.1574-6976.2008.00101.xCrossRefGoogle Scholar
  26. Lanzetta P.A., Alvarez L.J., Reinach P., Candia O.A. 1979. An improved assay for nanomole amounts of inorganic phosphate. Analytical Biochemistry, 100, 95–97CrossRefGoogle Scholar
  27. Livak K.J., Schmittgen T.D. 2001. Analysis of Relative Gene Expression Data Using Real-Time Quantitative PCR and the 2-ΔΔCT Method. Methods 25, 402–408. DOI: 10.1006/meth.2001.12CrossRefGoogle Scholar
  28. Lom J. 1976. Biology of the trypanosomes and trypanoplasms of fish. In: (Eds W. H. R. Lumsden, and D. A. Evans) Biology of the Kinetoplastida. Academic Press, London/New York/San Francisco. 269–337Google Scholar
  29. Lovell S.C., Davis I.W., Arendall W.B., de Bakker P.I., Word J.M., Prisant M.G., et al. 2003. Structure validation by C-alpha geometry: phi,psi and C-beta deviation. Proteins, 50, 437–450. DOI: 10.1002/prot.10286CrossRefGoogle Scholar
  30. Lowendorf H.S., Slayman C.L., Slayman C.W. 1974. Phosphate transport in Neurospora. Kinetic characterization of a constitutive, low-affinity transport system. Biochimica et Biophysica Acta, 373, 369–382CrossRefGoogle Scholar
  31. Margarane M., UniProt Consorsium. 2011. UniProt Knowledgebase: a hub of integrated protein data. Database (Oxford), 2011, bar009. DOI: 10.1093/database/bar009Google Scholar
  32. Martinez P., Persson B.L. 1998. Identification, cloning and characterization of a derepressible Na+-coupled phosphate transporter in Saccharomyces cerevisiae. Molecular Genetics and Genomics, 258, 628–638CrossRefGoogle Scholar
  33. Mason P.W., Carbone D.P., Cushman R.A., Waggoner A.S. 1981. The importance of inorganic phosphate in regulation of energy metabolism of Streptococcus lactis. Journal of Biological Chemistry, 256, 1861–1866.PubMedGoogle Scholar
  34. McGwire B.S., Satoskar A.R. 2014. Leishmaniasis: clinical syndromes and treatment. QJM, 107, 7–14. DOI: 10.1093/qjmed/hct116CrossRefGoogle Scholar
  35. Michels P., Bringaud F., Herman M., Hannaert V. 2006. Metabolic functions of glycosomes in trypanosomatids. Biochimica et Biophysica Acta 3- Molecular Cell Research, 1763, 1463–1477. DOI: 10.1016/j.bbamcr.2006.08.019CrossRefGoogle Scholar
  36. Oshima Y. 1997. The phosphatase system in Saccharomyces cerevisiae. Genes & Genetic Systems, 72, 323–334CrossRefGoogle Scholar
  37. Pabon M.A., Caceres A.J., Gualdron M., Quinones W., Avilan L., Concepcion J.L. 2007. Purification and characterization of hexokinase from Leishmania mexicana. Parasitology Research, 100, 803–810. DOI: 10.1007/s00436-006-0351-4CrossRefGoogle Scholar
  38. Persson B.L., Berhe A., Fristedt U., Martinez P., Pattison J., Petersson J., Weinander R. 1998. Phosphate permeases of Saccharomyces cerevisiae. Biochimica et Biophysica Acta, 1365, 23–30. DOI: 10.1016/S0005-2728(98)00037-1CrossRefGoogle Scholar
  39. Persson B.L., Petersson J., Fristedt U., Weinander R., Berhe A., Pattison J. 1999. Phosphate permeases of Saccharomyces cerevisiae: structure, function and regulation. Biochimica et Biophysica Acta, 1422, 255–272CrossRefGoogle Scholar
  40. Pillai A.D., Addo R., Sharma P., Nguitragool W., Srinivasan P., Desai S. A. 2013. Malaria parasites tolerate a broad range of ionic environments and do not require host cation remodeling. Molecular Microbiology, 88, 20–34. DOI: 10.1111/mmi.12159CrossRefGoogle Scholar
  41. Rai A.K., Kumar P., Saini S., Thakur C.P., Seth T., Marta D.K. 2016. Increased level of soluble adenosine deaminase in bone marrow of visceral leishmaniasis patients: an inverse relation with parasite load. Acta Parasitologica, 61, 645–649. DOI: 10.1515/ap-2016-0087CrossRefGoogle Scholar
  42. Rizzo S.C., Eckel R.E. 1966. Control of glycolysis in human erythrocytes by inorganic phosphate and sulfate. American Journal of Physiology, 211, 429–436PubMedGoogle Scholar
  43. Rosenberg H., Gerdes R.G., Chegwidden K. 1977. Two systems for the uptake of phosphate in Escherichia coli. Journal of Bacteriology, 131, 505–511PubMedPubMedCentralGoogle Scholar
  44. Roy A., Kucukural A., Zhang Y. 2010. I-TASSER: a unified platform for automated protein structure and function prediction. Nature Protocols, 5, 725–738. DOI: 10.1038/nprot.2010.5CrossRefGoogle Scholar
  45. Russo-Abrahao T., Alves-Bezerra M., Majerowicz D., Freitas-Mesquita A. L., Dick C. F., Gondim K. C., Meyer-Fernandes J. R. 2013. Transport of inorganic phosphate in Leishmania infantum and compensatory regulation at low inorganic phosphate concentration. Biochimica et Biophysica Acta, 1830, 2683–2689CrossRefGoogle Scholar
  46. Russo-Abrahao T., Koeller C.M., Steinmann M.E., Silva-Rito S., Marins-Lucena T., Alves-Bezerra M., et al. 2017. H+ dependent inorganic phosphate uptake in Trypanosoma brucei is influenced by myo-inositol transporter. Journal of Bioenergetics and Biomembranes, 49, 183–194. DOI: 10.1007/s10863-017-9695-yCrossRefGoogle Scholar
  47. Sacci J.B. Jr., Campbell T. A., Gottlieb M. 1990. Leishmania donovani: regulated changes in the level of expression of the surface 3’-nucleotidase/nuclease. Experimental Parasitology, 71, 158–168CrossRefGoogle Scholar
  48. Saitou N., Nei M. 1987. The neighbor-joining method: a new method for reconstructing phylogenetic trees. Molecular Biology and Evolution, 4, 406–425PubMedGoogle Scholar
  49. Saliba K.J., Martin R.E., Broer A., Henry R.I., McCarthy C.S., Downie M.J., Allen R.J., Mullin K.., McFadden G.I., Broer S., Kirk K. 2006. Sodium-dependent uptake of inorganic phosphate by the intracellular malaria parasite. Nature, 443, 582–585. DOI: 10.1038/nature05149PubMedGoogle Scholar
  50. Samira A., Philippe L. 2017. In vitro effects of purine and pyrimidine analogues on Leishmania donovani and Leishmania infantum promastigotes and intracellular amastigotes. Acta Parasitologica, 62, 582–588. DOI: 10.1515/ap-2017-0070Google Scholar
  51. Schmittgen T. D., Livak K. J. 2008. Analyzing real-time PCR data by the comparative CT method. Nature Protocols, 3, 1101–1108CrossRefGoogle Scholar
  52. Tasaki Y., Kamiya Y., Azwan A., Hara T., Joh T. 2002. Gene expression during Pi deficiency in Pholiota nameko: accumulation of mRNAs for two transporters. Bioscience, Biotechnology, and Biochemistry, 66, 790–800. DOI: 10.1271/bbb.66.790CrossRefGoogle Scholar
  53. Tiwari P., Verma P., Kureel A. K., Saini S., Rai A. K. 2016. Pi inhibits intracellular accumulation of methylglyoxal in promastigote form of L. donovani. Molecular and Biochemical Parasitology, 207, 89–95. DOI: 10.1016/j.molbiopara.2016.06.005CrossRefGoogle Scholar
  54. Tsirigos K. D., Peters C., Shu N., Kall L., Elofsson A. 2015. The TOPCONS web server for combined membrane protein topology and signal peptide prediction. Nucleic Acids Research, 43, W401–W407. DOI:10.1093/nar/gkv485CrossRefGoogle Scholar
  55. Tyler P., Sudhandiran G., Hobbs S. B., Seyfang A. 2004. Substrate specificity of the Leishmania donovani myo-inositol transporter: critical role of inositol C-2, C-3 and C-5 hydroxyl groups. Molecular & Biochemical Parasitology 135, 133–141. DOI: 10.1016/j.molbiopara.2004.01.015CrossRefGoogle Scholar
  56. Versaw W. K., Metzenberg R. L. 1995. Repressible cation-phosphate symporters in Neurospora crassa. Proceedings of the National Academy of Sciences, 92, 3884–3887CrossRefGoogle Scholar
  57. Vieira D. P., Paletta-Silva R., Saraiva E. M., Lopes A. H., Meyer-Fernandes J. R. 2011. Leishmania chagasi: an ecto-3’-nucleotidase activity modulated by inorganic phosphate and its possible involvement in parasite-macrophage interaction. Experimental Parasitology, 127, 702–707. DOI: 10.1016 /j.exppara.2010.11.003CrossRefGoogle Scholar
  58. Vieira B.R., Gomes-Vieira A.L., Carvalho-Kelly L.F., Russo A.T., Meyer Frenandes J.R. 2017. The biochemcial chacracterization of two phosphate transport system in Phytomonas serpens. Experimental Parasitology, 173, 1–8. DOI: 10.1016/j.exppara.2016.12.007CrossRefGoogle Scholar
  59. Wallner B., Elofsson A. 2003. Can correct protein models be identified? Protein Science, 12, 1073–1086. DOI: 10.1110/ps.0236803CrossRefGoogle Scholar
  60. Ward J. J., Sodhi J. S., McGuffin L. J., Buxton B. F., Jones D. T. 2004. Prediction and functional analysis of native disorder in proteins from the three kingdoms of life. Journal of Molecular Biology, 337, 635–645. DOI: 10.1016/j.jmb.2004.02.002CrossRefGoogle Scholar
  61. Ward J. J., McGuffin L. J., Bryson K., Buxton B. F., Jones D. T. 2004. The DISOPRED server for the prediction of protein disorder. Bioinformatics, 20, 2138–2139. DOI: 10.1093/bioinformatics/bth195CrossRefGoogle Scholar
  62. Wass M. N., Kelley L. A., Sternberg M. J. 2010. 3DLigandSite: predicting ligand-binding sites using similar structures. Nucleic Acids Research, 38, W476–473. DOI: 10.1093/nar/gkq406CrossRefGoogle Scholar
  63. WHO 2015. Kala-Azar elimination programme: report of a WHO consultation of partners, Geneva, Switzerland, 10–11 February 2015Google Scholar
  64. Willsky G. R., Bennett R. L., Malamy M. H. 1973. Inorganic Phosphate Transport in Escherichia coli: Involvement of Two Genes Which Play a Role in Alkaline Phosphatase Regulation. Journal of Bacteriology 113, 529–539PubMedPubMedCentralGoogle Scholar
  65. Willsky G. R., Malamy M. H. 1980. Characterization of two genetically separable inorganic phosphate transport systems in Escherichia coli. Journal of Bacteriology, 144, 356–365PubMedPubMedCentralGoogle Scholar
  66. Yang J., Zhang Y. 2015. I-TASSER server: new development for protein structure and function predictions. Nucleic Acids Research, 43, 174–181. DOI: 10.1093/nar/gkv342CrossRefGoogle Scholar
  67. Zhang K., Hsu F. F., Scott D. A., Docampo R., Turk J., Beverley S. M. 2005. Leishmania salvage and remodelling of host sphingolipids in amastigote survival and acidocalcisome biogenesis. Molecular Microbiology, 55, 1566–1578. DOI: 10.1111/j.1365-2958.2005.04493.xCrossRefGoogle Scholar
  68. Zhang Y. 2008. I-TASSER server for protein 3D structure prediction. BMC Bioinformatics, 9, 40. DOI: 10.1186/1471-2105-9-40CrossRefGoogle Scholar
  69. Zuckerkandl E., Pauling L. 1965. Evolutionary divergence and convergence in proteins. Evolving Genes and Proteins, 97–166. DOI:10.1016/B978-1-4832-2734-4.50017-6CrossRefGoogle Scholar

Copyright information

© Witold Stefański Institute of Parasitology, Polish Academy of Sciences 2018

Authors and Affiliations

  • K. J. Sindhu
    • 1
  • Amit Kumar Kureel
    • 1
  • Sheetal Saini
    • 1
  • Smita Kumari
    • 1
  • Pankaj Verma
    • 1
  • Ambak Kumar Rai
    • 1
    Email author
  1. 1.Department of BiotechnologyMotilal Nehru National Institute of TechnologyIndia

Personalised recommendations