Characterisation of the pre-invasion glycophosphatidylinositol-anchored surface proteins of Plasmodium falciparum merozoites
Plasmodium falciparum is a protozoan parasite responsible for causing the most severe form of malaria in humans. This species is responsible for over 90% of malaria mortalities which occur predominantly in Africa. An increase in drug resistant parasites in recent years is threatening the progress made against malaria and thus new antimalarial drugs and vaccines are needed to combat this disease. During the intraerythrocytic phase, merozoites egress from mature schizonts to invade new uninfected erythrocytes. Glycophosphatidylinositol (GPI) -anchored proteins cover most of the exterior surface of the merozoite prior to invasion, while other GPI-anchored proteins are released onto the merozoite surface through apical organelle secretions. These proteins are involved in interactions with erythrocytes and are thought to be vital to erythrocyte invasion. GPI-anchored proteins have also been implicated as a cause of pathogenic symptoms and activation of immune components. These proteins are then released or cleaved to enable merozoite entry into the erythrocyte. Several enzymes are thought to be involved in their cleavage including the serine proteases subtilisin-like proteases (SUB) 1 and 2, and phosphatidylinositol-phospholipase C (PIPLC); GPI-anchored proteins are also generally sensitive to phospholipase A2 (PLA2). Cleaved proteins are released into the host blood system, while uncleaved proteins are carried into the erythrocyte during invasion. Merozoites have a limited period in which they retain invasive capacity. A previous lack of available techniques that are specifically adapted to merozoite analysis has resulted in an incomplete understanding of invasion and GPI-anchored protein involvement in invasion. This study aimed to determine how GPI-anchored proteins on the merozoite surface are altered in the invasive phase, and explore the possibility of using merozoite GPI-anchored proteins as potential drug targets to block erythrocyte invasion. Optimised methods of in vitro parasite culturing which produce highly synchronised merozoites was essential to this study. Parasite culturing techniques were optimised by utilising low haematocrit cultures with frequent culture splitting and optimised synchronisation. The “Malarwheel” is a tool that was developed for this research to provide a means for scheduling sorbitol treatments and MACs isolations. This tool and optimised culturing methods enabled large volumes of highly synchronised invasive merozoites to be harvested. Four compounds (vanadate, edelfosine, dioctyl sodium sulfosuccinate (DSS), and gentamicin) suspected to interfere with GPIanchored cleavage or processes were screened on intraerythrocytic stages and merozoites. Antimalarial and anti-invasive properties of these compounds were screened by modified malaria SYBR Green I-based fluorescence (MSF) assay and merozoite invasion assays (MIA) respectively. DSS and gentamicin showed limited potential as antimalarials or as anti-invasive agents. Vanadate and edelfosine both showed antimalarial and anti-invasive activity, while edelfosine was the most potent anti-invasive agent at physiological concentrations. The merozoite GPI-anchored proteome was analysed by sodium dodecyl sulphatepolyacrylamide gel electrophoresis (SDS-PAGE) followed by complete gel lane analyses conducted by liquid chromatography-tandem mass spectrometry (LC-MS/MS) on soluble and pelleted merozoite proteins in samples from either invasive or non-invasive merozoites. Thirteen known or predicted GPI-anchored proteins were identified in samples. Several changes were identified in merozoite GPI-anchored proteins between the invasive phase and after its completion, and minor differences were observed following treatment with edelfosine. Edelfosine showed partial inhibition of erythrocyte invasion, however, the primary cause of inhibition cannot be directly related to interferences with GPI-anchored proteins. These results suggest that GPIanchored proteins are controlled by various complex processes, and are cleaved or processed by diverse mechanisms during the invasive phase. These mechanisms may be controlled by multiple signals which effect proteins or groups of proteins in specific ways. These signals may be influenced by “checkpoints” during invasion processes including the time period after egress from schizonts, and possibly the recognition of erythrocyte targets. These methods and results provide a foundation for future research to enable culturing of P. falciparum parasites specifically for merozoite research, and to identify merozoite proteins active during the invasive phase. These results confirm and challenge previous ideas reported in literature on the GPI-anchored processes of merozoites and further characterise less studied GPIanchored proteins. The results suggest that the processes controlling GPI-anchored proteins may be more complex than previously thought. These results form a basis to further identify and characterise GPI-anchored proteins in the aim to develop antimalarial medications and vaccines that target merozoites and their GPI-anchored processes.
Malaria, Plasmodium falciparum, Merozoite, Erythrocyte invasion, Cell culture, MACs isolation, MSF assay, Merozoite invasion assay, SDS-PAGE, Electrophoresis, LC-MS/MS, Proteomics
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