Functional characterisation of putative Plasmodium falciparum invasion ligands and their homologues

Abstract

The apicomplexan parasite Plasmodium falciparum is a major threat to global health, accounting for the majority of malaria-related morbidities and mortalities, totalling to cause ~400,000 deaths per year of which the lives of children under the age of 5 are the most impacted (WHO 2020). This is exacerbated by the onset of resistance against our frontline antimalarials and the lack of an effective licensed vaccine against malaria. Studies which investigate novel interventions against P. falciparum host infection are critical in order produce clinically relevant outcomes. In this thesis, I focussed my studies on the blood stage lifecycle of P. falciparum which is responsible for all clinical symptoms of malaria. More specifically, I examined the molecular mechanisms by which the invasive form of the parasite, the merozoite, uses to invade host red blood cells. The point in time in which merozoites invade red blood cells is defined as a vital crux for the blood stage lifecycle to proceed, whereby blocking invasion prevents further downstream parasite growth and the resulting pathologies. Uncovering the proteins which govern merozoite invasion will aid in informing effective vaccine and antimalarial design in the field. As such, we have identified several conserved genes in P. falciparum which encode proteins with a potential function in merozoites for functional characterisation. We first undertook a study on cytosolically-exposed rhoptry leaflet interacting protein 2 (PfCERLI2), expressed from a duplicated gene of its paralogue PfCERLI1 which is essential for merozoite invasion. Using super-resolution microscopy platforms and Western blot-based membrane assays, we discerned that PfCERLI2 localises to the cytosolic face of the rhoptry, a key invasion organelle, and is more specifically attached to the rhoptry bulb membrane. Our attempts to knock out Pfcerli2 have failed, suggesting it as essential for blood stage growth. To investigate its function, we generated glucosamine-inducible knockdown parasite lines for PfCERLI2 which revealed it as essential for merozoite invasion. Moreover PfCERLI2 appears to be important for establishing proper rhoptry organellar development. Lastly, to determine which proteins may be working in tandem with both PfCERLI1 and PfCERLI2, we utilised dimerization induced quantitative proximity-dependent biotin identification (DiQ-BioID) and quantitative mass spectrometry. The results of this study have revealed the new unexplored biology linked to PfCERLI1 and PfCERLI2’s function in merozoites. Concurrently, we performed a functional characteirsation of P. falciparum C3H1 zinc finger proteins 1 and 2 (PfCZIF1 (Pf3D7_1468400), PfCZIF2 (Pf3D7_0818100)). Our studies have shown PfCZIF1 and PfCZIF2 to be peripherally-attached to a membrane facing the cytosol, with peak expression in schizonts at the point of merozoite development. Functional data in protein truncation and knockout parasite lines have revealed that neither protein in isolation is essential for blood-stage growth, however both cannot be knocked out in tandem suggesting that they fulfil compensatory roles. PfCZIF2 has revealed potential links with the protein PfKAHRP, which is involved in host cell modification. This study has provided the first insights into the functional interplay between PfCZIF1 and PfCZIF2.