Deciphering parasite-specific factors involved in retrograde trafficking and virulence of Toxoplasma gondii – APISORTING

Molecular biology, cell biology, molecular genetics.

We directed our first investigations towards the achievement of Tasks 1-3 of the APISORTING project.
Task 1: We have developed a quantitative LC-SRM approach on six selected proteins that specifically co-immunopurified with the retromer proteins (Vps26, Vps29 and Vps35).
Task 2: In 2012, we have developed a vectorial proteomics approach to determine the proteome of the retrograde trafficking route in a given cell type. Using ANR funding for a proof of concept study, we have now identified alpha5beta1 integrin as a new retrograde cargo protein. This work has been published in 2016 in Nature Cell Biology.
Task 3: To study the biogenesis of secretory organelles in Toxoplasma gondii, we have focused on two proteins: the clathrin adaptor AP1, and the Vps35 protein of retromer. We found that AP1 regulates the differential sorting of ROP and MIC proteins. This data are currently submitted for publication. We have also characterized the retromer complex protein Vps35. These findings have recently been published in Nature Communications.

To transpose our advances to the retrograde trafficking process of Toxoplasma gondii, a post-doctoral fellow (Maika Deffieu) has been recruited with ANR funding for a duration of 24 months. She will start in September 2016.

Sangaré LO, Alayi TD, Westermann B, Hovasse A, Sindikubwabo F, Callebaut I, Werkmeister E, Lafont F, Slomianny C, Hakimi MA, Van Dorsselaer A, Schaeffer-Reiss C, Tomavo S. Unconventional endosome-like compartment and retromer complex in Toxoplasma gondii govern parasite integrity and host infection. Nat Commun. 2016; 7:11191

Shafaq-Zadah M, Gomes-Santos CS, Bardin S, Maiuri P, Maurin M, Iranzo J, Gautreau A, Lamaze C, Caswell P, Goud B, and Johannes L. 2016. Persistent cell migration and adhesion rely on retrograde transport of beta1 integrin. Nat Cell Biol 18: 54-64

The phylum Apicomplexa comprises a large group of obligate intracellular parasites of wide human and agricultural significance. Most notable is Plasmodium spp., the causative agents of malaria, and Toxoplasma gondii, one of the most common infectious agents of humans, responsible for disease of the developing fetus and immune compromised individuals. The extracellular forms of these parasites are perfectly tailored for the recognition and invasion of host cells and can be identified by the apical complex consisting of secretory organelles named micronemes (MIC) and rhoptries (ROP). These organelles are released sequentially during host cell entry and invasion. In addition to these parasite-specific organelles, the canonical eukaryotic organelles such as a central nucleus surrounded by an extensive endoplasmic reticulum (ER) and a single Golgi situated immediately anterior to the nucleus are also present in these parasites. T. gondii ROP and MIC proteins navigate through the ER, Golgi and endosome-like organelles prior to being packaged into their respective apical secretory organelles. Although it is known that in higher eukaryotic cells protein trafficking beyond the Golgi can be influenced by residues in the cytoplasmic tail of type I transmembrane receptors, the basis for initial segregation of proteins between the Golgi and the parasite secretory organelles remains unclear. We have reported that TgSORTLR, a type I transmembrane receptor localized in the Golgi cisternae and proximal vesicles of T. gondii acts as a cargo receptor likely involved in retrograde and/or anterograde transport that is necessary for the biogenesis of secretory organelles. Further, TgSORTLR is essential for host cell egress, gliding motility, cell invasion and in vivo infection, likely because of the crucial roles for the biogenesis and/or functioning of apical secretory organelles. Our findings reveal an indispensable role for T. gondii sortilin, which is the first indication that, unlike yeast and higher eukaryotes, lower branching eukaryotes such as apicomplexan parasites do not have redundant mechanisms of protein trafficking. Yet, we aim to elucidate the biological functions of retromer transport in protein trafficking and post-secretory vesicle biogenesis in T gondii. The functional networks involving the classical retromer proteins and their associated novel parasite-specific proteins will be studied by three complementary teams that are headed as follows: S. Tomavo (Team 1 at the Pasteur Institute Lille: molecular, cell biology and genetics of Apicomplexa parasites), L. Johannes (Team 2 at the Curie Institute in Paris: retrograde traffic, signalling and unconventional clathrin-independent endocytosis), and A. Van Dorsselaer (Team 3 at IPHC in Strasbourg: proteomics and biochemistry of proteins). First, we will refine the first protein network of the endolysosomal pathways and will determine how these pathways are required for organelle biogenesis in T. gondii and likely in other apicomplexan parasites. With genetically engineered parasites, we will biochemically analyze the proteome of the retrograde transport route, using a vectorial proteomics approach, a cell and model membrane reconstitution strategy, and highly sensitive quantitative proteomics, respectively. In addition, we are currently using reverse genetics to generate conditional knock out mutants for novel parasite-specific proteins that specifically bound to retromer VPS35. These mutants will be exploited to elucidate the importance of TgSORTLR and its retromer-associated components in retrograde protein trafficking and post-secretory vesicle formation. We will precisely decipher how endolysosomal-like vesicle formation and transport can be exploited to understand the biogenesis of key parasite-specific secretory organelles that are essential for parasite virulence and pathogenesis.