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Intracellular Trafficking and Biological Impact of Leishmania Virulence Factors on Macrophages


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Arango Duque, Guillermo (2018). Intracellular Trafficking and Biological Impact of Leishmania Virulence Factors on Macrophages Thèse. Québec, Université du Québec, Institut national de la recherche scientifique, doctorat en immunologie et virologie, 357 p.

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Leishmania parasites cause a spectrum of debilitating diseases found worldwide. The Leishmania life cycle is digenetic, and starts with sandflies that inoculate metacyclic promastigotes into a vertebrate host. Promastigotes are internalized by tissue phagocytes where they transform into amastigotes. Phagosomes mature into highly microbicidal phagolysosomes via membrane exchanges with lysosomes, and with organelles in the secretory pathway. Remarkably, Leishmania remodels phagolysosomes into parasitophorous vacuoles that promote parasite growth. To achieve this feat, the parasite employs an armament of abundant surface-bound glycoconjugates that include the GP63 metalloprotease and lipophosphoglycan. GP63 cleaves multiple host substrates, thereby enabling Leishmania to subvert phagocyte functions such as transcription, translation, lipid metabolism, cytokine secretion, LC3-associated phagocytosis and antigen cross-presentation. Lipophosphoglycan, a complex glycophospholipid, promotes parasite survival by inhibiting phagolysosomal maturation, hence quenching the microbicidal power of the phagosome.

The effector functions of a macrophage rely on a very active endomembrane trafficking system that regulates how the cell responds to environmental stimuli. Vesicle fusion is regulated by members of the soluble N-ethylmaleimide-sensitive factor activating protein receptor, and Synaptotagmin families. In this work, we characterized Syt XI, an inhibitory member of the Synaptotagmin family whose function had not been reported. We discovered that Syt XI is a recycling endosome and lysosome-associated protein that controls phagocytosis, as well as the phagosome’s killing capacity [primary article no. 1]. We found that Syt XI dampens TNF and IL-6 secretion. Importantly, Syt XI is degraded by GP63 and excluded from parasitophorous vacuoles via lipophosphoglycan. Additionally, Leishmania-infected macrophages were found to secrete TNF and IL-6 in a GP63-dependent fashion. To demonstrate that this release depends on Syt XI degradation, siRNA knockdown of Syt XI before infection revealed that the effects of siRNA knockdown and GP63 degradation are not cumulative. To bring these findings into the in vivo context, we showed that injection of GP63-containing parasites into mice also leads to increased TNF and IL-6 secretion and to an augmented influx of neutrophils and inflammatory monocytes to the infection site [primary article no. 2].

Unlike bacteria, Leishmania is not known to inject its virulence factors across the phagosome membrane. This raises the question of how the parasite’s virulence effectors reach their targets. Due to the reported importance of the host’s secretory pathway on parasite survival, we hypothesized that Leishmania’s virulence factors co-opt this pathway in order to egress from the phagosome into the host cell cytoplasm. Using biochemistry and microscopy-based assays, we demonstrated that GP63 and the phosphoglycans are rapidly redistributed throughout the cytoplasm in vesicles containing markers of the endoplasmic reticulum, and of the endoplasmic reticulum-Golgi intermediate compartment. Importantly, chemical inhibition of the secretory pathway hinders the redistribution of GP63 and the phosphoglycans, thereby impeding the cleavage of GP63 targets. This prompted us to study the role of Sec22b, which regulates endoplasmic reticulum-Golgi trafficking, on the intracellular trafficking of GP63 and phosphoglycans. We found that Sec22b promotes the redistribution of these virulence factors and enables GP63 to reach host proteins [primary article no. 3].

In sum, the work presented in this dissertation sheds light into how Leishmania‘s virulence molecules enter the host cell in order to exert their functions. We also revealed that GP63 induces TNF and IL-6 release in vitro and in vivo, thereby contributing to the accrual of inflammatory phagocytes to the infection site. Remarkably, GP63 and the phosphoglycans access their targets by hijacking cellular organelles and their resident vesicle fusion molecules. These findings provide important insight into how Leishmania sabotages macrophage biology.

Type de document: Thèse Thèse
Directeur de mémoire/thèse: Descoteaux, Albert
Mots-clés libres: -
Centre: Centre INRS-Institut Armand Frappier
Date de dépôt: 08 mars 2019 15:31
Dernière modification: 08 mars 2019 15:31
URI: http://espace.inrs.ca/id/eprint/7882

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