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 Plasmodium 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 (antigenic variation). These molecules are transported to the erythrocyte surface via unique, parasite-specific, secretory pathways. Another line is study of the biological role of the nuclear architecture and gene relocation in differential expression of gene families. These studies already have given important insights into the biology of telomeres and virulence factor genes located in subtelomeric regions.
Cytoadhesion molecules and malaria pathogenesis:
Benoit Gamain, Nicki Viebig, Pablo Fernandez, 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). One of the main objectives of the BIHP unit is to define the interactions involved during pregnancy associated malaria (PAM) in order to identify the optimal antigens for inclusion in a vaccine that will prevent malaria during pregnancy. Chondroitin sulfate A (CSA) ; present in the placental intervillous blood spaces, is the main receptor involved in the massive sequestration of P. falciparum parasitized erythrocytes (PE) to the placenta. In order to investigate for the parasite's molecules associated with the CSA adhesion phenotype, we recently established in the laboratory the human trophoblastic BeWo cell line as a quick and easy model for selecting P. falciparum CSA binding parasites (photo 1A). The adhesion molecule, which mediates binding to CSA, is encoded by a member of the var gene family (var2CSA). We have performed KO parasites for the var2CSA gene. Given that FCR3var2-CSA disruptant mutants do not recover the CSA binding phenotype, even after multiple rounds of panning selection, we conclude that no other parasite gene can compensate for the loss of function under the experimental CSA selection conditions of our work. 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. Our results demonstrate the central role of var2CSA in placental adhesion and support var2CSA as a leading vaccine candidate aiming to protect pregnant women and their foetuses. Attempts to complement the FCR3var2-CSA mutants using synthetic GFP tagged mini-var2CSA genes are underway (photo 1B). These mutant parasites will be an excellent tool to investigate if other host receptors are involved during PAM. In order to study putative factors that can induce the CSA-phenotype during pregnancy we investigate if serum factors enhance switching to var2CSA. In parallel, we analyse signal transduction molecules that could be involved in var gene switching.
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 attached to 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". In order to test this hypothesis, we have initiated deformability studies of RSP2-tagged erythrocytes (in collaboration with Thérèse Cynober, Hôpital de Bicêtre). Preliminary studies show that RSP2-tagged erythrocytes are less deformable than control culture of erythrocytes. As a consequence, these red blood cells could be destroyed, possibly leading to anemia. Further studies are under way to investigate this process in more detail and its biological implication in malaria
2. Molecular mechanisms of antigenic variation:
Stuart Ralph, Alisson Gontijo, Ana Paola Rojas-Meza, Catherine Keeling, Neha Issar, Jose Juan Lopez Rubio, Rosaura Hernandez-Rivas, 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 form 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 (chromatin immunoprecipation assays) 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 hyper-acetylation. Another epigenetic factor appears to play a role in telomere-associated var gene activation. Switching 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. We focus now on two key questions in antigenic variation: what determines mono-allelic exclusion and genetic imprinting of active var genes. To this end we investigate epigenetic changes associated with the chromatin of var genes (silent and active state). Several chromatin marks have been associated with active var genes and will be explored further.
3. P. falciparum subnuclear compartments: telomere and nucleolus:
Liliana Mancio da Silva, Artur Scherf
Epigenetic silencing may not be limited to antigenically variant gene families at telomeres but could also control transcription of rDNA genes, which are dispersed on 7 out of 14 chromosomes. A number of specific chromatin factors such as PfSir2 co-localises to telomeric regions and the nucleolus. We are investigating the role of these factors using ChIP, FISH and KO of specific genes such as PfSir2. The nuclear distribution of individual rDNA genes has been investigated and indicates that these loci have a preferential location in the nuclear periphery (Photo 2).
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:
Jose Manuel Lima Nogueira, Emeric Roux, Denise Mattei
Sequestration of P. falciparum-infected erythrocytes (IE) is strongly implicated in the pathogenesis of cerebral malaria and pregnancy-associated malaria and is the target of new intervention strategies. Our laboratory investigates the trafficking process of cytoadherence-linked molecule to the surface of IE in parasite lines selected for binding to different endothelial adhesion receptors, such as CD36 and CSA. Previous studies suggested that the clag9 (cytoadherence linked asexual gene 9) gene located on the right arm of chromosome 9 is directly involved in the binding of IE to CD36. Our results show that clag9 is expressed at very late stages in rhoptries of parasites selected for binding to CD36 and CSA and in the parasitophorous vacuole in ring stages, but not at the onset of sequestration in young trophozoites. The analysis of clag9 mutant parasites (T9-96 and D10 lines) revealed that parasites express a member of the var gene family at the surface of IE but these IE are unable to adhere to common adhesion receptors (CD36, ICAM-1, CSA, etc.). Our data indicate that clag9 is not itself an adhesion molecule but modulates (by a yet unknown process) the correct assembly of the adhesive complex at the surface of IE.
Photo 1 : A.) Cytoadhesion of infected red blood cells to CSA of BEWO cells (trophoblast cell line). B.) Parasites transfected with a plasmid carrying a min-var2CSA gene fused to GFP. The var-GFP fusion protein is transported to the Maurer s clefts.
Photo 2 : Nuclear distribution of rDNA genes that are dispersed on 7 chromosomes of P. falciparum. A.) FISH (Fluorescent in situ hybridization) image obtained with a probe specific to all 7 rDNA units. B.) Superposition of the FISH signal with the nucleus stained with DAPI.
Keywords: malaria, protein trafficking, antigenic variation, cytoadhesion, placental malaria, telomere