PfCERLI1 is a conserved rhoptry associated protein essential for Plasmodium falciparum merozoite invasion of erythrocytes

Abstract

The disease-causing blood-stage of the Plasmodium falciparum lifecycle begins with invasion of human erythrocytes by merozoites. Many vaccine candidates with key roles in binding to the erythrocyte surface and entry are secreted from the large bulb-like rhoptry organelles at the apical tip of the merozoite. Here we identify an essential role for the conserved protein P. falciparum Cytosolically Exposed Rhoptry Leaflet Interacting protein 1 (PfCERLI1) in rhoptry function. We show that PfCERLI1 localises to the cytosolic face of the rhoptry bulb membrane and knockdown of PfCERLI1 inhibits merozoite invasion. While schizogony and merozoite organelle biogenesis appear normal, biochemical techniques and semi-quantitative superresolution microscopy show that PfCERLI1 knockdown prevents secretion of key rhoptry antigens that coordinate merozoite invasion. PfCERLI1 is a rhoptry associated protein identified to have a direct role in function of this essential merozoite invasion organelle, which has broader implications for understanding apicomplexan invasion biology.

Pfcerli2, a duplicated gene in the malaria parasitePlasmodium falciparumessential for invasion of erythrocytes as revealed by phylogenetic and cell biological analysis

“…Pfcerli1 and Pfcerli2 across Plasmodium spp. had Ka/Ks of 0.091 and 0.29 respectively, indicating strong negative selection (favouring low sequence diversity), which is consistent with their essentiality for P. falciparum 9,10,13 and P. berghei 14 blood-stage survival. This negative selection in both lineages may be indicative of subfunctionalisation in Apicomplexa, where both paralogues have maintained a subset of the function of the ancestral gene.…”

“…PfCERLI2 predominantly solubilised in the carbonate fraction, a distinctive feature of peripheral membrane proteins 19 . Combining the localisation analyses with the proteinase protection and solubility assays suggest that PfCERLI2 localises to the cytosolic face of the rhoptry bulb membrane, a subcellular localisation akin to that reported for PfCERLI1 9 .…”

“…We and Suarez et al had previously identified the rhoptry bulb protein PfCERLI1 and determined that it played an essential role in merozoite invasion and rhoptry secretion 9,10 .…”

“…As the gene structure of the cerli homologue of Cryptosporidium is highly similar to that of cerli1 across other Apicomplexa, we suggest that cerli1 is most similar to the ancestral gene that underwent duplication and gave rise to the more divergent cerli2 gene. From studies of both PfCERLI1 and PfCERLI2, it is clear that both proteins have functional similarities due to their stage of expression and localisation, along with their knockout and knockdown phenotypes 9,10,13 . Additionally, in Toxoplasma gondii it has been reported that TgRASP1 and TgRASP2 form a complex 10 , but it not clear if this occurs in other Apicomplexa.…”

“…To determine whether PfCERLI2 was cytosolically exposed or compartmentalised in an organelle, a proteinase K protection assay modified from a previous study was performed 7,9 .…”