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
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.