Perfil transcricional e proteômico de leveduras de P. brasiliensis presentes em lesões granulomatosas crônicas de camundongos C57BL/6
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Data
2023-08-24
Autores
Borges, Bruno Montanari [UNIFESP]
Orientadores
Loures, Flávio Vieira [UNIFESP]
Tipo
Tese de doutorado
Título da Revista
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Resumo
Granulomas são estruturas imunológicas envolvidas na defesa de hospedeiros contra o fungo Paracoccidioides brasiliensis, principal agente etiológico da Paracoccidioidomicose (PCM), micose sistêmica granulomatosa endêmica na América Latina. Estudos com transcritos em lesões granulomatosas podem revelar importantes genes que atuam no granuloma, permitindo uma melhor compreensão de mecanismos da relação fungo-hospedeiro. Igualmente, um proteoma das leveduras granulomatosas e estudos das vesículas extracelulares (EVs) produzidas por esses fungos podem apontar importantes mecanismos de virulência expressos sob as condições de estresse resultantes da atividade do sistema imunológico no granuloma. Portanto, camundongos C57BL/6 foram infectados com leveduras do P. brasiliensis e após 8 e 12 semanas de infecção, as lesões granulomatosas foram obtidas e o RNA fúngico e murino extraídos. Proteínas fúngicas também foram obtidas. Ademais, leveduras presentes nas lesões foram posteriormente cultivadas e submetidas a outro ensaio proteômico e à extração de suas EVs, as quais foram utilizadas no desafio de macrófagos e células dendríticas, e no estudo da resposta adaptativa celular desenvolvida por linfócitos em cocultivo com as células inatas. Os transcritos evidenciaram atividade gênica relaciona a neutrófilos, macrófagos e linfócitos, além de uma resposta Th1/Th17 presente nas lesões. Transcritos para o CTLA-4, PD-1 e arginase-1, evidenciam a ação de mecanismos regulatórios. Os transcritos fúngicos apontam para um aumento da produção de sideróforos para captura de ferro, mecanismo de virulência fúngico nunca observado em granulomas. Adicionalmente, transcritos e proteoma apontam para uma repressão da atividade fúngica dentro do granuloma. Contudo, foi visto que após o cultivo dessas leveduras extraídas das lesões granulomatosas o metabolismo do fungo foi induzido, apresentando maior abundância de proteínas relacionadas ao metabolismo energético e fatores de virulência, mostrando que após a passagem do fungo pelo granuloma a virulência é aumentada, confirmação feita por ensaios de sobrevida. Além disso, foi visto pelo proteoma das EVs que elas auxiliam na indução do metabolismo e virulência fúngica, sendo capazes inclusive de modular uma resposta imunológica. Do fato, em resposta às EVs, células apresentadoras de antígeno tiveram menor ativação quando comparado ao estímulo dado por EVs controles. Ademais, as EVs do fungo recuperado do granuloma induziram respostas adaptativas do tipo Th1/Tc1, Th2/Tc2 e Th17/Tc17 mais exacerbadas, além de induzirem uma maior frequência de linfócitos T regulares, mostrando pela primeira vez a capacidade de EVs fúngicas em modular uma resposta adaptativa celular.
Granulomas are immunological structures involved in host defense against the fungus Paracoccidioides brasiliensis, the main etiological agent of Paracoccidioidomycosis (PCM), a granulomatous systemic mycosis endemic in Latin America countries. Studies with transcripts produced in granulomatous lesions may reveal crucial genes that act in the granuloma, allowing a better understanding of the mechanisms of the fungus-host interaction. Likewise, a proteome of granulomatous yeasts and studies with extracellular vesicles (EVs) produced by these fungi may reveal important virulence mechanisms expressed under stress conditions due to the activity of the immune system in the granuloma. Therefore, C57BL/6 WT mice were infected with P. brasiliensis yeasts and after 8 and 12 weeks of infection, the granulomatous lesions were obtained, and fungal and murine RNA were extracted. Fungal proteins were also obtained. Furthermore, the yeasts present in the lesions were later cultivated and subjected to another proteomic assay and the extraction of their EVs. The vesicles were used to challenge macrophages and dendritic cells, and in the study of the adaptive cellular response developed by lymphocytes in co-culture with innate cells. The transcripts showed gene activity related to neutrophils, macrophages, and lymphocytes, and indicate a Th1/Th17 response in granulomatous lesions. Transcripts to CTLA-4, PD-1 and arginase-1 reveal the presence of immunosuppressive mechanisms. Regarding the fungal data, the transcripts suggest an increase in the production of siderophores for iron uptake, a fungal virulence mechanism never observed in granulomas. Additionally, transcripts and proteomes indicate an repression of fungal activity within the granuloma. However, it was observed that after the culture of the yeast extracted from granulomatous lesions, the fungal metabolism was induced, with a higher abundance of proteins related to energy metabolism and virulence factors. This demonstrates that after the passage of the fungus through the granuloma, the virulence is increased, a confirmation made by survival assays. Furthermore, it was observed by the proteome of the EVs that they assist in inducing fungal metabolism and virulence and are even capable to modulate an immune response. In fact, in response to the EVs, antigen-presenting cells showed less activation when compared to the stimulus given by control EVs. Moreover, EVs derived from the fungus recovered from the granuloma induced a more exacerbated Th1/Tc1, Th2/Tc2, and Th17/Tc17 adaptive responses, in addition to inducing a higher frequency of regular T lymphocytes, showing for the first time the capacity of fungal EVs to modulate a cellular adaptive response.
Granulomas are immunological structures involved in host defense against the fungus Paracoccidioides brasiliensis, the main etiological agent of Paracoccidioidomycosis (PCM), a granulomatous systemic mycosis endemic in Latin America countries. Studies with transcripts produced in granulomatous lesions may reveal crucial genes that act in the granuloma, allowing a better understanding of the mechanisms of the fungus-host interaction. Likewise, a proteome of granulomatous yeasts and studies with extracellular vesicles (EVs) produced by these fungi may reveal important virulence mechanisms expressed under stress conditions due to the activity of the immune system in the granuloma. Therefore, C57BL/6 WT mice were infected with P. brasiliensis yeasts and after 8 and 12 weeks of infection, the granulomatous lesions were obtained, and fungal and murine RNA were extracted. Fungal proteins were also obtained. Furthermore, the yeasts present in the lesions were later cultivated and subjected to another proteomic assay and the extraction of their EVs. The vesicles were used to challenge macrophages and dendritic cells, and in the study of the adaptive cellular response developed by lymphocytes in co-culture with innate cells. The transcripts showed gene activity related to neutrophils, macrophages, and lymphocytes, and indicate a Th1/Th17 response in granulomatous lesions. Transcripts to CTLA-4, PD-1 and arginase-1 reveal the presence of immunosuppressive mechanisms. Regarding the fungal data, the transcripts suggest an increase in the production of siderophores for iron uptake, a fungal virulence mechanism never observed in granulomas. Additionally, transcripts and proteomes indicate an repression of fungal activity within the granuloma. However, it was observed that after the culture of the yeast extracted from granulomatous lesions, the fungal metabolism was induced, with a higher abundance of proteins related to energy metabolism and virulence factors. This demonstrates that after the passage of the fungus through the granuloma, the virulence is increased, a confirmation made by survival assays. Furthermore, it was observed by the proteome of the EVs that they assist in inducing fungal metabolism and virulence and are even capable to modulate an immune response. In fact, in response to the EVs, antigen-presenting cells showed less activation when compared to the stimulus given by control EVs. Moreover, EVs derived from the fungus recovered from the granuloma induced a more exacerbated Th1/Tc1, Th2/Tc2, and Th17/Tc17 adaptive responses, in addition to inducing a higher frequency of regular T lymphocytes, showing for the first time the capacity of fungal EVs to modulate a cellular adaptive response.