Abstracts Category : Other

Characterisation of the pre-invasionglycophosphatidylinositol-anchored surface proteins of Plasmodiumfalciparum merozoites

by Tarryn Lee Venter

Plasmodium falciparum is a protozoan parasiteresponsible for causing the most severe form of malaria in humans.This species is responsible for over 90% of malaria mortalitieswhich occur predominantly in Africa. An increase in drug resistantparasites in recent years is threatening the progress made againstmalaria and thus new antimalarial drugs and vaccines are needed tocombat this disease. During the intraerythrocytic phase,merozoites egress from mature schizonts to invade new uninfectederythrocytes. Glycophosphatidylinositol (GPI) -anchored proteinscover most of the exterior surface of the merozoite prior toinvasion, while other GPI-anchored proteins are released onto themerozoite surface through apical organelle secretions. Theseproteins are involved in interactions with erythrocytes and arethought to be vital to erythrocyte invasion. GPI-anchored proteinshave also been implicated as a cause of pathogenic symptoms andactivation of immune components. These proteins are then releasedor cleaved to enable merozoite entry into the erythrocyte. Severalenzymes are thought to be involved in their cleavage including theserine proteases subtilisin-like proteases (SUB) 1 and 2, andphosphatidylinositol-phospholipase C (PIPLC); GPI-anchoredproteins are also generally sensitive to phospholipase A2 (PLA2).Cleaved proteins are released into the host blood system, whileuncleaved proteins are carried into the erythrocyte duringinvasion. Merozoites have a limited period in which they retaininvasive capacity. A previous lack of available techniques thatare specifically adapted to merozoite analysis has resulted in anincomplete understanding of invasion and GPI-anchored proteininvolvement in invasion. This study aimed to determine howGPI-anchored proteins on the merozoite surface are altered in theinvasive phase, and explore the possibility of using merozoiteGPI-anchored proteins as potential drug targets to blockerythrocyte invasion. Optimised methods of in vitro parasiteculturing which produce highly synchronised merozoites wasessential to this study. Parasite culturing techniques wereoptimised by utilising low haematocrit cultures with frequentculture splitting and optimised synchronisation. The Malarwheelis a tool that was developed for this research to provide a meansfor scheduling sorbitol treatments and MACs isolations. This tooland optimised culturing methods enabled large volumes of highlysynchronised invasive merozoites to be harvested. Four compounds(vanadate, edelfosine, dioctyl sodium sulfosuccinate (DSS), andgentamicin) suspected to interfere with GPIanchored cleavage orprocesses were screened on intraerythrocytic stages and merozoites.Antimalarial and anti-invasive properties of these compounds werescreened by modified malaria SYBR Green I-based fluorescence (MSF)assay and merozoite invasion assays (MIA) respectively. DSS andgentamicin showed limited potential as antimalarials or asanti-invasive agents. Vanadate and edelfosine both showedAdvisors/Committee Members: Cromarty, Allan Duncan (advisor), Birkholtz, Lyn-Marie (coadvisor).