טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentTsigankov Polina
SubjectPhosphoproteomic and Molecular Analysis of Leishmania
donovani Development
DepartmentDepartment of Biology
Supervisor Professor Emeritus Dan Zilberstein
Full Thesis textFull thesis text - English Version


Abstract

Protists of the genus Leishmania are obligatory intracellular parasites that cause a wide range of cutaneous, mucocutaneous, and visceral diseases in humans. They cycle between phagolysosomes of mammalian macrophages and sand flies midgut, proliferating as intracellular amastigotes and extracellular promastigotes, respectively. Exposure to lysosomal environment, i.e. acidic pH and body temperature, signals promastigotes to differentiate into amastigotes. Promastigote-to-amastigote differentiation is divided into four morphologically distinct phases: I, signal perception (0-5 hours after exposure); II, movement cessation and aggregation (5-10 hours); III, amastigote morphogenesis (10-24 hours); and IV, maturation (24-120 hours). Time course analyses indicated that Leishmania differentiation is a highly regulated and coordinated process. However, the role of posttranslational events such as protein phosphorylation in this process is still unknown.

Herein, we compared the phosphoproteomes of L. donovani amastigotes and promastigotes and analyzed changes in protein phosphorylation abundance during the course of differentiation using an axenic hostfree system that simulates parasite differentiation. The analysis indicated that the majority of the phosphorylations are stage-specific. In addition, serine phosphorylation in a previously identified trypanosomatid-specific “SF” motif was significantly enriched in amastigotes.

The results demonstrated that Leishmania contained proteins with multiple phosphorylation sites that were phosphorylated at distinct stages of the life cycle and showed different kinetic trends. For over half of the phosphorylation events, changes in phosphoprotein abundance did not positively correlate with changes in protein abundance, suggesting functional regulation. Our analysis revealed that phosphorylation was predominating during phase I and III, whereas phase II and IV were characterized by greater dephosphorylation.

Several proteins (including a protein kinase) were phosphorylated in phase I after exposure to the complete differentiation signal (i.e. temperature and pH); but not after either of the physical parameters separately. Several other protein kinases (including regulatory subunits) and phosphatases also showed changes in phosphorylation during differentiation. This prompted us to further investigate the role of proteins that potentially regulate differentiation using molecular and cell biology approaches.

This work constitutes the first genome-scale interrogation of phosphorylation dynamics in a parasitic protozoa; revealing the outline of a signaling pathway during Leishmania differentiation.