|M.Sc Student||Saadon Ilona|
|Subject||Molecular Characterization of Arginine Transport|
Regulation in the Parasitic Protozoan
|Department||Department of Biology||Supervisor||Professor Emeritus Dan Zilberstein|
|Full Thesis text - in Hebrew|
The parasitic protozoa Leishmania donovani are the causative agents of kala-azar in humans. The parasites cycle between the intracellular amastigotes in the human macrophage phagolysosomes, an amino acids and fatty acids-rich environment and the extracellular promastigotes in the sand fly vector digestive tract, which is rich with amino acids and sugar. The parasite’s survival in these environments is thus largely attributed to its ability to regulate its intracellular contents, in part through the modulation of transport systems. Leishmania cannot synthesize arginine and therefore need to uptake it. LdAAP3 is a monospeciifc, high affinity arginine transporter that had been cloned and characterized in our laboratory. We hypothesized that its activity is critical for maintaining cellular arginine homeostasis in promastigotes. We assessed the relationships between LdAAP3 activity and the polyamine pathway, for which arginine is the sole precursor. We used ornithine decarboxylase (ΔODC) and spermidine synthase (ΔSpdS) null auxotroph mutants of L. donovani which can only survive when supplemented with polyamines. Mutant promastigotes exhibited lower arginine uptake levels compared to wild type promastigotes due to a decrease in the abundance of LdAAP3p. Ectopic expression of the wild type gene in ΔODC partially recovered arginine uptake and the abundance of LdAAP3p. Wild type promastigotes grown in the presence of excess putrescine exhibited lower levels of arginine uptake compared to untreated cells. We concluded that arginine transport via LdAAP3 is regulated by the polyamine pathway. Next, we assessed the effect of amino acids availability on LdAAP3 activity. Amino acid starvation induced arginine uptake in a time-dependant manner. This up-regulation depended on an external supply of either glucose or L-proline and was due to de-novo synthesis of new transporters. We examined the effect of starvation on LdAAP3 at the RNA and found an increase in expression of LdAAP3 copy 2. We did not observe a significant increase in L-lysine and L-proline uptake during 4 hours of amino acids starvation, indicating the specificity of the phenomenon to the arginine transport system. Amino acid starvation caused a 25% increase in arginine concentration, indicating that arginine homeostasis is largely uninterrupted during this treatment. External addition of some amino acids during starvation, including arginine, inhibited LdAAP3 up-regulation and arginine uptake. We conclude that cellular arginine homeostasis is tightly controlled by regulating arginine uptake via LdAAP3.