Cryptococcus neoformans: papel da proteína Vps34 sobre os fatores de virulência e resistência a estresses osmótico, salino e oxidativo
Data
2023-12-06
Autores
Sousa, Guilherme Oliveira de 
Orientadores
Vallim, Marcelo Afonso
Tipo
Trabalho de conclusão de curso
Título da Revista
ISSN da Revista
Título de Volume
Resumo
A criptococose é uma doença de grande importância clínica, quando se diz respeito a doenças infecciosas oportunistas. Dados afirmam que há cerca de 250.000 casos por ano, sendo que as taxas de mortalidade chegam a 60%. Sendo assim, o estudo dos mecanismos que conferem virulência ao fungo causador da doença devem ser analisados, a fim de se explorar novas possibilidades de alvos terapêuticos para tratamentos mais eficazes e que não sejam tóxicos ao paciente. Contudo, a colonização do hospedeiro pela levedura gera a necessidade do microorganismo se adaptar ao ambiente e poder explorá-lo de forma a obter nutrientes que garantem o seu estabelecimento e proliferação. Entretanto, para se estabelecer até obter os nutrientes externos, as células devem realizar processos que reciclam seus componentes disponibilizando nutrientes para a mesma, o que é crucial nesta etapa. A via de autofagia é um desses mecanismos importantes para a reciclagem de nutrientes. Previamente, nosso grupo de pesquisa mostrou que proteínas que fazem parte desta via possuem relação com a virulência deste fungo, por meio de mutantes que demonstraram ser avirulentos e mais sensíveis aos medicamentos existentes para o tratamento da criptococose. Assim, este trabalho teve como objetivo aprofundar os estudos sobre a relação de autofagia e virulência avaliando o papel da ausência da proteína Vps34 em C. neoformans. Essa proteína é responsável pela formação de dois complexos protéicos, um capaz de localizar e regular o tráfego de proteínas intracelulares, através de eventos de fosforilação de proteínas e lipídios para o vacúolo de degradação, e outro capaz de contribuir para a formação do autofagossomo pelo recrutamento de outras proteínas estruturais. Portanto, foi avaliado o mutante vps34Δ, o qual teve o gene selvagem (VPS34) deletado para aferir o impacto da ausência de Vps34 sobre a patogênese de C. neoformans. A partir dele, foram realizados testes como resistência a estresses com meios de cultura suplementados com agentes estressores para cada tipo de estresse, crescimento à temperatura fisiológica humana a partir de curvas de crescimento e à produção de fatores de virulência com meios de cultura contendo os precursores para a síntese desses fatores, os quais são alterados no mutante vps34Δ. Assim, foi possível observar que este mutante apresenta grande sensibilidade aos fatores estressantes e baixa produção dos fatores de virulência, tendo virulência atenuada em modelo animal. Deste modo, podemos concluir que a proteína Vps34 é um fator importante para a sobrevivência de C. neoformans em condições adversas o que torna essa proteína um alvo possível para o desenvolvimento de novas opções terapêuticas.
Cryptococcosis is a disease of great clinical importance when it comes to opportunistic infectious diseases. Data states that there are around 250,000 cases per year, and in 60% of these cases the patient dies. Therefore, the study of the mechanisms that confer virulence to the fungus that causes the disease must be analyzed in order to explore new possibilities for therapeutic targets for more effective treatments that are not toxic to the patient. However, the colonization of the host by yeast creates the need for the microorganism to adapt to the environment and be able to explore it in order to obtain nutrients that guarantee its establishment and proliferation. However, to establish itself and obtain external nutrients, cells must carry out processes that recycle their components, making nutrients available to them, which is crucial at this stage. The autophagy pathway is one of these important mechanisms for nutrient recycling. Previously, our research group showed that proteins that are part of this pathway are related to the virulence of this fungus, through mutants that demonstrated to be avirulent and more sensitive to existing drugs for the treatment of cryptococcosis. Thus, this work aimed to deepen studies on the relationship between autophagy and virulence by evaluating the role of the absence of the Vps34 protein in C. neoformans. This protein is responsible for the formation of two protein complexes, one capable of localizing and regulating the trafficking of intracellular proteins, through protein and lipid phosphorylation events to the degradation vacuole, and the other capable of contributing to the formation of the autophagosome through recruitment of other structural proteins. Therefore, the vps34Δ mutant was evaluated, in which the wild-type gene (VPS34) was knocked out, to assess the impact of the absence of Vps34 on the pathogenesis of C. neoformans. From it, tests were carried out such as resistance to stress with culture media supplemented with stressors for each type of stress, growth at human physiological temperature based on growth curves and the production of virulence factors with culture media containing the precursors for the synthesis of these factors, which are altered in the vps34Δ mutant. Thus, it was possible to observe that this mutant presents great sensitivity to stress factors and low production of virulence factors, with attenuated virulence in an animal model. Therefore, we can conclude that the Vps34 protein is an important factor for the survival of C. neoformans in adverse conditions, which makes this protein a possible target for the development of new therapeutic options.
Cryptococcosis is a disease of great clinical importance when it comes to opportunistic infectious diseases. Data states that there are around 250,000 cases per year, and in 60% of these cases the patient dies. Therefore, the study of the mechanisms that confer virulence to the fungus that causes the disease must be analyzed in order to explore new possibilities for therapeutic targets for more effective treatments that are not toxic to the patient. However, the colonization of the host by yeast creates the need for the microorganism to adapt to the environment and be able to explore it in order to obtain nutrients that guarantee its establishment and proliferation. However, to establish itself and obtain external nutrients, cells must carry out processes that recycle their components, making nutrients available to them, which is crucial at this stage. The autophagy pathway is one of these important mechanisms for nutrient recycling. Previously, our research group showed that proteins that are part of this pathway are related to the virulence of this fungus, through mutants that demonstrated to be avirulent and more sensitive to existing drugs for the treatment of cryptococcosis. Thus, this work aimed to deepen studies on the relationship between autophagy and virulence by evaluating the role of the absence of the Vps34 protein in C. neoformans. This protein is responsible for the formation of two protein complexes, one capable of localizing and regulating the trafficking of intracellular proteins, through protein and lipid phosphorylation events to the degradation vacuole, and the other capable of contributing to the formation of the autophagosome through recruitment of other structural proteins. Therefore, the vps34Δ mutant was evaluated, in which the wild-type gene (VPS34) was knocked out, to assess the impact of the absence of Vps34 on the pathogenesis of C. neoformans. From it, tests were carried out such as resistance to stress with culture media supplemented with stressors for each type of stress, growth at human physiological temperature based on growth curves and the production of virulence factors with culture media containing the precursors for the synthesis of these factors, which are altered in the vps34Δ mutant. Thus, it was possible to observe that this mutant presents great sensitivity to stress factors and low production of virulence factors, with attenuated virulence in an animal model. Therefore, we can conclude that the Vps34 protein is an important factor for the survival of C. neoformans in adverse conditions, which makes this protein a possible target for the development of new therapeutic options.