Big Discoveries in Small Parasites: Expansion Microscopy Reveals Plasmodium's Hidden Biology

Abstract

Malaria, caused by the intracellular Plasmodium parasites, remains a global health crisis, affecting an estimated 263 million people and causing 597,000 deaths in 2023. The efficacy of current antimalarial strategies is diminishing due to rising resistance to all major drugs developed over the past six decades and the absence of a highly effective, affordable vaccine. My work employs Ultrastructure Expansion Microscopy (U-ExM) to investigate critical aspects of organelle biogenesis, spatial organization, and dynamics during the Plasmodium blood-stage life cycle. Through U-ExM, I have gained insights into the biogenesis, fission, and spatial organization of the mitochondrion and the apicoplast, two essential metabolic organelles. Further, analysis of invasion organelles revealed the function of PfRON11 in the formation of rhoptry organelles. Investigating the mode of action of a novel antimalarial candidate, GNF179, revealed that treated parasites display an expansion of ER folds and a mislocalization of the Golgi Apparatus, leading to rapid parasite death. U-ExM analysis showed similar ER expansion in PfSEY1 knockdown parasites, suggesting that GNF179 targets this protein. Further, I analyzed the assembly and dynamics of the essential nuclear pore complexes (NPCs). U-ExM enabled the visualization and quantification of NPCs during the blood-stage life cycle. Employing a recombination-induced tag exchange (RITE) system, I demonstrated the dynamics of NPC assembly and recycling. Finally, I characterized a previously unstudied protein, PfAnchor, establishing its essential role in apicoplast fission during the blood stage. Using U-EXM and biochemical assays, I demonstrated that PfAnchor associates with the cytoplasmic side of the apicoplast membrane. Live microscopy of PfAnchor-deficient parasites revealed daughter parasites remained clustered together, and U-ExM analysis confirmed a failure in apicoplast fission. Importantly, treatment with azithromycin disrupted the apicoplast's branching structure, resulting in a vesiculated morphology, which rescued growth defects in PfAnchor-deficient parasites. This finding underscores PfAnchor's essential role in apicoplast fission and its impact on parasite replication. Protein pulldown assays showed an interaction between PfAnchor and PfDyn2, recently implicated in apicoplast fission. Collectively, my work advances our understanding of Plasmodium cell biology and establishes U-ExM as a transformative tool for malaria research. These findings can open new avenues for therapeutic interventions against Plasmodium parasites.