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Abstract
Grant Number: 1R01AI051507-01A1 Project Title: Nucleoside Transporters of Plasmodium falciparum
PI Information: Name Title ULLMAN, BUDDY ullmanb@ohsu.edu PROFESSOR Abstract: DESCRIPTION (provided by the applicant): 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 PfNT1has also been functionally over-expressed 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 immuno-electron microscopy. Antibodies against PfNT2 have also been generated. These reagents are the cornerstones of the three specific aims in this proposal. The multi-component 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 immuno-locatization 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.
Public Health Relevance:
This Public Health Relevance is not available.Thesaurus Terms:
gene expression, intracellular parasitism, intracellular transport, membrane transport protein, protein structure function, purine nucleoside
genetic regulation
Plasmodium falciparum, Xenopus oocyte, clinical research, confocal scanning microscopy, gene targeting, genetic screening, human tissue, immunocytochemistry, immunoelectron microscopy, nucleic acid sequence, polymerase chain reaction, transfection
Institution: OREGON HEALTH AND SCIENCE UNIVERSITY 3181 SW Sam Jackson Pk Rd PORTLAND, OR 972393098 Fiscal Year: 2003 Department: BIOCHEMISTRY AND MOLECULAR BIOLOGY Project Start: 01-JAN-2003 Project End: 31-DEC-2007 ICD: NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES IRG: TMP