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 interesting potential therapeutic targets in the parasites.

 

Research project #1. The apicoplast as a drug target in the context of acute toxoplasmosis

As several other medically important parasites of the phylum Apicomplexa (ie malaria-causing Plasmodium), T. gondii contains an unusual organelle called the apicoplast. This organelle is a startling peculiarity that not only highlights the diversity of eukaryotic cell biology, but can also potentially be leveraged for therapeutic development. This four-membrane plastid was acquired by an unusual secondary endosymbiosis, in which an alga was engulfed by another eukaryote forming a new secondary plastid in the host. Although the apicoplast has lost its photosynthetic function, it houses several important metabolic pathways for generating: iron/sulfur cluster, hem, fatty acids (FASII), isoprenoids (Fig. 1).

 

Figure 1. Schematic representation of a Toxoplasma tachyzoite, with a four-membrane apicoplast and the main biochemical pathways it hosts.


Although the apicoplast is known as an important metabolic hub for many species of apicomplexan parasites, the metabolic pathways which are absolutely essential for parasite viability may vary depending on the parasite or the developmental stage. For instance, T. gondii has a complex life cycle involving several developmental stages that develop in felids (the definitive hosts, where sexual reproduction occurs), but also divide asexually in the many species of warm-blooded animals that can act as intermediate hosts. The two developmental forms found in intermediate hosts are the tachyzoite and the bradyzoite. Tachyzoites are rapidly dividing forms associated with the acute phase of toxoplasmosis. Upon control by a competent immune system, however, the parasites can differentiate into slowly-growing bradyzoites, establishing within tissue cysts and responsible for the chronic phase of toxoplasmosis.

The apicoplast is already targeted by anti-parasitic drugs (for instance through several prokaryotic translation inhibitors) and apicoplast-hosted hem, fatty acid and isoprenoid synthesis pathways have all been shown to contribute to the fitness of the tachyzoite stage. However, several metabolic pathways remain unexplored and unexploited as potential drug targets.  

This research project aims at:

► identifying novel apicoplast-hosted parasite-specific functions

► assessing if they can be exploited to target the tachyzoite stage

Latest publication related to this project:

Differential contribution of two organelles of endosymbiotic origin to iron-sulfur cluster synthesis in Toxoplasma Sarah Pamukcu, Aude Cerutti, Sonia Hem, Valérie Rofidal, Sébastien Besteiro. bioRxiv 2021.01.28.428257; doi: https://doi.org/10.1101/2021.01.28.428257



Research project #2. Contribution of the apicoplast to Toxoplasma persistence

As mentioned above, 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).



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)

Latest publication related to this project:

Cerutti A, Blanchard N, Besteiro S. Pathogens. (2020) The Bradyzoite: A Key Developmental Stage for the Persistence and Pathogenesis of Toxoplasmosis. Cerutti A, Blanchard N, Besteiro S. Pathogens. 2020 Mar 21;9(3):234. doi: 10.3390/pathogens9030234. Pubmed



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, as well as canonical and non-canonical functions of the autophagy machinery.

This includes:

► elucidating the physiological roles of parasite autophagy in the context of acute and chronic toxoplasmosis (collaboration with the lab of Vern Carruthers, Univ. Michigan, USA)

► identifying novel parasite-specific functions for the autophagy-related machinery at the apicoplast

                                                                              
Publications

Toxoplasma TgATG9 is critical for autophagy and long-term persistence in tissue cysts David Smith, Geetha Kannan, Isabelle Coppens, Fengrong Wang, Hoa Mai Nguyen, Aude Cerutti, Tracey L. Schultz, Patrick A. Rimple, Manlio Di Cristina, Sébastien Besteiro, Vern B. Carruthers bioRxiv 2020.05.13.093401; doi: https://doi.org/10.1101/2020.05.13.093401

The role of host autophagy machinery in controlling Toxoplasma infection. Besteiro S. (2019) Virulence. Dec;10(1):438-447. doi: 10.1080/21505594.2018.1518102. Epub 2018 Sep 29. Pubmed

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 parasitesCurr 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

 

Other:

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


Group members

Theme leader


Sebastien Besteiro

Research Associate (CR) INSERM
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LPHI  Laboratory of Pathogen Host Interactions
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