Publicação
Fluorescent tag Sporothrix brasiliensis: a tool for host-pathogen interaction studies
| Resumo: | Sporotrichosis is considered an emerging health problem, being the world's most prevalent subcutaneous mycosis, occurring on mammal hosts, which facilitates zoonotic transmission. Agrobacterium tumefaciens-mediated transformation (ATMT) is a broadly technique used for both plants and fungus transformation. Here, we report the establishing of an ATMT system for S. brasiliensis, considering different strains of A. tumefaciens and S. brasiliensis and several conditions of co-cultivation. Our results point to 72h of co-cultivation at 26°C, the AGL-1 bacterial strain, and the 2:1 ratio (bacteria:fungi) as ideal conditions for a high number of transformants. In these conditions, we obtained 3179 ± 1171 mutants/co-cultivation. PCR analysis showed that all Hygromycin B resistant clones tested harboured a copy of the HPH gene. FACS analysis showed high mitotic stability of the GFP gene in pGAPDH::GFP mutants. The S. brasiliensis pGAPDH::GFP and pGAPDH::H2A::GFP strains were respectively used to evaluate the phagocytosis index and fungicidal activity,. pGAPDH::GFP cytoplasm expression of GFP allowed a strong fluorescence visualization inside monocytes/macrophages in both microscopic and FACS analysis. The phagocytosis index was 64.25 ± 9.96% at the MOI of one peripheral blood mononuclear cell (PBMC) to five pGAPDH::GFP yeast cells, with two hours of infection. The pGAPDH::H2A::GFP S. brasiliensis strain failed to provide a correlation between loss of fungal viability or fungal death and loss of GFP fluorescence in all fungicidal experiments performed. Furthermore, its GFP fluorescence was not visible upon monocytes/macrophages engulfment in both microscopic and FACS analysis. Our results showed, firstly, an efficient genetic toolbox to create large-scale transformant libraries for S. brasiliensis, and secondly, the possibility to use the pGAPDH::GFP strain in phagocytosis assays without fluorophore stain prior to infection. Together, these results can help provide new insights to better understand the host-pathogen interactions and virulence mechanism for the Sporothrix spp.. |
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| Autores principais: | Sousa Filho, Jorge Carlos Dias de |
| Assunto: | ATMT Fluorescent tag strains Sporotrichosis Sporothrix brasiliensis Esporotricose Marcadores fluorescentes Ciências Médicas::Ciências da Saúde |
| Ano: | 2023 |
| País: | Portugal |
| Tipo de documento: | dissertação de mestrado |
| Tipo de acesso: | acesso aberto |
| Instituição associada: | Universidade do Minho |
| Idioma: | inglês |
| Origem: | RepositóriUM - Universidade do Minho |
| Resumo: | Sporotrichosis is considered an emerging health problem, being the world's most prevalent subcutaneous mycosis, occurring on mammal hosts, which facilitates zoonotic transmission. Agrobacterium tumefaciens-mediated transformation (ATMT) is a broadly technique used for both plants and fungus transformation. Here, we report the establishing of an ATMT system for S. brasiliensis, considering different strains of A. tumefaciens and S. brasiliensis and several conditions of co-cultivation. Our results point to 72h of co-cultivation at 26°C, the AGL-1 bacterial strain, and the 2:1 ratio (bacteria:fungi) as ideal conditions for a high number of transformants. In these conditions, we obtained 3179 ± 1171 mutants/co-cultivation. PCR analysis showed that all Hygromycin B resistant clones tested harboured a copy of the HPH gene. FACS analysis showed high mitotic stability of the GFP gene in pGAPDH::GFP mutants. The S. brasiliensis pGAPDH::GFP and pGAPDH::H2A::GFP strains were respectively used to evaluate the phagocytosis index and fungicidal activity,. pGAPDH::GFP cytoplasm expression of GFP allowed a strong fluorescence visualization inside monocytes/macrophages in both microscopic and FACS analysis. The phagocytosis index was 64.25 ± 9.96% at the MOI of one peripheral blood mononuclear cell (PBMC) to five pGAPDH::GFP yeast cells, with two hours of infection. The pGAPDH::H2A::GFP S. brasiliensis strain failed to provide a correlation between loss of fungal viability or fungal death and loss of GFP fluorescence in all fungicidal experiments performed. Furthermore, its GFP fluorescence was not visible upon monocytes/macrophages engulfment in both microscopic and FACS analysis. Our results showed, firstly, an efficient genetic toolbox to create large-scale transformant libraries for S. brasiliensis, and secondly, the possibility to use the pGAPDH::GFP strain in phagocytosis assays without fluorophore stain prior to infection. Together, these results can help provide new insights to better understand the host-pathogen interactions and virulence mechanism for the Sporothrix spp.. |
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