Caracterização das proteínas parceiras de Ape4, Ams1 e Atg8 durante o processo de autofagia em Cryptococcus neoformans: estudo sobre os impactos na virulência e sensibilidade aos antifúngicos
Data
2019-10-23
Autores
Roberto, Thiago Nunes 
Orientadores
Vallim, Marcelo Afonso
Tipo
Tese de doutorado
Título da Revista
ISSN da Revista
Título de Volume
Resumo
A criptococose é uma micose de caráter sistêmico de grande importância clínica. A
doença é causada, sobretudo, pelo complexo Cryptococcus neoformans, na qual,
está associado principalmente a pacientes imunodeficientes. O tratamento dessa
micose compreende o uso de fármacos que, geralmente, são tóxicos para o
paciente. Assim sendo, futuras pesquisas precisam ser desenvolvidas visando
identificar novos alvos/estratégias de tratamento e controle da doença. Por sua vez,
a elucidação das vias autofágicas em fungos pode fornecer novos entendimentos
sobre as relações estabelecidas para garantir o processo de infecção. A autofagia é
um sistema de degradação e reciclagem intracelular de macromoléculas e
organelas, essencial para a sobrevivência da célula em condições adversas. Em
Saccharomyces cerevisiae, durante o processo de autofagia do tipo Cvt (Cytoplasm
to vacuole targeting) há a participação de cinco principais proteínas, sendo que, a
espécie C. neoformans é capaz de codificar apenas três dessas proteínas, isto é:
Ape4, Ams1 e Atg8. Por esta razão, este projeto teve como objetivo analisar quais
proteínas interagem com Ape4, Ams1 e Atg8 em C. neoformans, verificando seus
respectivos papéis no mecanismo da autofagia e, também, em eventos não
autofágicos. Utilizando a metodologia de dois-híbridos, conseguimos identificar e
confirmar a interação de Atg8 com a proteína Os-9. Os resultados mostraram que a
deleção do gene OS-9 não interferiu no desenvolvimento celular para as condições
analisadas. Além disso, confirmamos experimentalmente para C. neoformans a
interação física descrita na literatura entre as proteínas Atg8 e Atg3, Atg4 e Atg7.
Ainda, os resultados mostraram que os genes ATG4 e ATG8 são fundamentais para
a sobrevivência da célula em privação nutricional; o transporte e proteólise
intravacuolar da proteína Atg8 é dependente de Atg4; os mutantes atg4 e atg8 são
mais tolerantes ao estresse oxidativo, sensíveis ao corante vermelho de Congo e ao
inibidor de proteassoma bortezomibe (BTZ). Concluímos assim que as interações de
Atg8 para a sua conjugação com fosfatidiletanolamina (PE) e regulação da via da
autofagia encontram-se conservadas em C. neoformans.
Cryptococcosis is a systemic mycosis of medical importance. The disease is mainly caused by the complex Cryptococcus neoformans, which is prevalent in immunodeficient patients. The treatment of this mycosis comprises the use of drugs that are mostly toxic to the patient. Therefore, future researches need to be developed to identify new targets/strategies for treatment and control of the disease. The elucidation of autophagic pathways in fungi may provide new insights into the relationship established to ensure the infection. Autophagy is a mechanism of intracellular degradation and recycling of macromolecules and organelles, essential for cell survival under adverse conditions. In Saccharomyces cerevisiae, during the autophagy process called Cvt (Cytoplasm to vacuole targeting), there are the participation of five major proteins, however, the C. neoformans genome can encode only three of them: Ape4, Ams1 and Atg8. For this reason, this project aimed to analyze which proteins interact with Ape4, Ams1 and Atg8 in C. neoformans, verifying their respective roles in the autophagic and non-autophagic events. Using the two-hybrid methodology, we were able to identify and confirm the interaction between Atg8 and Os-9. The results showed that OS-9 gene deletion did not affect the cellular development for the conditions analyzed. In addition, we experimentally confirmed in C. neoformans the physical interaction described in the literature for the autophagy-related proteins Atg8 and Atg4. Furthermore, the results showed that the genes ATG4 and ATG8 are essential for the cell survival under nutritional deprivation; Atg8 intravacuolar transport and proteolysis is Atg4-dependent; the mutants atg4 and atg8 are more tolerant to oxidative stress, sensitive to Congo red and to the proteasome inhibitor bortezomib (BTZ). We conclude that the interactions of Atg8 for its conjugation with phosphatidylethanolamine (PE) and autophagy pathway regulation are conserved in C. neoformans.
Cryptococcosis is a systemic mycosis of medical importance. The disease is mainly caused by the complex Cryptococcus neoformans, which is prevalent in immunodeficient patients. The treatment of this mycosis comprises the use of drugs that are mostly toxic to the patient. Therefore, future researches need to be developed to identify new targets/strategies for treatment and control of the disease. The elucidation of autophagic pathways in fungi may provide new insights into the relationship established to ensure the infection. Autophagy is a mechanism of intracellular degradation and recycling of macromolecules and organelles, essential for cell survival under adverse conditions. In Saccharomyces cerevisiae, during the autophagy process called Cvt (Cytoplasm to vacuole targeting), there are the participation of five major proteins, however, the C. neoformans genome can encode only three of them: Ape4, Ams1 and Atg8. For this reason, this project aimed to analyze which proteins interact with Ape4, Ams1 and Atg8 in C. neoformans, verifying their respective roles in the autophagic and non-autophagic events. Using the two-hybrid methodology, we were able to identify and confirm the interaction between Atg8 and Os-9. The results showed that OS-9 gene deletion did not affect the cellular development for the conditions analyzed. In addition, we experimentally confirmed in C. neoformans the physical interaction described in the literature for the autophagy-related proteins Atg8 and Atg4. Furthermore, the results showed that the genes ATG4 and ATG8 are essential for the cell survival under nutritional deprivation; Atg8 intravacuolar transport and proteolysis is Atg4-dependent; the mutants atg4 and atg8 are more tolerant to oxidative stress, sensitive to Congo red and to the proteasome inhibitor bortezomib (BTZ). We conclude that the interactions of Atg8 for its conjugation with phosphatidylethanolamine (PE) and autophagy pathway regulation are conserved in C. neoformans.