L'épigénétique joue un rôle majeur dans le développement érythrocytaire de Plasmodium falciparum, tels que variation antigénique, pathogenèse, différenciation sexuée. Jusqu'à présent, ces éléments n'ont jamais été décrits chez les sporozoïtes. Pour caractériser la régulation épigénétique au niveau des sporozoïtes de P. falciparum, nous avons étudié les principaux régulateurs épigénétiques PfHP1 (P. falciparum hétérochromatine Protein 1) ainsi que PfSET6 et PfSET7 (méthyltransférases histone lysine). J'ai établi une cartographie génomique des marques épigénétiques répressives associées à l'hétérochromatine, et actives associées à l'euchromatine. J'ai identifié un nouveau mécanisme stade-spécifique de contrôle de l'expression génique, qui réprimés plusieurs gènes codant pour des protéines exportées. Ce mécanisme repose sur une expansion d'hétérochromatine. De plus, je démontre qu'un membre de la famille des gènes var, qui code pour le facteur de virulence PfEMP1 des stades sanguins, est exprimé à la surface des sporozoïtes. Cette localisation contraste avec les stades sanguins, où PfEMP1 est transporté à la surface des érythrocytes et participe à cytoadhérence. L'ensemble de ces résultats ouvre de nouvelles questions biologiques: quels sont les facteurs qui régulent la formation d'hétérochromatine chez les sporozoïtes? Quelle est la fonction de PfEMP1 sur la surface d'un sporozoïte? Mes conclusions indiquent un rôle putatif de PfEMP1 lors de la migration des sporozoïtes. En outre, l'expression, à la surface du sporozoïte, d'un antigène polymorphique et spécifique de souche pourrait expliquer la réponse immunitaire souche-spécifique, induite par les sporozoïtes atténués. / Epigenetic mechanisms control key processes during Plasmodium falciparum blood stage development such as antigenic variation, malaria pathogenesis and sexual commitment. However, the epigenetic landscape has not been reported for the sporozoites stage. To characterize epigenetic regulation in sporozoites, we tested the major epigenetic regulators P. falciparum Heterochromatin Protein 1 (PfHP1) and the histone lysine methyltransferases (PfSET6 and PfSET7) in P. falciparum sporozoites. I obtained a reliable genome-wide occupancy data for repressive heterochromatin and active euchromatin marks. Notably, I discovered an unprecedented stage specific mechanism of silencing, which represses several hundreds of genes, encoding parasite surface exported proteins. This is based on an expansion of facultative heterochromatin boundaries in sporozoites. Moreover, I demonstrate that a single member of the polymorphic var gene family, encoding the blood stage virulence factor PfEMP1, is expressed at the surface of sporozoites. This is in contrast to blood stages where PfEMP1 is transported to the erythrocyte surface participating in cytoadhesion. Overall, my findings rise new biological questions including what are the factors that regulate heterochromatin boundaries and what is the function of a virulence-associated surface antigen in sporozoites stage. My findings point to a putative function of this adhesion molecule in sporozoites migration. Moreover, the expression of a highly polymporphic and strain-specific antigen on the surface of sporozoites might provide a molecular explanation for the strain-specific protective immune response induced by attenuated sporozoites.

Platelet-mediated clumping of Plasmodium falciparum-infected erythrocytes (IEs) is a common property of field isolates associated with severe disease (Pain, Ferguson et al. 2001). Platelet receptors CD36 (Pain, Ferguson et al. 2001), P-Selectin (Wassmer, Taylor et al. 2008) and gC1qR (Biswas, Hafiz et al. 2007) mediate clumping. To characterize the molecular specificities of the clumping phenotype, I cloned clumping parasite line IT/C10 by limiting dilution. I characterized var gene expression in the IT/C10 clones using generic primers for the DBL tag region (Bull, Berriman et al. 2005). Clumping assays were conducted in the presence of specific reagents to delineate host factors hypothesized to contribute to development of the clumping phenotype. Finally, I conducted a clinical study with isolates from children with malaria in Kilifi, Kenya. This study shows that in parasite line IT/C10, platelet-mediated clumping is associated with Itvar30 suggesting a prominent role for the PfEMP-1 encoded by this var gene in development of platelet-mediated clumping. For IT/C10 parasites, platelet activation appears to be involved in platelet-mediated clumping. Platelet P-Selectin appears to mediate clumping using lectin-dependent interactions. To further elucidate the mechanisms that mediate clumping by host platelets, I have used a panel of platelet antagonists to delineate specific platelet activation pathways. Our results show that platelet activation plays an important role in platelet-mediated clumping. Finally, in this study, platelet-mediated clumping was associated with parasitaemia, but not with disease severity.

Erythrocytes infected by mature forms of the Plasmodium falciparum parasite adhere to other components of the vascular space, a behavior considered critical to the pathogenesis of severe malaria. Adhesion is mediated by the P. falciparum erythrocyte membrane protein 1 (PfEMP1), a highly variant antigen expressed by the parasite and subject to switching during the course of an infection. The host ligands of PfEMP1 include CD36, ICAM-1 and the ABO antigens. By employing a series of population- and family-based association studies from multiple African populations, we examined whether variation in the genes underlying these molecules affects susceptibility to severe malaria. Our results suggest that a common frameshift mutation in the ABO glycosyltransferase, responsible for blood group O, is associated with protection from severe malarial phenotypes (P=2x10⁻⁷), particularly severe malarial anaemia. However, we found no significant disease associations with variation in either the ICAM1 or CD36 genes. We focused on two particular functional polymorphisms, the missense ICAM-1Kilifi and the CD36 nonsense mutation T1264G. We genotyped both markers in around 10,000 individuals, but neither demonstrated an association with severe malarial phenotypes. Malaria has been a profound selection pressure shaping human genetic diversity. The last decade has seen the development of several haplotype-based methods to detect signatures of recent positive evolutionary selection. These techniques are potentially invaluable tools in our hunt for genetic variants that protect from life threatening malaria. We used simulations and empirical data from the International HapMap Project to demonstrate the validity of searching for long regions of haplotype homozygosity, as an approach to finding alleles undergoing selective sweeps. We analysed genetic data from a range of populations, particularly those utilized by HapMap, to investigate whether our candidate genes were associated with signals of recent positive selection. We characterized the distribution of a selection event associated with the CD36 1264G allele, focused in Central-West Africa, and demonstrated a novel signal of low population differentiation at the ABO gene, suggestive of longstanding balancing selection. Our work confirms that variation in the host ligands of PfEMP1 modulates severe malaria susceptibility, and highlights the value of using signals of selection, along with functional experiments and genetic association studies, to dissect the biology of severe malaria.