Biology subjects

Gruschow, S.

Publications and source records attributed to Gruschow, S..

7 recordsLinked to original sources

bioRxiv2026

Cyclic tri-adenylate signalling by a Panoptes anti-phage guard system with a CARF-TM effector

Cyclic nucleotide second messengers are used in all domains of life to amplify viral infection signals and activate cellular defences. In prokaryotes, CBASS (cyclic nucleotide based antiphage signalling system) and type III CRISPR-Cas systems generate a range of cyclic nucleotides which bind and allosterically activate effector proteins to mount an anti-viral response. Viruses have evolved counter-measures to antagonise these signalling pathways in the form of cyclic nucleotide sponges and phosphodiesterases that sequester or degrade these molecules to subvert immunity. Recently, the Panoptes system was shown to function as a guard against these viral tactics. The type I Panoptes polymerase, mCpol, generates cyclic dinucleotides as decoy molecules that, when sequestered by phage proteins, results in the activation of the membrane-permeabilising effector 2TM{beta} to halt the phage infection cycle. Here, we investigate the type II Panoptes system, demonstrating that it generates cyclic tri-adenylate (cA3) to maintain a CRISPR-associated Rossmann fold-transmembrane (CARF-TM) effector in an inactive, dimeric state. When cA3 is sequestered or degraded, the CARF-TM protein oligomerises, resulting in increased outer membrane permeability and growth arrest. These findings expand our understanding of the guard systems that constitute a fascinating component of the bacterial immune system.

biochemistry↗

bioRxiv2024

Diversity and abundance of ring nucleases in type III CRISPR-Cas loci

Most type III CRISPR-Cas systems facilitate immune responses against invading mobile genetic elements such as phages by generating cyclic oligoadenylates (cOAs). Downstream effectors activated by cOAs are typically non-specific proteins that induce damage to essential cellular components, thereby preventing phage epidemics. Due to these toxic effects, it is crucial that the production and concentration of cOAs remain under tight regulatory control during infection-free periods or when deactivating the immune response after clearing an infection. Type III CRISPR loci often encode enzymes known as ring nucleases (RNs) that bind and degrade specific cOAs, while some effectors are auto-deactivating. Despite the discovery of several classes of RNs, a comprehensive bioinformatic analysis of type III CRISPR-Cas loci in this context is lacking. Here, we examined 38,742 prokaryotic genomes to provide a global overview of type III CRISPR loci, focussing on the known and predicted RNs. The candidate RNs Csx16 and Csx20 are confirmed as active enzymes, joining Crn1-3. Distributions and patterns of co-occurrence of RNs and associated effectors are explored, allowing the conclusion that a sizeable majority of type III CRISPR systems regulate cOA levels by degrading the signalling molecules, which has implications for cell fate following viral infection.

bioinformatics↗

bioRxiv2024

Cas10 based 7SL-sRNA diagnostic for the detection of active trypanosomiasis

Animal Trypanosomosis (AT) is a significant disease affecting cattle across sub-Saharan Africa, Latin America, and Asia, posing a major threat to economic productivity and animal welfare. The absence of reliable diagnostic tests has led to an over-reliance on widespread pre-emptive drug treatments, which not only compromise animal health but also heighten the risk of drug resistance. The chronic nature of AT, characterized by cyclical low or undetectable parasite levels, and the necessity for field-applicable tests that can distinguish between active infection and prior exposure, present considerable challenges in developing effective diagnostics. In previous work, we identified a parasite-specific small RNA, 7SL-sRNA, which is detectable in the serum of infected cattle, even during the chronic stages of infection. However, existing methods for detecting sRNA require specialized equipment, making them unsuitable for field use. In this study, we have developed a lateral flow diagnostic test utilizing Cas10 technology for the detection of 7SL-sRNA. This assay demonstrates high sensitivity and specificity, effectively identifying active infections in cattle, including during chronic phases (with positive signals observed up to the 63 days post-infection end point). This is the first instance of Cas10 being used for small RNA detection, paving the way for a cost-effective, user-friendly, and field-deployable diagnostic test for AT, while establishing Cas10 technology for the detection of small RNAs in general. Author summaryAnimal Trypanosomosis (AT) is a parasitic disease that affects livestock, causing major economic losses and harm to animal health. Cheap effective diagnostics that can be easily used in the field would have a major impact on the disease burden by enabling better treatment practices. However, parasites causing the disease - trypanosomes - are often undetectable via conventional methods, especially during chronic disease stages. More accurate diagnostics are not suitable for use in the field and are therefore unavailable to farmers. This has led to the overuse of drug treatments that can harm animals and lead to the emergence of drug resistance. We previously discovered a trypanosome-specific small RNA, called 7SL-sRNA, that can be detected in the blood of infected cattle, even during the chronic stages of AT when parasites are not visible via microscopy. However, detecting this RNA has required complex equipment, limiting its use in field settings. Building on this work, we have now developed a field applicable test for trypanosomosis that uses Cas10 technology to detect 7SL-sRNA via a simple lateral flow assay. This test is easy to use and accurately identifies active infections, including during the chronic phase of the disease. This is the first time Cas10 technology has been used to detect small RNAs, offering a promising, cost-effective tool for diagnosing AT and potentially other neglected parasitic diseases.

microbiology↗

bioRxiv2023

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

CRISPR 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 a N-terminal transmembrane -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.

microbiology↗

bioRxiv2023

Antiviral Type III CRISPR signalling via conjugation of ATP and AdoMet

CRISPR systems are widespread in the prokaryotic world, providing adaptive immunity against mobile genetic elements (MGE) 1, 2. Type III CRISPR systems, with the signature gene cas10, use CRISPR RNA (crRNA) to detect non-self RNA, activating the enzymatic Cas10 subunit to defend the cell against MGE either directly, via the integral HD nuclease domain 3-5 or indirectly, via synthesis of cyclic oligonucleotide (cOA) second messengers to activate diverse ancillary effectors 6-9. A subset of type III CRISPR systems encode an uncharacterised CorA-family membrane protein and an associated NrN family phosphodiesterase 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 MGE when expressed inE. coli. However, B. fragilis Cmr does not synthesise cOA species on activation, instead generating a previously undescribed sigalling molecule, SAM-AMP (3-adenylyl-AdoMet) by conjugating ATP to S-adenosyl methionine via a phosphodiester bond. Once synthesised, SAM-AMP binds to the CorA effector, presumably leading to cell death by disruption of the membrane integrity. SAM-AMP is degraded by CRISPR associated phosphodiesterases or a SAM-AMP lyase, providing an "off switch" analogous to cOA specific ring nucleases 10. SAM-AMP thus represents a new class of second messenger for antiviral signalling, which may function in different roles in diverse cellular contexts.

microbiology↗

bioRxiv2021

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.

biochemistry↗

bioRxiv2019

A viral ring nuclease anti-CRISPR subverts type III CRISPR immunity

The CRISPR system provides adaptive immunity against mobile genetic elements in bacteria and archaea. On detection of viral RNA, type III CRISPR systems generate a cyclic oligoadenylate (cOA) second messenger1-3, activating defence enzymes and sculpting a powerful antiviral response that can drive viruses to extinction4,5. Cyclic nucleotides are increasingly implicated as playing an important role in host-pathogen interactions6,7. Here, we identify a widespread new family of viral anti-CRISPR (Acr) enzymes that rapidly degrade cyclic tetra-adenylate (cA4). The viral ring nuclease (AcrIII-1) is the first Acr described for type III CRISPR systems and is widely distributed in archaeal and bacterial viruses, and proviruses. The enzyme uses a novel fold to bind cA4 specifically and utilizes a conserved active site to rapidly cleave the signalling molecule, allowing viruses to neutralise 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. This study highlights the crucial role of cyclic nucleotide signalling in the conflict between viruses and their hosts.

biochemistry↗