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.