JCJC SVSE 3 - JCJC - SVSE 3 - Microbiologie, immunologie, infectiologie

Functional analysis of the autophagic pathway in Toxoplasma gondii – Autophago-Toxo

Unexpected functions for self-digesting machinery in an early-diverging eukaryote

The autophagy (self-digesting process) machinery is very conserved from Man to unicellular eukaryotes. Toxoplasma is a eukaryotic pathogen in which almost nothing is known about the functions of this machinery. We have thus started deciphering the particular roles of autophagy-related proteins in this parasite.

Elucidating the importance of the autophagy machinery for a family of human pathogens

Very few things are known about autophagy in the parasite Toxoplasma gondii and other members of the phylum Apicomplexa (which includes other pathogens of considerable importance for human health such as Plasmodium, the causative agent of Malaria). We have started, a few years ago, characterising the functions of the autophagy machinery in this parasite by combining functional genomics and imaging approaches. The essentiality of several of the proteins of this pathway (called ATG proteins) in this parasite has demonstrated there is an important role played by several of these proteins in the survival of the parasite. This role seems nevertheless to be very different from the canonical autophagic function found in other eukaryotes, including in mammals which are intermediate hosts of the parasite. This opens new perspectives in the discovery of potential novel therapeutic target within this biological pathway.

Toxoplasma is a model which is much more amenable to genetic manipulation than other apicomplexan parasites.Thus we can generate transgenic parasites in order to express an auto-fluorescent copy of a protein, or to specifically down-regulate the expression of a gene in a conditional way. This way we could gererate several ATG mutant cell lines. The ability to tag proteins with auto-fluorescent molecules allows us to follow the dynamics of proteins such as TgATG8 during the cell cycle by time lapse live imaging. We have also used proteins biochemistry-based methods for isolating and identifying partners of ATG proteins in order to gain insights into the original functions of these proteins.

We have discovered an original function for a part of the autophagy machinery in maintaining the homeostasis of an organelle called the apicoplast. The apicoplast is a non-photosynthetic plastid found in most apicomplexan parasites, which contains metabolic pathways that are essential to parasite survival. The TgATG8 protein, as well as proteins regulating its membrane association (TgATG3, TgATG4), are essential for maintaining the plastid in the parasite, and thus for its survival. TgATG8 localises to the outer membrane of the plastid, where we suppose it could play a role in positioning the organelle, or establishing a link with cytoskeleton elements, during cell division.

We are also currently charcterising the canonical, catabolic, autophagy pathway in the parasite. To this day, there is no clear demonstration of a catabolic autophagic pathway in Apicomplexa. Autophagic vesicles are formed upon stresses such as starvation, for example, but there is no proof they are degraded and their content is recycled. We are currently setting up an assay for measuring proteolysis that we could apply to autophagy mutants. We are also generating mutants for the early machinery for forming autophagic vesicles, to specifically impact the canonical autophagy pathway and not the apicoplast-linked function.

Studying the canonical autophagic pathway in Toxoplasma is of interest from a fundamental research point of view, as it is an early-diverging eukaryote with a reduced machinery. It could then provide information on the minimal machinery required for the autophagic process.
However, quite clearly, our priority is to pursue studying the original function of TgATG8 regarding the apicoplast. This unexpected role is potentially interesting for discovering new therapeutic targets, as it concerns a critical function for an organelle which is essential to the survival of the parasite.

1 article in a peer-reviewed journal
1 book chapter
3 oral communications at international meetings or seminars
1 oral communications at a national meeting
4 poster communications at national meetings

In eukaryotic cells, autophagy is a usually reparative and life-sustaining process by which cytoplasmic components are sequestered in double-membrane vesicles called autophagosomes and degraded after fusion with a lysosomal compartment. Autophagy can be triggered when cells are in need of nutrients in order to recycle cellular material, but might also be involved in cell remodelling during normal eukaryotic development. Toxoplasma gondii is an obligate intracellular parasitic protist that is virtually able to infect all species of warm-blooded animals. This parasite is a member of the phylum Apicomplexa, which also includes several other notable human pathogens such as Plasmodium and Cryptosporidium.
The genome of T. gondii seems to contain genes coding for the core machinery necessary for the autophagic process. Using GFP-fused TgATG8 as an autophagosomal membrane marker in order to quantify autophagy in T. gondii tachyzoites, we have shown that autophagy can be induced in vitro in extracellular parasites by starving the cells in amino acid-depleted medium. However, we have also followed autophagy across normal development of the parasite within the host cell and showed that it is occurring consistently, yet transiently, during cell division.
Direct knock-out strategies to interfere with TgATG8 function have proven to be unsuccessful, suggesting a crucial role for this protein for the multiplication of Toxoplasma. Instead, we have produced conditional knock out mutants for autophagy-related genes TgATG3 and TgATG4 (1, 2), whose products are important for regulating TgATG8 association with the membrane of the autophagosomes. Both mutant cell lines showed a severe growth defect and a several organellar defects such as fragmentation of their mitochondrial network or loss of their apicoplast (a relict, non-photosynthetic, yet metabolically important plastid found in these parasites). These converging phenotypes suggest that TgATG8-related machinery is essential for the normal development of the parasite, and seems to be more specifically involved in maintaining organellar homeostasis.
The objectives of this proposal are to decipher the cellular functions of autophagy in T. gondii and their subsequent implications in the survival of these parasites.
Our first aim is to explore the dynamics of the interactions between the autophagosomes and cellular organelles, such as the mitochondrial network or the apicoplast, during cell division. The second goal is to use autophagosomal markers to delineate the terminal lytic compartment responsible for degradation of the autophagic material and, more precisely, the hydrolases involved. Parallel to this, we aim to discover new parasite-specific autophagosomal proteins and targets. Finally, recent data suggest that autophagy could be involved in mediating cell death in parasites exposed to severe stress conditions, although the specificity and role of this is currently unknown; we will thus screen for conditions that can trigger autophagy and whether this can be integrated in a global stress or cell death response.
There is a fundamental interest in studying a parasitic protist with an apparently reduced machinery, to bring information on autophagy as a mechanism. Beyond this, we have already validated autophagy as an essential mechanism for parasite growth and our long term objectives are now to elucidate the molecular actors involved and identify putative targets that could be interfered with to modulate this cellular function. They might later be validated in other medically important Apicomplexa.

1. Besteiro S, Brooks CF, Striepen B, Dubremetz J-F (2011). PLoS Pathog. 7: e1002416.
2. Kong-Hap MA, Mouammine A, Daher W, Berry L, Lebrun M, Dubremetz JF, Besteiro S (2013). Autophagy. in revision

Project coordinator

Monsieur Sébastien Besteiro (UMR5235, Dynamique des Interactions Membranaires Normales et Pathologiques)

The author of this summary is the project coordinator, who is responsible for the content of this summary. The ANR declines any responsibility as for its contents.

Partner

DIMNP UMR5235, Dynamique des Interactions Membranaires Normales et Pathologiques

Help of the ANR 210,000 euros
Beginning and duration of the scientific project: December 2013 - 42 Months

Useful links

Explorez notre base de projets financés

 

 

ANR makes available its datasets on funded projects, click here to find more.

Sign up for the latest news:
Subscribe to our newsletter