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.
