Efeito do pH ácido na modulação celular e nos atributos de virulência do Paracoccidioides brasiliensis
Data
2022-12-13
Autores
Silva, Rafael de Souza 
Orientadores
Batista, Wagner Luiz
Tipo
Tese de doutorado
Título da Revista
ISSN da Revista
Título de Volume
Resumo
Fungos do gênero Paracoccidoides respondam a diferentes estresses, como: altas temperaturas, ROS/RNS, hipóxia e limitação de macro e micronutrientes. Entretanto, pouco se sabe sobre como esses fungos respondem a baixos níveis de pH. Principalmente quando ocorrem condições de pH mais baixo no tecido do hospedeiro, no interior das células fagocíticas, formação de granulomas e privação de nutrientes. Além disso, proteases ácidas têm sido descritas como tendo papel importante no processo de invasão do hospedeiro e modulação da resposta imune em muitos microrganismos patogênicos. No presente estudo avaliamos a adaptação de Paracoccidioides brasiliensis em resposta a ambientes com baixo pH e caracterizamos a modulação de proteases ácidas. Leveduras cultivadas em pH ácido apresentam diminuição de sua capacidade proliferativa, o qual foi confirmado através de ensaios de proteômica. O pH ácido regula a expressão de genes envolvidos nas vias de síntese de melanina e estimula a melanização das células de P. brasiliensis. A melanização pode ser observada visualmente no fungo e foi confirmada pela atividade da Lacase. Leveduras pigmentadas apresentaram aumento nos níveis de quitina e uma diminuição de manana. Também demonstramos que as células leveduriformes cultivadas em pH ácido apresentaram menor índice fagocítico e baixos níveis de marcadores pró-inflamatórios em células RAW 264.7. Além disso, detectamos quatro aspartil proteases em P. brasiliensis com alta homologia à protease aspártica Pep4 de Saccharomyces cerevisiae. Demonstramos que as proteases aspárticas são um importante regulador da transição dimórfica (Micélio → Levedura) em P. brasiliensis. Observamos que os genes das aspartil proteases foram modulados durante o termodimorfismo (transição M→L), durante a exposição a diferentes fontes de carbono e nitrogênio, e no estresse em pH ácida. Por fim, verificamos aumento da atividade das proteases aspárticas secretadas de P. brasiliensis quando cultivadas em pH 4. Juntos, esses achados contribuem para o entendimento de como P. brasiliensis é capaz de sobreviver ao estresse de pH ácido, bem como entender os atributos de virulência das proteases aspárticas neste fungo.
Fungi of the genus Paracoccidoides respond to different stresses, such as high temperatures, ROS/RNS, hypoxia, and the limitation of macro and micronutrients. However, little is known about how these fungi respond to low pH levels. Especially when conditions of lower pH occur in the host tissue, inside the phagocytic cells, granuloma formation, and nutrient deprivation. Furthermore, acid proteases have been described as having an important role in the host invasion process and modulation of the immune response in many pathogenic microorganisms. In the present study, we evaluated the adaptation of Paracoccidioides brasiliensis in response to low pH environments and characterized the modulation of acid proteases. Yeasts grown at acidic pH showed a decrease in their proliferative capacity, which was confirmed by proteomic assays. Acidic pH regulates gene expression in melanin synthesis pathways and stimulates the melanization of P. brasiliensis cells. Melanization can be visually observed in the fungus and was confirmed by Lacasse activity. Pigmented yeasts showed an increase in chitin levels and a decrease in mannan. We also demonstrated that yeast cells cultured at acidic pH had a lower phagocytic index and lower levels of pro-inflammatory markers in RAW 264.7 cells. Furthermore, we detected four aspartyl proteases in P. brasiliensis with high homology to the aspartic protease Pep4 from Saccharomyces cerevisiae. We demonstrate that aspartic proteases are an important regulator of the dimorphic transition (Mycelium → Yeast) in P. brasiliensis. We observed that aspartyl protease genes were modulated during thermodimorphism (M→L transition), exposure to different carbon and nitrogen sources, and stress at acidic pH. Finally, we verified an increase in the activity of the secreted aspartic proteases of P. brasiliensis when cultivated at pH 4. Together, these findings contribute to the understanding of how P. brasiliensis can survive acidic pH stress, as well as to understand the attributes of virulence of aspartic proteases in this fungus.
Fungi of the genus Paracoccidoides respond to different stresses, such as high temperatures, ROS/RNS, hypoxia, and the limitation of macro and micronutrients. However, little is known about how these fungi respond to low pH levels. Especially when conditions of lower pH occur in the host tissue, inside the phagocytic cells, granuloma formation, and nutrient deprivation. Furthermore, acid proteases have been described as having an important role in the host invasion process and modulation of the immune response in many pathogenic microorganisms. In the present study, we evaluated the adaptation of Paracoccidioides brasiliensis in response to low pH environments and characterized the modulation of acid proteases. Yeasts grown at acidic pH showed a decrease in their proliferative capacity, which was confirmed by proteomic assays. Acidic pH regulates gene expression in melanin synthesis pathways and stimulates the melanization of P. brasiliensis cells. Melanization can be visually observed in the fungus and was confirmed by Lacasse activity. Pigmented yeasts showed an increase in chitin levels and a decrease in mannan. We also demonstrated that yeast cells cultured at acidic pH had a lower phagocytic index and lower levels of pro-inflammatory markers in RAW 264.7 cells. Furthermore, we detected four aspartyl proteases in P. brasiliensis with high homology to the aspartic protease Pep4 from Saccharomyces cerevisiae. We demonstrate that aspartic proteases are an important regulator of the dimorphic transition (Mycelium → Yeast) in P. brasiliensis. We observed that aspartyl protease genes were modulated during thermodimorphism (M→L transition), exposure to different carbon and nitrogen sources, and stress at acidic pH. Finally, we verified an increase in the activity of the secreted aspartic proteases of P. brasiliensis when cultivated at pH 4. Together, these findings contribute to the understanding of how P. brasiliensis can survive acidic pH stress, as well as to understand the attributes of virulence of aspartic proteases in this fungus.
Descrição
Citação
SILVA, Rafael de Souza. Efeito do pH ácido na modulação celular e nos atributos de virulência do Paracoccidioides brasiliensis. São Paulo, 2022. 134 f. Tese (Doutorado em Microbiologia e Imunologia) - Escola Paulista de Medicina (EPM), Universidade Federal de São Paulo (UNIFESP). São Paulo, 2022.