Using transgenic Plasmodium knowlesi to investigate the role of Plasmodium vivax DBP and RBP ligands in host cell tropism during erythrocyte invasion

Selection and invasion into host erythrocytes by Plasmodium merozoites is essential for blood-stage replication and is mediated by several conserved protein families including the reticulocyte binding-like (RBL) and Duffy binding-like (DBL) ligands. The expanded nature of these families and a lack of known host cell receptors have made them difficult to study, particularly in species which lack long term in vitro culture, such as P. vivax which causes millions of malaria cases annually. In this thesis, the role of RBL and DBL proteins were investigated using the closely-related and genetically tractable zoonotic species, P. knowlesi. Using a combination of orthologue replacement and conditional expression systems, P. knowlesi was modified to express the P. vivax RBL PvRBP2b, which is thought to be involved in the characteristic reticulocyte restriction of P. vivax. PvRBP2b did not functionally complement the P. knowlesi RBL, PkNBPXa, however its expression in addition to normal P. knowlesi ligands increased the growth rate in enriched reticulocytes, suggesting that PvRBP2b is involved in promoting reticulocyte invasion. In addition, this project explored how variations in a host cell DBL receptor, DARC, influence host cell tropism. Results show a clear binding preference of both P. knowlesi and P. vivax DARC-binding DBP orthologues for a common allelic variant, Fyb , over Fya . This work provides in vitro context to studies suggesting that the Fya allele may be mildly protective against vivax malaria, and evidence that a similar (or stronger) effect may be seen with P. knowlesi infections. Finally, antibody-mediated inhibition of both families was trialled, with polyclonal anti-PkNBPXa rabbit sera eliciting strong inhibition of P. knowlesi and a potentially synergistic effect when combined with antibodies targeting DBP. These orthologue replacement models offer new methods for dissecting the role of RBL and DBL proteins individually, and for validating inhibitory antibodies in vitro.