Conference Agenda

Almost all cells have a shape, size, and organisation specialised for their functions. During replication, malaria parasites are amorphous and disorganised. By contrast, their host-cell-invading ‘zoite’ stages exhibit extreme apical-basal polarity, with invasion-specialised organelles at their apical end. It is currently unclear when this transition begins, how it is initiated, or what proteins control it.

Using ultrastructure-expansion microscopy, we imaged blood-stage, liver-stage, and mosquito-stage malaria parasites from replication until the completion of zoite formation to identify how this disorganised to hyper-polarised transition occurs. In all three lifecycle stages, the first sign of polarity establishment in the parasite was the anchoring of a structure called the centriolar plaque to the parasite plasma membrane. Subsequently, the parasite would begin to build its invasion-specialised organelles at this site, suggesting that this anchoring event represents the establishment of apical-basal polarity. In blood-stage parasites we observed a dramatic repositioning of the Golgi following centriolar plaque anchoring, providing a potential mechanism for how this event triggers apical organelle biogenesis. Work is currently ongoing to define the centriolar plaque proteins that coordinate the establishment of polarity within the parasite. Collectively, this study provides new insights into the biology of daughter cell formation in malaria parasites.

Malaria parasites depend on the apicoplast, an intriguing organelle of algal origin, for survival throughout the life cycle. Transport of metabolites across the apicoplast membranes is poorly understood, and only 11 transporter proteins have been confirmed to localize to the organelle to date. We report apicoplast localization of two previously uncharacterized transporters in Plasmodium falciparum. Knockdown of apicoplast transporter 1 (at1) resulted in death of asexual blood-stage parasites. Knockout of at1 in PfMev parasites, which have a metabolic apicoplast bypass, resulted in disruption of apicoplast morphology and loss of the organellar genome, suggesting that AT1 is involved in apicoplast housekeeping. Knockout of apicoplast transporter 2 (at2) did not affect asexual blood-stage parasites, nor gametocyte and gamete formation. In the mosquito, however, oocyst size was significantly decreased and no sporozoites were observed in salivary glands up until day 21, phenocopying knockouts of fatty acid metabolism. Metabolomics, drug assays, transport assays in yeast, and protein modeling provided further information on candidate substrates for both transporters. Taken together, we identified two novel apicoplast transporters, with AT1 being essential for asexual blood stages by supporting apicoplast housekeeping, and AT2 being important for parasite growth in mosquitoes, possibly by facilitating fatty acid metabolism.

Type IV P-type ATPases (P4-ATPases) are critical regulators of membrane lipid asymmetry in eukaryotic cells. Plasmodium falciparum is predicted to encode six P4-ATPases, but their roles remain to be defined. Recently, amplification of the gene encoding one of these, PfATP2, was associated with resistance to the antiplasmodial compounds MMV007224 and MMV665794. Here, we show that PfATP2 is a plasma membrane localised P4-ATPase that functions as a phospholipid flippase and is important for parasite growth. Using genetically modified parasites, we found that the PfATP2 expression level of parasites correlates with the rate by which they internalise a fluorescent analogue of phosphatidylserine (NBD-PS). Overexpression of PfATP2 enhanced NBD-PS translocation, whereas conditional knockdown significantly impaired this process. Further, exposure of parasites to MMV007224 and MMV665794 gave rise to a reduction in NBD-PS internalisation. PfATP2 knockdown parasites were hypersensitive to growth inhibition by MMV007224 and MMV665794, while PfATP2 overexpressing parasites were resistant to the compounds. Taken together, these findings establish PfATP2 as a major contributor to ATP-dependent phosphatidylserine internalisation on the parasite plasma membrane and a potential target of MMV007224 and MMV665794. We are currently investigating whether a reduction in PfATP2-mediated phospholipid flipping affects the activities of other transporters on the parasite plasma membrane.

Despite global gains combating malaria, the increasing incidence of antimalarial drug resistance to front line therapeutics demands new drugs with novel targets be developed. Potential targets for the design of therapeutic drugs include channel proteins that are critical for the movement of essential cargo within the parasite. Here, the essentiality of the voltage dependent anion channel (VDAC) was investigated in the deadliest species of malaria P. falciparum, via protein knockdown and localisation studies, followed by mitochondrial drug sensitivity studies and metabolomic analysis. Knockdown of vdac led to a survival defect in the RBC stages. Furthermore, the failure to generate conventional knockouts indicated VDAC is essential for parasite survival. Immunofluorescent microscopy successfully localised VDAC to the mitochondria, while the knockdown of VDAC was shown to sensitise parasites to mitochondrial target drugs atovaquone and proguanil, providing further indication for a role at the parasite mitochondria. Analysis of the parasite metabolic profile following VDAC knockdown is currently being used to investigate a possible role in the pyrimidine biosynthesis pathway at the outer mitochondrial membrane. Whilst the precise role of VDAC at the mitochondria requires further investigation, this channel protein is an essential and unique target for the future design of novel antimalarial therapeutics.

Pfs16 is a 16 kDa protein expressed early in the process of gametocyte development in Plasmodium falciparum. It localises to the parasitophorous vacuole membrane (PVM) of gametocytes. Previous studies have focused exclusively on its monomeric form. However, AlphaFold modelling predicts that Pfs16 assembles into an oligomeric complex containing a membrane-spanning pore. Given the essential role of Pfs16 in parasite transmission, we aimed to characterise this oligomeric structure and its function, which could provide new insights for transmission-blocking strategies. A combination of molecular and structural biology techniques was employed, including chemical crosslinking, Western blotting, native gel electrophoresis, and surface biotinylation, to explore its oligomeric subunits. Its functional activity was characterised using electrophysiological analysis in the Xenopus oocyte expression system. Both in silico modelling and experimental data indicate that Pfs16 forms a pentameric complex. Electrophysiological analysis in Xenopus oocytes has demonstrated that the membrane-spanning pore exhibits ion-conducting activity, supporting the presence of a functional pore. These results provide evidence that Pfs16 assembles into an oligomeric, likely pentameric, ion channel. Given its essential role in gametocyte development and transmission, targeting this complex may represent a promising strategy for the development of transmission-blocking interventions.

The intracellular apicomplexan parasite Toxoplasma gondii relies on both nutrient scavenging and biosynthetic pathways to acquire amino acids required for growth and survival. Our previous work identified the plasma membrane transporter TgApiAT2 as the primary glutamine transporter and, through a CRISPR-based screen, revealed that multiple amino acid biosynthetic pathways become fitness-conferring upon TgApiAT2 disruption, suggesting functional redundancy between uptake and synthesis. Here, we experimentally validate this model by generating double mutants lacking TgApiAT2 alongside key enzymes in amino acid biosynthesis. These mutants exhibit severe growth defects, demonstrating that parasites depend on compensatory mechanisms to maintain amino acid homeostasis. To further characterise TgApiAT2 function, we performed radiolabeled uptake assays, confirming that TgApiAT2 mediates the uptake of numerous non-essential amino acids. Together, our findings provide direct functional evidence for redundancy between amino acid uptake and synthesis in T. gondii, highlighting the metabolic flexibility that underpins parasite adaptation to variable host nutrient environments.