3 - Functional analysis of the autophagic pathway in Toxoplasma

Besteiro photo equipe 2017

Sébastien Besteiro (Project leader), Hoa Mai Nguyen (post-doc), Ksenia Semenovskaya (PhD student)


Autophagy is a catabolic pathway that is highly conserved among eukaryotes and permits the degradation of cellular material. During the autophagic process, cytoplasmic components are sequestered in double-membrane vesicles called autophagosomes, and degraded after fusion with a degradative compartment (Fig. 1A). Autophagy is involved in multiple survival-promoting processes. It not only facilitates the maintenance of cell homeostasis by degrading long-lived proteins and damaged organelles, but it also plays a role in cell differentiation and cell development. The roles of autophagy and the autophagic machinery itself are poorly documented in protozoan parasites, especially in medically important apicomplexan parasites. We have shown that autophagosomes can be induced in Toxoplasma gondii in response to stress (Fig. 1B), in particular in extracellular parasites (Fig. 2A). This suggests the presence of a functional canonical autophagic pathway in this parasite. This pathway is likely important for surviving stress-generating environments, like when the parasites are under the immune pressure from the host, but seems not essential for intracellular in vitro growth in permissive conditions. Paradoxically, we have also demonstrated that several members of the autophagic machinery are necessary for parasite growth. In fact, the TgATG8 protein, which is a bona fide autophagosomal marker in many eukaryotic cells, also associates with a peculiar organelle during the intracellular development of the parasites, the apicoplast (Fig. 2B). The apicoplast is a non-photosynthetic plastid which is vital to the parasite's survival. It has been acquired by the ancestor of apicomplexan parasites through a double endosymbiosis event. Consequently, its outermost membrane might bear properties similar to phagosomal membranes, which also appear to be able to recruit ATG8 in other eukaryotic systems. The precise function of TgATG8 at the apicoplast is currently unknown, but it highlights essential, yet non-canonical, roles for the autophagic machinery in Apicomplexa.

 

figure 1

Figure 1. A) Schematic representation of the macroautophagy process. The text in red describes the different steps of autophagosome formation and recycling. Putative T. gondii homologs of proteins involved in the formation of autophagosomes and the regulation of this process are mentioned.
B) Typical cytoplasm-containing multi-membrane autophagosomes can be observed in starved Toxoplasma tachyzoites by electron microscopy. Scale bar= 0.5µM

 

 

Overall, the presence of a pathway that is
i) conserved in the phylum Apicomplexa,
ii) potentially essential for the survival of these pathogens iii) containing potentially druggable targets that could bear significant differences with their host’s counterparts, provides the rationale for further studies in the cellular functions of autophagy in these parasites.

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figure 2

 Figure 2. A) TgATG8 labels autophagosomal structures in starved extracellular parasites.
B) In dividing intracellular parasites (mother and daughter cells are labelled with inner membrane complex –IMC- staining), TgATG8 associates with the apicoplast. Scale bar= 5µM.

 

Our project focuses on:
► elucidating the physiological roles of parasite autophagy in the context of acute and chronic toxoplasmosis
► identifying which stimuli are responsible for inducing parasite autophagy, as well as mechanisms regulating the pathway
► identifying novel parasite-specific functions for the autophagy-related machinery

 

Funding:
► Over recent years, financial support for this project has been provided by the CNRS, the “Fondation pour la Recherche Médicale” and the “Agence Nationale de la Recherche”.

 

Our recent publications related to this project:

- Besteiro S. (2017) Autophagy in apicomplexan parasites. Curr Opin Microbiol. 40:14-20 Pubmed

- Nguyen HM, Berry L, Sullivan WJ Jr, Besteiro S. (2017) Autophagy participates in the unfolded protein response in Toxoplasma gondii. FEMS Microbiol Lett. 15;364(15). Pubmed

- Di Cristina M, Dou Z, Lunghi M, Kannan G, Huynh MH, McGovern OL, Schultz TL, Schultz AJ, Miller AJ, Hayes BM, van der Linden W, Emiliani C, Bogyo M, Besteiro S, Coppens I, Carruthers VB. (2017) Toxoplasma depends on lysosomal consumption of autophagosomes for persistent infection. Nat Microbiol. Jun 19;2:17096. Pubmed

- Nguyen HM, El Hajj H, El Hajj R, Tawil N, Berry L, Lebrun M, Bordat Y, Besteiro S. (2017)  Toxoplasma gondii autophagy-related protein ATG9 is crucial for the survival of parasites in their host.  Cell Microbiol. In press. Pubmed

- Latré de Laté P, Pineda M, Harnett M, Harnett W, Besteiro S, Langsley G. (2017) Apicomplexan autophagy and modulation of autophagy in parasite-infected host cells. Biomed J. 40(1):23-30. Pubmed

- Harnett MM, Pineda MA, Latré de Laté P, Eason RJ, Besteiro S, Harnett W, Langsley G. (2017). From Christian de Duve to Yoshinori Ohsumi: More to autophagy than just dining at home. Biomed J. 40(1):9-22. Pubmed

- Lévêque MF, Nguyen HM, Besteiro S. (2016) Repurposing of conserved autophagy-related protein ATG8 in a divergent eukaryote. Commun Integr Biol. 9(4):e1197447. Pubmed

- Lévêque MF, Berry L, Cipriano MJ, Nguyen HM, Striepen B, Besteiro S. (2015) Autophagy-related protein ATG8 has a noncanonical function for Apicoplast inheritance in Toxoplasma gondii. MBio.;6(6):e01446-15. Pubmed

- Kong-Hap MA, Mouammine A, Daher W, Berry L, Lebrun M, Dubremetz JF, Besteiro S. (2013) Regulation of ATG8 membrane association by ATG4 in the parasitic protist Toxoplasma gondii. Autophagy. 9(9):1334-48 Pubmed

- Besteiro S. (2012) Which roles for autophagy in Toxoplasma gondii and related apicomplexan parasites? Mol Biochem Parasitol. 184(1):1-8 Pubmed

- Besteiro S. (2012) Role of Atg3 in the parasite Toxoplasma gondii : autophagy in an early branching eukaryote. Autophagy. 8 (3):435-7 Pubmed

- Besteiro S, Brooks CF, Striepen B, Dubremetz JF. (2011) Autophagy protein Atg3 is essential for maintaining mitochondrial integrity and for normal intracellular development of Toxoplasma gondii tachyzoites. PLoS Pathog. 7(12):e100241 Pubmed

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Other research projects:
► We are also interested in other aspects of the cell biology of Toxoplasma, including specific aspects of the cell division process, the cytoskeleton, and apicoplast homeostasis.


For example see:
- Lévêque MF, Berry L, Yamaryo-Botté Y, Nguyen HM, Galera M, Botté CY, Besteiro S. (2017) TgPL2, a patatin-like phospholipase domain-containing protein, is involved in the maintenance of apicoplast lipids homeostasis in Toxoplasma. Mol Microbiol. In press. Pubmed

- Lévêque MF, Berry L, Besteiro S. (2016) An evolutionarily conserved SSNA1/DIP13 homologue is a component of both basal and apical complexes of Toxoplasma gondii. Sci Rep.;6:27809. Pubmed.