Trafficking of Plasmodium Falciparum Proteins: A Novel Anti-Malaria Drug Target?

Malaria continues to be a global health problem and in 2015 the World Health Organisation estimated that there were ~214 million cases of the disease and ~438 000 deaths. Despite a marked reduction in mortality and morbidity over the last 15 years, these hard fought gains are threatened by resistance of the Anopheles mosquito vector to insecticides and resistance of the malaria parasite to artemisinin, currently the main frontline drug used in treatment of the disease. There is no effective vaccine and thus new drug targets are needed to fuel the development of compounds that kill parasites. Plasmodium falciparum is the most lethal of the malaria parasites, but our knowledge of the fundamental biology of this parasite is still incomplete and ~60% of the proteome is uncharacterised.

This study aims to identify unique proteins/pathways involved in internal trafficking of parasite proteins. Clinical symptoms of malaria are caused by the intra-erythrocytic phase of the parasite life cycle and 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 the apical organelles. Three micronemal proteins, apical membrane antigen-1 (PfAMA-1), subtilisin 2 (PfSUB2) and Erythrocyte Binding Antigen 181 (PfEBA-181) were investigated. Bioinformatic analysis revealed that the proteins contain N-terminal signal peptides, which direct the newly synthesised invasion proteins to the endoplasmic reticulum (ER). Transmembrane domains and cytoplasmic tails are located in the C-terminal ends and various functional domains are present in the central sections.

To identify the domains responsible for targeting the micronemes, selected domains were amplified by PCR or RT-PCR from P. falciparum genomic DNA or RNA and used to create 7 mini-genes by overlap extension PCR. All products contained the N-terminal signal peptide that is required for transport through the ER. A pARL-mCherry plasmid (kindly donated by Jude Przyborski) was modified to replace the constitutive Pfcrt promoter with a Pfama-1 promoter that is only active during the schizont stage of parasite development when micronemes are formed. The plasmid contains a positive selection cassette that codes for human dihydrofolate reductase, which is resistant to WR99210 drug. The mini-genes were cloned into the modified plasmid and these constructs were used to transfect P. falciparum parasites by electroporation. Transgenic parasites were selected by WR99210 drug pressure and the expression of red fluorescent mCherry-tagged chimeric proteins was visualised in live parasites. Co-localisation studies were performed with green fluorescent PfEBA-175, a microneme marker, to assess if the transgenic mini-proteins reached their destination. In line with other EBAs, PfEBA-181 required the cysteine-rich ectodomain for correct localisation, whereas the prodomain of PfAMA-1 and transmembrane domain of PfSUB2 were essential for microneme targeting.

Since these 3 invasion proteins all require different domains for transport to the micronemes, the possibility of a protein escorter was explored. The PfAMA-1 prodomain was expressed as a recombinant histidine tagged "bait" protein and immobilised onto Nickel-coated beads, which were exposed to a P. falciparum phage display library for 4 rounds of biopanning. Phage that bound to the bait were isolated and the parasite inserts were sequenced. Comparison of the sequences to the Plasmodium database revealed 2 binding partners: a putative chaperone binding protein and a putative P. falciparum formin 2. The human orthologue of formin has several functions in the actin cytoskeleton, including intracellular vesicle transport along the actin fibres. These binding partners are currently being expressed as GST-tagged recombinant proteins, which will be used in in vitro binding assays to verify their interaction with the prodomain of PfAMA-1. To ascertain if formin 2 and chaperone binding protein are common transporters for micronemal invasion proteins, their interaction with the targeting domains of PfSUB2 and PfEBA-181 will be investigated. Since humans don't have apical organelles, these trafficking mechanisms are unique to P. falciparum and may be exploited as novel anti-malaria drug targets.