Combining reverse genetics and live microscopy to dissect Plasmodium knowlesi invasion of red blood cells

Plasmodium knowlesi is one of six Plasmodium species responsible for causing malaria in humans. Symptoms of the disease arise from the blood stage of the parasite’s life cycle, when a form of the parasite, known as a merozoite, invades a red blood cell (RBC), develops and multiplies within it, and then bursts (egresses) from the host cell, releasing more invasive merozoites. Invasion progresses through several key steps, which can be visualised using live microscopy: weak parasite-host cell interactions, stronger interactions causing cell deformation and parasite re-orientation, and finally parasite entry and resealing of the host cell. Throughout invasion, the parasite secretes numerous proteins from specialised secretory organelles, called micronemes and rhoptries, which facilitate each step. Two protein families that have been shown to be essential for merozoite invasion are the Duffy binding protein (DBP) and reticulocyte binding like (RBL) proteins. In P. falciparum, these families consist of multiple members, with significant redundancy, which has led to difficulty assigning specific functions to each member, or even family. P. knowlesi, on the other hand, has a smaller repertoire of proteins within each family consisting of three DBPs (PkDBPα, PkDBPβ, and PkDBPγ) and two RBLs, the Normocyte binding proteins Xa and Xb (PkNBPXa and PkNBPXb). Of these proteins, only two are required for human red cell invasion: PkDBPα and PkNBPXa. The first part of this study investigates how wild type parasites invade human erythrocytes, taking advantage of the large size and distinctly polarised shape of P. knowlesi merozoites to dissect the morphological steps of invasion in much greater detail than previously achieved for P. falciparum. Live microscopy has clarified the order of early events leading up to internalisation and revealed a new preinternalisation step, gliding motility, which is required for deformation and enhances parasite-host cell interactions. The second part of this study uses this new understanding of invasion as a framework to investigate the roles of PkDBPα and PkNBPXa by performing live microscopic analysis of tagged and conditional knockout (cKO) parasite lines. Multi- tagged lines show both proteins share a similar localisation pre egress, but exhibit distinct localisations once released from apical organelles. Finally, live imaging has revealed distinct roles for both families, showing that PkNBPXa is essential for host cell deformation, while PkDBPα performs a function downstream of this process.