Analysis of the Leishmania donovani transcriptome reveals an ordered progression of transient and permanent changes in gene expression during differentiation

Abstract

Leishmania donovani is an intracellular protozoan parasite that causes kala-azar in humans. During infection the extracellular insect forms (promastigotes) undergo rapid differentiation to intracellular amastigotes that proliferates in phagolysosomes of mammalian macrophages. We used microarraybased expression profiling to investigate the time-course of changes in RNA abundance during promastigote-to-amastigote differentiation in a host-free system that mimics this process. These studies revealed that several hundred genes underwent an ordered progression of transient or permanent up-and down-regulation during differentiation. Genes that were permanently upregulated in amastigotes were enriched for transporters and surface proteins, but under-represented in genes involved in protein and other metabolism. Most of these changes occurred late in the differentiation process, when morphological differentiation was essentially complete. Downregulated genes were over-represented in those involved in cell motility, growth and/or maintenance, and these changes generally occurred earlier in the process. Genes that were transiently up-or downregulated during differentiation included those encoding heat shock proteins, ubiquitin hydrolases, RNA binding proteins, protein kinases, a protein phosphatase, and a histone deacetylase. These results suggest that changes in mRNA abundance may be important in signal transduction, as well as protein and mRNA turnover, during differentiation. In addition to these mRNA changes, other transcripts including one or more rRNAs and snoRNAs, and non-coding RNAs from several telomeres, also showed substantial changes in abundance during the differentiation process. This paper provides the first genome-scale quantitative analysis of gene expression during the transition from promastigotes to amastigotes and demonstrates the utility of the host-free differentiation system.

Phosphorylation of Translation Initiation Factor 2-Alpha in Leishmania donovani under Stress Is Necessary for Parasite Survival

“…3E and F). This is consistent with many previous reports showing that elevated temperature and/or acidic pH induced the mRNA expression of several stress-responsive genes, including LdApx and sHSP20 (1,(25)(26)(27), favored by reduced translation during amastigote differentiation, allowing parasites to conserve energy while reconfiguring the expression of specific sets of genes necessary for their survival in the mammalian host (28). It has been shown that elevated temperature and acidic pH induce the phosphorylation of eIF2␣ and trigger the differentiation of L. infantum by decreasing global translation and enhancing the relative expression levels of stress-responsive proteins, enabling the parasite to adapt according to persisting physiological conditions inside the phagolysosomal compartment of host macrophages …”

“…Stress-induced transformation inside mammalian macrophages adapts the parasite to thrive in the harsh phagolysosomal environment, achieved by modulating gene expression at both the transcriptional and posttranscriptional levels (1)(2)(3)(4).…”

Regulation Dynamics of Leishmania Differentiation: Deconvoluting Signals and Identifying Phosphorylation Trends

“…Based on cell morphology, Barak et al divided differentiation into four phases: phase I (the first 5 h after exposure of promastigotes to the differentiation signal), which is dedicated to signal perception; phase II (5-10 h after signal exposure), during which the parasites cease movement and start to aggregate; phase III (10 -24 h), when cells undergo morphological change into amastigote-shaped cells; and phase IV (24 -120 h), during which the amastigotes undergo maturation (8). Significant changes in mRNA (9,10) and protein abundance (11), as well as in the rate of translation (12), allow the parasites to adjust to an amastigote lifestyle by re-tooling their energy metabolism from glycolysis to fattyacid-and amino-acid-based catabolism. For example, the transition is accompanied by an increase in protein abundance and activity rates of gluconeogenic enzymes, as it becomes an essential pathway in amastigotes (11,13,14).…”

T7 polymerase-driven transcription is downregulated in metacyclic promastigotes and amastigotes of Leishmania mexicana

“…The mCherry, T7 polymerase and Tet-repressor genes were all integrated into the 18S rRNA locus. Previous studies showed a 5 to 20-fold decrease in transcription of several RNAs derived from this locus during Leishmania donovani (Laveran et Mesnil, 1903) differentiation (Saxena et al 2007). To exclude an influence of the rRNA locus-dependent regulation on the reporter genes expression, we compared mCherry expression profile normalised to the 18S and LmxM.07.0510 genes ( Fig.…”