טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentAdele Goldman
SubjectArginine Availability Pathway in Leishmania Donovani
DepartmentDepartment of Biology
Supervisor Professor Emeritus Zilberstein Dan
Full Thesis textFull thesis text - English Version


Abstract

Arginine is an essential amino acid for the human pathogen Leishmania, but not its host. Thus, the mechanism by which this protozoan parasite regulates cellular homeostasis of arginine is critical for its survival and virulence. In this work we establish that LdAAP3, a high affinity arginine transporter in L. donovani, plays a major role in maintaining cellular arginine homeostasis, particularly under arginine deprivation conditions.  Starving promastigotes for amino acids decreased the cellular level of most amino acids including arginine and increased the abundance of both LdAAP3 mRNA and protein and up-regulated arginine transport activity. We then established that it was the deprivation of arginine alone that caused this up-regulation and no other amino acid deprivation had a similar effect. Genetic obliteration of the polyamine biosynthesis pathway for which arginine is the sole precursor caused a significant decrease in the rate of arginine transport and cellular content. Cumulatively, we observed that expression and activity of LdAAP3 as well as the cellular level of arginine are tightly regulated by the ability of cells to utilize arginine and by arginine availability. Arginine availability is sensed externally, rather than the signal being internal arginine content.

 In light of our results we suggest that arginine deprivation activates a signaling pathway in L.donovani promastigotes that senses external arginine and subsequently regulates LdAAP3 expression and activity. A phosphorproteomic approach was used to study the pathway itself and established that the response of L. donovani to arginine deprivation is regulated to a large extent by mRNA abundance. We also show that LdAAP3 localizes to the glycosomes as well as to the plasma membrane of promastigotes. Interestingly, the response of LdAAP3 to amino acid availability is identical to that of the mammalian cationic amino acid transporter 1 (CAT1), implying that Leishmania mimic host response to amino acid availability. However, it was still unclear when these parasites encounter arginine deprivation in their natural life cycle. Based on our finding that in-vitro infection of macrophages with Leishmania triggers starvation response in the parasites, we suggest that it is within the macrophage that parasites experience arginine deprivation and utilize the arginine starvation pathway for survival. We therefore suggest a role for starvation response in the Leishmanial virulence capacity.