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. / Dissertation (MSc)--University of Pretoria, 2017. / Pharmacology / MSc / Unrestricted
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:up/oai:repository.up.ac.za:2263/63040 |
| Date | January 2017 |
| Creators | Venter, Tarryn Lee |
| Contributors | Cromarty, Allan Duncan, tarrynlee07@gmail.com, Birkholtz, Lyn-Marie |
| Publisher | University of Pretoria |
| Source Sets | South African National ETD Portal |
| Language | English |
| Detected Language | English |
| Type | Dissertation |
| Rights | © 2017 University of Pretoria. All rights reserved. The copyright in this work vests in the University of Pretoria. No part of this work may be reproduced or transmitted in any form or by any means, without the prior written permission of the University of Pretoria. |
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