Determination of Acr-mediated immunosuppression in Pseudomonas aeruginosa

• Published in: MethodsX Vol. 10; p. 101941
• Main Authors: Pons, Benoit J., Westra, Edze R., van Houte, Stineke
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01-01-2023; Elsevier
• Subjects: Anti-CRISPR; CRISPR-Cas; Determination of Acr-mediated immunosuppression in Pseudomonas aeruginosa; Method; Transformation efficiency; CRISPR-Cas; Anti-CRISPR; Determination of Acr-mediated immunosuppression in Pseudomonas aeruginosa; Transformation efficiency
• ISSN: 2215-0161; 2215-0161
• DOI: 10.1016/j.mex.2022.101941

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.
[Display omitted]