Avaliação da influência dos micronutrientes metálicos Ferro e Zinco na formação do biofilme e na virulência de Histoplasma capsulatum
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Data
2023-03-01
Autores
Pires, Ana Carolina Moreira da Silva
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Universidade Estadual Paulista (Unesp)
Resumo
Histoplasmose é uma doença fúngica ocasionada pela inalação de microconídios do fungo Histoplasma capsulatum presentes em solos contaminados. Após a inalação, para que o fungo se instale dentro dos macrófagos do hospedeiro são necessários mecanismos de virulência e adesão. Estudos mostram alguns mecanismos utilizados pelo fungo como a formação de biofilme e a utilização de adesinas. Mais recentemente, estudos têm mostrados a habilidade do fungo capturar íons metálicos essenciais que auxiliam na sua multiplicação no interior da célula. Dentre os íons requisitados, o ferro e o zinco são metais que influenciam em diversas atividades celulares de organismos eucarióticos. A partir disso, o objetivo do trabalho foi avaliar a influência dos íons metálicos ferro e zinco em células planctônicas, bem como na formação e nos mecanismos de virulência do biofilme de H. capsulatum. As cepas utilizadas para o trabalho foram a Panamense G186A (ATCC 26029) e a oriunda de morcego EH-315 (BAC1). Os quelantes TPEN e DTPA foram selecionados para mimetizar ambientes com escassez de íons, e as soluções de sulfato ferroso e sulfato de zinco para adição de mais íons ao meio HAM-F12. Os resultados mostraram que na presença dos quelantes tanto as células planctônicas quanto a formação do biofilme foram prejudicadas. Na presença do zinco, a cepa G186A teve um aumento na sua atividade metabólica e densidade celular no crescimento planctônico, ao contrário da EH-315 que não teve esses parâmetros alterados em relação ao crescimento controle. Na presença do ferro ambas as cepas apresentaram aumento de atividade metabólica e densidade celular. Em relação a formação do biofilme, na presença do zinco só a cepa G186A mostrou maior atividade metabólica, enquanto na presença do ferro ambas as cepas tiveram esse parâmetro aumentado. Não houve influência na formação de biomassa e da matriz extracelular para as duas cepas na presença dos íons ou dos quelantes. As microscopias eletrônicas de varredura e confocal mostraram que na presença dos íons foram formados biofilmes mais densos e robustos após 144 horas para ambas as cepas, sendo que com o ferro foram formados biofilmes predominantemente de hifas. O quelante TPEN, principalmente, inibiu a formação de aglomerados celulares tanto em 24 quanto em 144 horas de cultivo. Os ensaios com o corante congo red mostraram que na presença dos quelantes houve maior susceptibilidade das cepas ao corante, principalmente a cepa EH-315, indicando possíveis alterações na parede celular fúngica. Para análise da virulência, o modelo animal alternativo Galleria mellonella foi utilizado. Não houve maior sobrevivência das larvas infectadas após crescimento em meios com quelantes em comparação com as larvas infectadas com leveduras da condição controle. Entretanto, as larvas infectadas com as leveduras oriundas dos biofilmes inicial e maduro cultivados em meio com zinco e ferro da cepa G186A apresentaram menor sobrevivência. A cepa EH-315 não apresentou diferenças estatísticas na sobrevivência das larvas infectadas com leveduras oriundas dos meios com os íons. De maneira geral, os resultados trazem informações inéditas em relação a morfologia e a influência de íons em biofilmes do fungo Histoplasma spp. A adição dos íons metálicos influenciou na atividade metabólica e na viabilidade celular das cepas, principalmente para a cepa G186A. Os biofilmes mostraram maior robustez na presença de mais íons metálicos além de mudanças morfológicas em ambas as cepas. Além disso, a cepa G186A mostrou-se mais virulenta na presença dos íons indicando que os íons contribuem para seus mecanismos de virulência. O uso de quelantes podem contribuir para novas opções terapêuticas anti- H. capsulatum, visto que podem diminuir a viabilidade e a formação dos biofilmes do fungo.
Histoplasmosis is a fungal disease caused by the inhalation of microconidia of the fungus Histoplasma capsulatum present in contaminated soils. After inhalation, virulence and adhesion mechanisms are required for the fungus to install itself within the host’s macrophages. Studies show some mechanisms used by the fungus, such as biofilm formation and the use of adhesins. More recently, studies have shown the ability of the fungus to capture essential metal ions that help in its multiplication inside the cell. Among the required ions, iron and zinc are metals that influence various cellular activities of eukaryotic organisms. From this, the objective of this work was to evaluate the influence of iron and zinc in planktonic cells, as well as in the formation and mechanisms of virulence of the biofilm of H. capsulatum. The strains used for the work were the Panamanian G186A (ATCC 26029) and the bat strain EH-315 (BAC1). The TPEN and DTPA chelators were selected to mimic environments with ion scarcity, and the ferrous sulfate and zinc sulfate solutions to add more ions to the HAM-F12 medium. The results showed that in the presence of chelators, both planktonic cells and biofilm formation were impaired. In the presence of zinc, the G186A strain had an increase in its metabolic activity and cell density in planktonic growth, unlike EH-315, which did not have these parameters altered in relation to the control growth. In the presence of iron, both strains showed an increase in metabolic activity and cell density. Regarding biofilm formation, in the presence of zinc, only the G186A strain showed greater metabolic activity, while in the presence of iron, both strains had this parameter increased. There was no influence on the formation of biomass and extracellular matrix for both strains in the presence of ions or chelators. Scanning and confocal electron microscopy showed that in the presence of ions denser and more robust biofilms were formed after 144 hours for both strains, and with iron predominantly hyphal biofilms were formed. The chelator TPEN, mainly, inhibited the formation of cell clusters both in 24 and 144 hours of culture. Assays with the congo red dye showed that in the presence of chelators there was greater susceptibility of the strains to the dye, mainly the EH-315 strain, indicating possible alterations in the fungal cell wall. For virulence analysis, the alternative animal model Galleria mellonella was used. There was no greater survival of infected larvae after growth in chelating media compared to larvae infected with yeast in the control condition. However, larvae infected with yeasts from the initial and mature biofilms grown in a medium with zinc and iron of the G186A strain showed lower survival. The EH-315 strain did not show statistical differences in the survival of larvae infected with yeasts from the media with ions. In general, the results bring unprecedented information regarding the morphology and influence of ions in biofilms of the fungus Histoplasma spp. The addition of metal ions influenced the metabolic activity and cell viability of the strains, mainly for the G186A strain. Biofilms showed greater robustness in the presence of more metal ions in addition to morphological changes in both strains. Furthermore, the G186A strain was more virulent in the presence of ions, indicating that ions contribute to its virulence mechanisms. The use of binders can contribute to new anti-H. capsulatum therapeutic options, as they can decrease the fungus's viability and biofilm formation.
Histoplasmosis is a fungal disease caused by the inhalation of microconidia of the fungus Histoplasma capsulatum present in contaminated soils. After inhalation, virulence and adhesion mechanisms are required for the fungus to install itself within the host’s macrophages. Studies show some mechanisms used by the fungus, such as biofilm formation and the use of adhesins. More recently, studies have shown the ability of the fungus to capture essential metal ions that help in its multiplication inside the cell. Among the required ions, iron and zinc are metals that influence various cellular activities of eukaryotic organisms. From this, the objective of this work was to evaluate the influence of iron and zinc in planktonic cells, as well as in the formation and mechanisms of virulence of the biofilm of H. capsulatum. The strains used for the work were the Panamanian G186A (ATCC 26029) and the bat strain EH-315 (BAC1). The TPEN and DTPA chelators were selected to mimic environments with ion scarcity, and the ferrous sulfate and zinc sulfate solutions to add more ions to the HAM-F12 medium. The results showed that in the presence of chelators, both planktonic cells and biofilm formation were impaired. In the presence of zinc, the G186A strain had an increase in its metabolic activity and cell density in planktonic growth, unlike EH-315, which did not have these parameters altered in relation to the control growth. In the presence of iron, both strains showed an increase in metabolic activity and cell density. Regarding biofilm formation, in the presence of zinc, only the G186A strain showed greater metabolic activity, while in the presence of iron, both strains had this parameter increased. There was no influence on the formation of biomass and extracellular matrix for both strains in the presence of ions or chelators. Scanning and confocal electron microscopy showed that in the presence of ions denser and more robust biofilms were formed after 144 hours for both strains, and with iron predominantly hyphal biofilms were formed. The chelator TPEN, mainly, inhibited the formation of cell clusters both in 24 and 144 hours of culture. Assays with the congo red dye showed that in the presence of chelators there was greater susceptibility of the strains to the dye, mainly the EH-315 strain, indicating possible alterations in the fungal cell wall. For virulence analysis, the alternative animal model Galleria mellonella was used. There was no greater survival of infected larvae after growth in chelating media compared to larvae infected with yeast in the control condition. However, larvae infected with yeasts from the initial and mature biofilms grown in a medium with zinc and iron of the G186A strain showed lower survival. The EH-315 strain did not show statistical differences in the survival of larvae infected with yeasts from the media with ions. In general, the results bring unprecedented information regarding the morphology and influence of ions in biofilms of the fungus Histoplasma spp. The addition of metal ions influenced the metabolic activity and cell viability of the strains, mainly for the G186A strain. Biofilms showed greater robustness in the presence of more metal ions in addition to morphological changes in both strains. Furthermore, the G186A strain was more virulent in the presence of ions, indicating that ions contribute to its virulence mechanisms. The use of binders can contribute to new anti-H. capsulatum therapeutic options, as they can decrease the fungus's viability and biofilm formation.
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Palavras-chave
Histoplasma capsulatum, biofilme, ferro, zinco