Caracterização do biofilme de Histoplasma capsulatum em diferentes condições de crescimento in vitro e identificação de proteínas na interação desses biofilmes com macrófagos alveolares murinos
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Data
2021-07-27
Autores
Gonçalves, Larissa Naiara Carvalho
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Universidade Estadual Paulista (Unesp)
Resumo
A histoplasmose é uma micose sistêmica, causada pelo fungo dimórfico Histoplasma capsulatum. Biofilmes são comunidades sésseis de microrganismos, que se agrupam de maneira organizada a um substrato ou que se aderem uns aos outros, e estão envolvidos por uma matriz extracelular que eles mesmos produzem. A capacidade desses fungos em formar biofilmes, um fenótipo que pode induzir resistência e aumentar a virulência, foi descrita. Além disso, também foi estabelecida uma correlação entre o modo de infecção de H. capsulatum e a formação dessas comunidades. O presente estudo teve como objetivo aprofundar o estudo dos biofilmes formados por diferentes cepas de H. capsulatum (EH-315, G186A e G217B), por meio da verificação da influência de quatro meios de cultura (BHI, HAM-F12, DMEM e RPMI) e diferentes atmosferas de oxigênio (microaerofilia e aerobiose) no desenvolvimento dessas comunidades. Adicionalmente, também objetivou-se estudar a interação entre células planctônicas e derivadas de biofilme após a infecção em macrófagos alveolares, verificando seu comportamento e sua capacidade de infecção, bem como averiguar a formação de biofilme em ambiente ácido, mimetizando o ambiente intracelular no macrófago e realizando uma análise proteômica por técnica de shotgun, a fim de identificar proteínas exclusivas nessas condições para a futura busca de alvos antifúngicos. A padronização do crescimento fúngico foi conduzida a partir da construção de curvas de crescimento, por meio de contagem de colônias, verificação da viabilidade celular com azul de Tripan e densidade óptica. A formação dos biofilmes foi caracterizada utilizando técnicas de microscopia óptica, e colorimétricas, como o ensaio de redução de XTT, coloração por cristal violeta e safranina, além de microscopia eletrônica de varredura (MEV) para observação das topografias. A interação com os macrófagos alveolares foi observada por microscopia de fluorescência e confocal para posterior análise proteômica em espectrômetro de massas. Nos ensaios de caracterização dos biofilmes, verificou-se que, embora todos os meios de cultura tenham estimulado a maturação das comunidades, o HAM-F12 proporcionou o melhor desenvolvimento de biomassa e material polissacarídico. Em relação às atmosferas de O2, ambas estimularam um excelente desenvolvimento das comunidades, porém, em condições de baixo O2, foi observada uma quantidade exuberante de matriz extracelular quando comparada aos biofilmes formados em aerobiose, principalmente no meio HAM-F12. Nos biofilmes formados em BHI, RPMI e DMEM observou-se uma reversão significativa do fenótipo de levedura para hifa, exigindo uma investigação mais aprofundada. Na interação com macrófagos, observou-se a formação de aglomerados fúngicos em macrófagos. No entanto, a formação dos aglomerados, bem como a fluorescência da marcação com o anticorpo anti-Hsp60 e o sofrimento celular foram mais expressivos na infecção com as células derivadas de biofilme. Além disso, as células derivadas de biofilme exibiram maior taxa de infecção após 1, 5 e 24 horas comparada as células planctônicas. O ambiente ácido não foi impeditivo para a formação de biofilmes maduros. Nos ensaios de proteômica, observou-se diferentes perfis proteicos nas condições testadas, com 72 proteínas exclusivamente expressas pelo biofilme quando comparado ao crescimento planctônico. A grande maioria dessas proteínas é relacionada a funções citoplasmáticas. Os dados proteômicos das células derivadas de biofilme também revelaram um aumento de proteínas envolvidas no estresse, defesa, virulência e na obtenção de energia e uma redução de proteínas envolvidas na síntese proteica e no processo metabólico. No proteoma da infecção pelas células derivadas do biofilme foram identificadas 6 proteínas exclusivas comparada ao controle (biofilme), a maioria classificadas como nucleares, permitindo um entendimento parcial sobre as vias associadas à sobrevivência e adaptação do fungo ao ambiente do hospedeiro. Esses resultados apresentam avanços no campo dos biofilmes e contribuem para estudos futuros que podem provar o papel dessas comunidades na interação patógeno-hospedeiro. Além disso, abrem novas possibilidades para o estudo de alvos com o intuito de estabelecer novas estratégias terapêuticas para histoplasmose.
Histoplasmosis is a systemic mycosis, caused by the dimorphic fungus Histoplasma capsulatum. Biofilms are sessile communities of microorganisms, which group together in an organized way a substrate or adhere to others and are surrounded by an extracellular matrix of their own. Fungal abilities to form biofilms, a phenotype that can induce resistance and increase virulence, was made available. Furthermore, a correlation between the mode of infection of H. capsulatum and a formation of these communities has also been published. This study aimed to deepen the study of biofilms formed by different strains of H. capsulatum (EH-315, G186A and G217B), by verifying the influence of four culture media (BHI, HAM-F12, DMEM and RPMI) and different oxygen atmospheres (microaerophilia and aerobiosis) in the development of these communities. Additionally, it was also aimed to study an interaction between planktonic and biofilm-derived cells after infection in alveolar macrophages, verifying their behavior and their capacity for infection, as well as verifying the formation of biofilm in an acidic environment, mimicking the intracellular environment in the macrophage and performing a proteomic analysis by shotgun technique, in order to identify unique proteins in these conditions for a future search for antifungal targets. The standardization of fungal growth was conducted from the construction of growth, through colony counting, verification of cell viability with Trypan blue and optical density. The formation of biofilms was characterized using optical and colorimetric microscopy techniques, such as the XTT reduction assay, crystal violet and safranin staining, in addition to scanning electron microscopy (SEM) for observation of topographies. The interaction with alveolar macrophages was observed by fluorescence and confocal microscopy for further proteomic analysis in a mass spectrometer. In biofilm characterization tests, it was found that, although all culture media have stimulated the maturation of communities, HAM-F12 provided the best development of biomass and polysaccharide material. In relation to the O2 atmospheres, both stimulated an excellent development of the communities, however, under low O2 conditions, an exuberant amount of extracellular matrix was observed when compared to the biofilms formed in aerobiosis, mainly in the HAM-F12 media. In biofilms formed in BHI, RPMI and DMEM, an explained reversion of the phenotype from yeast to hypha was observed, requiring further investigation. In the interaction with macrophages, the formation of fungal clusters in macrophages was observed. However, the formation of clusters, as well as the fluorescence of the labeling with the anti-Hsp60 antibody and the cellular suffering were more expressive in the infection with cells derived from biofilm. Furthermore, biofilm-derived cells exhibited a higher infection rate after 1, 5 and 24 hours compared to planktonic cells. The acidic environment was not an impediment to the formation of mature biofilms. In proteomics assays, different protein profiles were observed under the conditions tested, with 72 specially expressed by the biofilm when compared to planktonic growth. The vast majority of these proteins are related to cytoplasmic functions. Proteomic data from biofilm-derived cells also revealed an increase in proteins involved in stress, defense, virulence and energy acquisition and a reduction in proteins involved in protein synthesis and the metabolic process. In the proteome of infection by biofilm-derived cells, 6 unique proteins were identified compared to the control (biofilm), most classified as nuclear, allowing a partial understanding of the pathways associated with the adaptation and adaptation of the fungus to the host's environment. These results present advances in the field of biofilms and contribute to future studies that can prove the role of these communities in the host-pathogen interaction. Furthermore, they open up new possibilities for the study of targets in order to establish new therapeutic strategies for histoplasmosis.
Histoplasmosis is a systemic mycosis, caused by the dimorphic fungus Histoplasma capsulatum. Biofilms are sessile communities of microorganisms, which group together in an organized way a substrate or adhere to others and are surrounded by an extracellular matrix of their own. Fungal abilities to form biofilms, a phenotype that can induce resistance and increase virulence, was made available. Furthermore, a correlation between the mode of infection of H. capsulatum and a formation of these communities has also been published. This study aimed to deepen the study of biofilms formed by different strains of H. capsulatum (EH-315, G186A and G217B), by verifying the influence of four culture media (BHI, HAM-F12, DMEM and RPMI) and different oxygen atmospheres (microaerophilia and aerobiosis) in the development of these communities. Additionally, it was also aimed to study an interaction between planktonic and biofilm-derived cells after infection in alveolar macrophages, verifying their behavior and their capacity for infection, as well as verifying the formation of biofilm in an acidic environment, mimicking the intracellular environment in the macrophage and performing a proteomic analysis by shotgun technique, in order to identify unique proteins in these conditions for a future search for antifungal targets. The standardization of fungal growth was conducted from the construction of growth, through colony counting, verification of cell viability with Trypan blue and optical density. The formation of biofilms was characterized using optical and colorimetric microscopy techniques, such as the XTT reduction assay, crystal violet and safranin staining, in addition to scanning electron microscopy (SEM) for observation of topographies. The interaction with alveolar macrophages was observed by fluorescence and confocal microscopy for further proteomic analysis in a mass spectrometer. In biofilm characterization tests, it was found that, although all culture media have stimulated the maturation of communities, HAM-F12 provided the best development of biomass and polysaccharide material. In relation to the O2 atmospheres, both stimulated an excellent development of the communities, however, under low O2 conditions, an exuberant amount of extracellular matrix was observed when compared to the biofilms formed in aerobiosis, mainly in the HAM-F12 media. In biofilms formed in BHI, RPMI and DMEM, an explained reversion of the phenotype from yeast to hypha was observed, requiring further investigation. In the interaction with macrophages, the formation of fungal clusters in macrophages was observed. However, the formation of clusters, as well as the fluorescence of the labeling with the anti-Hsp60 antibody and the cellular suffering were more expressive in the infection with cells derived from biofilm. Furthermore, biofilm-derived cells exhibited a higher infection rate after 1, 5 and 24 hours compared to planktonic cells. The acidic environment was not an impediment to the formation of mature biofilms. In proteomics assays, different protein profiles were observed under the conditions tested, with 72 specially expressed by the biofilm when compared to planktonic growth. The vast majority of these proteins are related to cytoplasmic functions. Proteomic data from biofilm-derived cells also revealed an increase in proteins involved in stress, defense, virulence and energy acquisition and a reduction in proteins involved in protein synthesis and the metabolic process. In the proteome of infection by biofilm-derived cells, 6 unique proteins were identified compared to the control (biofilm), most classified as nuclear, allowing a partial understanding of the pathways associated with the adaptation and adaptation of the fungus to the host's environment. These results present advances in the field of biofilms and contribute to future studies that can prove the role of these communities in the host-pathogen interaction. Furthermore, they open up new possibilities for the study of targets in order to establish new therapeutic strategies for histoplasmosis.
Descrição
Palavras-chave
Histoplasma capsulatum, Biofilme, Macrófagos, Infecção, Proteoma, Biofilm, Macrophages, Infection, Proteome