טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
M.Sc Thesis
M.Sc StudentBalno Caitlin
SubjectCharacterization of homeostatic response of arginine
transport in Leishmania donovani
DepartmentDepartment of Biology
Supervisor Professor Emeritus Dan Zilberstein
Full Thesis textFull thesis text - English Version


Abstract

The human pathogen, Leishmania, is a parasitic protozoan which resides in host phagocytic immune cells, such as macrophages. The amino acid, arginine, is essential for Leishmania, but not for its host. Leishmania utilizes arginine in biosynthetic pathways, such as polyamine synthesis resulting in trypanothione synthesis; therefore the availability of this amino acid is tightly regulated. Upon pathogen invasion, host macrophages utilize external arginine to supplement NO production, an antiparasitic response, or polyamine synthesis. Consequently, parasite invasion induces activity of host Arginase I enzyme, causing depletion of host arginine. Arginine is the source of a bottleneck between host and parasite. The crucial roles of arginine in both organisms create a resource competition for this amino acid.

We hypothesize the parasite responds to arginine competition upon macrophage invasion. The LdAAP3 transporter translocates arginine across the plasma and glycosomal membranes of Leishmania. Increased arginine uptake by the parasites results in suppression of host NO production and the weakening the host’s resistance to infection. Here we characterize a pathway, ADR (arginine deprivation response), which is induced as a consequence of arginine depletion in the parasite's axenic environment and upon host invasion. Omitting arginine from parasite growth medium induces rapid up-regulation of LdAAP3 expression (mRNA and protein) and arginine transport. We report lack of arginine in the extracellular environment is the cause of ADR activation. Within 15 minutes, mRNA stability and protein abundance increase by >2-fold for a dozen proteins, including an LdAAP3, an ion transporter, metabolic enzymes, and several proteins with yet unknown function. Here, we establish ADR is mediated by MPK2 and activated in Leishmania during macrophage invasion. Phosphoproteomic analysis upon arginine deprivation revealed MPK2 activation and enrichment of the “SP” substrate motif in 18.2% of phosphopeptides within 5 min of ADR onset. L.mexicana lacking the MPK2 gene was not sensitive to arginine deprivation. This pathway is activated upon parasite invasion of macrophages, as indicated by LdAAP3 expression increasing >3-fold during infection in Thp1 macrophages. We hypothesize an arginine-sensing molecule localized to the parasite’s surface detects lack of arginine and subsequently induces a MPK2-mediated signaling pathway. This research characterized a signaling pathway in response to arginine deprivation that may help further decipher the relationship between the pathogen and its host.