Putative gene regulatory functions of Csy1, a component of the CRISPR-Cas adaptive immune system, in the opportunistic pathogen Pseudomonas aeruginosa. (360G-Wellcome-109776_Z_15_Z)

It is becoming increasingly clear that CRISPR-Cas (Clustered Regularly Interspaced Short Palindromic Repeats; CRISPR associated) systems function beyond adaptive immunity. Preliminary data show that CRISPR-Cas deletion from Pseudomonas aeruginosa reduces both in vitro fitness and in vivo virulence. These phenotypes are caused by Csy1, a Cas protein thought to be involved in sequence-specific binding of the 5'-handle of crRNA. We hypothesise that Csy1 regulates expression of genes carrying sequen ce motifs similar to the 5'-handle. We will test this hypothesis by (1) transcriptome analysis of WT and Csy1 deletion mutants, (2) RIP-seq analysis of RNA co-purifying with Csy1 and (3) EMSA to measure sequence specificity of Csy1-RNA interactions. This project will reveal whether Csy1 of the P. aeruginosa CRISPR-Cas system is involved in a gene regulation function and will identify specific RNA motifs targeted by Csy1. If successful, the data from this seed project will serve as a basis for a full grant proposal aimed at (1) validating the Csy1-motif interactions in vivo (2) generalising Csy1-mediated gene regulation across P. aeruginosa strains and (3) testing the impact of Csy1 on P. aeruginosa virulence in other, more relevant, infection models, such as cell culture and thermally injured mouse infection models.