The objective of this proposal is to characterize at the molecular level several transporters from Leishmania and trypanosomes that mediate the uptake of important nutrients from the hosts. These nutrients include glucose which is an major source of metabolic energy in the insect stage of the life cycle, purines which are essential nutrients that the parasites cannot synthesize de novo, and amino acids which are both catabolized to generate energy and utilized in anabolic pathways such as protein synthesis. In the first aim, the physiological functions of the LdGT1 and LdGT2 glucose transporter isoforms of L. donovani will be probed by generating null mutants and analyzing their phenotypes. These studies may elucidate the functional differences between the flagellar membrane LdGT1 and the pellicular plasma membrane LdGT2. In the second aim, the permeation pathway of the L. donovani adenosine/pyrimidine nucleoside transporter LdNT1.1 will be probed by a combination of cysteine-scanning mutagenesis and sulfhydryl group chemical modification. These studies have been guided by the detection of two distinct point mutations that inactivate LdNT1.1, possibly by altering amino acids that line the permeation pathway. The TbNT8 transporter from Trypanosoma brucei, whose mRNA is expressed exclusively in insect stage parasites, is closely related in sequence to other well characterized nucleoside transporters, but it's function remains to be defined.
This third aim will characterize a strongly developmentally regulated nucleoside transporter and allow its comparison to other bloodstream-specific nucleoside transporters.
The final aim will functionally characterize a novel amino acid transporter from L. major that is related in sequence to mamrnalian N-type amino acid permeases that mediate the uptake of glutamine, and histidine, essential amino acids for growth of Leishmania parasites. These studies will initiate the molecular analysis of amino acid transporters in Leishmania.
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