טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentFischer-Weinberger Renana
SubjectA Divergent Regulatory Subunit of PKA has a Role in
Leishmania Development
DepartmentDepartment of Biology
Supervisors Professor Michael Glickman
Professor Emeritus Dan Zilberstein
Full Thesis textFull thesis text - English Version


Abstract

Protozoan parasites of the genus Leishmania belong to Trypanosomatidae family and are the causative agents of leishmaniasis. During their life, parasites cycle between two major forms, in two distinct environments. They start as extracellular flagellated promastigotes and differentiate into round, non-motile, intracellular amastigotes as they enter the macrophages phagolysosomes.

A host-free system was developed to enable comparative biochemical analysis of large population of homogeneous parasite stages. The system simulates differentiation by exposing parasites to lysosome-like environment: acidic pH (5.5), elevated temperature (37°C), 5% CO2 and a few additional modifications in the medium. The process of axenic differentiation is divided into four phases based on morphology: phase I - signal perception and processing (0-4 h), phase II - movement cessation and aggregation (5-10 h), phase III - morphological changes (11-24 h), and phase IV - amastigotes maturation (25-120 h). The signal transduction pathway that initiates the differentiation has not yet been identified. Quantitative phosphoproteomics analysis during differentiation revealed protein kinase A regulatory subunit (PKAR') that was phosphorylated specifically by the differentiation signal at the beginning of differentiation. Omitting PKAR' had a profound phenotype, the promastigotes differentiated slower and died after 4 days. Moreover, Δpkar’ were chubbier with shorter flagella than WT promastigotes. These results imply that LdPKAR' has an important role in Leishmania development.

In this work, LdPKAR' was characterized. It was shown that PKAR' is anchored to the sub-pellicular microtubule at the cell cortex. Binding to the microtubules is through an SH2 domain at the N-terminus of PKAR'. In promastigotes R’ is localized to the proximal region near the flagellar pocket, and in amastigotes it surrounds the entire cell cortex. The process of migration initiates 2.5 hours after the exposure to the differentiation signal and ends after 5 hours. Migration of R' correlates with promastigote morphogenesis to amastigote-shaped cells. These changes are also observed in intracellular parasites. PKAR' associates with the catalytic subunits PKAC1/C3 and regulates them in two ways: (1) R’ association with C subunits inactivates them and therefore PKA holoenzyme is inactivated (2) R' association with C is necessary to PKA activity. In the latter, R' anchors C to the microtubules and has a role in morphogenesis.

RNA-seq analysis comparing between axenic and intracellular parasites, revealed high similarity (~95%) in gene expression. Metabolite transporters were among the genes that were up-regulated only in intracellular amastigotes or down regulated only in axenic amastigotes. To better mimic the phagolysosomes milieu, the amino acids and glucose content in phagolysosomes were determined.  Valine and lysine were the most abundant amino acids in phagolysomes. Glucose level was undetectable.  It is thus conjectured that using a new differentiation medium, with the concentration of amino acids like in the phagolysosomes and without glucose, will improve the axenic differentiation.


In this work we show, for the first time, that LdPKAR’ is a real regulatory subunit of PKA, playing an important role in Leishmania morphogenesis.