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 immune escape strategies. These molecules are transported to the erythrocyte surface via unique, parasite specific, secretory pathways. Another recently established line is the structural organisation of the nucleus. This has given important insights into the biology of telomere adjacent region and virulence factor genes. A further line of research is a proteomic approach focussed on serin proteases of the merozoite. Two serine proteases crucial for the process of red cell invasion are studied in detail and validated as novel drug targets.
Cytoadhesion molecules and malaria pathogenesis: We study parasite-encoded adhesion molecules, which are inserted into the erythrocyte membrane during intracellular blood stage development (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 (photo1). In a previous study, we had identified the CSA-binding domain of PfEMP1 (encoded by the varCSA gene) and have now expressed this cysteine-rich domain in insect cells. Analysis of the antibodies raised against 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. Knock out mutations of the varCSA 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. Consequently, both varCSA genes seem essential components for the development of a vaccine that could protect pregnant women from severe malaria. 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 leading possibly to a rigidification of RSP2 tagged erythrocytes. Studies are under way to investigate this process more in detail and its biological implication during malaria pathophysiology.
Molecular mechanisms of immune escape: 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 (photo 1). 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. Preliminary data from our laboratory indicate that specific proteins bind to rep20 repeats, and are involved in the association P. falciparum chromosome ends. Despite the significant difference in their subtelomeric organisation all six Plasmodium species analysed for far present chromosome end clusters. The telomere position effect (TPE) brings about repression of genes placed near yeast telomere repeats and proteins essential for silencing are found to be concentrated in the telomeric regions. P. falciparum orthologues to most yeast telomere-associated proteins have been identified including the genes coding for the "silent information regulators". This finding implies that P. falciparum might use a similar mechanism to silence telomere-associated var genes. Gene inactivation studies are in progress to analyse the role of these telomere-associated molecules in P. falciparum.
Plasmodial telomerase: 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 nulceolus in P. falciparum (photo 2). Gene inactivation experiments indicate that telomerase is essential during the highly proliferative blood stage development defining this enzyme as a novel target for the development of anti-parasite therapies.
Intracellular trafficking: 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 the phosphorylation of proteins exported to the erythrocyte membrane and the implications 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. Furthermore, we observed that CLAG9 is localised in the rhoptries (photo 3) but not on the surface of infected erythrocytes. Furthermore, we demontrated (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 the cytoadherence remains puzzling.
Enzymes implicated in merozoite release or red cell invasion : The propagation of multi-drug resistant P. falciparum parasites and insecticide-resistant Anopheles mosquitoes has largely impaired the treatment and control of malaria. Consequently, new anti-malarial drugs are urgently needed and the identification of novel targets essential for the parasite life cycle is a first step in the development of new chemotherapeutic strategies. Our efforts are focused on the process of erythrocyte invasion since the molecules involved should provide ideal targets. The delivery of these molecules is regulated via interaction with the host cell, by exocytosis of parasite organelles. The biogenesis of these secretory organelles has only been approached by electron microscopy, their contents are only partially identified and the mechanism of their exocytosis is unknown.
We have recently engaged proteomic studies of these secretory organelles of the parasite, addressing to their site of action the enzymes implicated in merozoite release and red cell invasion. Using a serine protease specific probe, we have identified 4 novel proteins, including one with trypsin-like features, the molecular and functional characterisation of which is under way. Moreover, we have clarified the interaction between one of these compartments (the Maurer's clefts) and the host cell plasma membrane: this interaction involves the parasite trans-membrane protein PfSBP1 and a red cell trans-membrane receptor, the LANCL1-like receptor, and seems to be regulated by the state of phosphorylation of SBP1. Results from a global proteomic approach of the Maurer's clefts provide valuable pieces of information concerning the biological function of this compartment.
We have further characterised the merozoite specific serine protease, SUB2, from two human (P. falciparum and P. vivax ) and two rodent (P. berghei and P. yoelii ) malaria species and shown that it is expressed by all invasive stages of the parasite. Using genetic tools, we have demonstrated that sub2 is an essential gene for the parasite erythrocytic cycle and generated recombinant P. berghei parasites expressing Pbsub2 (but at a higher level) or Pfsub2. Their phenotypic characterisation is under way. In collaboration with J. Martinez (University of Montpellier), we are developing specific inhibitors of both SUB2 and Pfgp76, a merozoite-specific serine protease implicated in red cell invasion. Their activity is evaluated both in vitro and in vivo, in order to identify lead compounds with anti-malarial activity.
Photo 1 : FISH analysis of the nuclear architecture of P. falciparum reveals a highly organised nucleus with chromosome ends forming clusters' in the nuclear periphery.
Photo 2 : P. falciparum telomerase (PfTERT) localizes to a subcompartment of the nucleolus (PfNOP1) of P. falciparum.
Photo 3 : Localization of clag9 in the rhoptries de P. falciparum.
Keywords: invasion of erythrocytes, protein trafficing, antigenic variation, cytoadhesion, malaria