1-Host cell invasion by apicomplexan parasites

Maryse

 

 Maryse Lebrun (Project leader)

 

Bienvenu-Maynadier Marjorie (research assistant)
Guérin Amandine (PhD student)
Lamarque Mauld (Assistant Professor)
Peñarete-Vargas Diana (permanent researcher)
Ruivo Margarida (PhD student)
Suarez Catherine (post-doc)

Being obligatory intracellular organisms, Apicomplexa have developed a unique invasion mechanism that is conserved across the phylum. It involves a tight interaction formed between the host cell and the parasite surfaces called the 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.

Movie 1 : fibroblast invasion by a Toxoplasma tachyzoite : the moving junction correspond to the slight constriction of the invading parasite

Windows Media - 2.1 Mo

Our research focuses essentially on host cell membrane attachment and MJ creation, as well as PV formation. Although the morphological details of the MJ process have been described 30 years ago, the MJ was, up to a recent period, completely uncharacterized at the molecular level. We have shown that the actors of the MJ are proteins unique to the phylum and conserved between most members. MJ proteins are secreted from the micronemes and the rhoptries. MJ formation relies on secretion and export in the host cell of a rhoptry neck proteins (RONs) complex, including at least RONs 2, 4, and 5 (an additional partner RON8 is present in coccidian parasites). RON2 is integrated into the host plasma membrane, exposing its N-terminal domain within the cytosol of the host cell, likely retaining RONs 4, 5 and 8 near the cytosolic face of this membrane. RON2 also exposes an ectodomain on the cell surface that serves as a receptor for the secreted micronemal apical membrane antigen 1 (AMA1) which is displayed on the parasite cell surface. The organization of this AMA1-RON complex points towards a model whereby the parasite secretes and inserts the interacting components on both sides of the MJ, i.e. the plasma membranes of the host (RON2) and the parasite (AMA1) (Fig. 1). In this model, RON4/5/8 would allow the anchoring of the junction complex to the host cell cytoskeleton and/or restrict the access of host cell membrane proteins to the PVM.

Fig-1 copieweb.jpg schematic representation

Fig. 1 Schematic representation of the MJ organisation model. AMA1 is secreted from the micronemes at the surface of the parasite, whereas the RONs are secreted within the host cell and RON2 serves as a receptor for AMA1 (left). Right : Detailed view of the structure between AMA1 and a synthetic peptide RON2.

 

We demonstrated that the AMA1-RON2 interaction is equally important for the invasive process by 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. Interestingly, although the AMA1 and RON2 primary sequences differ among Apicomplexa, the AMA1-RON2 interaction is evolutionarily conserved. By solving the co-structures of both Toxoplasma and P. falciparum 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.

Our project focuses on :

► extending 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 ► capitalizing on our recent structural findings to design low molecular weight drugs capable of disrupting the AMA1-RON2 assembly and thereby invasion. ►deciphering the conserved rhoptry proteome in Apicomplexa ►characterizating molecules and signaling events involved in triggering rhoptry secretion

Expected outcomes :

► We wish to establish the core mechanisms of MJ establishment and inhibition. ► We wish to establish the core machinery of rhoptry secretion. ►The expected outcomes 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.

Project financed through a grant by the French National Research Agency ANR.

Recent references related to the project :

 Parker ML, Penarete-Vargas DM, Hamilton PT, Guérin A, Dubey JP, Perlman SJ, Spano F, Lebrun M, Boulanger MJ. Dissecting the interface between apicomplexan parasite and host cell: Insights from a divergent AMA-RON2 pair. Proc Natl Acad Sci U S A. 2016 Jan 12;113(2):398-403.

 Lentini G, Kong-Hap M, El Hajj H, Francia M, Claudet C, Striepen B, Dubremetz JF, Lebrun M. Identification and characterization of Toxoplasma SIP, a conserved apicomplexan cytoskeleton protein involved in maintaining the shape, motility and virulence of the parasite. Cell Microbiol. 2015 Jan;17(1):62-78.

 Lamarque MH, Roques M, Kong-Hap M, Tonkin ML, Rugarabamu G, Marq JB, Penarete-Vargas DM, Boulanger MJ, Soldati-Favre D, Lebrun M. Plasticity and redundancy among AMA-RON pairs ensure host cell entry of Toxoplasma parasites. Nat Commun. 2014 Jun 17;5:4098.

 Vulliez-Le Normand B, Tonkin ML, Lamarque MH, Langer S, Hoos S, Roques M, Saul FA, Faber BW, Bentley GA, Boulanger MJ, Lebrun M. Structural and functional insights into the malaria parasite moving junction complex. PLoS Pathog. 2012;8(6):e1002755.

 Lamarque MH, Papoin J, Finizio AL, Lentini G, Pfaff AW, Candolfi E, Dubremetz JF, Lebrun M. Identification of a new rhoptry neck complex RON9/RON10 in the Apicomplexa parasite Toxoplasma gondii. PLoS One. 2012;7(3):e32457.

 Tonkin*, Roques*, Lamarque, Pugnière, Douguet, Crawford, Lebrun*, Boulanger*. (2011) Host cell invasion by Apicomplexan parasites : Insights from the structure of AMA1 in complex with a RON2 peptide. Science, *equal contribution. 22 ;333(6041):463-7.

 Besteiro S, Dubremetz JF, Lebrun M. (2011) The moving junction of apicomplexan parasites : a key structure for invasion. Cell Microbiol. 13(6):797-805

 Lamarque, Besteiro, Papoin, Roques, Vulliez-Le Normand, Morlon-Guyot, Dubremetz, Fauquenoy, Tomavo, Faber, Kocken, Thomas, Boulanger, Bentley, and M. Lebrun. (2011). The RON2-AMA1 interaction is a critical step in moving junction-dependent invasion by Apicomplexan parasites. Plos Pathogen. e1001276

 S Besteiro, A Michelin, J Poncet, JF Dubremetz, M. Lebrun (2009). Export of a Toxoplasma gondii rhoptry neck protein complex at the host cell membrane to form the moving junction during invasion. Plos Pathogen. 5(2):e1000309.

 H El Hajj, J Papoin , O Cérède , N Garcia-Réguet, M Soête, JF Dubremetz and M Lebrun (2008) Molecular signals in the trafficking of the Toxoplasma gondii protein MIC3 to the micronemes Eukaryotic Cell 7:1019-1028

 S Besteiro, J Bertrand-Michel, M Lebrun, H Vial, JF Dubremetz.(2008) Lipidomic analysis of Toxoplasma gondii tachyzoites rhoptries : further insights into the role of cholesterol. Biochemical Journal, 415:87-96

 H El Hajj, M Lebrun, ST Arold, H Vial, G Labesse, JF Dubremetz (2007) ROP18 Is a Rhoptry Kinase Controlling the Intracellular Proliferation of Toxoplasma gondii. PLoS Pathogen, 3:e14.

 H El Hajj , E Demey , J Poncet , M Lebrun , B Wu , N Galéotti , MN Fourmaux , O Mercereau-Puijalon, H Vial , G Labesse, JF Dubremetz (2007) The ROP2 family of Toxoplasma gondii rhoptry proteins : Proteomic and genomic characterization and molecular modelling. Proteomics, 6:5773-5784

 El Hajj H, Lebrun M, Fourmaux MN, Vial H, Dubremetz JF. (2007) Inverted topology of the Toxoplasma gondii ROP5 rhoptry protein provides new insights into the association of the ROP2 protein family with the parasitophorous vacuole membrane. Cellular Microbiology 9:54-64.

 Ismael AB, Dimier-Poisson I, Lebrun M, Dubremetz JF, Bout D, Mevelec MN.(2006) Mic1-3 knockout of Toxoplasma gondii is a successful vaccine against chronic and congenital toxoplasmosis in mice. Journal of Infectious Disease, 194:1176-1183.

 Lebrun M, Michelin A, El Hajj H, Poncet J, Bradley PJ, Vial H, Dubremetz JF. (2005) The rhoptry neck protein RON4 re-localizes at the moving junction during Toxoplasma gondii invasion. Cellular Microbiology, 7:1823-33.

 O. Cérède, J. F. Dubremetz, M. Soête, D. Deslée, H. Vial, D. Bout, M. Lebrun (2005). Synergistic role of micronemal proteins in Toxoplasma gondii virulence. Journal of Experimental Medecine 201:453-463.

 O. Cérède, JF Dubremetz, D. Bout, M. Lebrun (2002) The Toxoplasma gondii protein MIC3 requires pro-peptide cleavage and dimerization to function as adhesin. EMBO Journal, 21:2526-2536.

 N. Garcia-Reguet* , M. Lebrun* , Fourmaux M.N., O. Mercereau-Puijalon, C. Beckers, B. Samyn, J. Van Beemen, D. Bout, J. F. Dubremetz (2000) The microneme protein MIC3 of Toxoplasma gondii is a secretory adhesin which binds to both the surface of the host cells and the surface of the parasite. Cellular Microbiology, 2:353-364

Patent :

M. Lebrun, M Roques, M Tonkin, M Boulanger. Identification of region of RON2 of Apicomplexan parasites suitable for targeting by vaccines and drugs. European Patent Application. May 6, 2011. EP 11305540.4