Padronização do isolamento de vesículas extracelulares de Paracoccidioides brasiliensis e do modelo murino de Paracoccidioidomicose

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Data

2024-11-29

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Universidade Federal de Goiás

Resumo

Paracoccidioidomycosis is an important systemic mycosis in Brazil, being the fungus of the genus Paracoccidioides spp. the etiological agents and Paracoccidioides brasiliensis species, the most prevalent in the country. The infection occurs through inhalation of infective propagules, which settle in the lungs and change the morphology to parasitic form. In addition to the action of the innate immune system, nutritional immunity is also considered a relevant reinforcement to control the disease by restricting the availability of several nutrients, including metals such as iron, which is important for the metabolic functions of the fungus. Consequently, the pathogen has developed strategies to circumvent the limitation of this metal, and extracellular vesicles are strategic structures that may be involved in the transference of virulence factors between fungal cells. Therefore, this study aims to standardize methods for isolating and characterizing extracellular vesicles produced by P. brasiliensis and to standardize a murine model of infection by the fungus. These approaches are to conduct future studies to understand how extracellular vesicles influence the pathogenicity of the disease. For this purpose, P. brasiliensis cells were cultured in solid modified minimal medium (MVM) under iron deprivation and sufficiency conditions for subsequent extraction of vesicles, which had their profile analyzed by Nanosight and the quantification of protein content was performed using the bicinchoninic acid method. For the assay with a murine model, 12 male Balb/c mice (CEUA 065/2022) were infected with 1x10⁶ P. brasiliensis 18 cells per animal, followed by euthanasia at 24, 48 and 72 hours after infection. For the murine model assay, 12 male Balb/c mice (CEUA 065/2022) were infected with 1x10⁶ P. brasiliensis 18 cells per animal, followed by euthanasia at 24, 48, and 72 hours after infection. At the determined times, bronchoalveolar lavage (BAL) was obtained to analyze the profile of recruited cells and the lungs were also removed, macerated in 1x phosphate-buffered saline (PBS), and cultured in supplemented brain-heart infusion (BHI) medium to assess the fungal load. The assay demonstrated that there was no statistically relevant difference in the characterization of the extracellular vesicles and in the quantification of their proteins. For the murine model, an increase in the total leukocyte count and an increase in the fungal load in the lung tissue were observed, even with the presence of inflammatory infiltrate. These results indicate that the standardization of the murine model would be an important advance for future studies on the pathogenicity of the disease and the use of extracellular vesicles as a mechanism of fungal virulence.

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Palavras-chave

Paracoccidioides brasiliensis, Padronização, Vesículas extracelulares, Modelo murino, Ferro, Standardization, Extracellular vesicles, Murine model, Iron

Citação

MELO, Ana Paula Martins de. Padronização do isolamento de vesículas extracelulares de Paracoccidioides brasiliensis e do modelo murino de Paracoccidioidomicose. 2024. 52 f. Trabalho de Conclusão de Curso (Bacharelado em Biomedicina) - Instituto de Ciências Biológicas, Universidade Federal de Goiás, Goiânia, 2024.