Maria Fernanda Vogado de Mesquita1, Paula Marian Vieira Goulart1,2, Lucas Silva de Oliveira1, Frédéric Fercoq2, Coralie Martin2, Philippe Grellier2, Izabela Marques Dourado Bastos Charneau3, Sébastien Olivier Charneau1
Leishmaniases are parasitic diseases caused by protozoa of the genus Leishmania, transmitted by phlebotomine sandflies that infect mammalian macrophages, where the parasite proliferates as an intracellular amastigote. The main clinical forms include cutaneous leishmaniasis (CL), mucocutaneous leishmaniasis (MCL), and visceral leishmaniasis (VL), the latter being the most severe, associated with L. donovani and L. infantum. According to the World Health Organization, the disease occurs in 98 countries, affecting over 1 billion people in endemic areas. The absence of effective vaccines and the use of drugs with high toxicity, elevated cost, and increasing resistance underscore the need to identify new therapeutic targets.
In this context, essential organelles such as glycosomes and the nucleus stand out as promising sources for the discovery of new molecular targets. Glycosomes are organelles unique to trypanosomatids, containing an enzymatic repertoire vital for parasite survival, participating in metabolic pathways such as the pentose phosphate pathway, gluconeogenesis, and β-oxidation of fatty acids. The nucleus, meanwhile, displays unique features, including gene expression regulatory proteins that differ significantly from those found in other eukaryotes. Despite their relevance, proteomic studies of these organelles in L. infantum remain scarce, and classical purification methods face limitations such as cross-contamination between cellular compartments.
Innovative techniques such as proximity labeling in live cells have emerged as effective alternatives for subcellular mapping. Among them, APEX2 (engineered ascorbate peroxidase) has gained prominence due to its ability to rapidly and specifically biotinylate neighboring proteins. In the presence of hydrogen peroxide, APEX2 catalyzes the oxidation of biotin-phenol, generating short-lived and highly reactive biotin-phenoxyl radicals that covalently label nearby proteins, allowing their subsequent purification via streptavidin and identification by mass spectrometry.
In this study, genetic constructs containing the APEX2 sequence fused to the PTS1 (glycosome-targeting) or NLS (nucleus-targeting) signal peptides were used, inserted into the pLEXSY-hyg2 vector (Jena Bioscience) for expression in L. infantum. The results showed that both fusion proteins were well expressed and correctly localized to the parasite’s target organelles (glycosomes or nucleus). However, the efficiency of protein labeling exhibited important limitations: when using biotin-phenol, detection of biotinylated proteins was restricted to assays with cell lysates, suggesting limited intracellular diffusion of the substrate and possible impermeability of the plasma membrane to biotin-phenol.
Recent studies indicate that desthiobiotin-phenol, a structural analog of biotin-phenol with greater cellular permeability, may overcome these limitations, enabling more efficient in vivo labeling. Therefore, the present project proposes a comparison between both reagents to optimize the proximity labeling protocol in Leishmania, ensuring enhanced coverage and specificity in the capture of nuclear and glycosomal proteins.
It is expected that the data obtained will contribute to the identification of proteins essential for parasite viability, pathogenicity, and metabolic adaptation, potentially revealing new therapeutic targets against leishmaniasis.
Agradecimentos: The author(s) would like to acknowledge the financial support provided by the funding agencies, which was essential for the development of this study. We are especially grateful to CNPq, CAPES e FAPDF for the scholarships and research grants that made this work possible. Their continued investment in scientific research and innovation is fundamental to the advancement of knowledge. We also thank the University of Brasília (UnB) for providing the institutional support and research infrastructure that enabled the execution of this project.