Autofagia não canônica em macrófagos infectados com isolados clínicos de Cryptococcus neoformans do Distrito Federal

Dissertação (mestrado)—Universidade de Brasília, Faculdade de Medicina, Pós-graduação em Medicina Tropical, 2018.A criptococose é uma micose sistêmica causada por fungos do gênero Cryptococcus que acomete principalmente pessoas com Aids. A principal manifestação clínica da doença é uma meningoencefalite severa. O tratamento desta doença é baseado atualmente em drogas intravenosas bastante tóxicas ou caras, que necessitam de internação hospitalar para administração e requerem acompanhamento laboratorial constante para manejo dos efeitos colaterais. Apesar da existência deste tratamento, se estima que aproximadamente 80% dos 223 mil casos anuais da doença levem à morte. Há evidências de que a infecção pelo fungo ocorra em todas as pessoas durante a infância; o que parece determinar a doença, portanto, é o surgimento de imunossupressão da resposta imunitária celular, o que indica que novas terapias possam ser criadas com alvo na resposta imunitária do hospedeiro. Um dos mecanismos possíveis para manipulação com fins terapêuticos da resposta do hospedeiro é a autofagia, um processo de reciclagem de material intracelular que tem sido demonstrado em experimentos in vitro e em modelos animais como importante também na resposta imunitária a fungos. Tendo em vista essa possibilidade de manipulação da autofagia, é necessário determinar se a autofagia é importante também na resposta imunitária humana, assim como os mecanismos pelos quais a autofagia exerce atividade antifúngica e como Cryptococcus evade da resposta autofágica. Para estudar o papel da autofagia na interação patógeno-hospedeiro da criptococose, nós utilizamos uma coleção de isolados clínicos de C. neoformans obtidos como parte do trabalho da Rede Criptococose Brasil no DF (RCB-DF). Esses isolados foram utilizados em testes in vitro para determinação de sua capacidade de evasão da autofagia do hospedeiro. Os resultados foram correlacionados com dados clínicos dos pacientes dos quais os isolados foram obtidos. Devido possivelmente ao grande número de isolados que seriam necessários para detectar diferenças, não conseguimos observar nenhuma. No entanto, correlações com outros fenótipos desses isolados trouxeram dados muito importantes sobre como C. neoformans causa doenças. Entre eles, o mais importante é a relação direta entre a capacidade de melanização de um isolado e sua capacidade de evadir da autofagia do hospedeiro, o que sugere um novo papel para a melnanina de C. neoformans: evadir da resposta autofágica.Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) e Fundação de Apoio à Pesquisa do Distrito Federal (FAP-DF).Cryptococcosis is a systemic mycosis caused by fungi of the genus Cryptococcus that mainly affects people with AIDS. The main clinical manifestation of the disease is a severe meningoencephalitis. The treatment is currently based on very toxic or expensive intravenous drugs that require hospital admission for administration and constant laboratory monitoring to manage side effects. Despite the existence of this treatment, it is estimated that approximately 80% of the 223,000 annual cases of the disease lead to death. There is evidence that fungal infection occurs in all people during childhood; what appears to determine the disease, therefore, is the emergence of immunosuppression of the cellular immune response, which indicates that novel therapies can be created targeting the immune response of the host. One of the possible mechanisms for therapeutic manipulation of host response is autophagy, an intracellular material recycling process that has been demonstrated in in vitro experiments and in animal models as a relevant tool in the immune response to fungi. In view of this possibility to manipulate the autophagy process, it is necessary to determine whether this is equally significant in the human immune response, as well as the mechanisms by which autophagy exerts antifungal activity and how Cryptococcus evades the autophagic response

Laccase affects the rate of Cryptococcus neoformans nonlytic exocytosis from macrophages

Nonlytic exocytosis is a process in which previously ingested microbes are expelled from host phagocytes with the concomitant survival of both cell types. This process has been observed in the interaction of Cryptococcus spp. and other fungal cells with phagocytes as distant as mammalian, bird, and fish macrophages and ameboid predators. Despite a great amount of research dedicated to unraveling this process, there are still many questions about its regulation and its final benefits for host or fungal cells. During a study to characterize the virulence attributes of Brazilian clinical isolates of C. neoformans, we observed great variability in their rates of nonlytic exocytosis and noted a correlation between this process and fungal melanin production/laccase activity. Flow cytometry experiments using melanized cells, nonmelanized cells, and lac1Δ mutants revealed that laccase has a role in the process of nonlytic exocytosis that seems to be independent of melanin production. These results identify a role for laccase in virulence, independent of its role in pigment production, that represents a new variable in the regulation of nonlytic exocytosis

Faster Cryptococcus Melanization Increases Virulence in Experimental and Human Cryptococcosis

Cryptococcus spp. are human pathogens that cause 181,000 deaths per year. In this work, we systematically investigated the virulence attributes of Cryptococcus spp. clinical isolates and correlated them with patient data to better understand cryptococcosis. We collected 66 C. neoformans and 19 C. gattii clinical isolates and analyzed multiple virulence phenotypes and host–pathogen interaction outcomes. C. neoformans isolates tended to melanize faster and more intensely and produce thinner capsules in comparison with C. gattii. We also observed correlations that match previous studies, such as that between secreted laccase and disease outcome in patients. We measured Cryptococcus colony melanization kinetics, which followed a sigmoidal curve for most isolates, and showed that faster melanization correlated positively with LC3-associated phagocytosis evasion, virulence in Galleria mellonella and worse prognosis in humans. These results suggest that the speed of melanization, more than the total amount of melanin Cryptococcus spp. produces, is crucial for virulence

Faster cryptococcus melanization increases virulence in experimental and human cryptococcosis

Cryptococcus spp. are human pathogens that cause 181,000 deaths per year. In this work, we systematically investigated the virulence attributes of Cryptococcus spp. clinical isolates and correlated them with patient data to better understand cryptococcosis. We collected 66 C. neoformans and 19 C. gattii clinical isolates and analyzed multiple virulence phenotypes and host–pathogen interaction outcomes. C. neoformans isolates tended to melanize faster and more intensely and produce thinner capsules in comparison with C. gattii. We also observed correlations that match previous studies, such as that between secreted laccase and disease outcome in patients. We measured Cryptococcus colony melanization kinetics, which followed a sigmoidal curve for most isolates, and showed that faster melanization correlated positively with LC3-associated phagocytosis evasion, virulence in Galleria mellonella and worse prognosis in humans. These results suggest that the speed of melanization, more than the total amount of melanin Cryptococcus spp. produces, is crucial for virulence