The effect of Quorum sensing inhibitors on the evolution of CRISPR-based phage immunity in Pseudomonas aeruginosa

Abstract

Quorum sensing controls the expression of a wide range of important traits in the opportunistic pathogen Pseudomonas aeruginosa, including the expression of virulence genes and its CRISPR-cas immune system, which protects from bacteriophage (phage) infection. This finding has led to the speculation that synthetic quorum sensing inhibitors could be used to limit the evolution of CRISPR immunity during phage therapy. Here we use experimental evolution to explore if and how a quorum sensing inhibitor influences the population and evolutionary dynamics of P. aeruginosa upon phage DMS3vir infection. We find that chemical inhibition of quorum sensing decreases phage adsorption rates due to downregulation of the Type IV pilus, which causes delayed lysis of bacterial cultures and favours the evolution of CRISPR immunity. Our data therefore suggest that inhibiting quorum sensing may reduce rather than improve the therapeutic efficacy of pilus-specific phage, and this is likely a general feature when phage receptors are positively regulated by quorum sensing.

Fig. 1. Inhibiting QS results in reduced phage sensitivity and prolonged phage persistence at low phage densities.

Observed resistance mechanism evolution in P. aeruginosa PA14 WT or surface mutant (sm) supplemented with DMSO (dark grey bars/points) or 100 µM QS inhibitor Baicalein (light grey bars/points) and at 3 days post infection with (A) 10⁴ pfu (B) 10⁷ pfu (C) 10⁹ pfu of phage DMS3vir. Bars represent mean proportions of each of the different bacterial phenotypes (i.e., the proportion of bacteria in the population that evolved CRISPR immunity, acquired surface mutations or that remained sensitive), error bars indicate 95% confidence interval, points represent individual replicates, N = 6.

Fig. 2. Phage are less able to infect hosts when QS is inhibited.

A Phage adsorption over time (3–50 min) when WT P. aeruginosa is infected with phage when supplemented with DMSO (control) (dark grey line) or 100 µM QS inhibitor Baicalein (light grey line). Samples were taken at 3, 6, 9, 12, 15, 20, 25, 30, 40 and 50 min post infection. Lines represent mean % of free phage particles detected, N = 3 (B) Titre of phage remaining at 24hpi of a CRISPR resistant host possessing two unique spacers targeting the infecting phage (BIM2) when initially infected with 10⁹ phage. Bars represent mean titre of phage present at 24hpi, error bars indicate 95% confidence interval, points represent individual replicates, N = 6, Y axis is log¹⁰ scale.

Fig. 3. Inhibiting QS increases the fitness of P. aeruginosa in the presence of 10⁶ pfu of phage.

A Relative fitness of sensitive CRISPR-KO strain during competition with the phage resistant surface mutant (sm). B Relative fitness of BIM2 during competition with a phage resistant surface mutant (sm). C Relative fitness of BIM2 during competition with a sensitive CRISPR-KO strain. Box plots show the median, 25th and 75th percentile, and the interquartile range. Raw values from each replicate are shown as points, N = 6. Dotted line in panels (B and C) indicates the point at which competing strains are equally fit (when relative fitness = 1).