Conference Agenda

PfCSC1 is a protein found in Plasmodium falciparum that belongs to a family of osmosensitive cation channels, some members of which have been found to double as scramblases (ATP-independent phospholipid transporters). Mutations in PfCSC1 or a putative rhomboid protease (PfROM8) are associated with resistance against specific compounds, termed PfROM8/PfCSC1-linked compounds (R/CLCs). To understand the mode of action of these compounds, the function of PfCSC1 must be understood. Previous research shows that PfCSC1 is an essential ion channel that can be activated by R/CLCs, with Na+ being one of its substrates.

Here, I present evidence that PfCSC1 doubles as a scramblase, capable of phospholipid transport in the parasite plasma membrane. The internalisation of a fluorescent phospholipid analogue (NBD-PS) was measured in ATP-depleted parasites under several conditions. Knockdown of PfCSC1 did not have a significant effect on NBD-PS internalisation. However, upon exposure to hypotonic conditions or R/CLCs – both predicted to activate PfCSC1 – parasites expressing a normal level of PfCSC1 displayed a significant increase in NBD-PS internalisation, whereas the response of parasites in which PfCSC1 was knocked down was less pronounced. The data suggest that while PfCSC1 is capable of phospholipid scrambling, it is likely not always active under physiological conditions.

The formation of the nucleus is one of the most significant paradigm shifts in the evolution of species. The nucleus allows eukaryotes to transcribe with higher fidelity and better regulate the expression of their genes, resulting in highly specialised cells; the caveat being that eukaryotes must transport their RNA cargo into the cytoplasm to re-couple the partitioned transcription and translation. Most eukaryotes use an RanGTP‑dependent system for nucleocytoplasmic transport including for the export of non-coding RNA. In addition, fungi and metazoans have evolved specialised RanGTP-independent pathways to export most Poly‑A+ mRNA.

The mechanisms of mRNA export in protist parasites are relatively understudied. Proteins that have conserved sequences to proteins involved in RanGTP‑independent mRNA export in humans and yeasts have been identified in Plasmodium spp. Toxoplasma gondii, and Cryptosporidium spp based on conserved sequences. However it is unclear if these parasites have mechanisms of mRNA export that are analogous to the RanGTP‑independent metazoan mechanisms or if they have innovated parasite specific mechanisms of mRNA export.

I aim to determine if apicomplexan parasites have evolved parasite specific processes of mRNA export by identifying key molecules involved nucleocytoplasmic transport.

Malaria remains a global health burden and with ongoing resistance across all classes of antimalarials, advancing our understanding of Plasmodium’s biology could help identify novel therapeutic targets. During continuous in vitro culture, mutations commonly arise within the parasite genome, although these are often phenotypically silent. However, we have identified a sorbitol-resistant Pf3D7 strain that no longer undergoes haemolysis when exposed to isotonic concentrations of sorbitol, indicating a disruption in nutrient acquisition via new permeability pathways (NPPs) formed by parasites to facilitate enhanced nutrient uptake. In contrast to other sorbitol-resistant lines, this occurs without growth defects under standard culture conditions. Whole genome sequencing of clonal parasites identified a recombinant CLAG3 gene (CLAG3n) with a missense mutation at the recombination region. CLAG3 is a component of the RhopH complex, a trimeric protein complex essential for NPP formation. To characterise the phenotype of our CLAG3n line, we assessed parasite growth under normal and nutrient-deprived conditions and performed osmotic lysis assays using a range of solutes to evaluate changes in substrate selectivity. Additionally, we conducted proteome-wide analyses to investigate alterations in protein expression and aim to conduct localisation studies to determine the impact of the recombination and mutation on protein export and NPP function.