Publicação
The role of carboxylic acids in pathogenicity of Candida albicans
| Resumo: | In recent decades, fungal pathogens have become a global health problem. Candida albicans, a common opportunistic commensal microorganism, is the most frequent cause of systemic candidiasis and nosocomial bloodstream fungal infections. C. albicans can colonize various parts of the host under abnormal conditions, especially in immunocompromised patients. The main feature of C. albicans that has made it a serious pathogen is its ability to adapt to different host niches and utilize alternative carbon sources, like carboxylic acids, in glucose-poor settings, such as the colon and vagina. Yeast plasma membrane carboxylate transporters belonging to the AceTr Transporter family, known as Atos (Acetate Transporter Orthologs), seem to play a crucial role in the uptake of these substrates in C. albicans. Previous work identified the orthologs of the Saccharomyces cerevisiae Ato1 (Ady2) monocarboxylate transporter in several Candida species. In C. albicans, eight Ato orthologs were detected, whose functions are still unknown. The purpose of this thesis was to characterize the function and structure of selected Ato family members and Jen transporters in C. albicans by heterologous expression in S. cerevisiae. We constructed 5 plasmids by Gap repair containing selected ATO genes under the control of the GPD promoter. Additionally, using the CRISPR-Cas9 method, we generated C. albicans strains with GFP fusions at the 3’ terminal of ATO and JEN genes to investigate the expression and subcellular localization of the selected transporters. Fluorescence microscopy analyses revealed that CaAto1- GFP and CaAto2-GFP were localized at the plasma membrane in the presence of lactate and acetate, suggesting these transporters play an important role in the utilization of these substrates. Moreover, an in-silico analysis of Ato proteins was performed to determine their three dimensional structure and docking with substrates. We found that the NPAPLGL(M/S), and FLY regions are conserved in selected Atos. Additionally, molecular docking showed that all selected C. albicans Ato proteins have four acetate-binding sites. Also, a comparison of the predicted pore radius and 3D structure of CaAto proteins with ScAto1 suggests that CaAto1 and CaAto2 have the same pore size and structure as ScAto1, which may indicate their identical roles, whereas CaAto3, CaAto4, and CaAto5 were dissimilar and thus might present a different specificity. |
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| Autores principais: | Ghasemi, Faezeh |
| Assunto: | Alternative carbon sources Candida albicans Carboxylic acids Plasma membrane transporters Ácidos carboxílicos Fonte de carbono alternativas Transportadores de membrana plasmática |
| 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: | In recent decades, fungal pathogens have become a global health problem. Candida albicans, a common opportunistic commensal microorganism, is the most frequent cause of systemic candidiasis and nosocomial bloodstream fungal infections. C. albicans can colonize various parts of the host under abnormal conditions, especially in immunocompromised patients. The main feature of C. albicans that has made it a serious pathogen is its ability to adapt to different host niches and utilize alternative carbon sources, like carboxylic acids, in glucose-poor settings, such as the colon and vagina. Yeast plasma membrane carboxylate transporters belonging to the AceTr Transporter family, known as Atos (Acetate Transporter Orthologs), seem to play a crucial role in the uptake of these substrates in C. albicans. Previous work identified the orthologs of the Saccharomyces cerevisiae Ato1 (Ady2) monocarboxylate transporter in several Candida species. In C. albicans, eight Ato orthologs were detected, whose functions are still unknown. The purpose of this thesis was to characterize the function and structure of selected Ato family members and Jen transporters in C. albicans by heterologous expression in S. cerevisiae. We constructed 5 plasmids by Gap repair containing selected ATO genes under the control of the GPD promoter. Additionally, using the CRISPR-Cas9 method, we generated C. albicans strains with GFP fusions at the 3’ terminal of ATO and JEN genes to investigate the expression and subcellular localization of the selected transporters. Fluorescence microscopy analyses revealed that CaAto1- GFP and CaAto2-GFP were localized at the plasma membrane in the presence of lactate and acetate, suggesting these transporters play an important role in the utilization of these substrates. Moreover, an in-silico analysis of Ato proteins was performed to determine their three dimensional structure and docking with substrates. We found that the NPAPLGL(M/S), and FLY regions are conserved in selected Atos. Additionally, molecular docking showed that all selected C. albicans Ato proteins have four acetate-binding sites. Also, a comparison of the predicted pore radius and 3D structure of CaAto proteins with ScAto1 suggests that CaAto1 and CaAto2 have the same pore size and structure as ScAto1, which may indicate their identical roles, whereas CaAto3, CaAto4, and CaAto5 were dissimilar and thus might present a different specificity. |
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