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Determination of Acr-mediated immunosuppression in Pseudomonas aeruginosa
Bacteria have a broad array of defence mechanisms to fight bacteria-specific viruses (bacteriophages, phages) and other invading mobile genetic elements. Among those mechanisms, the ‘CRISPR-Cas’ (Clustered Regularly Interspaced Short Palindromic Repeats – CRISPR-associated) system keeps record of pr...
Autores principales: | , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Elsevier
2022
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9731895/ https://www.ncbi.nlm.nih.gov/pubmed/36504499 http://dx.doi.org/10.1016/j.mex.2022.101941 |
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author | Pons, Benoit J. Westra, Edze R. van Houte, Stineke |
author_facet | Pons, Benoit J. Westra, Edze R. van Houte, Stineke |
author_sort | Pons, Benoit J. |
collection | PubMed |
description | Bacteria have a broad array of defence mechanisms to fight bacteria-specific viruses (bacteriophages, phages) and other invading mobile genetic elements. Among those mechanisms, the ‘CRISPR-Cas’ (Clustered Regularly Interspaced Short Palindromic Repeats – CRISPR-associated) system keeps record of previous infections to prevent re-infection and thus provides acquired immunity. However, phages are not defenceless against CRISPR-based bacterial immunity. Indeed, they can escape CRISPR systems by encoding one or several anti-CRISPR (Acr) proteins. Acr proteins are among the earliest proteins produced upon phage infection, as they need to quickly inhibit CRISPR-Cas system before it can destroy phage genetic material. As a result, Acrs do not perfectly protect phage from the CRISPR-Cas system, and infection often fails. However, even if the infection fails, Acr can induce a lasting inactivation of the CRISPR-Cas system. The method presented here aims to assess the lasting CRISPR-Cas inhibition in Pseudomonas aeruginosa • Infecting the P. aeruginosa strain with a phage carrying an acr gene. • Making the cell electrocompetent while eliminating the phage; • Transforming the cells with a plasmid targeted by the CRISPR-Cas system and a non-targeted one to measure the relative transformation efficiency of the plasmids. This method can be adapted to measure which parameters influence Acr-induced immunosuppression in different culture conditions. |
format | Online Article Text |
id | pubmed-9731895 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2022 |
publisher | Elsevier |
record_format | MEDLINE/PubMed |
spelling | pubmed-97318952022-12-10 Determination of Acr-mediated immunosuppression in Pseudomonas aeruginosa Pons, Benoit J. Westra, Edze R. van Houte, Stineke MethodsX Method Article Bacteria have a broad array of defence mechanisms to fight bacteria-specific viruses (bacteriophages, phages) and other invading mobile genetic elements. Among those mechanisms, the ‘CRISPR-Cas’ (Clustered Regularly Interspaced Short Palindromic Repeats – CRISPR-associated) system keeps record of previous infections to prevent re-infection and thus provides acquired immunity. However, phages are not defenceless against CRISPR-based bacterial immunity. Indeed, they can escape CRISPR systems by encoding one or several anti-CRISPR (Acr) proteins. Acr proteins are among the earliest proteins produced upon phage infection, as they need to quickly inhibit CRISPR-Cas system before it can destroy phage genetic material. As a result, Acrs do not perfectly protect phage from the CRISPR-Cas system, and infection often fails. However, even if the infection fails, Acr can induce a lasting inactivation of the CRISPR-Cas system. The method presented here aims to assess the lasting CRISPR-Cas inhibition in Pseudomonas aeruginosa • Infecting the P. aeruginosa strain with a phage carrying an acr gene. • Making the cell electrocompetent while eliminating the phage; • Transforming the cells with a plasmid targeted by the CRISPR-Cas system and a non-targeted one to measure the relative transformation efficiency of the plasmids. This method can be adapted to measure which parameters influence Acr-induced immunosuppression in different culture conditions. Elsevier 2022-11-28 /pmc/articles/PMC9731895/ /pubmed/36504499 http://dx.doi.org/10.1016/j.mex.2022.101941 Text en © 2022 The Author(s) https://creativecommons.org/licenses/by/4.0/This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). |
spellingShingle | Method Article Pons, Benoit J. Westra, Edze R. van Houte, Stineke Determination of Acr-mediated immunosuppression in Pseudomonas aeruginosa |
title | Determination of Acr-mediated immunosuppression in Pseudomonas aeruginosa |
title_full | Determination of Acr-mediated immunosuppression in Pseudomonas aeruginosa |
title_fullStr | Determination of Acr-mediated immunosuppression in Pseudomonas aeruginosa |
title_full_unstemmed | Determination of Acr-mediated immunosuppression in Pseudomonas aeruginosa |
title_short | Determination of Acr-mediated immunosuppression in Pseudomonas aeruginosa |
title_sort | determination of acr-mediated immunosuppression in pseudomonas aeruginosa |
topic | Method Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9731895/ https://www.ncbi.nlm.nih.gov/pubmed/36504499 http://dx.doi.org/10.1016/j.mex.2022.101941 |
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