|Ph.D Student||Lahav Tamar|
|Subject||Multiple Level of Gene Regulation Mediate Differenitation of|
the Intracellular Pathogen Leishmania donovani
|Department||Department of Biology||Supervisors||Professor Emeritus Dan Zilberstein|
|Dr. Hanne Volpin|
|Full Thesis text|
Protozoan parasites of the genus Leishmania are the causative agents of a visceral and cutaneous diseases in humans. During its lifecycle, the parasite undergoes differentiation from promastigote to amastigote. Here, we used a host-free, synchronized differentiation system and a high-throughput gene expression analysis to investigate the changes in transcript abundance during promastigote-to-amastigote differentiation. The major findings of our microarray analyses were that the expression of a substantial number of genes is transiently up- or down-regulated during differentiation. Thus, there appears to be an ordered progression of specific changes in gene expression after the exposure of L. donovani promastigotes to the differentiation signal. To better understand the molecular events occurring during differentiation, we analyzed the correlation between mRNA and protein abundance. We compared the microarray results with iTRAQ data recently published by Rosenzweig et al. (2008). Total RNA and soluble proteins used in this analysis were extracted from the same batches of L. donovani cells. A comparative analysis indicated that changes in protein abundance are much greater than changes in mRNA, suggesting a limited involvement of mRNA in gene expression regulation or that small changes in mRNA might lead to greater changes in protein abundance. A high correlation between mRNA and protein abundance was also found. Interestingly, we found that about 30% of L. donovani genes showed a high positive significant correlation between developmental-dependent changes in mRNA and protein accumulation. These genes are likely regulated by mRNA abundance. For 23% of the genes, the protein levels were up-regulated during differentiation while the mRNA was down-regulated or did not change; these genes are likely to be regulated at the translation level. The rest of the genes are likely to show translational and/or post-translational regulation. Next, we observed a burst in SL RNA transcription followed by a transient but significant increase in the steady-state level of SL RNA in the parasites’ cell nucleus. In addition, we found evidence that the [AC]13 motif in the 5`UTR of the genes might be involved in the control of trans-splicing in Leishmania.
In conclusion, we found that Leishmania cells can regulate the mRNA level, and this level could influence the final protein concentration in the cell. We suggested that trans-splicing might regulate the mRNA level and that a specific motif found in the trans-splicing area might interrupt the trans-splicing of specific genes. This study constitutes the first comprehensive transcriptome/proteome comparative analysis of gene expression during differentiation of an intercellular pathogen.