Publicação
Application of antisense therapy to control Candida glabrata biofilms
| Resumo: | The incidence of fungal infections, commonly caused by Candida species, has increased in recent decades and is associated with high morbidity and mortality rates. Candida glabrata is considered the second most isolated Candida species and its virulence is related to its resistance to antifungal agents and its ability to form biofilms. Conventional therapies are limited in the treatment of these infections, so it is important to develop alternative therapies. Therefore, based on antisense thecnology, the main aim of this work was to validate the use of antisense oligonucleotides (ASOs) as therapeutic molecules to control C. glabrata biofilm formation. Indeed, three genes involved in C. glabrata adhesion (PDR1, EPA3 and EPA6) were selected and ASOs were designed and synthesized with LNA modification to hybridize with the corresponding mRNA to degrade it and thus block its conversion into protein. The effect of pH (pH 4 and pH 7) on gene expression and on the adhesion ability of C. glabrata was evaluated. It was observed that the pH did not affect the adhesion of C. glabrata, although there was an increase in the level of gene expression, in particular of PDR1 and EPA6, at pH 7. No cytotoxicity of ASOs was observed in vitro. Therefore, the effect of ASOs was evaluated in terms of adhesion reduction and gene expression inhibition. At pH 4, it was observed that only anti-EPA3 and anti-EPA6 ASOs, at a concentration of 100 nM, were able to reduce cell adhesion and gene expression. At pH 7, however, none of the ASOs were able to reduce cell adhesion, but they were able to significantly reduce gene expression for all genes. In vivo, using the Galleria mellonella model, no toxicity was observed and although there was no significant improvement in the survival of larvae infected with C. glabrata with the administration of anti-EPA3 and anti-EPA6 ASOs, a 20% increase was observed with the application of a single dose of anti-PDR1 ASO. In conclusion, the possibility of using ASOs as a new strategy to control C. glabrata virulence factors and consequently C. glabrata infections has been demonstrated. |
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| Autores principais: | Ribeiro, Tânia Coelho |
| Assunto: | Antisense Oligonucleotides Candidiasis Galleria mellonella Virulence factor Candidíase Fatores de virulência Oligonucleótidos Antisense Ciências Naturais::Ciências Biológicas |
| 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: | The incidence of fungal infections, commonly caused by Candida species, has increased in recent decades and is associated with high morbidity and mortality rates. Candida glabrata is considered the second most isolated Candida species and its virulence is related to its resistance to antifungal agents and its ability to form biofilms. Conventional therapies are limited in the treatment of these infections, so it is important to develop alternative therapies. Therefore, based on antisense thecnology, the main aim of this work was to validate the use of antisense oligonucleotides (ASOs) as therapeutic molecules to control C. glabrata biofilm formation. Indeed, three genes involved in C. glabrata adhesion (PDR1, EPA3 and EPA6) were selected and ASOs were designed and synthesized with LNA modification to hybridize with the corresponding mRNA to degrade it and thus block its conversion into protein. The effect of pH (pH 4 and pH 7) on gene expression and on the adhesion ability of C. glabrata was evaluated. It was observed that the pH did not affect the adhesion of C. glabrata, although there was an increase in the level of gene expression, in particular of PDR1 and EPA6, at pH 7. No cytotoxicity of ASOs was observed in vitro. Therefore, the effect of ASOs was evaluated in terms of adhesion reduction and gene expression inhibition. At pH 4, it was observed that only anti-EPA3 and anti-EPA6 ASOs, at a concentration of 100 nM, were able to reduce cell adhesion and gene expression. At pH 7, however, none of the ASOs were able to reduce cell adhesion, but they were able to significantly reduce gene expression for all genes. In vivo, using the Galleria mellonella model, no toxicity was observed and although there was no significant improvement in the survival of larvae infected with C. glabrata with the administration of anti-EPA3 and anti-EPA6 ASOs, a 20% increase was observed with the application of a single dose of anti-PDR1 ASO. In conclusion, the possibility of using ASOs as a new strategy to control C. glabrata virulence factors and consequently C. glabrata infections has been demonstrated. |
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