The CRISPR effector Cam1 mediates membrane depolarization for phage defence.

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.

DNA glycosylases provide antiviral defence in prokaryotes.

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.

Cas9 interaction with the tracrRNA nexus modulates the repression of type II-A CRISPR-cas genes.

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.

A bacterial TIR-based immune system senses viral capsids to initiate defense.

Toll/interleukin-1 receptor (TIR) domains are present in immune systems that protect prokaryotes from viral (phage) attack. In response to infection, TIRs can produce a cyclic adenosine diphosphate-ribose (ADPR) signaling molecule, which activates an effector that depletes the host of the essential metabolite NAD+ to limit phage propagation. How bacterial TIRs recognize phage infection is not known. Here we describe the sensing mechanism for the staphylococcal Thoeris defense system, which consists of two TIR domain sensors, ThsB1 and ThsB2, and the effector ThsA. We show that the major capsid protein of phage Φ80α forms a complex with ThsB1 and ThsB2, which is sufficient for the synthesis of 1"-3' glycocyclic ADPR (gcADPR) and subsequent activation of NAD+ cleavage by ThsA. Consistent with this, phages that escape Thoeris immunity harbor mutations in the capsid that prevent complex formation. We show that capsid proteins from staphylococcal Siphoviridae belonging to the capsid serogroup B, but not A, are recognized by ThsB1/B2, a result that suggests that capsid recognition by Sau-Thoeris and other anti-phage defense systems may be an important evolutionary force behind the structural diversity of prokaryotic viruses. More broadly, since mammalian toll-like receptors harboring TIR domains can also recognize viral structural components to produce an inflammatory response against infection, our findings reveal a conserved mechanism for the activation of innate antiviral defense pathways.

The Cas10 nuclease activity relieves host dormancy to facilitate spacer acquisition and retention during type III-A CRISPR immunity.

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.