This project will probe the role of purine nucleoside and nucleobase transporters (NTs) in the biology of Leishmania parasites. All parasitic protozoa examined to date are unable to synthesize purines and must salvage these essential nutrients from their hosts. The first step in salvage is the uptake of purine nucleosides or nucleobases across the parasite plasma membrane by NTs. There are three Specific Aims. The first Specific Aim is to study the structure and function of a model nucleoside transporter from L. donovani, LdNT1.1, to elucidate how nucleoside transporters function in Leishmania and in other parasitic protozoa. The first component of this Specific Aim is to analyze 18 previously identified missense mutants of LdNT1.1 that are non-functional in transport or have altered substrate specificity. These mutants will be examined for those that are non-functional because they fail to bind substrate and those that are still able to bind substrate but unable to translocate it across the plasma membrane. These experiments will identify amino acids that play crucial roles in substrate recognition and binding or in substrate translocation and will thus define components of the permease that promote its two essential mechanistic functions. The second component of Specific Aim 1 is to define extracellular and intracellular gating residues for LdNT1.1, functionally important residues that interact to close off the permeation pore in the inward-facing and outward-facing orientation respectively. The second Specific Aim entails the creation of a structural model of LmaNT4, a unique nucleobase transporter from L. major that functions optimally at pH 5, the intracellular pH of the macrophage phagolysosome where the parasite resides in the mammalian host, and the genetic dissection of key residues that likely control this unusual pH optimum. The third Specific Aim is to examine the molecular mechanisms L. donovani employs to adapt to purine starvation. Preliminary evidence reveals that the LdNT2 protein is dramatically upregulated upon purine starvation whereas the level of its mRNA is unaffected. Planned experiments will determine whether this regulation operates at the level of translation, protein turnover, or both. The potential regulation of the other LdNTs by purine limitation will also be examined.

Public Health Relevance

The overall objective of this proposal is to study in detail the structure, function, and regulation of purine nucleoside transporters in the parasite Leishmania donovani that causes the fatal disease visceral leishmaniasis. Uptake of purines is essential for Leishmania and all parasitic protozoa, underscoring the crucial role of purine nucleoside/nucleobase transporters in these microorganisms, and these transporters also mediate the uptake of several drugs or experimental drugs employed against Leishmania or other parasitic protozoa. The essential roles that purine nucleoside/nucleobase transporters play in parasite viability further suggests selective inhibition of these transporters could be exploited to develop novel drugs against parasitic diseases.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI044138-13
Application #
8240075
Study Section
Pathogenic Eukaryotes Study Section (PTHE)
Program Officer
Rogers, Martin J
Project Start
1999-03-01
Project End
2015-03-31
Budget Start
2012-04-01
Budget End
2013-03-31
Support Year
13
Fiscal Year
2012
Total Cost
$381,150
Indirect Cost
$133,650
Name
Oregon Health and Science University
Department
Microbiology/Immun/Virology
Type
Schools of Medicine
DUNS #
096997515
City
Portland
State
OR
Country
United States
Zip Code
97239
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
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
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
Landfear, Scott M (2011) Nutrient transport and pathogenesis in selected parasitic protozoa. Eukaryot Cell 10:483-93
Landfear, S M (2010) Transporters for drug delivery and as drug targets in parasitic protozoa. Clin Pharmacol Ther 87:122-5
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
Ortiz, Diana; Sanchez, Marco A; Koch, Hans P et al. (2009) An acid-activated nucleobase transporter from Leishmania major. J Biol Chem 284:16164-9
Ortiz, Diana; Sanchez, Marco A; Quecke, Paula et al. (2009) Two novel nucleobase/pentamidine transporters from Trypanosoma brucei. Mol Biochem Parasitol 163:67-76

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