Artemisinin (ART) resistance in Plasmodium is threatening the artemisinin combination therapies-the first line of defence against malaria. ART resistance has been established to be mediated by the Plasmodium Kelch13 (PfK13) protein. For the crucial role of PfK13 in multiple pathways of the Plasmodium life cycle and ART resistance, it is imperative that we investigate its interacting partners. We recombinantly expressed PfK13-p (Bric a brac/Poxvirus and zinc finger and propeller domains), generating anti-PfK13-p antibodies to perform co-immunoprecipitation assays and probed PfK13 interacting partners. Surface plasmon resonance and pull-down assays were performed to establish physical interactions of representative proteins with PfK13-p. The co-immunoprecipitation assays identified 17 proteins with distinct functions in the parasite life cycle- protein folding, cellular metabolism, and protein binding and invasion. In addition to the overlap with previously identified proteins, our study identified 10 unique proteins. Fructose-biphosphate aldolase and heat shock protein 70 demonstrated strong biophysical interaction with PfK13-p, with K_D values of 6.6 µM and 7.6 µM, respectively. Additionally, Plasmodium merozoite surface protein 1 formed a complex with PfK13-p, which is evident from the pull-down assay. This study adds to our knowledge of the PfK13 protein in mediating ART resistance by identifying new PfK13 interacting partners. Three representative proteins-fructose-biphosphate aldolase, heat shock protein 70, and merozoite surface protein 1-demonstrated clear evidence of biophysical interactions with PfK13-p. However, elucidation of the functional relevance of these physical interactions are crucial in context of PfK13 role in ART resistance.

Iron homeostasis is essential for both the Plasmodium parasite and its hosts, the mosquito and human; however, the molecular mechanisms governing intracellular iron trafficking remain poorly understood. In mammals, poly(rC)-binding proteins (PCBPs) function as major iron chaperones by delivering Fe ²⁺ to ferritin and other iron-dependent proteins, yet their role in insects has not been investigated. Here, we provide the first structural and functional characterization of the PCBP from a major malaria vector, Anopheles stephensi . Comparative structural and evolutionary analyses showed that insect PCBPs are most closely related to human PCBP3. Coordinated expression and co-localization of As PCBP with ferritin following blood feeding suggested a conserved role in mosquito iron homeostasis. Functional analyses demonstrated that KH2-KH3 linker and KH3 domain of PCBP, plays an important role in the As PCBP-ferritin interaction and that deletion of these regions, unexpectedly increased ferritin binding affinity but impaired iron delivery, indicating that these regions are dispensable for complex formation but essential for efficient iron transfer. Collectively, our results identify PCBP as a key component of mosquito iron homeostasis that could be exploited for the development of novel antimalarial strategies.

Iron metabolism is essential for both mosquito reproductive fitness and Plasmodium development within the vector, yet this axis remains insufficiently explored as a potential target for malaria control. Ferritin, the major iron-storage protein and a central regulator of iron homeostasis, has not previously been biochemically or functionally characterized in any hematophagous insect. Our computational and phylogenetic analyses reveal that mosquito ferritin represents a unique case, combining conserved iron-binding motifs with distinctive haem-coordinating residues that confer an exceptionally high haem-binding affinity exceeding that of bacterioferritin. In addition, through a screen of multiple iron chelators in Anopheles larvae, we identified potent candidates with potential utility for vector control. Together, these findings establish mosquito ferritin as an evolutionarily distinct and physiologically essential protein, and they highlight iron homeostasis as a promising dual-target axis for next-generation vector control strategies.