Laura Barbosa Rodrigues¹, Maria Eduarda Portela Ferreira¹, Lucas Silva de Oliveira¹, Marcos Rodrigo Alborghetti², Philippe Grellier³, Izabela Marques Dourado Bastos¹, Sébastien Charneau¹

Malaria is a major global public health challenge caused by protozoan parasites of the Plasmodium genus, with P. falciparum being the most prominent species. The infection causes fever, chills, sweating, headache, nausea, and weakness, and can progress to severe and potentially fatal complications such as cerebral malaria and kidney failure. Given the millions of cases reported annually, new therapeutic and vaccine strategies are needed to control the disease. The parasites rely on the invasion of human erythrocytes for survival, and this process is facilitated by specialized secretory organelles—rhoptries, micronemes, and dense granules—located within a distinct structure known as the apical complex. The largest of these organelles, the rhoptry, undergoes rapid and profound physiological changes as it secretes its contents during erythrocyte invasion by merozoites. These structures release essential proteins required for the formation of the moving junction and modulation of the host cell. However, many aspects of rhoptry biogenesis and molecular composition remain poorly understood. This project aims to map the rhoptry subproteome through in vivo proximity labeling and biotinylation of proteins using the engineered ascorbate peroxidase, known as APEX2. This enzyme catalyzes the oxidation of biotin-phenol into a short-lived biotin-phenoxyl radical in the presence of low concentrations of hydrogen peroxide. This radical reacts with electron-rich amino acids, such as tyrosine, in neighboring proteins, resulting in their biotinylation. Therefore, APEX2 must be specifically targeted to the rhoptries. Parasites of the 3D7 strain were transfected by electroporation with episomal plasmids—non-integrative to the genome—encoding APEX2 fused to the signal sequence of RAP1 (rhoptry-associated protein 1). APEX2 expression and localization will be validated by immunofluorescence, Western blotting, and transmission electron microscopy. Biotinylated proteins will be enriched using streptavidin-coated beads and identified by mass spectrometry using an LC-MS/MS system coupled to an Orbitrap analyzer. Peptides will be compared to the P. falciparum database (PlasmoDB), and only those presenting the characteristic spectral signatures of biotin-phenol labeling will be considered rhoptry-derived proteins. This approach will allow the identification of novel molecular components of rhoptries, contributing to a better understanding of rhoptry biogenesis and parasite invasion mechanisms, with potential applications in the development of new therapeutic targets.

Agradecimentos: FAPDF, CNPq, CAPES, UNB