ABSTRACT
Immune responses need to be regulated to prevent autoimmunity. CRISPR-Cas systems provide adaptive immunity in prokaryotes through the acquisition of short DNA sequences from invading viruses (bacteriophages), known as spacers. Spacers are inserted into the CRISPR locus and serve as templates for the transcription of guides used by RNA-guided nucleases to recognize complementary nucleic acids of the invaders and start the CRISPR immune response. In type II-A CRISPR systems, Cas9 uses the guide RNA to cleave target DNA sequences in the genome of infecting phages, and the tracrRNA to bind the promoter of cas genes and repress their transcription. We previously isolated a Cas9 mutant carrying the I473F substitution that increased the frequency of spacer acquisition by 2-3 orders of magnitude, leading to a fitness cost due to higher levels of autoimmunity. Here, we investigated the molecular basis underlying these findings. We found that the I473F mutation decreases the association of Cas9 to tracrRNA, limiting its repressor function, leading to high levels of expression of cas genes, which in turn increase the strength of the type II-A CRISPR-Cas immune response. We obtained similar results for a related type II-A system, and therefore our findings highlight the importance of the interaction between Cas9 and its tracrRNA cofactor in tuning the immune response to balanced levels that enable phage defense but avoid autoimmunity.
ABSTRACT
Bacteria have adapted to phage predation by evolving a vast assortment of defence systems1. Although anti-phage immunity genes can be identified using bioinformatic tools, the discovery of novel systems is restricted to the available prokaryotic sequence data2. Here, to overcome this limitation, we infected Escherichia coli carrying a soil metagenomic DNA library3 with the lytic coliphage T4 to isolate clones carrying protective genes. Following this approach, we identified Brig1, a DNA glycosylase that excises α-glucosyl-hydroxymethylcytosine nucleobases from the bacteriophage T4 genome to generate abasic sites and inhibit viral replication. Brig1 homologues that provide immunity against T-even phages are present in multiple phage defence loci across distinct clades of bacteria. Our study highlights the benefits of screening unsequenced DNA and reveals prokaryotic DNA glycosylases as important players in the bacteria-phage arms race.
Subject(s)
Bacteria , Bacteriophage T4 , DNA Glycosylases , Bacteria/classification , Bacteria/enzymology , Bacteria/genetics , Bacteria/immunology , Bacteria/virology , Bacteriophage T4/growth & development , Bacteriophage T4/immunology , Bacteriophage T4/metabolism , DNA Glycosylases/genetics , DNA Glycosylases/metabolism , Escherichia coli/genetics , Escherichia coli/virology , Gene Library , Metagenomics/methods , Soil Microbiology , Virus ReplicationABSTRACT
A hallmark of CRISPR immunity is the acquisition of short viral DNA sequences, known as spacers, that are transcribed into guide RNAs to recognize complementary sequences. The staphylococcal type III-A CRISPR-Cas system uses guide RNAs to locate viral transcripts and start a response that displays two mechanisms of immunity. When immunity is triggered by an early-expressed phage RNA, degradation of viral ssDNA can cure the host from infection. In contrast, when the RNA guide targets a late-expressed transcript, defense requires the activity of Csm6, a non-specific RNase. Here we show that Csm6 triggers a growth arrest of the host that provides immunity at the population level which hinders viral propagation to allow the replication of non-infected cells. We demonstrate that this mechanism leads to defense against not only the target phage but also other viruses present in the population that fail to replicate in the arrested cells. On the other hand, dormancy limits the acquisition and retention of spacers that trigger it. We found that the ssDNase activity of type III-A systems is required for the re-growth of a subset of the arrested cells, presumably through the degradation of the phage DNA, ending target transcription and inactivating the immune response. Altogether, our work reveals a built-in mechanism within type III-A CRISPR-Cas systems that allows the exit from dormancy needed for the subsistence of spacers that provide broad-spectrum immunity.
ABSTRACT
Prokaryotic type III CRISPR-Cas systems provide immunity against viruses and plasmids using CRISPR-associated Rossman fold (CARF) protein effectors1-5. Recognition of transcripts of these invaders with sequences that are complementary to CRISPR RNA guides leads to the production of cyclic oligoadenylate second messengers, which bind CARF domains and trigger the activity of an effector domain6,7. Whereas most effectors degrade host and invader nucleic acids, some are predicted to contain transmembrane helices without an enzymatic function. Whether and how these CARF-transmembrane helix fusion proteins facilitate the type III CRISPR-Cas immune response remains unknown. Here we investigate the role of cyclic oligoadenylate-activated membrane protein 1 (Cam1) during type III CRISPR immunity. Structural and biochemical analyses reveal that the CARF domains of a Cam1 dimer bind cyclic tetra-adenylate second messengers. In vivo, Cam1 localizes to the membrane, is predicted to form a tetrameric transmembrane pore, and provides defence against viral infection through the induction of membrane depolarization and growth arrest. These results reveal that CRISPR immunity does not always operate through the degradation of nucleic acids, but is instead mediated via a wider range of cellular responses.
Subject(s)
Bacteriophages , CRISPR-Cas Systems , Membrane Potentials , Staphylococcus aureus , Bacteriophages/immunology , Bacteriophages/metabolism , CRISPR-Associated Proteins/metabolism , CRISPR-Cas Systems/genetics , CRISPR-Cas Systems/immunology , Nucleotides, Cyclic/metabolism , RNA, Guide, CRISPR-Cas Systems , Second Messenger Systems , Staphylococcus aureus/cytology , Staphylococcus aureus/genetics , Staphylococcus aureus/immunology , Staphylococcus aureus/virologyABSTRACT
Cyclic oligonucleotide-based antiphage signalling systems (CBASS) protect prokaryotes from viral (phage) attack through the production of cyclic oligonucleotides, which activate effector proteins that trigger the death of the infected host1,2. How bacterial cyclases recognize phage infection is not known. Here we show that staphylococcal phages produce a structured RNA transcribed from the terminase subunit genes, termed CBASS-activating bacteriophage RNA (cabRNA), which binds to a positively charged surface of the CdnE03 cyclase and promotes the synthesis of the cyclic dinucleotide cGAMP to activate the CBASS immune response. Phages that escape the CBASS defence harbour mutations that lead to the generation of a longer form of the cabRNA that cannot activate CdnE03. As the mammalian cyclase OAS1 also binds viral double-stranded RNA during the interferon response, our results reveal a conserved mechanism for the activation of innate antiviral defence pathways.
Subject(s)
Bacteria , Nucleotidyltransferases , RNA, Viral , Staphylococcus Phages , Animals , 2',5'-Oligoadenylate Synthetase/metabolism , Bacteria/enzymology , Bacteria/immunology , Evolution, Molecular , Immunity, Innate , Nucleotidyltransferases/metabolism , Oligonucleotides/immunology , Oligonucleotides/metabolism , RNA, Viral/immunology , RNA, Viral/metabolism , Signal Transduction/immunology , Staphylococcus Phages/genetics , Staphylococcus Phages/immunologyABSTRACT
The Streptococcus pyogenes type II-A CRISPR-Cas systems provides adaptive immunity through the acquisition of short DNA sequences from invading viral genomes, called spacers. Spacers are transcribed into short RNA guides that match regions of the viral genome followed by a conserved NGG DNA motif, known as the PAM. These RNA guides, in turn, are used by the Cas9 nuclease to find and destroy complementary DNA targets within the viral genome. While most of the spacers present in bacterial populations that survive phage infection target protospacers flanked by NGG sequences, there is a small fraction that target non-canonical PAMs. Whether these spacers originate through accidental acquisition of phage sequences and/or provide efficient defense is unknown. Here we found that many of them match phage target regions flanked by an NAGG PAM. Despite being scarcely present in bacterial populations, NAGG spacers provide substantial immunity in vivo and generate RNA guides that support robust DNA cleavage by Cas9 in vitro; with both activities comparable to spacers that target sequences followed by the canonical AGG PAM. In contrast, acquisition experiments showed that NAGG spacers are acquired at very low frequencies. We therefore conclude that discrimination against these sequences occurs during immunization of the host. Our results reveal unexpected differences in PAM recognition during the spacer acquisition and targeting stages of the type II-A CRISPR-Cas immune response.
Subject(s)
Bacteriophages , CRISPR-Cas Systems , Streptococcus pyogenes , Bacteriophages/genetics , Clustered Regularly Interspaced Short Palindromic Repeats , CRISPR-Cas Systems/genetics , Nucleotide Motifs , Streptococcus pyogenes/physiology , Streptococcus pyogenes/virologyABSTRACT
Hoffmann et al. (2022) demonstrate that RNA-guided transposons are remarkably sequence specific due to the action of a AAA+ ATPase, TnsC, that recruits the transposase to the correct target site.
Subject(s)
Escherichia coli Proteins , Escherichia coli Proteins/genetics , Escherichia coli/genetics , DNA Transposable Elements/genetics , DNA, Bacterial , Clustered Regularly Interspaced Short Palindromic Repeats , DNA-Binding Proteins/genetics , Transposases/genetics , Transposases/metabolismABSTRACT
Prokaryotic organisms have developed multiple defense systems against phages; however, little is known about whether and how these interact with each other. Here, we studied the connection between two of the most prominent prokaryotic immune systems: restriction-modification and CRISPR. While both systems employ enzymes that cleave a specific DNA sequence of the invader, CRISPR nucleases are programmed with phage-derived spacer sequences, which are integrated into the CRISPR locus upon infection. We found that restriction endonucleases provide a short-term defense, which is rapidly overcome through methylation of the phage genome. In a small fraction of the cells, however, restriction results in the acquisition of spacer sequences from the cleavage site, which mediates a robust type II-A CRISPR-Cas immune response against the methylated phage. This mechanism is reminiscent of eukaryotic immunity in which the innate response offers a first temporary line of defense and also activates a second and more robust adaptive response.
Subject(s)
Bacteriophages , DNA, Viral , Bacteriophages/metabolism , CRISPR-Cas Systems , DNA Restriction Enzymes/genetics , DNA, Viral/genetics , Endonucleases/genetics , ImmunityABSTRACT
CRISPR-Cas systems have the ability to integrate invasive DNA sequences to build adaptive immunity in bacteria. In this issue Dimitriu et al. show bacteriostatic antibiotics prompt CRISPR acquisition events, illustrating how environmental conditions affect complex dynamics between host and virus and the corresponding biological and genetic arms race.
Subject(s)
Anti-Bacterial Agents , Viruses , Anti-Bacterial Agents/pharmacology , Bacteria/genetics , Base Sequence , CRISPR-Cas SystemsABSTRACT
CRISPR-Cas systems provide prokaryotic organisms with an adaptive defense mechanism that acquires immunological memories of infections. This is accomplished by integration of short fragments from the genome of invaders such as phages and plasmids, called 'spacers', into the CRISPR locus of the host. Depending on their genetic composition, CRISPR-Cas systems can be classified into six types, I-VI, however spacer acquisition has been extensively studied only in type I and II systems. Here, we used an inducible spacer acquisition assay to study this process in the type III-A CRISPR-Cas system of Staphylococcus epidermidis, in the absence of phage selection. Similarly to type I and II spacer acquisition, this type III system uses Cas1 and Cas2 to preferentially integrate spacers from the chromosomal terminus and free dsDNA ends produced after DNA breaks, in a manner that is enhanced by the AddAB DNA repair complex. Surprisingly, a different mode of spacer acquisition from rRNA and tRNA loci, which spans only the transcribed sequences of these genes and is not enhanced by AddAB, was also detected. Therefore, our findings reveal both common mechanistic principles that may be conserved in all CRISPR-Cas systems, as well as unique and intriguing features of type III spacer acquisition.
Subject(s)
Staphylococcus epidermidis/genetics , Bacteriophages/genetics , CRISPR-Associated Proteins/genetics , CRISPR-Associated Proteins/metabolism , CRISPR-Cas Systems , Plasmids/genetics , Staphylococcus epidermidis/metabolism , Staphylococcus epidermidis/virologyABSTRACT
CRISPR loci are composed of short DNA repeats separated by sequences, known as spacers, that match the genomes of invaders such as phages and plasmids. Spacers are transcribed and processed to generate RNA guides used by CRISPR-associated nucleases to recognize and destroy the complementary nucleic acids of invaders. To counteract this defence, phages can produce small proteins that inhibit these nucleases, termed anti-CRISPRs (Acrs). Here we demonstrate that the ΦAP1.1 temperate phage utilizes an alternative approach to antagonize the type II-A CRISPR response in Streptococcus pyogenes. Immediately after infection, this phage expresses a small anti-CRISPR protein, AcrIIA23, that prevents Cas9 function, allowing ΦAP1.1 to integrate into the direct repeats of the CRISPR locus, neutralizing immunity. However, acrIIA23 is not transcribed during lysogeny and phage integration/excision cycles can result in the deletion and/or transduction of spacers, enabling a complex modulation of the type II-A CRISPR immune response. A bioinformatic search identified prophages integrated not only in the CRISPR repeats, but also the cas genes, of diverse bacterial species, suggesting that prophage disruption of the CRISPR-cas locus is a recurrent mechanism to counteract immunity.
Subject(s)
Clustered Regularly Interspaced Short Palindromic Repeats , Prophages/physiology , Streptococcus Phages/physiology , Streptococcus pyogenes/immunology , Streptococcus pyogenes/virology , Lysogeny , Plasmids/genetics , Plasmids/metabolism , Prophages/genetics , Streptococcus Phages/genetics , Streptococcus pyogenes/genetics , Virus IntegrationABSTRACT
CRISPR-Cas systems provide immunity to bacteria by programing Cas nucleases with RNA guides that recognize and cleave infecting viral genomes. Bacteria and their viruses each encode recombination systems that could repair the cleaved viral DNA. However, it is unknown whether and how these systems can affect CRISPR immunity. Bacteriophage λ uses the Red system (gam-exo-bet) to promote recombination between related phages. Here, we show that λ Red also mediates evasion of CRISPR-Cas targeting. Gam inhibits the host E. coli RecBCD recombination system, allowing recombination and repair of the cleaved DNA by phage Exo-Beta, which promotes the generation of mutations within the CRISPR target sequence. Red recombination is strikingly more efficient than the host's RecBCD-RecA in the production of large numbers of phages that escape CRISPR targeting. These results reveal a role for Red-like systems in the protection of bacteriophages against sequence-specific nucleases, which may facilitate their spread across viral genomes.
Subject(s)
Bacteriophage lambda/genetics , CRISPR-Cas Systems , Escherichia coli/genetics , Mutation , Recombination, Genetic , Bacteriophage lambda/immunology , Bacteriophage lambda/physiology , Escherichia coli/immunology , Escherichia coli/virology , Escherichia coli Proteins/genetics , Escherichia coli Proteins/immunology , Exodeoxyribonuclease V/genetics , Exodeoxyribonuclease V/immunology , Host-Pathogen Interactions , Viral Proteins/genetics , Viral Proteins/immunologyABSTRACT
Horizontal gene transfer and mutation are the two major drivers of microbial evolution that enable bacteria to adapt to fluctuating environmental stressors1. Clustered, regularly interspaced, short palindromic repeats (CRISPR) systems use RNA-guided nucleases to direct sequence-specific destruction of the genomes of mobile genetic elements that mediate horizontal gene transfer, such as conjugative plasmids2 and bacteriophages3, thus limiting the extent to which bacteria can evolve by this mechanism. A subset of CRISPR systems also exhibit non-specific degradation of DNA4,5; however, whether and how this feature affects the host has not yet been examined. Here we show that the non-specific DNase activity of the staphylococcal type III-A CRISPR-Cas system increases mutations in the host and accelerates the generation of antibiotic resistance in Staphylococcus aureus and Staphylococcus epidermidis. These mutations require the induction of the SOS response to DNA damage and display a distinct pattern. Our results demonstrate that by differentially affecting both mechanisms that generate genetic diversity, type III-A CRISPR systems can modulate the evolution of the bacterial host.
Subject(s)
CRISPR-Cas Systems/genetics , CRISPR-Cas Systems/immunology , Mutagenesis , Mutation , Staphylococcus/genetics , Anti-Bacterial Agents/pharmacology , Bacteriophages/classification , Bacteriophages/physiology , CRISPR-Associated Proteins/metabolism , DNA, Single-Stranded/genetics , DNA, Single-Stranded/metabolism , Deoxyribonucleases/metabolism , Drug Resistance, Microbial/drug effects , SOS Response, Genetics/drug effects , Staphylococcus/drug effects , Staphylococcus/immunology , Staphylococcus/virology , Staphylococcus aureus/drug effects , Staphylococcus aureus/genetics , Staphylococcus aureus/virology , Staphylococcus epidermidis/drug effects , Staphylococcus epidermidis/genetics , Staphylococcus epidermidis/virology , Time FactorsABSTRACT
CRISPR-Cas9 is an RNA-guided DNA endonuclease involved in bacterial adaptive immunity and widely repurposed for genome editing in human cells, animals and plants. In bacteria, RNA molecules that guide Cas9's activity derive from foreign DNA fragments that are captured and integrated into the host CRISPR genomic locus by the Cas1-Cas2 CRISPR integrase. How cells generate the specific lengths of DNA required for integrase capture is a central unanswered question of type II-A CRISPR-based adaptive immunity. Here, we show that an integrase supercomplex comprising guide RNA and the proteins Cas1, Cas2, Csn2 and Cas9 generates precisely trimmed 30-base pair DNA molecules required for genome integration. The HNH active site of Cas9 catalyzes exonucleolytic DNA trimming by a mechanism that is independent of the guide RNA sequence. These results show that Cas9 possesses a distinct catalytic capacity for generating immunological memory in prokaryotes.
Subject(s)
CRISPR-Associated Protein 9/metabolism , CRISPR-Cas Systems , Integrases/metabolism , CRISPR-Associated Protein 9/chemistry , CRISPR-Associated Proteins/metabolism , Clustered Regularly Interspaced Short Palindromic Repeats , DNA/metabolism , Genome , Protein Domains , RNA/chemistry , RNA/metabolismABSTRACT
In 1944, the Journal of Experimental Medicine published the groundbreaking discovery that DNA is the molecule holding genetic information (1944. J. Exp. Med.https://doi.org/10.1084/jem.79.2.137). This seminal finding was the genesis of molecular biology and the beginning of an incredible journey to understand, read, and manipulate the genetic code.
Subject(s)
DNA/history , Gene Editing/history , Animals , CRISPR-Associated Protein 9/history , CRISPR-Cas Systems , Clustered Regularly Interspaced Short Palindromic Repeats/genetics , Codon/history , History, 20th Century , History, 21st Century , HumansABSTRACT
In the type III CRISPR-Cas immune response of prokaryotes, infection triggers the production of cyclic oligoadenylates that bind and activate proteins that contain a CARF domain1,2. Many type III loci are associated with proteins in which the CRISPR-associated Rossman fold (CARF) domain is fused to a restriction endonuclease-like domain3,4. However, with the exception of the well-characterized Csm6 and Csx1 ribonucleases5,6, whether and how these inducible effectors provide defence is not known. Here we investigated a type III CRISPR accessory protein, which we name cyclic-oligoadenylate-activated single-stranded ribonuclease and single-stranded deoxyribonuclease 1 (Card1). Card1 forms a symmetrical dimer that has a large central cavity between its CRISPR-associated Rossmann fold and restriction endonuclease domains that binds cyclic tetra-adenylate. The binding of ligand results in a conformational change comprising the rotation of individual monomers relative to each other to form a more compact dimeric scaffold, in which a manganese cation coordinates the catalytic residues and activates the cleavage of single-stranded-but not double-stranded-nucleic acids (both DNA and RNA). In vivo, activation of Card1 induces dormancy of the infected hosts to provide immunity against phage infection and plasmids. Our results highlight the diversity of strategies used in CRISPR systems to provide immunity.
Subject(s)
Adenine Nucleotides/metabolism , CRISPR-Cas Systems/immunology , DNA, Single-Stranded/metabolism , Deoxyribonucleases/metabolism , Endoribonucleases/metabolism , Oligoribonucleotides/metabolism , RNA/metabolism , Staphylococcus/enzymology , Staphylococcus/immunology , Adenine Nucleotides/immunology , Adenosine Triphosphate/metabolism , Bacteriophages/immunology , Bacteriophages/physiology , Biocatalysis , Catalytic Domain , Deoxyribonucleases/chemistry , Deoxyribonucleases/genetics , Endoribonucleases/chemistry , Endoribonucleases/genetics , Enzyme Activation , Ligands , Manganese/chemistry , Manganese/metabolism , Models, Molecular , Oligoribonucleotides/immunology , Plasmids/genetics , Plasmids/metabolism , Protein Multimerization , Rotation , Staphylococcus/growth & development , Staphylococcus/virology , Substrate SpecificityABSTRACT
Prokaryotes have developed numerous defense strategies to combat the constant threat posed by the diverse genetic parasites that endanger them. Clustered regularly interspaced short palindromic repeat (CRISPR)-Cas loci guard their hosts with an adaptive immune system against foreign nucleic acids. Protection starts with an immunization phase, in which short pieces of the invader's genome, known as spacers, are captured and integrated into the CRISPR locus after infection. Next, during the targeting phase, spacers are transcribed into CRISPR RNAs (crRNAs) that guide CRISPR-associated (Cas) nucleases to destroy the invader's DNA or RNA. Here we describe the many different molecular mechanisms of CRISPR targeting and how they are interconnected with the immunization phase through a third phase of the CRISPR-Cas immune response: primed spacer acquisition. In this phase, Cas proteins direct the crRNA-guided acquisition of additional spacers to achieve a more rapid and robust immunization of the population.
Subject(s)
Bacteria/genetics , CRISPR-Cas Systems/genetics , Immunity/genetics , Animals , DNA/genetics , RNA/geneticsABSTRACT
Athukoralage et al. (2020) identify a new anti-CRISPR (Acr) that degrades cA4, a cyclic oligo-adenylate second messenger produced during the type III CRISPR immune response. This provides an effective way by which invaders can bypass downstream CRISPR effectors that rely on this signaling molecule.
Subject(s)
Bacteriophages , Clustered Regularly Interspaced Short Palindromic Repeats , Adenine Nucleotides , CRISPR-Cas Systems , OligoribonucleotidesABSTRACT
The CRISPR RNA (crRNA)-guided nuclease Cas13 recognizes complementary viral transcripts to trigger the degradation of both host and viral RNA during the type VI CRISPR-Cas antiviral response. However, how viruses can counteract this immunity is not known. We describe a listeriaphage (ÏLS46) encoding an anti-CRISPR protein (AcrVIA1) that inactivates the type VI-A CRISPR system of Listeria seeligeri Using genetics, biochemistry, and structural biology, we found that AcrVIA1 interacts with the guide-exposed face of Cas13a, preventing access to the target RNA and the conformational changes required for nuclease activation. Unlike inhibitors of DNA-cleaving Cas nucleases, which cause limited immunosuppression and require multiple infections to bypass CRISPR defenses, a single dose of AcrVIA1 delivered by an individual virion completely dismantles type VI-A CRISPR-mediated immunity.