Understanding and Interfering with the Moving-Junction that drives Invasion in apicomplexan parasites – MOVINGJUNCTION-TARGET

We will produce and characterize conditional mutant parasites for the proteins involved in MJ formation. We will study the contribution of the host cell by searching for cellular partners associated with the MJ. We will use data we have previously acquired on molecular and cellular features of the MJ to undertake a virtual and a biochemical screen of compounds inhibiting the MJ

We expect elucidating the function of each protein of the MJ complex, characterizing host cell proteins associated to this parasitic structure and demonstrating its contribution in host cell invasion by Toxoplasma gondii. We expect characterizing chemical families able to inhibit the AMA1-RON2 interaction which is essential to the formation of the MJ

In addition to better understand the original molecular mechanisms of MJ formation and invasion, which have no counterpart out of the Apicomplexa phylum, this study will help developing new therapeutic approaches against malaria. This concept will be extended to other members of the phylum responsible for animal diseases of high economical impact (Babesia, Eimeria, Neospora)

This project lies on our previous works that pionneered the characterization of parasite molecules involved in the formation of the MJ, allowing to set up the fondamental bases of searching for invasion inhibitory molecules
Vuillez-le-normand et al., PlosPathogen 2012 ; Tonkin et al., Science 2011 ; Lamarque et al., PlosPathogen 2011 ; Besteiro et al., PlosPathogen 2009 ; Lebrun et al., Cell Microbiol 2005

The phylum Apicomplexa includes some of the most important pathogenic parasites of man and animals, the deadliest of which is the malaria parasite Plasmodium falciparum, responsible for a toll of one million human deaths per year. No vaccine currently exists against P. falciparum and parasites are becoming increasingly drug-resistant. Being obligatory intracellular organisms, Apicomplexa have developed a unique invasion mechanism that is conserved across the phylum involving a tight interaction formed between the host cell and the parasite surfaces called Moving Junction (MJ). The MJ appears as a punctuate focus at the apical tip of the parasite, then rapidly resolves into a ring that moves posteriorly over the parasite in conjunction with host membrane invagination and eventual engulfment of the invading parasite. The MJ is essential for this process, as it anchors the parasite to the host surface while the parasite’s actin-myosin motor provides forward motion into the host cell. We have previously shown that the MJ contains two key parasite components [1,2]: the surface protein Apical Membrane Antigen 1 (AMA1) and its receptor, the Rhoptry Neck Protein (RON) complex, the latter one being targeted to the host cell membrane during invasion. In particular, we demonstrated that the extra-cellular region of RON2, a transmembrane component of the RON complex, interacts directly with AMA1, providing a bridge between the parasite and its host cell that is crucial for successful invasion [1,3]. We have shown that the other members of the RON complex (RON4/5/8) are tethered to RON2 and exposed to the cytosolic face of the host cell membrane [1], suggesting a role in anchoring the MJ to the host cell cytoskeleton. We demonstrated that the AMA1-RON2 interaction is equally important for the invasive process in Toxoplasma and Plasmodium, as peptides derived from the ectodomain of RON2 are able to compete in the nanomolar range with the native RON2 for AMA1 interaction [5]. Interestingly, although the AMA1 and RON2 primary sequences differ among Apicomplexa, the AMA1-RON2 interaction is evolutionarily conserved [3]. By solving the co-structures of both Toxoplasma [4] and P. falciparum (Vuillez et al., Submitted) AMA1 complexed with a peptide derived from the ectodomain of RON2, we showed that the complex has an extensive buried surface area that very likely withstands the strong mechanical forces involved in host-cell invasion and highlighted the residues that contribute to the interaction and the species specificity.
By combining cell biology, structural biology and computational analysis approaches, our current scientific challenge is to extend the characterization of the MJ at the cellular, molecular and structural levels, with the aim to decipher the respective role of each component of the MJ and search for putative host partners. We will capitalize on our recent structural findings to design low molecular weight drugs capable of disrupting the AMA1-RON2 assembly and thereby invasion. Through our cross disciplinary approaches we will be able to establish the core mechanisms of MJ establishment and inhibition. The expected outcome of this project will have a positive economic and social impact on the fight against malaria, and bring a general concept for medicinal chemistry to be applied to the inhibition of the invasion of other apicomplexan parasites (veterinary as Eimeria, Babesia, Theileria…) that use the same mechanism to successfully infect their host.
1. Besteiro S, Michelin A, Poncet J, Dubremetz JF, Lebrun M (2009). PLoS Pathog 5: e1000309.
2. Lebrun M, Michelin A, El Hajj H, et al. (2005) Cell Microbiol 7: 1823-1833.
3. Lamarque M, Besteiro S, Papoin J, … Lebrun M. (2011) PLoS Pathog 7: e1001276.
4. Tonkin ML*, Roques M*, Lamarque MH, Pugniere M, Douguet D, Lebrun M*, Boulanger M*. (2011) Science 333: 463-467.
5. Lebrun M, Boulanger M, Roques M, Tonkin M. Patent Application. 2011. EP 11305540.4
* equally contributed to this work