Impacto da deleção de GPP2 sobre o processo de autofagia em Cryptococcus neoformans
Data
2021-09-30
Autores
Padilla, Adrian Alvarez Padilla [UNIFESP]
Orientadores
Vallim, Marcelo Afonso [UNIFESP]
Tipo
Dissertação de mestrado
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, pelas espécies dos complexos Cryptococcus neoformans e Cryptococcus gattii, esses fungos patogênicos, onipresentes e oportunistas causam infecção pulmonar, cutânea, renal, esplenomegalia e, mais importante dada a letariedade, a meningoencefalite. A criptococcose acomete, principalmente, pacientes com sistema imunológico debilitado. Durante a infecção, a levedura precisa se adaptar às condições desfavoráveis no hospedeiro como a alta temperatura (37° C) e a disponibilidade de nutrientes. O tratamento dessa micose compreende o uso prolongado de antifúngicos que, em sua maioria, são tóxicos para o paciente e o uso constante desses medicamentos induz o surgimento de cepas resistentes. Dessa forma, novas terapias antifúngicas são necessárias para aumentar as opções de tratamento. A autofagia atua como um sistema de degradação e reciclagem intracelular de macromoléculas e organelas que garante a homeostasia e a sobrevivência da célula diante de diferentes fatores de estresse. Análises de transcriptoma RNAseq em meio rico (YPD) em C. neoformans mostram que a deleção do gene GPP2 reprime e induz alguns genes chaves da autofagia tais como: ATG1, ATG2, ATG4, VPS15, ATG15, VPS30 e PEP4. Os nossos dados de qPCR validaram os resultados das análises de RNAseq. Trabalhos anteriores do nosso grupo mostraram que no mutante gpp2Δ ocorre a interrupção de várias vias biossintéticas e captação de aminoácidos que resulta na queda da viabilidade celular, incapacidade de lidar com vários tipos de estresses, redução da expressão de fatores de virulência, (melanina, fosfolipase e urease) e atenuação de virulência em Galleria mellonella, indicando que a biossíntese e absorção de aminoácidos sulfurosos é uma característica importante para a sobrevivência do patógeno in vivo e in vitro. Em Saccharomyces cerevisiae, o Gpp2 é induzido em resposta ao estresse hiperosmótico e oxidativo. Neste trabalho mostramos que em C. neoformans, Gpp2 é necessário para responder, principalmente, ao estresse osmótico, e que a via da autofagia encontra-se reprimida na presença do aminoácido metionina tanto na linhagem selvagem como no mutante gpp2Δ. Nossos dados em conjunto com os trabalhos anteriores, reportados pelo nosso grupo de pesquisa, sugerem que Gpp2 é um fator importante para a adaptação geral do patógeno a vários estresses e talvez possa ser um alvo interessante para uso terapêutico.
Cryptococcosis is a systemic mycosis of great clinical importance. The disease is mainly caused by species of the complexes Cryptococcus neoformans and Cryptococcus gattii, these pathogenic, ubiquitous and opportunistic fungi cause pulmonary, skin, renal, spleen and, more importantly, meningoencephalitis, given their lethargy. Cryptococcosis mainly affects patients with weakened immune systems. During infection, the yeast needs to adapt to unfavorable conditions in the host such as high temperature (37°C) and availability of nutrients. The treatment of this mycosis comprises the prolonged use of antifungals, which are mostly toxic to the patient, and the constant use of these drugs induces the emergence of resistant strains. Therefore, new antifungal therapies are needed to increase treatment options. Autophagy acts as a system of intracellular degradation and recycling of macromolecules and organelles that guarantees homeostasis and cell survival under different stress factors. RNAseq transcriptome analyzes in rich medium (YPD) in C. neoformans show that the deletion of the GPP2 gene represses and induces some key autophagy genes such as (ATG1, ATG2, ATG4, VPS15, ATG15, VPS30 and PEP4. Our qPCR data validated and validated RNAseq analysis results. Previous work by our group showed that in the gpp2Δ mutant there is an interruption of several biosynthetic pathways and amino acid uptake which results in a drop in cell viability, inability to deal with various types of stresses, reduced expression of virulence factors, (melanin, phospholipase and urease) and attenuation of virulence in Galleria mellonella. Indicating that the biosynthesis and absorption of sulfur amino acids is an important characteristic for the survival of the pathogen in vivo and in vitro. In Saccharomyces cerevisiae, Gpp2 is induced in response to hyperosmotic and oxidative stress. In this work, we show that in C. neoformans, Gpp2 is necessary to respond mainly to osmotic stress, and that the autophagy pathway is repressed in the presence of the amino acid methionine in both wild-type and mutant gpp2Δ strains. Our data together with previous work reported by our research group suggest that Gpp2 is an important factor in the pathogen's general adaptation to various stresses, and perhaps it could be an interesting target for therapeutic use.
Cryptococcosis is a systemic mycosis of great clinical importance. The disease is mainly caused by species of the complexes Cryptococcus neoformans and Cryptococcus gattii, these pathogenic, ubiquitous and opportunistic fungi cause pulmonary, skin, renal, spleen and, more importantly, meningoencephalitis, given their lethargy. Cryptococcosis mainly affects patients with weakened immune systems. During infection, the yeast needs to adapt to unfavorable conditions in the host such as high temperature (37°C) and availability of nutrients. The treatment of this mycosis comprises the prolonged use of antifungals, which are mostly toxic to the patient, and the constant use of these drugs induces the emergence of resistant strains. Therefore, new antifungal therapies are needed to increase treatment options. Autophagy acts as a system of intracellular degradation and recycling of macromolecules and organelles that guarantees homeostasis and cell survival under different stress factors. RNAseq transcriptome analyzes in rich medium (YPD) in C. neoformans show that the deletion of the GPP2 gene represses and induces some key autophagy genes such as (ATG1, ATG2, ATG4, VPS15, ATG15, VPS30 and PEP4. Our qPCR data validated and validated RNAseq analysis results. Previous work by our group showed that in the gpp2Δ mutant there is an interruption of several biosynthetic pathways and amino acid uptake which results in a drop in cell viability, inability to deal with various types of stresses, reduced expression of virulence factors, (melanin, phospholipase and urease) and attenuation of virulence in Galleria mellonella. Indicating that the biosynthesis and absorption of sulfur amino acids is an important characteristic for the survival of the pathogen in vivo and in vitro. In Saccharomyces cerevisiae, Gpp2 is induced in response to hyperosmotic and oxidative stress. In this work, we show that in C. neoformans, Gpp2 is necessary to respond mainly to osmotic stress, and that the autophagy pathway is repressed in the presence of the amino acid methionine in both wild-type and mutant gpp2Δ strains. Our data together with previous work reported by our research group suggest that Gpp2 is an important factor in the pathogen's general adaptation to various stresses, and perhaps it could be an interesting target for therapeutic use.