טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
M.Sc Thesis
M.Sc StudentLiburkin Dan Tatyana
SubjectExpression of the Leishmania Proline/Alanine Transporter
during Differentiation
DepartmentDepartment of Biology
Supervisor Professor Emeritus Dan Zilberstein
Full Thesis textFull thesis text - English Version


Abstract

Parasitic protozoa of the genus Leishmania are the causative agents of a wide range of cutaneous, mucocutaneous and visceral diseases in humans. These obligatory intracellular parasites cycle between the sand fly vector and the mammalian host, and therefore encounter major environmental changes in their life cycle. From the relatively alkaline, sugar- and amino acid-rich, sand fly gut, the parasites move to acidic, fatty- and amino acid-rich phagolysosomes of mammalian macrophages. Leishmania cells have developed mechanisms of adaptation that favor utilization of amino acids, which they use as osmolytes, alternative source of carbon, signaling, and more. For this reason, the expression level and activity of membrane transporters that regulate cellular amino acid content might be crucial for parasite survival. LdAAP24 is a proline/alanine transporter that regulates translocation and homoeostasis of proline, alanine, glutamate and arginine. LdAAP24 was previously shown to degrade in an early stage of axenic (host-free) differentiation, however, was later shown to be present in host-derived-amastigotes. Herein, we established that despite LdAAP24 presence in macrophage-derived amastigotes, the protein is degraded in both axenic and in-vivo cultures. We also show, with integration of additional data, that despite LdAAP24 degradation, LdAAP24 translation is ongoing in both axenic and in-vivo cultures. We shed light on some aspects of the trigger to the degradation process; we establish that the degradation initiation depends on co-existence of both components of the differentiation signal. We also ruled out the possibility of a molecular factor in the FCS which facilitates the complete LdAAP24 degradation in axenic differentiation. We show that early in axenic differentiation LdAAP24 mRNA reaches a new lower steady state that seems to correlate with the lower steady state of LdAAP24 protein in macrophage-derived parasites. With this result and integration of additional data, we propose that the difference between complete LdAAP24 degradation in axenic parasites and partial LdAAP24 degradation in intracellular parasites is due to the difference in mRNA abundance. Due to the LdAAP24 immunofluorescence pattern early in axenic differentiation, proteasome-mediated degradation is unlikely, and we therefore hypothesize that LdAAP24 degradation is mediated by the secretory system.