Publicação
Antisense therapy to control adhesion and filamentation of Candida albicans
| Resumo: | The incidence of fungal infections has increased significantly in the last years, contributing to the augment of morbidity and mortality in health care system. A raise in antimicrobial resistance, the number of immunosuppressive patients and the restricted number of antifungal drugs are the most common causes of these infections, where Candida species are the major responsible. The prevalence of these opportunistic infections, named candidiasis, has been attributed mostly to Candida albicans. Currently, conventional therapies, as antifungal drugs tend to be limitant in the control of these infections, and therefore antisense therapy (AST) arises as an alternative treatment. So, the main aim of this work was the development of a nano-drug, based in AST, for the control of virulence factors associated with Candida albicans infections. To accomplish the main goal, the C. albicans inter-strains variability concerning their ability to filament and adhere on abiotic surfaces, important virulence factors to be targeted by AST, was assessed. Then, an antisense oligonucleotide (ASO), capable of inhibiting the expression of HWP1 gene in C. albicans and reducing adhesion and filamentation was designed and optimized. The specificity and sensitivity of the probe was evaluated using C. albicans and other species by FISH assays. The ASO effects in the filamentation and adhesion was evaluated by microbiologic assays and the HWP1 expression levels determined by qRT-PCR. The results for inter-strains variability showed that strains SC5314 and 569322 had the highest capacity to adhere and filament and that 324LA/94 and 568426 were the strains with the lower capacity of filamentation. After the design of ASO and optimization of FISH conditions (37ºC as hybridization temperature, 4M of urea and 400 nM of ASO concentration), it was verified that the ASO was able to block 60% and 80% of the gene expression using concentrations of 40 nM and 200 nM, respectively, at 4h. On the other hand, the phenotype results demonstrated that the percentage of filamentation was only reduced by 3% and 6% for the 40 and 200 nM of ASO, respectively. However, the inclusion of urea, increased to 6 to 10% the reduction on filamentation. It was also possible to verify that the presence of urea inhibits the expression of this gene. In conclusion, it was demonstrated the possibility these nano-drugs, based in AST, as a novel strategy to control in future C. albicans virulence factors. |
|---|---|
| Autores principais: | Azevedo, Nuno Miguel Morais |
| Assunto: | Engenharia e Tecnologia::Outras Engenharias e Tecnologias |
| Ano: | 2017 |
| 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 has increased significantly in the last years, contributing to the augment of morbidity and mortality in health care system. A raise in antimicrobial resistance, the number of immunosuppressive patients and the restricted number of antifungal drugs are the most common causes of these infections, where Candida species are the major responsible. The prevalence of these opportunistic infections, named candidiasis, has been attributed mostly to Candida albicans. Currently, conventional therapies, as antifungal drugs tend to be limitant in the control of these infections, and therefore antisense therapy (AST) arises as an alternative treatment. So, the main aim of this work was the development of a nano-drug, based in AST, for the control of virulence factors associated with Candida albicans infections. To accomplish the main goal, the C. albicans inter-strains variability concerning their ability to filament and adhere on abiotic surfaces, important virulence factors to be targeted by AST, was assessed. Then, an antisense oligonucleotide (ASO), capable of inhibiting the expression of HWP1 gene in C. albicans and reducing adhesion and filamentation was designed and optimized. The specificity and sensitivity of the probe was evaluated using C. albicans and other species by FISH assays. The ASO effects in the filamentation and adhesion was evaluated by microbiologic assays and the HWP1 expression levels determined by qRT-PCR. The results for inter-strains variability showed that strains SC5314 and 569322 had the highest capacity to adhere and filament and that 324LA/94 and 568426 were the strains with the lower capacity of filamentation. After the design of ASO and optimization of FISH conditions (37ºC as hybridization temperature, 4M of urea and 400 nM of ASO concentration), it was verified that the ASO was able to block 60% and 80% of the gene expression using concentrations of 40 nM and 200 nM, respectively, at 4h. On the other hand, the phenotype results demonstrated that the percentage of filamentation was only reduced by 3% and 6% for the 40 and 200 nM of ASO, respectively. However, the inclusion of urea, increased to 6 to 10% the reduction on filamentation. It was also possible to verify that the presence of urea inhibits the expression of this gene. In conclusion, it was demonstrated the possibility these nano-drugs, based in AST, as a novel strategy to control in future C. albicans virulence factors. |
|---|