Amalgamating tools of genetics, molecular biology, biochemistry, and cell biology, this proposal offers an interdisciplinary analysis of the nucleoside transporters of Leishmania donovani and Trypanosoma brucei. As protozoan parasites are auxotrophic for purines, nucleoside transporters provide an important, if not vital, nutritional function for the parasite. These membrane carriers also mediate the translocation of melarsoprol and pentamidine, two anti-trypanosomal drugs, as well as allopurinol riboside and formycin B, two anti-trypanosomatid agents, into the parasite. Two nucleoside transporters have been genetically and biochemically defined for L. donovani and T. brucei. We have cloned, sequenced, and partially characterized the genes, LdNT1 and LdNT2, encoding the two L. donovani nucleoside transporters employing a functional rescue strategy of mutant nucleoside transport-deficient parasites. The sequences of LdNT1 and LdNT2 enable the subsequent isolation of two T.brucei nucleoside transport cDNAs, TbNT1 and TbNT2. These reagents serve as the molecular cornerstone of the three specific aims in this proposal.
Specific Aim 1 will be to characterize the adenosine/pyrimidine transporter, LdNT1. We will verify whether LdNT1 is a proton transporter by expression of LdNT1 in Xenopus oocytes, determine the subcellular location of LdNT1 by immunocytochemistry, and ascertain the nature of the genetic lesion(s) in the adenosine/pyrimidine transport-deficient strain, TUBA5.
Specific Aim II involves the characterization of LdNT2 will be completed. Next, LdNT2 function will be characterized after expression in both inosine/guanosine transport-deficient FBD5 parasites and oocytes. Whether LdNT2 is a proton supporter, the location of the protein within the parasite, and the nature of the mutation(s) in FBD5 cells will also be evaluated. The final specific aim will be to characterize TbNT1 and TbNT2 substrate specificities and ligand affinities by heterologous expression of the corresponding cDNAs in L. donovani and oocytes, to determine whether either transporter recognizes melarsoprol or pentamidine, and to discover whether three truncated versions of TbNT1 and TbNT2 encode functional nucleoside transporters.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI044138-02
Application #
6163967
Study Section
Tropical Medicine and Parasitology Study Section (TMP)
Program Officer
Fairfield, Alexandra
Project Start
1999-03-01
Project End
2004-02-29
Budget Start
2000-03-01
Budget End
2001-02-28
Support Year
2
Fiscal Year
2000
Total Cost
$242,845
Indirect Cost
Name
Oregon Health and Science University
Department
Microbiology/Immun/Virology
Type
Schools of Medicine
DUNS #
009584210
City
Portland
State
OR
Country
United States
Zip Code
97239
Martin, Jessica L; Yates, Phillip A; Soysa, Radika et al. (2014) Metabolic reprogramming during purine stress in the protozoan pathogen Leishmania donovani. PLoS Pathog 10:e1003938
Vald├ęs, Raquel; Elferich, Johannes; Shinde, Ujwal et al. (2014) Identification of the intracellular gate for a member of the equilibrative nucleoside transporter (ENT) family. J Biol Chem 289:8799-809
Soysa, Radika; Carter, Nicola S; Yates, Phillip A (2014) A dual luciferase system for analysis of post-transcriptional regulation of gene expression in Leishmania. Mol Biochem Parasitol 195:1-5
Arendt, Cassandra S (2013) Crithidia fasciculata adenosine transporter 1 (CfAT1), a novel high-affinity equilibrative nucleoside transporter specific for adenosine. Mol Biochem Parasitol 191:75-9
Boitz, Jan M; Ullman, Buddy; Jardim, Armando et al. (2012) Purine salvage in Leishmania: complex or simple by design? Trends Parasitol 28:345-52
Valdes, Raquel; Shinde, Ujwal; Landfear, Scott M (2012) Cysteine cross-linking defines the extracellular gate for the Leishmania donovani nucleoside transporter 1.1 (LdNT1.1). J Biol Chem 287:44036-45
Landfear, Scott M (2011) Nutrient transport and pathogenesis in selected parasitic protozoa. Eukaryot Cell 10:483-93
Carter, Nicola S; Yates, Phillip A; Gessford, Sarah K et al. (2010) Adaptive responses to purine starvation in Leishmania donovani. Mol Microbiol 78:92-107
Ortiz, Diana; Valdes, Raquel; Sanchez, Marco A et al. (2010) Purine restriction induces pronounced translational upregulation of the NT1 adenosine/pyrimidine nucleoside transporter in Leishmania major. Mol Microbiol 78:108-18
Arendt, Cassandra S; Ullman, Buddy (2010) Role of transmembrane domain 4 in ligand permeation by Crithidia fasciculata equilibrative nucleoside transporter 2 (CfNT2). J Biol Chem 285:6024-35

Showing the most recent 10 out of 40 publications