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.
