Quorum sensing controls the Pseudomonas aeruginosa CRISPR-Cas adaptive immune system

Abstract

CRISPR-Cas are prokaryotic adaptive immune systems that provide protection against bacteriophage (phage) and other parasites. Little is known about how CRISPR-Cas systems are regulated, preventing prediction of phage dynamics in nature and manipulation of phage resistance in clinical settings. Here, we show that the bacterium Pseudomonas aeruginosa PA14 uses the cell-cell communication process, called quorum sensing, to activate cas gene expression, to increase CRISPR-Cas targeting of foreign DNA, and to promote CRISPR adaptation, all at high cell density. This regulatory mechanism ensures maximum CRISPR-Cas function when bacterial populations are at highest risk for phage infection. We demonstrate that CRISPR-Cas activity and acquisition of resistance can be modulated by administration of pro- and antiquorum-sensing compounds. We propose that quorum-sensing inhibitors could be used to suppress the CRISPR-Cas adaptive immune system to enhance medical applications, including phage therapies.

Keywords: CRISPR; immunity; phage; phage defense; quorum sensing.

Fig. 1.

QS activates cas3 expression. (A) Relative cas3 expression normalized to 5S RNA measured by qRT-PCR in PA14 at low and high cell density (OD₆₀₀ = 0.1 and 1.0, respectively). (B) Relative cas3 expression at high cell density measured as in A for PA14 (WT) and the designated QS mutants. AI indicates 2 µM 3OC12-HSL + 10 µM C4-HSL. Error bars represent SD from n = 3 replicates (A) and n = 6 replicates (B).

Fig. S1.

QS activates cas and csy expression. Relative expression of cas1, cas3, and csy1–4. Relative expression of all cas and csy genes normalized to 5S RNA measured by qRT-PCR in WT PA14 (black) and the ∆lasI ∆rhlI double-AI synthase mutant (white). Strains were grown to OD₆₀₀ = 1. Error bars represent SD from n = 3 replicates.

Fig. 2.

QS regulates CRISPR-Cas activity. Retention of the control plasmid pHERD30T (A) and the CRISPR-targeted plasmid pCR2SP1 (B) in WT and in the ∆lasI ∆rhlI mutant during growth (100% denotes no plasmid loss). (C) EOT of WT PA14 and designated mutants at high cell density (OD₆₀₀ = 1) quantified as the percentage transformation by the CRISPR-targeted plasmid pCR2SP1 compared with that of the parent vector pHERD30T lacking the targeted sequence (23). Here, 100% denotes an EOT ratio of 1 for the two plasmids. In all panels, AI indicates 2 µM 3OC12-HSL + 10 µM C4-HSL. Error bars denote SD from n = 3 replicates.

Fig. S2.

Sequencing of the CRISPR-targeted protospacer and PAM in pCR2SP1. WT and ∆lasI ∆rhlI mutant cells were transformed with the CRISPR-targeted plasmid pCR2SP1 at OD₆₀₀ = 1, and colonies were allowed to form on selective medium. The region in pCR2SP1 encoding the targeted protospacer and PAM was sequenced in 10 WT and 10 ∆lasI ∆rhlI colonies. No mutations were discovered in the protospacer or PAM in any of the 20 plasmids. A representative sequencing result from a WT colony is shown. The protospacer sequence targeted by CRISPR 2 spacer 1 is underlined and the GG PAM sequence is highlighted in bold.

Fig. 3.

QS controls CRISPR-Cas–mediated immunity by increasing spacer acquisition. Integration of new CRISPR spacers into the CRISPR2 locus was measured by PCR of single colonies of WT PA14, the ∆cas3 mutant, and the ∆lasI ∆rhlI mutant. Each of the strains harbored the CRISPR-targeted plasmid, pCR2SP1 seed, containing a seed mutation to promote adaptation. Each adaptation event results in acquisition of a new spacer and CRISPR repeat, which is exhibited by a 60-bp expansion of the CRISPR locus. Quantitation of the spacer population present in each colony is shown below each lane of the gel. Data are shown for representative colonies. AI indicates 2 µM 3OC12-HSL + 10 µM C4-HSL and inhibitor indicates 100 µM Baicalein (36).

Fig. S3.

The QS inhibitor Baicalein represses pyocyanin production and cas3 expression in PA14. (A) Relative pyocyanin production measured at OD₆₉₅ in PA14 treated with the indicated concentrations of the QS inhibitor Baicalein normalized to the pyocyanin levels from PA14 treated with DMSO. (B) Relative cas3 expression normalized to 5S RNA measured by qRT-PCR in the ∆lasI ∆rhlI mutant at high cell density (OD₆₀₀ = 1.0). Additions: DMSO (control), 2 µM 3OC12-HSL + 10 µM C4-HSL (AI), or 2 µM 3OC12-HSL + 10 µM C4-HSL + 100 µM Baicalein (AI + inhibitor) (36). Error bars denote SD from n = 3 replicates.