Characterization of Plasmodium falciparum merozoite apical membrane antigen-1 protein changes prior to erythrocyte invasion

Abstract

Malaria is a global pandemic that affects millions of people each year. It is a parasitic infection caused by the Plasmodium family, with Plasmodium falciparum being the most virulent strain. Malaria is transmitted to humans by the female Anopheles mosquito. The parasite undergoes two different cycles of its life cycle within the human host: the liver and intraerythrocytic life cycle. The latter consists of an asexual and sexual cycle. The intraerythrocytic cycle is perhaps the most important stage of the parasite's life cycle as it promotes the spread of the disease within and between hosts. The focus of this investigation was aimed at the invasion process of the merozoites into the erythrocytes. The Plasmodium merozoite utilises a cascade of proteins during the erythrocyte invasion process, which is a swift action that takes place in approximately 30 seconds. A number of surface proteins are expressed during merozoite development and are distributed along the merozoite surfaces to assist with attachment and invasion, the most crucial being MSP-1, AMA-1 and RON-2. MSP-1 and AMA-1 are vital targets for the development of malaria vaccines. AMA-1 is the central target protein of this investigation as it plays an essential role in the invasion process. AMA-1 commits the merozoite to invade the erythrocyte, as it assists the RON proteins in the formation of an irreversible tight-junction with the membrane of the erythrocyte. Antibodies, specific to AMA-1, bind to the protein, which prevents the formation of the tight junction and inhibits the invasion of the merozoite into the erythrocyte, therefore preventing the spread of the disease. However, before invasion, AMA-1 undergoes a number of proteolytic processes. It is synthesized as an 83 kDa (AMA-183) precursor protein in the apical organelle of the merozoite. This is then cleaved at the N-terminus to give rise to a 66 kDa (AMA-166) fragment, which is secreted onto the surface of the merozoite. The AMA-166 fragment is then cleaved into either a 48 kDa (AMA-148) or 44 kDa (AMA-144) fragment. One of these three fragments is then used by the merozoite for erythrocyte invasion. The aim of this investigation was to isolate and characterise each of the fragments of the Plasmodium falciparum AMA-1 (PfAMA-1) protein using the 3D7 lab strain of P. falciparum and to visualise the merozoite-erythrocyte invasion process, to possibly identify which of the AMA-1 fragments are involved in the invasion process. In order to achieve this large clusters of merozoites from sorbitol-synchronised cultures were isolated. Schizonts were isolated from culture by magnetic separation and incubated with E64 to prevent the release of merozoites. Merozoites that were required for the isolation of PfAMA-1 were harvested from the schizonts by saponin lysis, then homogenised, separated by SDS-PAGE and digested for LC-MS/MS analysis. Merozoites that were required for the visualisation procedures were not incubated with E64, to allow natural egression from the erythrocyte. The transmission electron microscopy results produced clear images of the merozoiteerythrocyte invasion process and the positioning of PfAMA-1 on the merozoite, before and after schizont rupture, was visualised from results obtained from confocal microscopy. Then PfAMA-1 was identified in isolated merozoite samples by LC-MS/MS analysis. However, due to its low abundance, isolation of high enough concentrations of PfAMA-1 to characterise its different fragments was not achieved. Further investigation into the development of the culturing and isolating methods could help in future projects aimed at isolating higher concentrations of merozoite proteins from synchronised cultures with a lower merozoite egression window period, in order to accomplish detailed analysis on invading proteins for the future development of treatments against malaria.