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Contribution of the apicoplast to the metabolism and survival of T. gondii in the context of acute and chronic toxoplasmosis.


Toxoplasma gondii is a parasitic protist infecting a wide variety of warm-blooded animals and about one third of the human population. Although it remains essentially asymptomatic in immunocompetent individuals, this parasite can cause a life-threatening disease called toxoplasmosis in fetuses, newborns, and individuals with weakened immune systems. Our group studies how these obligate intracellular parasites multiply and persist in the host. We are targeting essential cellular processes, which are thus sometimes conserved among eukaryotes, but bearing enough differences to make them potential therapeutic targets in the parasites.

Research project #1. Elucidating the unusual function of TgATG8 in maintaining apicoplast homeostasis.


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. 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.
Over the recent years, we have been investigating autophagy in T. gondii tachyzoites, a highly invasive and fast replicating stage of the parasite responsible for the acute phase of toxoplasmosis. We have shown that autophagosomes can be induced in T. gondii in response to stress, in particular in extracellular tachyzoites (Fig. 1A). 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. 1B). 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.

 

 

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Figure 1. 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.

 

 

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.


This research project aims at:


► elucidating the physiological roles of parasite autophagy in the context of acute and chronic toxoplasmosis (collaboration with the lab ofVern Carruthers, Univ. Michigan, USA) ► identifying novel parasite-specific functions for the autophagy-related machinery at the apicoplast ► discovering drugs that specifically inhibit TgATG8 function at the apicoplast (collaboration with the lab of  Feng Tan, Wenzhou Medical University, China)


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 publications related to this project:


- Nguyen HM, Liu S, Daher W, Tan F, Besteiro S. (2018) Characterisation of two Toxoplasma PROPPINs homologous to Atg18/WIPI suggests they have evolved distinct specialised functions. PLoS One. Apr 16;13(4):e0195921. Pubmed


- Besteiro S. (2017) Autophagy in apicomplexan parasites. (2017)  Curr Opin Microbiol. Dec;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. 19(6). 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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Research project #2. Contribution of the apicoplast to Toxoplasma persistence.

 

Acute infection of intermediate hosts by T. gondii is associated with the rapid replication and spread of the tachyzoite forms within the body. This infection phase is often readily contained by the immune system. However, the parasites can differentiate into slowly growing bradyzoites, establishing within tissue cysts, primarily in the central nervous system and muscle. Although there are effective medicines available against tachyzoites, the persistent chronic form of the pathogen remains in the host throughout its life and can convert repeatedly back into tachyzoites, and hence lead to a severe pathology (i.e. encephalitis or retinitis) in the event of a weakened immune system. These bradyzoites forms are thus central to the pathology, yet there are no effective drugs against them so far.
In bradyzoites, where apicoplast function has been largely overlooked, we wish to determine the importance of the organelle for survival and persistence of this parasite stage. We are using stage-specific conditional knock-down or knock-out approaches to deplete apicoplast proteins in bradyzoites (Fig. 2).

 

 

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Figure 2.  Stage-specific depletion of an apicoplast marker (green) in in vitro-differentiated bradyzoites (arrowhead, cyst labelled by the Dolichos biflorus lectin –DBL, red-).

 

This research project aims at:


► generating genetic tools to investigate the function of essential genes in bradyzoites ► elucidating the contribution of the apicoplast to the persistence and reactivation of bradyzoites in vitro and in vivo (collaboration with the lab of Nicolas Blanchard, Université de Toulouse, France)

 

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.

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For examples 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. 105(1):158-174.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

 


Current group members.


Aude Cerrutti, Post-doctoral fellow. Project: Apicoplast function in bradyzoites.


Ksenia Semenovskaya, PhD Student. Project: Characterization of a Zinc finger protein involved in coordinating Toxoplasma cell cycle.


Sarah Z. PhD Student. Project: Non-canonical function of ATG8 at the apicoplast.

 

 

Alumni.


Hoa Mai Nguyen (Post-doctoral fellow 2014-2017)


Maude Lévêque (PhD student 2013-2016)