A Thesis submitted to the
Faculty of Health Sciences,
University of the Witwatersrand, Johannesburg
in the fulfillment of the requirements for the degree of
Doctor of Philosophy
Johannesburg, 2017. / Malaria continues to be a global health problem. Despite a marked reduction in mortality
over the last 15 years, these hard-fought gains are threatened by growing resistance of the
Plasmodium falciparum malaria parasite to artemisinin, the frontline drug used in treatment
of the disease. Clinical symptoms of malaria are caused by the intra-erythrocytic phase of
the parasite life cycle. Entry into the erythrocyte is accomplished by several specialised
invasion proteins, which are stored in unique apical secretory organelles known as
micronemes and rhoptries. Very little is known about the trafficking signals and transport
mechanisms of invasion proteins to these organelles.
Three micronemal proteins, Apical Membrane Antigen-1 (PfAMA-1), Subtilisin-like
protease 2 (PfSUB2) and Erythrocyte Binding Antigen 181 (PfEBA181) were investigated
with the aim of identifying domains responsible for targeting the micronemes. Selected
domains were amplified and mini-genes were created by overlap extension PCR. A pARLmCherry
plasmid containing a Pfama-1 stage-specific promoter that is only active during
the schizont stage of parasite development when micronemes are formed, was used to create
mCherry-tagged constructs. P. falciparum parasites were transfected by electroporation of
the plasmid constructs. Transgenic parasites were selected by drug pressure and the
expression of red fluorescent mCherry-tagged chimaeric proteins was visualised in live
parasites. Co-localisation studies were performed with a microneme marker to assess if the
transgenic mini-proteins reached their destination. Interestingly, all three proteins required
different domains to target the micronemes: PfEBA181 required an extended region of a
conserved cysteine-rich domain, PfAMA-1 required the prodomain, and PfSUB2 required
the transmembrane domain. Since no common targeting signal was identified, the possibility
of a protein escorter was explored. The PfAMA-1 prodomain was expressed as a
recombinant histidine-tagged protein and immobilised onto Nickel-coated beads, which
were exposed to a P. falciparum phage display library for four rounds of biopanning. Two
novel binding partners were identified: a putative Chaperone Binding Protein and a putative
Formin 2.
The identification of the molecular trafficking determinants of three invasion proteins, as
well as a potential protein escorter for microneme targeting, represent novel findings that
extend our knowledge of a fundamental biological process in the malaria parasite. This
pathway may be exploited for drug development and new malaria treatment strategies. / MT2017
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:wits/oai:wiredspace.wits.ac.za:10539/23177 |
| Date | January 2017 |
| Creators | Churchyard, Alisje |
| Source Sets | South African National ETD Portal |
| Language | English |
| Detected Language | English |
| Type | Thesis |
| Format | Online resource (xxv, 198 leaves), application/pdf, application/pdf |
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