Dissecting the role of the tight junction in Plasmodium merozoite invasion of the human erythrocyte

Abstract

Erythrocyte invasion by the malaria causing Plasmodium parasites is a highly coordinated process. Formation of a structure called the tight junction, composed of the AMA1-RON complex in P. falciparum, is a prerequisite for successful invasion and is thought to represent a conserved mechanism of invasion across Apicomplexan parasites. Plasmodium knowlesi, a zoonotic parasite causing severe-to-lethal malaria disease in humans, has only recently been adapted to continuous culture in human erythrocytes. In comparison to the most virulent human malaria, Plasmodium falciparum, there are, however, few cellular tools available to study its biology in particular direct investigation of erythrocyte invasion by blood-stage P. knowlesi merozoites. Work presented in this thesis reports a robust method for the isolation of viable and invasive P. knowlesi merozoites to high purity and yield. Purified merozoites were used to establish two important cellular applications: a P. knowlesi invasion inhibition assay; and high-resolution imaging of P. knowlesi merozoites captured during invasion. I use the new-found ability to isolate human-adapted P. knowlesi merozoites to explore comparative dissection of merozoite invasion revealing P. knowlesi can invade human erythrocytes by a Basigin independent mechanism. This raises important mechanistic questions towards the essential P. falciparum Rh5-Basigin interaction. Additionally, I utilise the larger, robust P. knowlesi merozoites as a model to investigate the tight junction during Plasmodium invasion and show the RON complex also plays a key conserved role in erythrocyte invasion by a second human infectious Plasmodium species. Finally, preliminary evidence presented here tentatively identifies an additional member of the megadalton PkRON complex, PkRALP1. Further work investigating this candidate might reveal mechanistic details of how the tight junction forms and facilitates erythrocyte invasion. In brief, the P. knowlesi merozoite platform validated here provides a valuable tool for cross-species comparative invasion biology analysis.