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...
| Main Authors: | , , |
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| Format: | Article |
| Language: | English |
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Elsevier
2023-01-01
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| Series: | MethodsX |
| Subjects: | |
| Online Access: | http://www.sciencedirect.com/science/article/pii/S2215016122003181 |
| _version_ | 1827917788096757760 |
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| author | Benoit J. Pons Edze R. Westra Stineke van Houte |
| author_facet | Benoit J. Pons Edze R. Westra Stineke van Houte |
| author_sort | Benoit J. Pons |
| collection | DOAJ |
| 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 induced by Acr proteins by: • 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. |
| first_indexed | 2024-03-13T03:33:27Z |
| format | Article |
| id | doaj.art-757e9f7dc9ff43dd9f4d319591514b79 |
| institution | Directory Open Access Journal |
| issn | 2215-0161 |
| language | English |
| last_indexed | 2024-03-13T03:33:27Z |
| publishDate | 2023-01-01 |
| publisher | Elsevier |
| record_format | Article |
| series | MethodsX |
| spelling | doaj.art-757e9f7dc9ff43dd9f4d319591514b792023-06-24T05:16:45ZengElsevierMethodsX2215-01612023-01-0110101941Determination of Acr-mediated immunosuppression in Pseudomonas aeruginosaBenoit J. Pons0Edze R. Westra1Stineke van Houte2Corresponding author.; Environment and Sustainability Institute, Biosciences, University of Exeter, Penryn, UKEnvironment and Sustainability Institute, Biosciences, University of Exeter, Penryn, UKEnvironment and Sustainability Institute, Biosciences, University of Exeter, Penryn, UKBacteria 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 induced by Acr proteins by: • 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.http://www.sciencedirect.com/science/article/pii/S2215016122003181Determination of Acr-mediated immunosuppression in Pseudomonas aeruginosa |
| spellingShingle | Benoit J. Pons Edze R. Westra Stineke van Houte Determination of Acr-mediated immunosuppression in Pseudomonas aeruginosa MethodsX 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 | Determination of Acr-mediated immunosuppression in Pseudomonas aeruginosa |
| url | http://www.sciencedirect.com/science/article/pii/S2215016122003181 |
| work_keys_str_mv | AT benoitjpons determinationofacrmediatedimmunosuppressioninpseudomonasaeruginosa AT edzerwestra determinationofacrmediatedimmunosuppressioninpseudomonasaeruginosa AT stinekevanhoute determinationofacrmediatedimmunosuppressioninpseudomonasaeruginosa |