Publicação
Selection of specific aptamers for mucoid Pseudomonas aeruginosa biofilms developed in the airways of cystic fibrosis patients
| Resumo: | Chronic infections are one of the main contributors for decline of the lung function, respiratory failure and premature death of cystic fibrosis (CF) patients. Pseudomonas aeruginosa is an opportunistic pathogen that commonly infects CF lungs and recognized as the main cause of chronic infections due to its ability of biofilm formation. Being associated with concerning mortality rates, a rapid detection and identification is necessary in order to perform an appropriate antimicrobial stewardship. Aptamers have emerged as promising biorecognition elements, constituting an innovative tool for a rapid, sensitive and specific identification of targets, which include the microorganisms that grow in biofilms. In this study, we aimed at selecting and characterizing, for the first time, specific aptamers for P. aeruginosa grown in biofilms, in the context of CF disease, to significantly improve the early diagnosis of infections and the effectiveness of current antimicrobial therapies. The cell-systematic evolution of ligands by exponential enrichment (cell-SELEX) technique was used to select DNA aptamers against mucoid P. aeruginosa, a distinguishing feature of bacteria grown in CF lungs, for a total of ten cycles. Three cycles constituted counter selection rounds with Staphylococcus aureus as a non-target cell, the second most common pathogen, with the specific purpose to ensure the recognition of P. aeruginosa target in the context of CF disease. Binding assays were performed to determine the affinity between aptamers and the target bacteria and to monitor the enrichment of specific aptamers throughout the selection process by analysing the aptamer-associated fluorescence through flow cytometry (protocol optimised for this study) and enzyme-linked immunosorbent assay (ELISA). The results indicated that the fluorescence signal with P. aeruginosa target was gradually increased after incubation with the enriched ssDNA pools. The most enriched pool was sent to sequencing through Illumina MiSeg NGS to identify the most promising ssDNA aptamer candidates against the P. aeruginosa target, aided by bioinformatic tools as Geneious. Overall, SELEX was a valuable tool for the identification of novel targeting aptamers even for pathogens grown in biofilms. |
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| Autores principais: | Carvalho, Maria Beatriz |
| Outros Autores: | Rodrigues, L. R.; Pereira, Maria Olívia; Ferreira, Débora; Sousa, Ana Margarida |
| Ano: | 2023 |
| País: | Portugal |
| Tipo de documento: | outro |
| Tipo de acesso: | acesso restrito |
| Instituição associada: | Universidade do Minho |
| Idioma: | inglês |
| Origem: | RepositóriUM - Universidade do Minho |
| Resumo: | Chronic infections are one of the main contributors for decline of the lung function, respiratory failure and premature death of cystic fibrosis (CF) patients. Pseudomonas aeruginosa is an opportunistic pathogen that commonly infects CF lungs and recognized as the main cause of chronic infections due to its ability of biofilm formation. Being associated with concerning mortality rates, a rapid detection and identification is necessary in order to perform an appropriate antimicrobial stewardship. Aptamers have emerged as promising biorecognition elements, constituting an innovative tool for a rapid, sensitive and specific identification of targets, which include the microorganisms that grow in biofilms. In this study, we aimed at selecting and characterizing, for the first time, specific aptamers for P. aeruginosa grown in biofilms, in the context of CF disease, to significantly improve the early diagnosis of infections and the effectiveness of current antimicrobial therapies. The cell-systematic evolution of ligands by exponential enrichment (cell-SELEX) technique was used to select DNA aptamers against mucoid P. aeruginosa, a distinguishing feature of bacteria grown in CF lungs, for a total of ten cycles. Three cycles constituted counter selection rounds with Staphylococcus aureus as a non-target cell, the second most common pathogen, with the specific purpose to ensure the recognition of P. aeruginosa target in the context of CF disease. Binding assays were performed to determine the affinity between aptamers and the target bacteria and to monitor the enrichment of specific aptamers throughout the selection process by analysing the aptamer-associated fluorescence through flow cytometry (protocol optimised for this study) and enzyme-linked immunosorbent assay (ELISA). The results indicated that the fluorescence signal with P. aeruginosa target was gradually increased after incubation with the enriched ssDNA pools. The most enriched pool was sent to sequencing through Illumina MiSeg NGS to identify the most promising ssDNA aptamer candidates against the P. aeruginosa target, aided by bioinformatic tools as Geneious. Overall, SELEX was a valuable tool for the identification of novel targeting aptamers even for pathogens grown in biofilms. |
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