Integrative proximity labeling proteomics of metabolic crosstalk between the apicoplast and mitochondrion during erythrocytic schizogony of Plasmodium falciparum

Maria Eduarda Portela Ferreira1, Lucas Silva de Oliveira1, Marcos Rodrigo Alborghetti2, Farah Camila Murtadha1, Izabela Marques Dourado Bastos1, Philippe Grellier3, Sébastien Charneau1

1. UnB, University of Brasília; Laboratory of Biochemistry and Protein Chemistry, Department of Cell Biology, Darcy Ribeiro University Campus, University of Brasília, Brasília, Federal District - 70910-900, Brazil.
2. CNPEM, National Center for Research in Energy and Materials; National Laboratory of Biosciences, National Center for Research in Energy and Materials, Campinas, São Paulo, Brazil.
3. MNHN, National Museum of Natural History; 57 Rue Cuvier, 75005 Paris, France

Malaria, caused by the protozoan parasite Plasmodium falciparum, remains a critical public health issue, particularly in tropical and subtropical regions, where it disproportionately affects vulnerable populations. Despite advances in control strategies, the emergence of drug-resistant strains continues to compromise treatment efficacy, reinforcing the need for novel therapeutic approaches grounded in a deeper understanding of parasite biology. This project focuses on elucidating the metabolic interplay between two essential organelles of P. falciparum — the mitochondrion and the apicoplast — during the intraerythrocytic schizogony stage, when the parasite undergoes intense replication and organelle biogenesis.

To address this, we employ an integrative proximity-labeling proteomic approach based on the engineered peroxidase APEX2, which enables the in situ biotinylation of proteins in the immediate vicinity of the targeted compartment. This strategy allows for the identification of proteins and potential transient interactions occurring within or near the organelles, offering spatially resolved insights into their functional connectivity.

So far, parasites from the 3D7 and FCB1 strains have been successfully cultured and transfected with constructs targeting APEX2 to the mitochondrion using a signal sequence from the P. falciparum thioredoxin reductase (TrxR), as well as with constructs containing the signal peptide from the acyl carrier protein (ACP) to direct APEX2 to the apicoplast. Subsequent steps include validation of APEX2 expression by Western blotting and immunofluorescence assays, ultrastructural analysis by transmission electron microscopy, and enrichment of biotinylated proteins using streptavidin-based affinity purification. The enriched proteomes will then be subjected to mass spectrometry to characterize the local proteomic landscape associated with the mitochondrion and, in future steps, with the apicoplast.

By revealing molecular players involved in the functional interaction between these two organelles, this study aims to uncover novel aspects of P. falciparum metabolic organization that may be essential for parasite survival. Given the unique evolutionary origin and metabolic roles of both the mitochondrion and apicoplast in apicomplexan parasites, these findings are expected to contribute to the broader field of parasite cell biology and may ultimately support the identification of new vulnerabilities exploitable in antimalarial drug development.

Agradecimentos: CAPES, CNPq, FAPDF