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


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          The cell–cell communication process, called quorum sensing, activates all three key aspects of the prokaryotic adaptive immune system (termed CRISPR-Cas): expression, activity, and adaptation in the pathogen Pseudomonas aeruginosa. We show that pro- and antiquorum-sensing compounds activate and repress CRISPR-Cas, respectively, suggesting the exciting possibility of a combination quorum-sensing–inhibition-phage therapy cocktail. In P. aeruginosa, quorum-sensing inhibitors repress virulence, making P. aeruginosa more susceptible to elimination by the human immune system, while simultaneously making P. aeruginosa more prone to killing by phage therapy through inhibition of the CRISPR-Cas defense mechanism. Finally, because we show that quorum sensing activates adaptation by the CRISPR-Cas immune system, a quorum-sensing inhibitor should also reduce acquisition of resistance against the administered phage.


          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.

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          Most cited references36

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          CRISPR interference limits horizontal gene transfer in staphylococci by targeting DNA.

          Horizontal gene transfer (HGT) in bacteria and archaea occurs through phage transduction, transformation, or conjugation, and the latter is particularly important for the spread of antibiotic resistance. Clustered, regularly interspaced, short palindromic repeat (CRISPR) loci confer sequence-directed immunity against phages. A clinical isolate of Staphylococcus epidermidis harbors a CRISPR spacer that matches the nickase gene present in nearly all staphylococcal conjugative plasmids. Here we show that CRISPR interference prevents conjugation and plasmid transformation in S. epidermidis. Insertion of a self-splicing intron into nickase blocks interference despite the reconstitution of the target sequence in the spliced mRNA, which indicates that the interference machinery targets DNA directly. We conclude that CRISPR loci counteract multiple routes of HGT and can limit the spread of antibiotic resistance in pathogenic bacteria.
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            Unravelling the structural and mechanistic basis of CRISPR-Cas systems.

            Bacteria and archaea have evolved sophisticated adaptive immune systems, known as CRISPR-Cas (clustered regularly interspaced short palindromic repeats-CRISPR-associated proteins) systems, which target and inactivate invading viruses and plasmids. Immunity is acquired by integrating short fragments of foreign DNA into CRISPR loci, and following transcription and processing of these loci, the CRISPR RNAs (crRNAs) guide the Cas proteins to complementary invading nucleic acid, which results in target interference. In this Review, we summarize the recent structural and biochemical insights that have been gained for the three major types of CRISPR-Cas systems, which together provide a detailed molecular understanding of the unique and conserved mechanisms of RNA-guided adaptive immunity in bacteria and archaea.
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              Molecular memory of prior infections activates the CRISPR/Cas adaptive bacterial immunity system.

              CRISPR/Cas (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated genes) is a small RNA-based adaptive prokaryotic immunity system that functions by acquisition of short fragments of DNA (mainly from foreign invaders such as viruses and plasmids) and subsequent destruction of DNA with sequences matching acquired fragments. Some mutations in foreign DNA that affect the match prevent CRISPR/Cas defensive function. Here we show that matching sequences that are no longer able to elicit defense, still guide the CRISPR/Cas acquisition machinery to foreign DNA, thus making the spacer acquisition process adaptive and leading to restoration of CRISPR/Cas-mediated protection. We present evidence suggesting that after initial recognition of partially matching foreign DNA, the CRISPR/Cas acquisition machinery moves along the DNA molecule, occasionally selecting fragments to be incorporated into the CRISPR locus. Our results explain how adaptive CRISPR/Cas immunity becomes specifically directed towards foreign DNA, allowing bacteria to efficiently counter individual viral mutants that avoid CRISPR/Cas defense.

                Author and article information

                Proc Natl Acad Sci U S A
                Proc. Natl. Acad. Sci. U.S.A
                Proceedings of the National Academy of Sciences of the United States of America
                National Academy of Sciences
                3 January 2017
                14 November 2016
                : 114
                : 1
                : 131-135
                [1] aDepartment of Molecular Biology, Princeton University , Princeton, NJ 08544;
                [2] bBiosciences, Environment and Sustainability Institute, University of Exeter , Penryn TR10 9FE, United Kingdom;
                [3] cDepartment of Microbiology and Immunology, University of California, San Francisco , CA 94158;
                [4] d Howard Hughes Medical Institute , Chevy Chase, MD 20815
                Author notes
                1To whom correspondence should be addressed. Email: bbassler@ 123456princeton.edu .

                Contributed by Bonnie L. Bassler, October 20, 2016 (sent for review September 22, 2016; reviewed by Magnus Lundgren and Luciano Marraffini)

                Author contributions: N.M.H.-K., J.P., S.M., and B.L.B. designed research; N.M.H.-K., J.P., and S.M. performed research; N.M.H.-K., J.P., and S.M. contributed new reagents/analytic tools; N.M.H.-K., J.P., S.M., J.B., E.W., J.B.-D., and B.L.B. analyzed data; and N.M.H.-K. and B.L.B. wrote the paper.

                Reviewers: M.L., Uppsala University; and L.M., The Rockefeller University.

                PMC5224376 PMC5224376 5224376 201617415
                Page count
                Pages: 5
                Funded by: HHMI
                Award ID: Na
                Funded by: HHS | NIH | National Institute of General Medical Sciences (NIGMS) 100000057
                Award ID: 2R37GM065859
                Funded by: NSF | BIO | Division of Molecular and Cellular Biosciences (MCB) 100000152
                Award ID: MCB-0343821
                Funded by: Danish National Research Foundation (Danmarks Grundforskningsfond) 501100001732
                Award ID: DFF-4090-00265
                Funded by: Jane Coffin Childs Memorial Fund for Medical Research 100001033
                Award ID: NA
                Funded by: Wellcome Trust 100004440
                Award ID: NA
                Funded by: HHS | National Institutes of Health (NIH) 100000002
                Award ID: DP5-OD021344
                Biological Sciences
                From the Cover

                phage defense,phage,immunity,CRISPR,quorum sensing
                phage defense, phage, immunity, CRISPR, quorum sensing


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