Functional Characterisation Of Novel Plasmodium falciparum Proteins And Their Role In Erythrocyte Invasion

Abstract

Malaria caused by Plasmodium falciparum is responsible for the deaths of hundreds of thousands of children every year, and imparts an overwhelming economic burden on the world’s poorest countries. All symptoms of malaria are caused during the asexual blood-stage of the P. falciparum lifecycle, which is reliant on merozoite invasion of host red blood cells (RBCs). Due to its essentiality for parasite survival, and its exposure to the host immune system, merozoite invasion is an attractive target for the development of antimalarial therapeutics. Merozoite invasion is coordinated by a series of secretory organelles, of which the largest and most well characterised are the rhoptries. Previous studies into rhoptry proteins have been strongly foccused on antigens that are secreted from the rhoptries during invasion; primarily due to their promise as vaccine candidates. As such, very little is known about proteins that coordinate rhoptry biogenesis, structure, or function. Prior to this study the P. falciparum proteins Pf3D7_0210600 and Pf3D7_0405200, hereafter referred to as P. falciparum Cytosolically Exposed Rhoptry Leaflet Interacting proteins (PfCERLI) 1 and 2, were largely uncharacterised proteins that previous studies had suggested may play a role in merozoite invasion. We hypothesised that PfCERLI1 and PfCERLI2 were rhoptry proteins that shared an evolutionary relationship and were both essential for merozoite invasion. We aimed to test these hypotheses through bioinformatic analyses, immunofluorescence microscopy, and gene disruption or inducible knockdown. Bioinformatic and phylogenetic analyses showed that cerli1 and cerli2 arose through an ancestral gene duplication of cerli1 that was present in the most recent common ancestor of haematozoa and coccidia. Analysis of the structure of CERLI proteins revealed they possess a conserved motif with the consensus sequence PHISE/DxxP that we have termed PHIS, along with conserved C2 and Pleckstrin homology (PH) domains that are likely ito have a role in membrane association. Using selection linked integration targeted gene disruption (SLI-TGD) we determined that both Pfcerli1 and Pfcerli2 were refractory to gene deletion and likely important for blood-stage growth. To assess their functions, we used an inducible protein knockdown system whereby the addition of glucosamine (GLCN) results in specific mRNA degradation prior to translation. Knockdown of either CERLI1 or CERLI2 resulted in growth inhibition caused by an inability of merozoites to invade RBCs. Immunofluorescnce microscopy and biochemical techniques revealed that both proteins are peripheral membrane proteins that localise to the cytosolic face of the rhoptry bulb. Rhoptry secretion assays showed that knockdown of PfCERLI1, but not PfCERLI2, leads to a defect in the secretion of key rhoptry antigens. By contrast, electron microscopy analysis of rhoptry size indicated a significant increase in rhoptry length following PfCERLI2 knockdown, but no change with PfCERLI1 knockdown. Semi-quantiative super-resolution microscopy analysis determined that knockdown of PfCERLI1 alters rhoptry antigen distribution, and it was shown that both PfCERLI1 and PfCERLI2 knockdown inhibit processing of key rhoptry antigens. The findings of these studies show that the previously uncharacterised proteins, PfCERLI1 and PfCERLI2, are related rhoptry proteins whose functions are essential for maintaining rhoptry morphology, rhoptry secretion, and rhoptry antigen processing.