Genetic Evidence for Erythrocyte Receptor Glycophorin B Expression Levels Defining a Dominant Plasmodium falciparum Invasion Pathway into Human Erythrocytes.

Plasmodium falciparum, the parasite that causes the deadliest form of malaria, has evolved multiple proteins known as invasion ligands that bind to specific erythrocyte receptors to facilitate invasion of human erythrocytes. The EBA-175/glycophorin A (GPA) and Rh5/basigin ligand-receptor interactions, referred to as invasion pathways, have been the subject of intense study. In this study, we focused on the less-characterized sialic acid-containing receptors glycophorin B (GPB) and glycophorin C (GPC). Through bioinformatic analysis, we identified extensive variation in glycophorin B (GYPB) transcript levels in individuals from Benin, suggesting selection from malaria pressure. To elucidate the importance of the GPB and GPC receptors relative to the well-described EBA-175/GPA invasion pathway, we used an ex vivo erythrocyte culture system to decrease expression of GPA, GPB, or GPC via lentiviral short hairpin RNA transduction of erythroid progenitor cells, with global surface proteomic profiling. We assessed the efficiency of parasite invasion into knockdown cells using a panel of wild-type P. falciparum laboratory strains and invasion ligand knockout lines, as well as P. falciparum Senegalese clinical isolates and a short-term-culture-adapted strain. For this, we optimized an invasion assay suitable for use with small numbers of erythrocytes. We found that all laboratory strains and the majority of field strains tested were dependent on GPB expression level for invasion. The collective data suggest that the GPA and GPB receptors are of greater importance than the GPC receptor, supporting a hierarchy of erythrocyte receptor usage in P. falciparum

The human GTPase Rac1 plays an important role in Plasmodium falciparum invasion and growth inside human erythrocytes

Malaria is the deadliest parasitosis worldwide, causing 216 million cases and 445.000 casualties in 2016. The disease is caused by Plasmodium parasites that develop and grow inside human erythrocytes. Among them, Plasmodium falciparum is the deadliest one. The human protein Rac1 is a GTPase involved in the development of several cancers and essential in the invasion of the host cell by many intracellular pathogens, including Toxoplasma gondii, which belongs to the same phylum as P. falciparum. Rac1 has been extensively studied and several cell permeable inhibitors of the GTPase have already been developed. In order to investigate a possible role of Rac1 in malaria infection, we analysed Rac1 subcellular localization in P. falciparum infected erythrocytes by immuno-fluorescence assays. We showed that during invasion of the host cell, Rac1 is recruited to the site of parasite entrance and is activated by the parasite. During the intraerythrocytic growth of the parasite, Rac1 is further recruited from the cell membrane to the parasitophorous vacuole membrane . These data suggest that Rac1 may play a role in P. falciparum infection of human erythrocytes, To confirm the contribution of Rac1 to the process of host cell invasion, we generated two different Rac1-KO erythroid cell lines, demonstrating that P. falciparum invasion rates significantly decreased in both the transgenic cell lines compared to wild type. We also performed invasion assays on wt erythrocytes from donor, in the presence of two different Rac1-specific inhibitors, further confirming the role of the GTPase in parasite invasion of the host cell.Finally, 7 different chemical inhibitors of Rac1 were tested on synchronous P. falciparum cultures. These reduced parasite growth with a half minimal inhibitory concentration (IC50) below 20 µM, indicating that Rac1 is a druggable target . Two among the inhibitors showed nanomolar IC50. Rac1 may be thus considered an interesting target for the development of novel anti-malarial drugs. with the advantage of being a well studied protein, with a known x-ray crystal structure and several specific inhibitors on commerce. Moreover, therapies targeting the host instead of the parasite reduce the probability of resistance insurgence, a critical issue for currently available drugs

Structural organization of erythrocyte membrane microdomains and their relation with malaria susceptibility

Cholesterol-rich microdomains are membrane compartments characterized by specific lipid and protein composition. These dynamic assemblies are involved in several biological processes, including infection by intracellular pathogens. This work provides a comprehensive analysis of the composition of human erythrocyte membrane microdomains. Based on their floating properties, we also categorized the microdomain-associated proteins into clusters. Interestingly, erythrocyte microdomains include the vast majority of the proteins known to be involved in invasion by the malaria parasite Plasmodium falciparum. We show here that the Ecto-ADP-ribosyltransferase 4 (ART4) and Aquaporin 1 (AQP1), found within one specific cluster, containing the essential host determinant CD55, are recruited to the site of parasite entry and then internalized to the newly formed parasitophorous vacuole membrane. By generating null erythroid cell lines, we showed that one of these proteins, ART4, plays a role in P. falciparum invasion. We also found that genetic variants in both ART4 and AQP1 are associated with susceptibility to the disease in a malaria-endemic population