ABSTRACT: RNA Analysis for Modulation of aneuploidy in Leishmania donovani during adaptation to different in vitro and in vivo environments, and its impact on gene expression
Project description:Purpose: Monitor modulation of aneuploidy in Leishmania donovani during adaptation to different in vitro and in vivo environments, and its impact on gene expression Importance: Aneuploidy is usually detrimental in multicellular organisms, but in several micro-organisms (fungi being the best-studied) it can be tolerated and even beneficial. Leishmania – a protozoan parasite killing more than 30,000 persons each year – is emerging as a new model for aneuploidy studies: unexpectedly high levels of aneuploidy are found in clinical isolates. Leishmania lacks classical regulation of transcription through inducible promoters, so aneuploidy could represent a major adaptive strategy of this parasite to modulate gene dosage in response to stressful environments. For the first time, we document the dynamics of aneuploidy throughout the life cycle of the parasite, in vitro and in vivo. We show its adaptive impact on transcription and its intertwinement with regulation. Besides offering a new model for aneuploidy studies, we show that further genomic studies should be done directly in clinical samples (without parasite isolation) and that adequate methods should be developed for this.
Project description:Monastrol treatment of Leishmania donovani infected macrophages Macrophages were infected with Leishmania donovani and treated with monastrol to look for signalling molecules
Project description:The mRNA expression of antimony resistant strains of Leishmania donovani was compared to the expression of the sensitive Leishmania donovani.
Project description:The mRNA expression of antimony resistant strains of Leishmania donovani was compared to the expression of the sensitive Leishmania donovani. The antimony resistant and sensitive Leishmania donovani were grown in complete M199 medium with 10% FCS and Penicillin streptomycin mixture. At stationary phase (5 day culture) cells were harvested in sterile Phosphate buffered saline and used for RNA isolation.
Project description:<p>Aneuploidy causes system-wide disruptions in the stochiometric balances of transcripts, proteins, and metabolites, often resulting in detrimental effects for the organism. The protozoan parasite Leishmania has an unusually high tolerance for aneuploidy, but the molecular and functional consequences for the pathogen remain poorly understood. Here, we addressed this question in vitro and present the first integrated analysis of the genome, transcriptome, proteome, and metabolome of highly aneuploid Leishmania donovani strains. Our analyses unambiguously establish that aneuploidy in Leishmania proportionally impacts the average transcript- and protein abundance levels of affected chromosomes, ultimately correlating with the degree of metabolic differences between strains. This proportionality was present in both proliferative and non-proliferative in vitro promastigotes. However, protein complex subunits and non-cytoplasmic proteins, showed dosage compensation, responding less or even not at all to aneuploidy-induced dosage changes. In contrast to other Eukaryotes, we did not observe the widespread regulation at the transcript level that typically modulates some of the negative effects of aneuploidy. Further, the majority of differentially expressed proteins between aneuploid strains were encoded by non-aneuploid chromosomes and were not driven by a significant underlying transcript change, suggesting that aneuploidy is accompanied by extensive post-transcriptional protein-level modulation. This makes Leishmania a unique Eukaryotic model for elucidating post-transcriptional protein-abundance modulation in the context of aneuploidy.</p><p><br></p><p><strong>Data availability:</strong></p><p>Proteomic data associated with this study are available in the PRIDE repository: accession number <a href='https://www.ebi.ac.uk/pride/archive/projects/PXD028521' rel='noopener noreferrer' target='_blank'>PXD028521</a>.</p>
Project description:Differential gene expression analysis was carried out in clinical isolates of Leishmania donovani (L. donovani) parasite derived from VL patient before miltefosine treatment, post treatment and patient that relpased after miltefosine treatment
