טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
M.Sc Thesis
M.Sc StudentRonit Witztum
SubjectAmastigote to Promastigote Differentiation in the Human
Pathogen Leishmania
DepartmentDepartment of Biology
Supervisor Professor Emeritus Zilberstein Dan
Full Thesis text - in Hebrew Full thesis text - Hebrew Version


Abstract

Protozoan parasites of the genus Leishmania are the causative agents of Leishmaniasis, a wide range of cutaneous and visceral diseases. Their life cycle includes two main stages. The first stage is the extracellular flagellated promastigote that resides in sand-fly mid gut. The second stage is the intracellular amastigote that resides inside phagolysosomes of human macrophages. The main goal of this work is to characterize amastigote to promastigote differentiation. The specific objectives are: 1. Determine the time course of morphological changes of differentiating cells. 2. Examine changes in protein expression profile of three protein markers. 3. Monitor cell growth rate during differentiation. An axenic differentiation system for L. donovani was used to investigate parasite development. In order to investigate the first objective we examined cells using scan electron microscopy (SEM) at 10 time points during differentiation. From these experiments we concluded that the morphological transformation is composed of two consecutive steps. The first step is cell elongation, the second step is the budding of the flagella. The morphological transformation was completed thirty-three hours after initiation of differentiation. Examining changes in protein expression profile of protein markers revealed that promastigote maturation started at 24 hours after initiation of differentiation. The first marker we examined was ascorbate dependent peroxidase (APX), which protects cells against oxidative stress. Leishmania cells express two variants of this enzyme that differ slightly in molecular mass. The heavier variant is specific to amastigotes. The lighter variant is more dominant in promastigotes, but it is also expressed in amastigotes. Twenty- four hours after differentiation is initiated, there is an isoform switch between the two variants. We found that in parallel, the expression of the second protein marker Para Flagellar Rod 2C (PFR2C) started to increase. PFR2C is a component of the Para Flagellar rod (PFR) which has a role in motility of trypanosomatids. Promastigote to amastigote differentiation is regulated by the cell cycle and is initiated at G1. We found that following initiation of amastigote to promastigote differentiation, cells ceased growth during the first 10 hours of differentiation. Subsequently, parasite cells resumed growth and doubled at 16 hours.  After 33 hours the growth rate decreased to values characterizing mature promastigotes. In order to validate these results we performed similar experiments using intracellular amastigotes isolated from the spleen of infected hamsters. Intracellular amastigotes exhibited a similar time course for the morphological change. This work is the first to characterize amastigote to promastigote differentiation.