|M.Sc Thesis||Department of Biology|
|Supervisor:||Prof. Zilberstein Dan|
Leishmania donovani are parasitic protozoa that cause kala-azar in humans. These organisms undergo a developmental program following changes in the environment, resulting in the reversible transformation between the extracellular promastigote form in sand flies (the vector) and the obligatory intracellular amastigote form in phagolysosomes of human macrophages. A host-free differentiation system for L. donovani was used to investigate the initial process of differentiation. Using promastigotes that express GFP the time course of differentiation was determined. At five hours after exposing promastigotes to differentiation conditions (pH 5.5, 37°C) they aggregate and start to round into amastigote-shaped cells, a process that is completed 19 hours later. Treating promastigotes with the proteasome inhibitor MG132 triggered rapid differentiation, even when cells were exposed to low pH at low temperature. Together with previous results, this supports the idea that protein misfolding might be involved in this process.
We employed DNA microarray-based expression profiling to investigate the molecular basis of this differentiation process. RNA was isolated at 5, 10 and 24 hours after exposure to the differentiation signal as well as from promastigotes and axenic amastigotes, and used to probe microarrays containing PCR-amplified DNA from a random-amplified genomic library of L. major Friedlin. Statistical analysis of the data after normalization revealed that several hundred genes were up- or down-regulated during differentiation. There were 121 promastigote-specific genes, 131 amastigote-specific genes, 171 genes were up-regulated and 113 down-regulated transiently during the differentiation process. In addition, the relative abundance of transcripts containing telomeric sequences appeared to be regulated during the differentiation process.