CRISPR antiphage defence mediated by the cyclic nucleotide-binding membrane protein Csx23.

CRISPR-Cas provides adaptive immunity in prokaryotes. Type III CRISPR systems detect invading RNA and activate the catalytic Cas10 subunit, which generates a range of nucleotide second messengers to signal infection. These molecules bind and activate a diverse range of effector proteins that provide immunity by degrading viral components and/or by disturbing key aspects of cellular metabolism to slow down viral replication. Here, we focus on the uncharacterised effector Csx23, which is widespread in Vibrio cholerae. Csx23 provides immunity against plasmids and phage when expressed in Escherichia coli along with its cognate type III CRISPR system. The Csx23 protein localises in the membrane using an N-terminal transmembrane α-helical domain and has a cytoplasmic C-terminal domain that binds cyclic tetra-adenylate (cA4), activating its defence function. Structural studies reveal a tetrameric structure with a novel fold that binds cA4 specifically. Using pulse EPR, we demonstrate that cA4 binding to the cytoplasmic domain of Csx23 results in a major perturbation of the transmembrane domain, consistent with the opening of a pore and/or disruption of membrane integrity. This work reveals a new class of cyclic nucleotide binding protein and provides key mechanistic detail on a membrane-associated CRISPR effector.

Many anti-viral defence systems generate a cyclic nucleotide signal that activates cellular defences in response to infection. Type III CRISPR systems use a specialised polymerase to make cyclic oligoadenylate (cOA) molecules from ATP. These can bind and activate a range of effector proteins that slow down viral replication. In this study, we focussed on the Csx23 effector from the human pathogen Vibrio cholerae ­ a trans-membrane protein that binds a cOA molecule, leading to anti-viral immunity. Structural studies revealed a new class of nucleotide recognition domain, where cOA binding is transmitted to changes in the trans-membrane domain, most likely resulting in membrane depolarisation. This study highlights the diversity of mechanisms for anti-viral defence via nucleotide signalling.

Antiviral type III CRISPR signalling via conjugation of ATP and SAM.

CRISPR systems are widespread in the prokaryotic world, providing adaptive immunity against mobile genetic elements1,2. Type III CRISPR systems, with the signature gene cas10, use CRISPR RNA to detect non-self RNA, activating the enzymatic Cas10 subunit to defend the cell against mobile genetic elements either directly, via the integral histidine-aspartate (HD) nuclease domain3-5 or indirectly, via synthesis of cyclic oligoadenylate second messengers to activate diverse ancillary effectors6-9. A subset of type III CRISPR systems encode an uncharacterized CorA-family membrane protein and an associated NrN family phosphodiesterase that are predicted to function in antiviral defence. Here we demonstrate that the CorA-associated type III-B (Cmr) CRISPR system from Bacteroides fragilis provides immunity against mobile genetic elements when expressed in Escherichia coli. However, B. fragilis Cmr does not synthesize cyclic oligoadenylate species on activation, instead generating S-adenosyl methionine (SAM)-AMP (SAM is also known as AdoMet) by conjugating ATP to SAM via a phosphodiester bond. Once synthesized, SAM-AMP binds to the CorA effector, presumably leading to cell dormancy or death by disruption of the membrane integrity. SAM-AMP is degraded by CRISPR-associated phosphodiesterases or a SAM-AMP lyase, potentially providing an 'off switch' analogous to cyclic oligoadenylate-specific ring nucleases10. SAM-AMP thus represents a new class of second messenger for antiviral signalling, which may function in different roles in diverse cellular contexts.

Cyclic nucleotide-induced helical structure activates a TIR immune effector.

Cyclic nucleotide signalling is a key component of antiviral defence in all domains of life. Viral detection activates a nucleotide cyclase to generate a second messenger, resulting in activation of effector proteins. This is exemplified by the metazoan cGAS-STING innate immunity pathway1, which originated in bacteria2. These defence systems require a sensor domain to bind the cyclic nucleotide and are often coupled with an effector domain that, when activated, causes cell death by destroying essential biomolecules3. One example is the Toll/interleukin-1 receptor (TIR) domain, which degrades the essential cofactor NAD+ when activated in response to infection in plants and bacteria2,4,5 or during programmed nerve cell death6. Here we show that a bacterial antiviral defence system generates a cyclic tri-adenylate that binds to a TIR-SAVED effector, acting as the 'glue' to allow assembly of an extended superhelical solenoid structure. Adjacent TIR subunits interact to organize and complete a composite active site, allowing NAD+ degradation. Activation requires extended filament formation, both in vitro and in vivo. Our study highlights an example of large-scale molecular assembly controlled by cyclic nucleotides and reveals key details of the mechanism of TIR enzyme activation.

Specificity and sensitivity of an RNA targeting type III CRISPR complex coupled with a NucC endonuclease effector.

Type III CRISPR systems detect invading RNA, resulting in the activation of the enzymatic Cas10 subunit. The Cas10 cyclase domain generates cyclic oligoadenylate (cOA) second messenger molecules, activating a variety of effector nucleases that degrade nucleic acids to provide immunity. The prophage-encoded Vibrio metoecus type III-B (VmeCmr) locus is uncharacterised, lacks the HD nuclease domain in Cas10 and encodes a NucC DNA nuclease effector that is also found associated with Cyclic-oligonucleotide-based anti-phage signalling systems (CBASS). Here we demonstrate that VmeCmr is activated by target RNA binding, generating cyclic-triadenylate (cA3) to stimulate a robust NucC-mediated DNase activity. The specificity of VmeCmr is probed, revealing the importance of specific nucleotide positions in segment 1 of the RNA duplex and the protospacer flanking sequence (PFS). We harness this programmable system to demonstrate the potential for a highly specific and sensitive assay for detection of the SARS-CoV-2 virus RNA with a limit of detection (LoD) of 2 fM using a commercial plate reader without any extrinsic amplification step. The sensitivity is highly dependent on the guide RNA used, suggesting that target RNA secondary structure plays an important role that may also be relevant in vivo.

The CRISPR ancillary effector Can2 is a dual-specificity nuclease potentiating type III CRISPR defence.

Cells and organisms have a wide range of mechanisms to defend against infection by viruses and other mobile genetic elements (MGE). Type III CRISPR systems detect foreign RNA and typically generate cyclic oligoadenylate (cOA) second messengers that bind to ancillary proteins with CARF (CRISPR associated Rossman fold) domains. This results in the activation of fused effector domains for antiviral defence. The best characterised CARF family effectors are the Csm6/Csx1 ribonucleases and DNA nickase Can1. Here we investigate a widely distributed CARF family effector with a nuclease domain, which we name Can2 (CRISPR ancillary nuclease 2). Can2 is activated by cyclic tetra-adenylate (cA4) and displays both DNase and RNase activity, providing effective immunity against plasmid transformation and bacteriophage infection in Escherichia coli. The structure of Can2 in complex with cA4 suggests a mechanism for the cA4-mediated activation of the enzyme, whereby an active site cleft is exposed on binding the activator. These findings extend our understanding of type III CRISPR cOA signalling and effector function.

Tetramerisation of the CRISPR ring nuclease Crn3/Csx3 facilitates cyclic oligoadenylate cleavage.

Type III CRISPR systems detect foreign RNA and activate the cyclase domain of the Cas10 subunit, generating cyclic oligoadenylate (cOA) molecules that act as a second messenger to signal infection, activating nucleases that degrade the nucleic acid of both invader and host. This can lead to dormancy or cell death; to avoid this, cells need a way to remove cOA from the cell once a viral infection has been defeated. Enzymes specialised for this task are known as ring nucleases, but are limited in their distribution. Here, we demonstrate that the widespread CRISPR associated protein Csx3, previously described as an RNA deadenylase, is a ring nuclease that rapidly degrades cyclic tetra-adenylate (cA4). The enzyme has an unusual cooperative reaction mechanism involving an active site that spans the interface between two dimers, sandwiching the cA4 substrate. We propose the name Crn3 (CRISPR associated ring nuclease 3) for the Csx3 family.

Bacteria protect themselves from infections using a system called CRISPR-Cas, which helps the cells to detect and destroy invading threats. The type III CRISPR-Cas system, in particular, is one of the most widespread and efficient at killing viruses. When a bacterium is infected, the CRISPR-Cas system takes a fragment of the genetic material of the virus, and copies it into a molecule. These molecular 'police mugshots' are then loaded into a complex of Cas proteins that patrol the cell, looking for a match and destroying any virus that can be identified. Some Cas proteins also produce alarm signals, called cyclic oligoadenylates (cOAs), which can trigger additional defences. However, this process can damage the genetic material of the bacterium, harming or even killing the cell. Enzymes known as ring nucleases can promptly degrade cOAs and turn off this defence system before it causes harm. However, ring nucleases have only been found in a few species to date; how most bacteria deal with cOA toxicity has remained unknown. Here, Athukoralage et al. set out to determine whether a widespread enzyme known as Csx3, which is often associated with type III CRISPR-Cas systems, could be an alternative off switch for cOA triggered defences. Initial 'test tube' experiments with purified Csx3 proteins confirmed that the enzyme could indeed break down cOAs. A careful dissection of Csx3's molecular structure, using biochemical and biophysical techniques, revealed that it worked by 'sandwiching' a cOA molecule between two co-operating portions of the enzyme. As a final test, Csx3 was introduced into strains of bacteria genetically engineered to have a fully functional Type III CRISPR-Cas system. In these cells, Csx3 successfully turned off the Type III immune response. These results reveal a new way that bacteria avoid the toxic side effects of their own immune defences. Ultimately, this could pave the way for the development of anti-bacterial drugs that work by blocking Csx3 or similar proteins.

The dynamic interplay of host and viral enzymes in type III CRISPR-mediated cyclic nucleotide signalling.

Cyclic nucleotide second messengers are increasingly implicated in prokaryotic anti-viral defence systems. Type III CRISPR systems synthesise cyclic oligoadenylate (cOA) upon detecting foreign RNA, activating ancillary nucleases that can be toxic to cells, necessitating mechanisms to remove cOA in systems that operate via immunity rather than abortive infection. Previously, we demonstrated that the Sulfolobus solfataricus type III-D CRISPR complex generates cyclic tetra-adenylate (cA4), activating the ribonuclease Csx1, and showed that subsequent RNA cleavage and dissociation acts as an 'off-switch' for the cyclase activity. Subsequently, we identified the cellular ring nuclease Crn1, which slowly degrades cA4 to reset the system (Rouillon et al., 2018), and demonstrated that viruses can subvert type III CRISPR immunity by means of a potent anti-CRISPR ring nuclease variant AcrIII-1. Here, we present a comprehensive analysis of the dynamic interplay between these enzymes, governing cyclic nucleotide levels and infection outcomes in virus-host conflict.

Regulation of the RNA and DNA nuclease activities required for Pyrococcus furiosus Type III-B CRISPR-Cas immunity.

Type III CRISPR-Cas prokaryotic immune systems provide anti-viral and anti-plasmid immunity via a dual mechanism of RNA and DNA destruction. Upon target RNA interaction, Type III crRNP effector complexes become activated to cleave both target RNA (via Cas7) and target DNA (via Cas10). Moreover, trans-acting endoribonucleases, Csx1 or Csm6, can promote the Type III immune response by destroying both invader and host RNAs. Here, we characterize how the RNase and DNase activities associated with Type III-B immunity in Pyrococcus furiosus (Pfu) are regulated by target RNA features and second messenger signaling events. In vivo mutational analyses reveal that either the DNase activity of Cas10 or the RNase activity of Csx1 can effectively direct successful anti-plasmid immunity. Biochemical analyses confirmed that the Cas10 Palm domains convert ATP into cyclic oligoadenylate (cOA) compounds that activate the ribonuclease activity of Pfu Csx1. Furthermore, we show that the HEPN domain of the adenosine-specific endoribonuclease, Pfu Csx1, degrades cOA signaling molecules to provide an auto-inhibitory off-switch of Csx1 activation. Activation of both the DNase and cOA generation activities require target RNA binding and recognition of distinct target RNA 3' protospacer flanking sequences. Our results highlight the complex regulatory mechanisms controlling Type III CRISPR immunity.

An anti-CRISPR viral ring nuclease subverts type III CRISPR immunity.

The CRISPR system in bacteria and archaea provides adaptive immunity against mobile genetic elements. Type III CRISPR systems detect viral RNA, resulting in the activation of two regions of the Cas10 protein: an HD nuclease domain (which degrades viral DNA)1,2 and a cyclase domain (which synthesizes cyclic oligoadenylates from ATP)3-5. Cyclic oligoadenylates in turn activate defence enzymes with a CRISPR-associated Rossmann fold domain6, sculpting a powerful antiviral response7-10 that can drive viruses to extinction7,8. Cyclic nucleotides are increasingly implicated in host-pathogen interactions11-13. Here we identify a new family of viral anti-CRISPR (Acr) enzymes that rapidly degrade cyclic tetra-adenylate (cA4). The viral ring nuclease AcrIII-1 is widely distributed in archaeal and bacterial viruses and in proviruses. The enzyme uses a previously unknown fold to bind cA4 specifically, and a conserved active site to rapidly cleave this signalling molecule, allowing viruses to neutralize the type III CRISPR defence system. The AcrIII-1 family has a broad host range, as it targets cA4 signalling molecules rather than specific CRISPR effector proteins. Our findings highlight the crucial role of cyclic nucleotide signalling in the conflict between viruses and their hosts.

Phenylalanine meta-Hydroxylase: A Single Residue Mediates Mechanistic Control of Aromatic Amino Acid Hydroxylation.

The rare nonproteinogenic amino acid, meta-l-tyrosine is biosynthetically intriguing. Whilst the biogenesis of tyrosine from phenylalanine is well characterised, the mechanistic basis for meta-hydroxylation is unknown. Herein, we report the analysis of 3-hydroxylase (Phe3H) from Streptomyces coeruleorubidus. Insights from kinetic analyses of the wild-type enzyme and key mutants as well as of the biocatalytic conversion of synthetic isotopically labelled substrates and fluorinated substrate analogues advance understanding of the process by which meta-hydroxylation is mediated, revealing T202 to play an important role. In the case of the WT enzyme, a deuterium label at the 3-position is lost, whereas in in the T202A mutant 75 % retention is observed, with loss of stereospecificity. These data suggest that one of two possible mechanisms is at play; direct, enzyme-catalysed deprotonation following electrophilic aromatic substitution or stereospecific loss of one proton after a 1,2-hydride shift. Furthermore, our kinetic parameters for Phe3H show efficient regiospecific generation of meta-l-tyrosine from phenylalanine and demonstrate the enzyme's ability to regiospecifically hydroxylate unnatural fluorinated substrates.

Cyclic oligoadenylate signalling mediates Mycobacterium tuberculosis CRISPR defence.

The CRISPR system provides adaptive immunity against mobile genetic elements (MGE) in prokaryotes. In type III CRISPR systems, an effector complex programmed by CRISPR RNA detects invading RNA, triggering a multi-layered defence that includes target RNA cleavage, licencing of an HD DNA nuclease domain and synthesis of cyclic oligoadenylate (cOA) molecules. cOA activates the Csx1/Csm6 family of effectors, which degrade RNA non-specifically to enhance immunity. Type III systems are found in diverse archaea and bacteria, including the human pathogen Mycobacterium tuberculosis. Here, we report a comprehensive analysis of the in vitro and in vivo activities of the type III-A M. tuberculosis CRISPR system. We demonstrate that immunity against MGE may be achieved predominantly via a cyclic hexa-adenylate (cA6) signalling pathway and the ribonuclease Csm6, rather than through DNA cleavage by the HD domain. Furthermore, we show for the first time that a type III CRISPR system can be reprogrammed by replacing the effector protein, which may be relevant for maintenance of immunity in response to pressure from viral anti-CRISPRs. These observations demonstrate that M. tuberculosis has a fully-functioning CRISPR interference system that generates a range of cyclic and linear oligonucleotides of known and unknown functions, potentiating fundamental and applied studies.

A Type III CRISPR Ancillary Ribonuclease Degrades Its Cyclic Oligoadenylate Activator.

Cyclic oligoadenylate (cOA) secondary messengers are generated by type III CRISPR systems in response to viral infection. cOA allosterically activates the CRISPR ancillary ribonucleases Csx1/Csm6, which degrade RNA non-specifically using a HEPN (Higher Eukaryotes and Prokaryotes, Nucleotide binding) active site. This provides effective immunity but can also lead to growth arrest in infected cells, necessitating a means to deactivate the ribonuclease once viral infection has been cleared. In the crenarchaea, dedicated ring nucleases degrade cA4 (cOA consisting of 4 AMP units), but the equivalent enzyme has not been identified in bacteria. We demonstrate that, in Thermus thermophilus HB8, the uncharacterized protein TTHB144 is a cA4-activated HEPN ribonuclease that also degrades its activator. TTHB144 binds and degrades cA4 at an N-terminal CARF (CRISPR-associated Rossman fold) domain. The two activities can be separated by site-directed mutagenesis. TTHB144 is thus the first example of a self-limiting CRISPR ribonuclease.

Investigation of the cyclic oligoadenylate signaling pathway of type III CRISPR systems.

Type III CRISPR effector complexes utilize a bound CRISPR RNA (crRNA) to detect the presence of RNA from invading mobile genetic elements in the cell. This RNA binding results in the activation of two enzymatic domains of the Cas10 subunit-the HD nuclease domain, which degrades DNA, and PALM/cyclase domain. The latter synthesizes cyclic oligoadenylate (cOA) molecules by polymerizing ATP, and cOA acts as a second messenger in the cell, switching on the antiviral response by activating host ribonucleases and other proteins. In this chapter, we focus on the methods required to study the biochemistry of this recently discovered cOA signaling pathway. We cover protein expression and purification, synthesis of cOA and its linear analogues, kinetic analysis of cOA synthesis and cOA-stimulated ribonuclease activity, and small molecule detection and identification with thin-layer chromatography and mass spectrometry. The methods described are based on our recent studies of the type III CRISPR system in Sulfolobus solfataricus, but are widely applicable to other type III systems.

Ring nucleases deactivate type III CRISPR ribonucleases by degrading cyclic oligoadenylate.

The CRISPR system provides adaptive immunity against mobile genetic elements in prokaryotes, using small CRISPR RNAs that direct effector complexes to degrade invading nucleic acids1-3. Type III effector complexes were recently demonstrated to synthesize a novel second messenger, cyclic oligoadenylate, on binding target RNA4,5. Cyclic oligoadenylate, in turn, binds to and activates ribonucleases and other factors-via a CRISPR-associated Rossman-fold domain-and thereby induces in the cell an antiviral state that is important for immunity. The mechanism of the 'off-switch' that resets the system is not understood. Here we identify the nuclease that degrades these cyclic oligoadenylate ring molecules. This 'ring nuclease' is itself a protein of the CRISPR-associated Rossman-fold family, and has a metal-independent mechanism that cleaves cyclic tetraadenylate rings to generate linear diadenylate species and switches off the antiviral state. The identification of ring nucleases adds an important insight to the CRISPR system.

Control of cyclic oligoadenylate synthesis in a type III CRISPR system.

The CRISPR system for prokaryotic adaptive immunity provides RNA-mediated protection from viruses and mobile genetic elements. When viral RNA transcripts are detected, type III systems adopt an activated state that licenses DNA interference and synthesis of cyclic oligoadenylate (cOA). cOA activates nucleases and transcription factors that orchestrate the antiviral response. We demonstrate that cOA synthesis is subject to tight temporal control, commencing on target RNA binding, and is deactivated rapidly as target RNA is cleaved and dissociates. Mismatches in the target RNA are well tolerated and still activate the cyclase domain, except when located close to the 3' end of the target. Phosphorothioate modification reduces target RNA cleavage and stimulates cOA production. The 'RNA shredding' activity originally ascribed to type III systems may thus be a reflection of an exquisite mechanism for control of the Cas10 subunit, rather than a direct antiviral defence.

Scope and potential of halogenases in biosynthetic applications.

A large and diverse series of halogenated natural products exist. In many of these compounds the halogen is important to biological activity and bioavailability. We now recognise that nature has developed many different halogenation strategies for which well-known enzyme classes such as haem oxidases or flavin-dependent oxidases have been adapted. Enzymes capable of halogenating all kinds of different chemical groups from electron-rich to electron-poor, from aromatic to aliphatic have been characterised. Given that synthetic halogenation reactions are not trivial transformations and that halogenated molecules possess pharmaceutical usefulness, it will be worth investing into further research of halogenating enzymes.

Isolating antifungals from fungus-growing ant symbionts using a genome-guided chemistry approach.

We describe methods used to isolate and identify antifungal compounds from actinomycete strains associated with the leaf-cutter ant Acromyrmex octospinosus. These ants use antibiotics produced by symbiotic actinomycete bacteria to protect themselves and their fungal cultivar against bacterial and fungal infections. The fungal cultivar serves as the sole food source for the ant colony, which can number up to tens of thousands of individuals. We describe how we isolate bacteria from leaf-cutter ants collected in Trinidad and analyze the antifungal compounds made by two of these strains (Pseudonocardia and Streptomyces spp.), using a combination of genome analysis, mutagenesis, and chemical isolation. These methods should be generalizable to a wide variety of insect-symbiont situations. Although more time consuming than traditional activity-guided fractionation methods, this approach provides a powerful technique for unlocking the complete biosynthetic potential of individual strains and for avoiding the problems of rediscovery of known compounds. We describe the discovery of a novel nystatin compound, named nystatin P1, and identification of the biosynthetic pathway for antimycins, compounds that were first described more than 60 years ago. We also report that disruption of two known antifungal pathways in a single Streptomyces strain has revealed a third, and likely novel, antifungal plus four more pathways with unknown products. This validates our approach, which clearly has the potential to identify numerous new compounds, even from well-characterized actinomycete strains.

Revealing the first uridyl peptide antibiotic biosynthetic gene cluster and probing pacidamycin biosynthesis.

There is an urgent need for new antibiotics with resistance continuing to emerge toward existing classes. The pacidamycin antibiotics possess a novel scaffold and exhibit unexploited bioactivity rendering them attractive research targets. We recently reported the first identification of a biosynthetic cluster encoding uridyl peptide antibiotic assembly and the engineering of pacidamycin biosynthesis into a heterologous host. We report here our methods toward identifying the biosynthetic cluster. Our initial experiments employed conventional methods of probing a cosmid library using PCR and Southern blotting, however it became necessary to adopt a state-of-the-art genome scanning  and in silico hybridization approach  to pin point the cluster. Here we describe our "real" and "virtual" probing methods and contrast the benefits and pitfalls of each approach.