Unit: Biology of Host-Parasite Interactions
Director: SCHERF Artur
The research activities of the different groups in the Unité are mostly based on the red blood cell stage of the P. falciparum life cycle that is responsible for clinical symptoms. A major line of research is devoted to the study of the virulence factors involved in malaria pathogenesis (especially maternal malaria and anemia) and the molecular basis of immune escape strategies. These molecules are transported to the erythrocyte surface via unique, parasite-specific, secretory pathways. Another line is the structural organisation of the nucleus. This has given important insights into the biology of telomere adjacent regions and virulence factor genes.
1. Cytoadhesion molecules and malaria pathogenesis:
Benoit Gamain, Nicki Viebig, Marta Nunes, Yvon Sterkers, Christine Scheidig, Artur Scherf
We study parasite-encoded adhesion molecules, which are inserted into the erythrocyte membrane during intracellular blood stage development or during the merozoite invasion of red blood cells (performed in close collaboration with the laboratory of Dr. Gysin, University of Marseille). The adhesion of infected erythrocytes (IE) to chondroitin sulfate A (CSA) in the placenta is an important event in malaria pathogenesis during pregnancy. In a previous study, we had identified the CSA-binding domain of PfEMP1 (encoded by the var1CSA gene) and have now expressed this cysteine-rich domain in insect cells. Analysis of the antibodies raised against the recombinant domain revealed surface reactivity with CSA-binding parasites from different geographic regions, indicating the potential use of this molecule for the development of a vaccine to protect women during pregnancy (photo 1). Knock out mutations of the var1CSA gene were generated (in collaboration with Dr. Lanzers' group University of Heidelberg) and though the parasites initially lost the CSA binding phenotype, it was eventually possible to recover parasites capable of binding to CSA. The adhesion molecule, which mediates binding to CSA in KO parasites is encoded by a member of the var gene family (var2CSA). CSA binding domains within var2CSA were identified and are now evaluated for the development of a vaccine that could protect pregnant women from severe malaria. We have performed KO parasites for the var2CSA gene. These mutant parasites will allow us to investigate if other parasite CSA-ligands exist. In parallel, we are investigating the factors influencing the switch to the CSA binding phenotype during pregnancy.
We have also characterised the PfEMP1-independant cytoadhesion of ring-stage IE. Using several monoclonal antibodies raised against the surface of ring stage IE, we have identified Ring Surface Protein 2 (RSP2) as a crucial molecule in the ring adhesion process (collaboration with J. Gysin). The RSP2 rhoptry protein is inserted into the membrane of infected and non-infected erythrocytes during the invasion process in a large number of erythrocytes. The discovery of non-infected erythrocytes which are "tagged" with RSP2 at their surface has led us to investigate if these tagged cells undergo a change in cellular "rigidification". As a consequence, these red blood cells could be destroyed possibly leading to anemia. In order to test this hypothesis, we have initiated deformability studies of RSP2-tagged erythrocytes (in collaboration with Thérèse Cynober, Hôpital de Bicêtre et Max Hardeman de l'Academisch Meisch Centrum d'Amsterdam). Preliminary studies show that RSP2-tagged erythrocytes are less deformable than control culture of erythcotyes. Further studies are under way to investigate this process in more detail and its biological implication during malaria pathophysiology.
2. Molecular mechanisms of antigenic variation:
Stuart Ralph, Alisson Gontijo, Ana Paola Rojas-Meza, Catherine Keeling, Artur Scherf
Antigenic Variation is a strategy employed by malaria species to outmanoeuvre the host defence mechanisms long enough for their progeny to spread. We have established that epigenetic factors are involved in var gene regulation. In previous studies we demonstrated that the chromosomes ends in blood stage P. falciparum parasites are physically grouped together into clusters (between 4 and 7 in number per haploid nuclei) and are located at the nuclear periphery. This subnuclear compartmentalisation seems to create an environment that allows the expansion and diversification of var gene families located at chromosome ends. Two independent studies pointed to a subtelomeric element called rep20 as a critical DNA region involved in cluster formation between chromosome ends. Despite the significant difference in their subtelomeric organisation, all six Plasmodium species analysed so far present chromosome end clusters. A "telomere position effect"- like mechanism brings about repression of antigenic variation genes located near telomere repeats. P. falciparum orthologues to several yeast telomere-associated proteins have been identified including the gene coding for the "silent information regulator" PfSir2. ChIP experiments have shown that PfSir2 is associated with subtelomeric DNA (telomere, rep20) and to the promoter region of telomeric var genes. However, upon activation of a var gene, PfSir2 is removed from the promoter region, leading to histone acetylation. Another epigenetic factor appears to play a role in telomere-associated var gene activation. Swtching is associated with var loci movement between subnuclear loci, suggesting that specific regions compatible with gene transcription exist in the nuclear periphery. Heterochromatin analysis of the nucleus indicates that a specific region is free of dense chromatin. We are exploring if this region coincides with the transcription of var genes (Photo 2).
3. P. falciparum subnuclear compartments: telomere and nucleolus:
Liliana Mancio da Silva, Artur Scherf
Epigenetic silencing may not be limited to antigenic variation gene families at telomeres but could also control transcription of rDNA genes. A number of specific chromatin factors such as PfSir2 co-localises to telomere regions and the nucleolus. We are investigating the role of these factors using chromatin immunoprecipation assays, FISH and KO of specific genes such as PfSir2.
A candidate for the P. falciparum telomerase catalytic subunit (TERT) has been identified in the genome database. We have localised TERT to a specific sub-compartment of the nucleolus in P. falciparum. The role of telomerase and the nucleolus in the elongation of telomeres is not well understood and will be further analysed.
4. Intracellular trafficking:
Ana Filipa de Novais Julio, Solange Touron, Denise Mattei
We investigate the intracellular traffic of parasite proteins targeted to the membrane of P.falciparum -infected erythrocytes. The identification and characterisation of Plasmodium specific mechanisms and secretory pathways are of fundamental interest and might lead to the identification of new drug targets that might interfere with parasite sequestration to host endothelium. We localised the parasite homologue to the GSK-3 (Glycogen Synthase Kinase-3) in association with membranous structures, the Maurer's clefts, in the cytoplasm of the infected red blood cell (collaboration with Dr. L. Meyer, Roscoff). We study the role PfGSK-3 in parasite cytoadherence. Modifications of the red blood cell membrane, induced by the parasite have been implicated in the cytoadherence. Antigen CLAG9 (Cytoadherence-Linked Asexual Gene) has been described as being an adhesion ligand mediating binding to the host receptor CD36. Our work shows that the clag9 gene is transcribed in parasites selected by their binding capacities on the receptors CD36 and CSA. Moreover, we observed that CLAG9 is localised in the rhoptries but not on the surface of infected erythrocytes. Furthermore, we demonstrated (in collaboration with Irene Ling et A. Holder, NIMR, Mill Hill, Londres, UK) that CLAG9 is part of the RhopH complex. This is in contrast to the previously proposed role of CLAG9 as CD36 ligand. Thus, its role in cytoadherence remains puzzling. The analysis of clag9 mutant parasites (T996 and D10 cloned lines) revealed that parasites still express a member of the var gene family on the red blood cell surface but are unable to adhere to common adhesion receptors (CD36, ICAM-1, E-selectin and CSA). This raises the possibility that CLAG9 controls (by a yet unknown process) the expression of a large subset of genes of the var gene family. Alternatively, these PfEMP1 molecules have a conformation at the erythrocyte surface that is incompatible with receptor binding.
Photo 1 : A) Cytoadhesion of CSA-binding parasites to CSA coated to plastic plates.
B) Immunofluorescence showing the reactivity with antibodies raised against the CSA ligand expressed at the surface of placental-binding infected red blood cells.
Photo 2 : A.) Electron microscopy of P. falciparum blood stage parasites reveals heterochromatin regions in the nuclear periphery. Anti-PfSir2 antibodies react with the perinuclear region. Reconstruction of a P. falciparum nucleus of serial ultrathin-sections. Heterochromatin regions are highlighted.
Keywords: malaria, protein trafficking, antigenic variation, cytoadhesion, placental malaria, telomere