The reticulocyte restriction: invasion ligand RBP1a of Plasmodium vivax targets human TfR1, prohibitin-2, and basigin

Plasmodium vivax is the most widespread malaria species with predominance in Latin America and Southeast Asia. Devising effective controls against P. vivax infections presents unique challenges due to the parasites exclusive invasion of reticulocytes, a transient developmental stage of red blood cells. The ephemeral nature and limited availability of reticulocytes complicate efforts to establish continuous in vitro cultures to study the parasites biology and receptor-ligand interactions. Here, the potential of the erythroid cell lines JK-1 and BEL-A as alternatives to reticulocytes was confirmed through successful parasite invasion, marking the first report of P. vivax entry to BEL-A cells. Furthermore, LC-MS was used to quantitatively compare the membrane proteomes of these cell lines with those of reticulocytes and mature erythrocytes. This analysis revealed significant similarities between the membrane proteomes of JK-1, BEL-A, and reticulocytes. In addition to the known Plasmodium receptors, transferrin receptor protein 1 (TfR1 or CD71), CD98hc, and basigin (BSG), potential receptor candidates involved in the parasites invasion pathway were identified, including prohibitin-2 (PHB2), CAT-1 (SLC7A1), ATB(0) (SLC1A5), CD36, integrin beta-1 (ITGB1), and Metal transporter CNNM3. Proximity labeling using TurboID enabled the identification of specific interactions between PvRBP1a158-650 and TfR1, BSG, and prohibitin-2 in the erythroid cell lines. Notably, this is the first report of prohibitin-2 as a receptor for P. vivax. These findings advance the understanding of P. vivax receptor-ligand interactions and underscore the potential of JK-1 and BEL-A cell lines as alternative models for the study of parasite biology. Author SummaryPlasmodium vivax is a major cause of malaria, particularly in Latin America and Southeast Asia. Unlike other malaria parasites, it infects only immature red blood cells (reticulocytes), which are rare and short-lived, making laboratory studies challenging. No continuous in vitro culture system currently exists for P. vivax, limiting research into its invasion mechanisms. In this study, we evaluated two erythroid cell lines, JK-1 and BEL-A, as potential reticulocyte surrogates. We demonstrate that P. vivax can invade both cell types, including BEL-A cells, which had not previously been tested. Comparative analysis revealed key differences in surface protein expression between these cell lines and reticulocytes, compared to mature red blood cells, identifying seven potential parasite-receptor proteins. We further applied a proximity-based protein tagging approach to identify host-parasite interactions mediated by a specific P. vivax protein ligand, RBP1a. This technique revealed a potential role for prohibitin-2, TfR1, and basigin in mediating P. vivax entry into red blood cells by RBP1a binding. The role of prohibitin-2 as a Plasmodium receptor is a novel Discovery. Our results highlight JK-1 and BEL-A cells as valuable tools for P. vivax research and suggest new avenues for investigating host-pathogen interactions, with implications for malaria treatment and vaccine development.