Multiple levels of gene regulation mediate differentiation of the intracellular pathogenLeishmania

Abstract

For many years, mRNA abundance has been used as the surrogate measure of gene expression in biological systems. However, recent genome-scale analyses in both bacteria and eukaryotes have revealed that mRNA levels correlate with steady-state protein abundance for only 50-70% of genes, indicating that translation and post-translation processes also play important roles in determining gene expression. What is not yet clear is whether dynamic processes such as cell cycle progression, differentiation, or response to environmental changes change the relationship between mRNA and protein abundance. Here, we describe a systems approach to interrogate promastigote-to-amastigote differentiation in the obligatory intracellular parasitic protozoan Leishmania donovani. Our results indicate that regulation of mRNA levels plays a major role early in the differentiation process, while translation and post-translational regulation are more important in the latter part. In addition, it appears that the differentiation signal causes a transient global increase in the rate of protein synthesis, which is subsequently down-regulated by phosphorylation of α-subunit of translation initiation factor 2. Thus, Leishmania dynamically changes the relationship between mRNA and protein abundance as it adapts to new environmental circumstances. It is likely that similar mechanisms play a more important role than previously recognized in regulation of gene expression in other organisms.

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

“…A phosphopeptide containing pS 157 from LinJ.1040 (ribosomal protein S10) showed a similar (but only ϳ1.5-fold) transient increase in phosphorylation during phases I and II before the decreasing to ϳ2-fold below promastigote levels (see supplemental Table S1). These changes are in agreement with the previous observation (10) that protein synthesis is transiently up-regulated early in promastigote-to-amastigote differentiation, but the translation machinery is ultimately down-regulated in amastigotes.…”

“…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).…”

“…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). These biochemical changes are initiated in the middle of the third phase of differentiation, while parasites undergo morphogenesis (15), and appear mostly regulated by changes in translation rate and/or post-translational processing (10). Indeed, post-translational modifications occur at all phases of promastigote-to-amastigote differentiation.…”

“…These include protein glycosylation, methylation, acetylation, and phosphorylation (16). In contrast, most changes in protein abundance at the beginning of differentiation are regulated by mRNA, and there are a number of transient changes in mRNA and protein abundance (10).…”

Metabolomic-Based Strategies for Anti-Parasite Drug Discovery

“…The life cycle problem offers a major hurdle for metabolic reconstruction based on genome data alone, as major metabolic changes are evident as parasites move between life cycle stages, requiring different gene sets to be turned on and off. [7][8][9] Transcriptomics goes some way to provide life cycle specificity, but environmental nutrients also shape the metabolome. Our ability to make direct measurements on cellular metabolomes from various organisms is now affecting how we can study metabolism in any cell type, including the parasitic protozoa responsible for diseases such as human African trypanosomiasis, the leishmaniases, and malaria.…”