Early control of mRNA abundance and translation efficiency in macrophages infected with the protozoan parasite Leishmania donovani

Chaparro, Visnu (2022). Early control of mRNA abundance and translation efficiency in macrophages infected with the protozoan parasite Leishmania donovani Thèse. Québec, Université du Québec, Institut national de la recherche scientifique, Doctorat en en immunologie et virologie, 380 p.

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Résumé

Macrophages are professional phagocytes and first responders of the innate immune system against infectious organisms. Armed with a battery of antimicrobial tools and numerous pathogen- and damage-recognition receptors, macrophages exhibit high plasticity and stress-tailored responses that rely (among others) on regulation of mRNA abundance and translation. Paradoxically, macrophages represent the replicative niche for different pathogens including sandfly-transmitted protozoan parasites of the genus Leishmania, which are causative agents of an array of diseases collectively known as leishmaniases. L. donovani infection leads to development of visceral leishmaniasis (VL), with an estimation of 200,000 to 400,000 cases and 20,000 - 40,000 fatalities globally per year. Following sandfly inoculation, macrophage-phagocytized promastigotes transform into the non-motile amastigote form, which disseminates to internal organs and tissues such as lymph nodes, liver, spleen, and bone marrow with concomitant development of fatal clinical symptoms when untreated. To favor its own survival, L. donovani parasites subvert macrophage immune and cellular processes including modulation of gene expression. Modulation of mRNA abundance has been extensively reported in macrophages infected with viruses, bacteria and parasites. However, the role of translation in the course of infections remains poorly explored especially for protozoan parasitic infections. High throughput in vitro studies indicate L. donovani infection induces widespread perturbation of mRNA and protein abundance, although the majority of these changes have been documented over 12 hours post infection non accounting for early events that could affect infection progress (i.e., modulation of parasitophorous vacuole formation, oxidative burst, transcription factor activity, apoptosis initiation) or they have been performed using the promastigote form instead of the clinically relevant amastigote. Additionally, the role of macrophage translational control during L. donovani infection is yet to be determined.

Herein, by using polysome profiling coupled with RNAseq quantification we generated host profiles of mRNA abundance and translation from macrophages infected or not with L. donovani amastigotes and promastigotes 6 hours post infection. Using a combination of bio-informatic and biochemical tools we identified a stage-specific regulation of macrophage mRNA abundance. Amastigote-driven changes where enriched in upregulated transcripts encoding proteins associated with DNA repair mechanisms, while those encoding antigen-presenting and macrophage activation factors were markedly downregulated. In parallel, upregulation of immune inhibitors as well as an antioxidant transcriptional signature associated with NRF2 activity were identified in promastigote infected datasets. Additionally, hierarchical clustering of mRNAs associated with IRF3 and IRF7 transcriptional activity suggests that macrophages activate antimicrobial pathways upon L. donovani promastigote infection. Conversely, translational efficiency changes were found to be similar in amastigote- and promastigote-infected datasets when compared to uninfected controls. Gene ontology analyses on translationally regulated transcripts showed an enrichment of upregulated categories associated with RNA metabolism (i.e., chromatin remodeling, transcription, splicing, transport, silencing, and translation) and -similarly to analysis of mRNA abundance- downregulation of macrophage immune activators. Notably, subsets of mTORC1- and eIF4A-sensitive transcripts were identified including PABPC1 and EIF2AK2, the expression of which was inhibited by rapamycin treatment and TGFB1, which it was found to be affected after incubation with the rocaglate silvestrol. Furthermore, the biological significance of mTORC1 and eIF4A activities during L. donovani infection was highlighted via in vitro intracellular survival analysis indicating parasite survival is favored or compromised in the presence of rapamycin or silvestrol respectively. In sum, L. donovani promastigote and amastigote infection leads to the early widespread yet selective alterations of macrophage gene expression including mRNA abundance and translation efficiency that can tailor protective and harmful responses to the host underscoring the therapeutic potential of the molecular mechanisms regulating these events.

Type de document:	Thèse Thèse
Directeur de mémoire/thèse:	Jaramillo, Maritza
Mots-clés libres:	-
Centre:	Centre INRS-Institut Armand Frappier
Date de dépôt:	02 juill. 2024 13:51
Dernière modification:	02 juill. 2024 13:51
URI:	https://espace.inrs.ca/id/eprint/15752

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