טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
M.Sc Thesis
M.Sc StudentBar Avraham Yael
SubjectInvestigating the Role of Protein Kinase A in
Differentiation and Identifying Members of the
Arginine Deprivation Response Pathway
Pathway Leishmania Parasites
DepartmentDepartment of Biology
Supervisor Professor Emeritus Dan Zilberstein
Full Thesis textFull thesis text - English Version


Abstract

Parasitic protozoan of the genus Leishmania are the causative agents of leishmaniasis. During their life, parasites cycle between the midgut of female sand flies, where they proliferate as extracellular flagellated promastigotes and the phagolysosome of mammalian macrophages, where they proliferate as intracellular amastigotes. Leishmania parasites undergo axenic differentiation by shifting the cells from a midgut-like environment (26°C, pH 7.4) into a phagolysosome-like environment (37°C, pH5.5, 5% 𝐶𝑂2). Promastigotes differentiation into amastigotes is initiated by a signaling pathway that enables the parasite to adapt to both host and vector environments. Previous experiments revealed that Protein Kinase A (PKA) regulatory subunit R' (PKAR') is crucial for differentiation, as PKAR' null mutant is unable to differentiate normally. In the first part of this work, we found that the leishmanial PKA regulatory subunit R1 (PKAR1) is also crucial for differentiation as PKAR1 null mutant is unable to differentiate normally. On the other hand, absence of the PKA catalytic subunit C3 has no effect on differentiation, while excess of this regulatory subunit does damage the differentiation process. PKA activity was previously found to decrease during differentiation. However, the regulatory and the catalytic subunits stay intact in both promastigotes and at 5-hour differentiating cells. We conclude that both PKA regulatory subunits inhibit and activate the catalytic subunits depending on morphological changes which occur during differentiation.

The amino acid arginine is an essential amino acid for Leishmania parasite but not for its host. Leishmania utilizes arginine in biosynthetic pathways such as polyamine synthesis by using external arginine source. Amino acid permease 3 (AAP3) is a high affinity arginine transporter in L. donovani that maintains the cellular arginine homeostasis. When cells are deprived of arginine, both mRNA and protein level of AAP3 are upregulated, resulting in increased arginine transport rate. Phosphoproteomic analysis previously performed in our lab, revealed proteins that undergo rapid changes in phosphorylation due to arginine deprivation such as eukaryotic initiation factors, kinases, phosphatases and RNA binding proteins. The cellular response for arginine deprivation including changes in mRNA and protein abundance along with phosphoproteomic changes, is called: Arginine Deprivation Response (ADR). In the second part of this work, we identified two RNA binding proteins that may be responsible for stabilizing LdAAP3 transcripts in arginine deprived cells. By using Leishmania-adapted CRISPR/Cas9, we raised a few potentially mutated colonies of a RNA binding protein and a KH domain containing protein that eventually revealed impaired ADR. In addition, we found that PKA activity is necessary for normal ADR, as PKA inhibitor prevents the upregulation of both LdAAP3 mRNA and protein level in arginine deprived cells. Because arginine starvation has been shown to cause apoptosis-like cell death in Leishmania, and apoptotic-like population in Leishmania is required for infectivity by inhibiting the autophagy defense of macrophages, we hypothesize that ADR is essential for virulence. By using Leishmania-adapted CRISPR-Cas9, we targeted the AAP3 ORF in order to generate mutants lacking ADR. We raised two mutated colonies lacking ADR eventually found to be a-virulent, establishing a new link between ADR and Leishmania virulence.