Nucleoside Transporters of Plasmodium falciparum

Summary

Principal Investigator: B Ullman
Abstract: Amalgamating tools of molecular biology, biochemistry, genetics, and immunocytochemistry, this proposal offers an interdisciplinary dissection of the nucleoside transporters of Plasmodium falciparum. As protozoan parasites are incapable of synthesizing purine nucleotides de novo, nucleoside transporters provide an important, if not obligatory, nutritional function for the parasite and present several therapeutic paradigms. Two nucleoside transporter genes, PfNT1 and PfNT2, have been identified within available P. falciparum databases, and both have been cloned and sequenced in this laboratory. PfNT1 activity has been characterized in a preliminary fashion after PfNT1 cRNA injection into Xenopus laevis oocytes, and PfNT1 has also been functionally overexpressed in nucleoside transport-deficient Leishmania donovani. In addition polyclonal antisera specific for PfNT1 have been raised in rabbits and used to localize PfNT1 to the parasite plasma membrane by confocal and immunoelectron microscopy. Antibodies against PfNT2 have also been generated. These reagents are the cornerstone of the three specific aims in this proposal. The multicomponent Specific Aim I will encompass: i., a thorough biochemical characterization of PfNT1 with respect to ligand specificity and affinities and sensitivities to inhibitors of mammalian nucleoside transport; ii., an assessment of whether PfNT2 is a functional nucleoside transporter, and if so, a preliminary molecular and biochemical characterization, including immunolocatization of the protein in P. falciparum-infected erythrocytes; and iii., a verification of whether PfNT1 and PfNT2 are electrogenic transporters using the Xenopus oocyte cRNA expression system. The second Specific Aim initiates a structure-function analysis of PfNTI. We propose to implement a genetic screen for loss-of-function mutants to identify in an unbiased fashion key amino acids in PfNT1 that are required for ligand permeation and/or ligand selectivity. The last Specific Aim will explore the physiological function of PfNT1 within the parasitized erythrocyte using transfection and gene targeting approaches. Specifically, we will attempt to create Apfntl knockouts in either wild type or genetically complemented P. falciparum in order to test whether PfNT1 function is essential to the intact parasite. We will then characterize the resultant transport and growth phenotypes.
Funding Period: 2003-01-01 - 2008-12-31
more information: NIH RePORT

Top Publications

  1. pmc Plasmodium falciparum phosphoethanolamine methyltransferase is essential for malaria transmission
    April M Bobenchik
    Department of Internal Medicine, Yale University School of Medicine, New Haven, CT 06520
    Proc Natl Acad Sci U S A 110:18262-7. 2013
  2. pmc (1)H, (13)C, and (15)N chemical shift assignments for PfPMT, a phosphoethanolamine methyltransferase from Plasmodium falciparum
    Irina Bezsonova
    Department of Molecular, Microbial and Structural Biology, University of Connecticut Health Center, 263 Farmington Ave, Farmington, CT 6030 3305, USA
    Biomol NMR Assign 7:17-20. 2013
  3. pmc Gene selective mRNA cleavage inhibits the development of Plasmodium falciparum
    Yoann Augagneur
    Department of Internal Medicine, Yale University School of Medicine, New Haven, CT 06510, USA
    Proc Natl Acad Sci U S A 109:6235-40. 2012
  4. pmc PG12, a phospholipid analog with potent antimalarial activity, inhibits Plasmodium falciparum CTP:phosphocholine cytidylyltransferase activity
    Patricia Gonzalez-Bulnes
    Research Unit on Bioactive Molecules, Departament de Química Biomèdica, Instituto de Química Avanzada de Cataluña IQAC, CSIC, Jordi Girona 18 26, 08034 Barcelona, Spain
    J Biol Chem 286:28940-7. 2011
  5. pmc Phosphoethanolamine methyltransferases in phosphocholine biosynthesis: functions and potential for antiparasite therapy
    April M Bobenchik
    Department of Internal Medicine, Yale University School of Medicine, New Haven, CT 06510 3221, USA
    FEMS Microbiol Rev 35:609-19. 2011
  6. pmc PfNT2, a permease of the equilibrative nucleoside transporter family in the endoplasmic reticulum of Plasmodium falciparum
    Megan J Downie
    Section of Infectious Diseases, Yale School of Medicine, New Haven, CT 06512, USA
    J Biol Chem 285:20827-33. 2010
  7. pmc Identification of inhibitors of Plasmodium falciparum phosphoethanolamine methyltransferase using an enzyme-coupled transmethylation assay
    April M Bobenchik
    Department of Internal Medicine, Section of Infectious Diseases, Yale School of Medicine, 333 Cedar St, New Haven 06052, USA
    BMC Biochem 11:4. 2010
  8. pmc Disruption of the Plasmodium falciparum PfPMT gene results in a complete loss of phosphatidylcholine biosynthesis via the serine-decarboxylase-phosphoethanolamine-methyltransferase pathway and severe growth and survival defects
    William Harold Witola
    Department of Genetics and Developmental Biology, University of Connecticut Health Center, Farmington, Connecticut 06030, USA
    J Biol Chem 283:27636-43. 2008
  9. pmc Genetic evidence for the essential role of PfNT1 in the transport and utilization of xanthine, guanine, guanosine and adenine by Plasmodium falciparum
    Kamal El Bissati
    Department of Genetics and Developmental Biology, University of Connecticut Health Center, Farmington, CT 06030, USA
    Mol Biochem Parasitol 161:130-9. 2008
  10. pmc Purine salvage pathways in the intraerythrocytic malaria parasite Plasmodium falciparum
    Megan J Downie
    Department of Genetics and Developmental Biology, University of Connecticut Health Center, Farmington, CT 06030, USA
    Eukaryot Cell 7:1231-7. 2008

Scientific Experts

  • Jennifer M Reynolds
  • April M Bobenchik
  • Choukri Ben Mamoun
  • Kamal El Bissati
  • Megan J Downie
  • Yoann Augagneur
  • Buddy Ullman
  • Irina Bezsonova
  • Patricia Gonzalez-Bulnes
  • Kiaran Kirk
  • William Harold Witola
  • Rachel Zufferey
  • Cassandra S Arendt
  • Jeffrey C Hoch
  • Haribabu Arthanari
  • Iulian Rujan
  • Bing Hao
  • Vitaliy Gorbatyuk
  • Oksana Gorbatyuk
  • Mark W Maciejewski
  • Hyun Seop Tae
  • Donna Wesolowski
  • Sidney Altman
  • Henri J Vial
  • Rachel Cerdan
  • Amadeu Llebaria
  • Alexander Amerik
  • Laura Nic Lochlainn
  • Nicola Carter
  • Changan Xie
  • Paul D Roepe
  • Gabriella Pessi
  • Seong Kyoun Kim
  • Michael Horowitz
  • Phillip A Yates
  • Keirei Ri
  • Nicola S Carter
  • William H Witola

Detail Information

Publications14

  1. pmc Plasmodium falciparum phosphoethanolamine methyltransferase is essential for malaria transmission
    April M Bobenchik
    Department of Internal Medicine, Yale University School of Medicine, New Haven, CT 06520
    Proc Natl Acad Sci U S A 110:18262-7. 2013
    ..These studies set the stage for further optimization of NSC-158011 for development of a class of dual activity antimalarials to block both intraerythrocytic asexual replication and gametocytogenesis. ..
  2. pmc (1)H, (13)C, and (15)N chemical shift assignments for PfPMT, a phosphoethanolamine methyltransferase from Plasmodium falciparum
    Irina Bezsonova
    Department of Molecular, Microbial and Structural Biology, University of Connecticut Health Center, 263 Farmington Ave, Farmington, CT 6030 3305, USA
    Biomol NMR Assign 7:17-20. 2013
    ..In addition to enabling determination of the solution structure of the apoenzyme, the assignments will facilitate additional investigations into the interaction of PfPMT with its substrates and inhibitors...
  3. pmc Gene selective mRNA cleavage inhibits the development of Plasmodium falciparum
    Yoann Augagneur
    Department of Internal Medicine, Yale University School of Medicine, New Haven, CT 06510, USA
    Proc Natl Acad Sci U S A 109:6235-40. 2012
    ..The potency, selectivity, and predicted safety of PMO conjugates make this approach attractive for the development of a unique class of target-specific antimalarials and for large-scale functional analysis of the malarial genome...
  4. pmc PG12, a phospholipid analog with potent antimalarial activity, inhibits Plasmodium falciparum CTP:phosphocholine cytidylyltransferase activity
    Patricia Gonzalez-Bulnes
    Research Unit on Bioactive Molecules, Departament de Química Biomèdica, Instituto de Química Avanzada de Cataluña IQAC, CSIC, Jordi Girona 18 26, 08034 Barcelona, Spain
    J Biol Chem 286:28940-7. 2011
    ..The potent antimalarial of this compound, its low cytotoxicity profile, and its established mode of action make it an excellent lead to advance for further drug development and efficacy in vivo...
  5. pmc Phosphoethanolamine methyltransferases in phosphocholine biosynthesis: functions and potential for antiparasite therapy
    April M Bobenchik
    Department of Internal Medicine, Yale University School of Medicine, New Haven, CT 06510 3221, USA
    FEMS Microbiol Rev 35:609-19. 2011
    ....
  6. pmc PfNT2, a permease of the equilibrative nucleoside transporter family in the endoplasmic reticulum of Plasmodium falciparum
    Megan J Downie
    Section of Infectious Diseases, Yale School of Medicine, New Haven, CT 06512, USA
    J Biol Chem 285:20827-33. 2010
    ..This study provides the first evidence of an intracellular purine permease in apicomplexan parasites and suggests a novel biological function for the parasite endoplasmic reticulum during malaria infection...
  7. pmc Identification of inhibitors of Plasmodium falciparum phosphoethanolamine methyltransferase using an enzyme-coupled transmethylation assay
    April M Bobenchik
    Department of Internal Medicine, Section of Infectious Diseases, Yale School of Medicine, 333 Cedar St, New Haven 06052, USA
    BMC Biochem 11:4. 2010
    ..In P. falciparum, this activity is a limiting step in the pathway of synthesis of phosphatidylcholine from serine and plays an important role in the development, replication and survival of the parasite within human red blood cells...
  8. pmc Disruption of the Plasmodium falciparum PfPMT gene results in a complete loss of phosphatidylcholine biosynthesis via the serine-decarboxylase-phosphoethanolamine-methyltransferase pathway and severe growth and survival defects
    William Harold Witola
    Department of Genetics and Developmental Biology, University of Connecticut Health Center, Farmington, Connecticut 06030, USA
    J Biol Chem 283:27636-43. 2008
    ..Interestingly, loss of PfPMT resulted in significant defects in parasite growth, multiplication, and viability, suggesting that this gene plays an important role in the pathogenesis of intraerythrocytic Plasmodium parasites...
  9. pmc Genetic evidence for the essential role of PfNT1 in the transport and utilization of xanthine, guanine, guanosine and adenine by Plasmodium falciparum
    Kamal El Bissati
    Department of Genetics and Developmental Biology, University of Connecticut Health Center, Farmington, CT 06030, USA
    Mol Biochem Parasitol 161:130-9. 2008
    ..Taken together these results demonstrate that, in addition to hypoxanthine, inosine and adenosine, PfNT1 is essential for the transport and utilization of xanthine, guanine and guanosine...
  10. pmc Purine salvage pathways in the intraerythrocytic malaria parasite Plasmodium falciparum
    Megan J Downie
    Department of Genetics and Developmental Biology, University of Connecticut Health Center, Farmington, CT 06030, USA
    Eukaryot Cell 7:1231-7. 2008
  11. ncbi Biochemical and genetic analysis of the phosphoethanolamine methyltransferase of the human malaria parasite Plasmodium falciparum
    Jennifer M Reynolds
    Department of Genetics and Developmental Biology, University of Connecticut Health Center, Framington, CT 06030, USA
    J Biol Chem 283:7894-900. 2008
    ..Our analyses identify the importance of these residues in PfPMT activity and set the stage for advanced structural understanding of this class of enzymes...
  12. pmc Antimalarial activity of the anticancer and proteasome inhibitor bortezomib and its analog ZL3B
    Jennifer M Reynolds
    Department of Genetics and Developmental Biology, University of Connecticut Health Center, 263 Farmington Ave, Farmington, CT 06030 3301, USA
    BMC Clin Pharmacol 7:13. 2007
    ....
  13. pmc Genetic selection for a highly functional cysteine-less membrane protein using site saturation mutagenesis
    Cassandra S Arendt
    Department of Biochemistry and Molecular Biology, Oregon Health and Science University, Portland, OR 97239, USA
    Anal Biochem 365:185-93. 2007
    ..This combination of an improved site saturation mutagenesis technique and positive genetic selection provides a simple and efficient means to identify functional and perhaps unexpected amino acid variants at a desired position...
  14. pmc The plasma membrane permease PfNT1 is essential for purine salvage in the human malaria parasite Plasmodium falciparum
    Kamal El Bissati
    Department of Genetics and Developmental Biology, University of Connecticut Health Center, Farmington, CT 06030 3301, USA
    Proc Natl Acad Sci U S A 103:9286-91. 2006
    ..The phenotype of the conditionally lethal mutant establishes PfNT1 as a critical component of purine salvage in P. falciparum and validates PfNT1 as a potential therapeutic target...