1. Enzymes involved in the entry of the parasite into and its exit from the erythrocyte (C. Braun-Breton)
We are interested in characterisation of the secretory compartments of P. falciparum involved in the invasion of erythrocytes or release of the form of the parasite that infects erythrocytes, the merozoite. This study makes use of the techniques of proteomics and should enable us to determine which proteins constitute these compartments and the biological role of the various compartments. In particular, we are seeking to identify enzyme activities crucial for these key steps in parasite development, which could be the targets of specific inhibitors.
We have already demonstrated the activities of several proteases and phospholipases, the characterisation of which is underway. We are particularly interested in characterisation of the parasite serine protease responsible for the last step in the processing of MSP1 (the major surface protein of merozoites), which is essential for the entry of the parasite into the erythrocyte. Our work suggests that this processing is carried out by the product of the sub2 gene, which we have recently characterised in P. falciparum and in a species of Plasmodium that infects rodents, P. berghei. In collaboration with the laboratory of A. Waters (Leiden, the Netherlands) and using parasite transgenesis, we have shown that sub2 is a gene essential for the erythrocyte cycle of Plasmodium. We have also characterised the sub2 gene of another major causal agent of human malaria, P. vivax. Finally, we have developed tools for the determination of SUB2 activity. In collaboration with the laboratory of Jean Martinez (Montpellier University), we are developing specific inhibitors of PfSUB2 and of another parasite serine protease, Pfgp96, which we have shown to be involved in the entry of the parasite into red blood cells. Inhibitors active against these enzymes and that block the invasion of erythrocytes at micromolar concentrations have been obtained. We are currently trying to increase their efficacy.
2.1 Erythrocyte surface molecules of P. falciparum and their role in malaria pathogenesis and immune evasion (A. Scherf)
In natural P. falciparum infections, parasitized erythrocytes (PE) circulate in the peripheral blood for a period corresponding roughly to the first part of the erythrocytic life cycle (ring stage). Later in blood-stage development, parasite-encoded adhesion molecules are inserted into the erythrocyte membrane, preventing the circulation of the PE. The principal molecule mediating PE adhesion is P. falciparum erythrocyte membrane protein 1(PfEMP1), encoded by the polymorphic var gene family. Members of the var gene family are subject to clonal antigenic variation and we have recently shown that a single antigen variant is expressed at the PE surface, in an exclusive manner. In addition, our data demonstrate its role in immune evasion and switches in PfEMP1 expression may be associated with fundamental changes in parasite tissue tropism in malaria patients. A switch from CD36-binding to chondroitin sulfate A (CSA)-binding may lead to extensive sequestration of PE in placenta syncytiotrophoblasts. This is probably a key event in malaria pathogenesis during pregnancy. We have identified the CSA binding domain of the PfEMP1 molecule and are investigating its potential for new intervention strategies to protect women during pregnancy. The CSA-binding phenotype of mature PE is linked to another distinct adhesive phenotype: the recently described CSA-independent cytoadhesion of ring-stage PE. Thus, a subpopulation of PE that sequentially displays these two different phenotypes, may bind to an individual endothelial cell or syncytiotrophoblast throughout the asexual blood stage-cycle. This suggests that non-circulating (cryptic) parasite subpopulations are present in malaria patients.
2.2. Telomeres, are they the Achilles Heel' of Plasmodium ? (A. Scherf)
We have been studying different aspects of telomere biology of malaria parasites. In single-celled organisms, telomere length needs to be maintained within a minimal size range, to ensure the survival of the cell. However, due to the incomplete replication of linear chromosomes, there is a net loss of telomeric DNA with each successive cell division. To compensate for this loss, new telomeric repeats are added onto chromosome ends in a reaction catalysed by a specialised reverse transcriptase called telomerase. In the absence of telomerase activity, telomeres shrink with each replication cycle and the erosion is so extensive that with time the telomere capping function becomes severely compromised, leading to growth arrest and eventually senescence. We have shown that the mean telomere length of P. falciparum is maintained at a constant average size during blood stage proliferation and telomerase activity has been detected in protein extracts of cells at this stage. A candidate for the P. falciparum telomerase catalytic subunit has been identified in the genome database. Knock out experiments are in process to verify that this is an essential gene for the survival of the parasite and can be considered as a new target for the development of anti-parasite therapies.
New data on the organization of plasmodial telomeres has been recently obtained in our laboratory. Telomeres form clusters of 4 to 7 heterologous chromosome ends at the nuclear periphery in asexual and sexual parasite stages. This subnuclear compartment promotes gene conversion between members of subtelomeric virulence factor genes in heterologous chromosomes resulting in diversity of antigenic and adhesive phenotypes. This has important implications for parasite survival.
3. Trafficking of parasite proteins to the membrane of parasitised erythrocytes (D. Mattei)
"Knobs", protuberances resulting from modifications to the erythrocyte membrane induced by the parasite, are observed at points of adhesion to endothelial cells and are involved in the phenomenon of parasite sequestration. Several proteins of parasite origin are present in the "knobs": PfHRPI (histidine-rich protein I), the major structural component of "knobs", and the variant antigen PfEMP1 (erythrocyte membrane protein 1), a parasite ligand that interacts with endothelial cells. PfEMP1 is anchored to the surface of the "knobs" via PfHRPI.
Studies of cytoadhesion in vitro, in flux conditions similar to those of the bloodstream, showed that parasites devoid of "knobs" adhere only weakly to endothelial cells. The mechanisms of transport and protein targeting in P. falciparum are poorly understood. Our work has demonstrated the existence of two different secretion pathways: the classical pathway, via the endoplasmic reticulum (ER) and the Golgi apparatus, and an alternative pathway insensitive to brefeldin A (BFA). We found that, in strain FCR3, the transport of PfHRPI seemed to be insensitive to the action of BFA. The C-terminal extremity of PfHRPI presents a potential isoprenylation site preceded by a sequence of several basic amino acids. The association of these two motifs constitutes a signal for targeting to the plasma membrane and may mediate the secretion of PfHRPI into the cytoplasm of the red blood cell. Experiments involving metabolic labelling with a radioactive isoprenylation precursor, followed by immunoprecipitation, suggest that PfHRPI is probably isoprenylated. Characterisation of the secretion pathways specific to the parasite may lead to the identification of new targets of potential importance for the development of drugs active against the adhesion phenotype of parasitised erythrocytes.
Our recent co-localisation studies with several secreted proteins in BFA-treated parasites suggest that the ER is composed of different domains. It is possible that the properties of the polypeptides, such as their solubility or affinity for membranes, protein-protein interactions or post-translational modifications, determine their compartmentalisation'. Our result suggest that the targeting of P. falciparum proteins to the various compartments of the infected erythrocyte begins within the ER.