Target DNA-dependent activation mechanism of the prokaryotic immune system SPARTA.

In both prokaryotic and eukaryotic innate immune systems, TIR domains function as NADases that degrade the key metabolite NAD+ or generate signaling molecules. Catalytic activation of TIR domains requires oligomerization, but how this is achieved varies in distinct immune systems. In the Short prokaryotic Argonaute (pAgo)/TIR-APAZ (SPARTA) immune system, TIR NADase activity is triggered upon guide RNA-mediated recognition of invading DNA by an unknown mechanism. Here, we describe cryo-EM structures of SPARTA in the inactive monomeric and target DNA-activated tetrameric states. The monomeric SPARTA structure reveals that in the absence of target DNA, a C-terminal tail of TIR-APAZ occupies the nucleic acid binding cleft formed by the pAgo and TIR-APAZ subunits, inhibiting SPARTA activation. In the active tetrameric SPARTA complex, guide RNA-mediated target DNA binding displaces the C-terminal tail and induces conformational changes in pAgo that facilitate SPARTA-SPARTA dimerization. Concurrent release and rotation of one TIR domain allow it to form a composite NADase catalytic site with the other TIR domain within the dimer, and generate a self-complementary interface that mediates cooperative tetramerization. Combined, this study provides critical insights into the structural architecture of SPARTA and the molecular mechanism underlying target DNA-dependent oligomerization and catalytic activation.

Type III CRISPR-Cas Systems: Deciphering the Most Complex Prokaryotic Immune System.

The emergence and persistence of selfish genetic elements is an intrinsic feature of all living systems. Cellular organisms have evolved a plethora of elaborate defense systems that limit the spread of such genetic parasites. CRISPR-Cas are RNA-guided defense systems used by prokaryotes to recognize and destroy foreign nucleic acids. These systems acquire and store fragments of foreign nucleic acids and utilize the stored sequences as guides to recognize and destroy genetic invaders. CRISPR-Cas systems have been extensively studied, as some of them are used in various genome editing technologies. Although Type III CRISPR-Cas systems are among the most common CRISPR-Cas systems, they are also some of the least investigated ones, mostly due to the complexity of their action compared to other CRISPR-Cas system types. Type III effector complexes specifically recognize and cleave RNA molecules. The recognition of the target RNA activates the effector large subunit - the so-called CRISPR polymerase - which cleaves DNA and produces small cyclic oligonucleotides that act as signaling molecules to activate auxiliary effectors, notably non-specific RNases. In this review, we provide a historical overview of the sometimes meandering pathway of the Type III CRISPR research. We also review the current data on the structures and activities of Type III CRISPR-Cas systems components, their biological roles, and evolutionary history. Finally, using structural modeling with AlphaFold2, we show that the archaeal HRAMP signature protein, which heretofore has had no assigned function, is a degenerate relative of Type III CRISPR-Cas signature protein Cas10, suggesting that HRAMP systems have descended from Type III CRISPR-Cas systems or their ancestors.

Bacterial death and TRADD-N domains help define novel apoptosis and immunity mechanisms shared by prokaryotes and metazoans.

Several homologous domains are shared by eukaryotic immunity and programmed cell-death systems and poorly understood bacterial proteins. Recent studies show these to be components of a network of highly regulated systems connecting apoptotic processes to counter-invader immunity, in prokaryotes with a multicellular habit. However, the provenance of key adaptor domains, namely those of the Death-like and TRADD-N superfamilies, a quintessential feature of metazoan apoptotic systems, remained murky. Here, we use sensitive sequence analysis and comparative genomics methods to identify unambiguous bacterial homologs of the Death-like and TRADD-N superfamilies. We show the former to have arisen as part of a radiation of effector-associated α-helical adaptor domains that likely mediate homotypic interactions bringing together diverse effector and signaling domains in predicted bacterial apoptosis- and counter-invader systems. Similarly, we show that the TRADD-N domain defines a key, widespread signaling bridge that links effector deployment to invader-sensing in multicellular bacterial and metazoan counter-invader systems. TRADD-N domains are expanded in aggregating marine invertebrates and point to distinctive diversifying immune strategies probably directed both at RNA and retroviruses and cellular pathogens that might infect such communities. These TRADD-N and Death-like domains helped identify several new bacterial and metazoan counter-invader systems featuring underappreciated, common functional principles: the use of intracellular invader-sensing lectin-like (NPCBM and FGS), transcription elongation GreA/B-C, glycosyltransferase-4 family, inactive NTPase (serving as nucleic acid receptors), and invader-sensing GTPase switch domains. Finally, these findings point to the possibility of multicellular bacteria-stem metazoan symbiosis in the emergence of the immune/apoptotic systems of the latter.

Prokaryotic viperins produce diverse antiviral molecules.

Viperin is an interferon-induced cellular protein that is conserved in animals1. It has previously been shown to inhibit the replication of multiple viruses by producing the ribonucleotide 3'-deoxy-3',4'-didehydro (ddh)-cytidine triphosphate (ddhCTP), which acts as a chain terminator for viral RNA polymerase2. Here we show that eukaryotic viperin originated from a clade of bacterial and archaeal proteins that protect against phage infection. Prokaryotic viperins produce a set of modified ribonucleotides that include ddhCTP, ddh-guanosine triphosphate (ddhGTP) and ddh-uridine triphosphate (ddhUTP). We further show that prokaryotic viperins protect against T7 phage infection by inhibiting viral polymerase-dependent transcription, suggesting that it has an antiviral mechanism of action similar to that of animal viperin. Our results reveal a class of potential natural antiviral compounds produced by bacterial immune systems.

Diverse CRISPR-Cas Complexes Require Independent Translation of Small and Large Subunits from a Single Gene.

CRISPR-Cas adaptive immune systems provide prokaryotes with defense against viruses by degradation of specific invading nucleic acids. Despite advances in the biotechnological exploitation of select systems, multiple CRISPR-Cas types remain uncharacterized. Here, we investigated the previously uncharacterized type I-D interference complex and revealed that it is a genetic and structural hybrid with similarity to both type I and type III systems. Surprisingly, formation of the functional complex required internal in-frame translation of small subunits from within the large subunit gene. We further show that internal translation to generate small subunits is widespread across diverse type I-D, I-B, and I-C systems, which account for roughly one quarter of CRISPR-Cas systems. Our work reveals the unexpected expansion of protein coding potential from within single cas genes, which has important implications for understanding CRISPR-Cas function and evolution.

Evolutionary Ecology and Interplay of Prokaryotic Innate and Adaptive Immune Systems.

Like many organisms, bacteria and archaea have both innate and adaptive immune systems to defend against infection by viruses and other parasites. Innate immunity most commonly relies on the endonuclease-mediated cleavage of any incoming DNA that lacks a specific epigenetic modification, through a system known as restriction-modification. CRISPR-Cas-mediated adaptive immunity relies on the insertion of short DNA sequences from parasite genomes into CRISPR arrays on the host genome to provide sequence-specific protection. The discovery of each of these systems has revolutionised our ability to carry out genetic manipulations, and, as a consequence, the enzymes involved have been characterised in exquisite detail. In comparison, much less is known about the importance of these two arms of the defence for the ecology and evolution of prokaryotes and their parasites. Here, we review our current ecological and evolutionary understanding of these systems in isolation, and discuss the need to study how innate and adaptive immune responses are integrated when they coexist in the same cell.

Detection of CRISPR adaptation.

Prokaryotic adaptive immunity is built when short DNA fragments called spacers are acquired into CRISPR (clustered regularly interspaced short palindromic repeats) arrays. CRISPR adaptation is a multistep process which comprises selection, generation, and incorporation of prespacers into arrays. Once adapted, spacers provide immunity through the recognition of complementary nucleic acid sequences, channeling them for destruction. To prevent deleterious autoimmunity, CRISPR adaptation must therefore be a highly regulated and infrequent process, at least in the absence of genetic invaders. Over the years, ingenious methods to study CRISPR adaptation have been developed. In this paper, we discuss and compare methods that detect CRISPR adaptation and its intermediates in vivo and propose suppressing PCR as a simple modification of a popular assay to monitor spacer acquisition with increased sensitivity.

Atypical organizations and epistatic interactions of CRISPRs and cas clusters in genomes and their mobile genetic elements.

Prokaryotes use CRISPR-Cas systems for adaptive immunity, but the reasons for the frequent existence of multiple CRISPRs and cas clusters remain poorly understood. Here, we analysed the joint distribution of CRISPR and cas genes in a large set of fully sequenced bacterial genomes and their mobile genetic elements. Our analysis suggests few negative and many positive epistatic interactions between Cas subtypes. The latter often result in complex genetic organizations, where a locus has a single adaptation module and diverse interference mechanisms that might provide more effective immunity. We typed CRISPRs that could not be unambiguously associated with a cas cluster and found that such complex loci tend to have unique type I repeats in multiple CRISPRs. Many chromosomal CRISPRs lack a neighboring Cas system and they often have repeats compatible with the Cas systems encoded in trans. Phages and 25 000 prophages were almost devoid of CRISPR-Cas systems, whereas 3% of plasmids had CRISPR-Cas systems or isolated CRISPRs. The latter were often compatible with the chromosomal cas clusters, suggesting that plasmids can co-opt the latter. These results highlight the importance of interactions between CRISPRs and cas present in multiple copies and in distinct genomic locations in the function and evolution of bacterial immunity.

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.

2'-O-methylation within prokaryotic and eukaryotic tRNA inhibits innate immune activation by endosomal Toll-like receptors but does not affect recognition of whole organisms.

Bacterial RNA has emerged as an important activator of innate immune responses by stimulating Toll-like receptors TLR7 and TLR8 in humans. Guanosine 2'-O-methylation at position 18 (Gm18) in bacterial tRNA was shown to antagonize tRNA-induced TLR7/8 activation, suggesting a potential role of Gm18 as an immune escape mechanism. This modification also occurs in eukaryotic tRNA, yet a physiological immune function remained to be tested. We therefore set out to investigate the immune modulatory role of Gm18 in both prokaryotic and eukaryotic microorganisms, Escherichia coli and Saccharomyces cerevisiae, and in human cells. Using RiboMethSeq analysis we show that mutation of trmH in E. coli, trm3 in S. cereviase, and CRISPR/Cas9-induced knockout of TARBP1 in H. sapiens results in loss of Gm18 within tRNA. Lack of Gm18 across the kingdoms resulted in increased immunostimulation of peripheral blood mononuclear cells when activated by tRNA preparations. In E. coli, lack of 2'-O-methyltransferase trmH also enhanced immune stimulatory properties by whole cellular RNA. In contrast, lack of Gm18 in yeasts and human cells did not affect immunostimulation by whole RNA preparations. When using live E. coli bacteria, lack of trmH did not affect overall immune stimulation although we detected a defined TLR8/RNA-dependent gene expression signature upon E. coli infection. Together, these results demonstrate that Gm18 is a global immune inhibitory tRNA modification across the kingdoms and contributes to tRNA recognition by innate immune cells, but as an individual modification has insufficient potency to modulate recognition of the investigated microorganisms.

Do the Microbiota Influence Vaccines and Protective Immunity to Pathogens? Issues of Sovereignty, Federalism, and Points-Testing in the Prokaryotic and Eukaryotic Spaces of the Host-Microbial Superorganism.

In contrast to live attenuated vaccines, which are designed to induce immunity through a time-limited bloom in systemic tissues, the microbiota is a persistent feature of body surfaces, especially the intestine. The immune responses to the microbiota are idiosyncratic depending on the niche intimacy of different taxa and generally adapt the host to avoid overgrowth and maintain mutualism rather than to eliminate the organisms of that taxon. Both the microbiota and the host have so much molecular cross talk controlling each other, that the prokaryotic and the eukaryotic spaces of the host-microbial superorganism are federal rather than sovereign. This molecular cross talk is vital for the immune system to develop its mature form. Nevertheless, the microbiota/host biomass spaces are rather well separated: The microbiota also limits colonization and penetration of pathogens through intense metabolic competition. Immune responses to those members of the microbiota mutually adapted to intimate association at mucosal surfaces have attractive potential durability, but for clinical use as persistent vehicles they would require personalization and engineered reversibility to manage the immune context and complications in individual human subjects.

Optimal number of spacers in CRISPR arrays.

Prokaryotic organisms survive under constant pressure of viruses. CRISPR-Cas system provides its prokaryotic host with an adaptive immune defense against viruses that have been previously encountered. It consists of two components: Cas-proteins that cleave the foreign DNA and CRISPR array that suits as a virus recognition key. CRISPR array consists of a series of spacers, short pieces of DNA that originate from and match the corresponding parts of viral DNA called protospacers. Here we estimate the number of spacers in a CRISPR array of a prokaryotic cell which maximizes its protection against a viral attack. The optimality follows from a competition between two trends: too few distinct spacers make host vulnerable to an attack by a virus with mutated corresponding protospacers, while an excessive variety of spacers dilutes the number of the CRISPR complexes armed with the most recent and thus most useful spacers. We first evaluate the optimal number of spacers in a simple scenario of an infection by a single viral species and later consider a more general case of multiple viral species. We find that depending on such parameters as the concentration of CRISPR-Cas interference complexes and its preference to arm with more recently acquired spacers, the rate of viral mutation, and the number of viral species, the predicted optimal number of spacers lies within a range that agrees with experimentally-observed values.

Evolution of RNA- and DNA-guided antivirus defense systems in prokaryotes and eukaryotes: common ancestry vs convergence.

Complementarity between nucleic acid molecules is central to biological information transfer processes. Apart from the basal processes of replication, transcription and translation, complementarity is also employed by multiple defense and regulatory systems. All cellular life forms possess defense systems against viruses and mobile genetic elements, and in most of them some of the defense mechanisms involve small guide RNAs or DNAs that recognize parasite genomes and trigger their inactivation. The nucleic acid-guided defense systems include prokaryotic Argonaute (pAgo)-centered innate immunity and CRISPR-Cas adaptive immunity as well as diverse branches of RNA interference (RNAi) in eukaryotes. The archaeal pAgo machinery is the direct ancestor of eukaryotic RNAi that, however, acquired additional components, such as Dicer, and enormously diversified through multiple duplications. In contrast, eukaryotes lack any heritage of the CRISPR-Cas systems, conceivably, due to the cellular toxicity of some Cas proteins that would get activated as a result of operon disruption in eukaryotes. The adaptive immunity function in eukaryotes is taken over partly by the PIWI RNA branch of RNAi and partly by protein-based immunity. In this review, I briefly discuss the interplay between homology and analogy in the evolution of RNA- and DNA-guided immunity, and attempt to formulate some general evolutionary principles for this ancient class of defense systems. REVIEWERS: This article was reviewed by Mikhail Gelfand and Bojan Zagrovic.

SnapShot: Class 1 CRISPR-Cas Systems.

Class 1 CRISPR-Cas systems are characterized by effector modules consisting of multiple subunits. Class 1 systems comprise about 90% of all CRISPR-Cas loci identified in bacteria and archaea and can target both DNA and RNA.

United we stand: big roles for small RNA gene clusters.

Prokaryotes and eukaryotes evolved relatively similar RNA-based molecular mechanisms to fight potentially deleterious nucleic acids coming from phages, transposons, or viruses. Short RNAs guide effector complexes toward their targets to be silenced or eliminated. These short immunity RNAs are transcribed from clustered loci. Unexpectedly and strikingly, bacterial and eukaryotic immunity RNA clusters share substantial functional and mechanistic resemblances in fighting nucleic acid intruders.

[The CRISPR case, « ready-made ¼ mutations and Lamarckian evolution of an adaptive immunity system]. / Le cas CRISPR, mutations « ready-made ¼ et évolution lamarckienne d'un système immunitaire adaptatif.

Since genetics has shown that mutation predates selection, biology has developed within the Darwinian paradigm framework. However, a mechanism that produces favorable mutations preferentially in response to adaptive constraints has been recently identified. This mechanism, the CRISPR-Cas adaptive immunity system, is considered as a bona fide example of Lamarckian evolution, even if it only reflects loosely Lamarck's ideas. This unusual evolutionary process is made possible by two prokaryotic properties: i) somatic and germinal cells are not distinct sets of cells; ii) Archae and Bacteria very frequently integrate DNA fragments from the environment, and they therefore have access to a source of "ready-made" useful genetic information. The CRISPR-Cas is a defense system against viruses and plasmids that is based on the integration of genomic fragments of these infectious agents into the host genome, and that protects the host against subsequent infections. Therefore, this mechanism does produce advantageous mutations by integrating DNA from the environment and allowing its transmission to descendants. In conclusion, most of the time evolution relies on purely Darwinian processes, i.e. mutations occurring at random, but in a small minority of cases the occurrence of mutations is more or less biased, and is therefore more or less Lamarckian. Although they are rare, such processes are nevertheless important to our understanding of the plurality of modes of evolution.

Adaptation in CRISPR-Cas Systems.

Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) proteins constitute an adaptive immune system in prokaryotes. The system preserves memories of prior infections by integrating short segments of foreign DNA, termed spacers, into the CRISPR array in a process termed adaptation. During the past 3 years, significant progress has been made on the genetic requirements and molecular mechanisms of adaptation. Here we review these recent advances, with a focus on the experimental approaches that have been developed, the insights they generated, and a proposed mechanism for self- versus non-self-discrimination during the process of spacer selection. We further describe the regulation of adaptation and the protein players involved in this fascinating process that allows bacteria and archaea to harbor adaptive immunity.

Harnessing the Prokaryotic Adaptive Immune System as a Eukaryotic Antiviral Defense.

Clustered, regularly interspaced, short palindromic repeats - CRISPR-associated (CRISPR-Cas) systems - are sequence-specific RNA-directed endonuclease complexes that bind and cleave nucleic acids. These systems evolved within prokaryotes as adaptive immune defenses to target and degrade nucleic acids derived from bacteriophages and other foreign genetic elements. The antiviral function of these systems has now been exploited to combat eukaryotic viruses throughout the viral life cycle. Here we discuss current advances in CRISPR-Cas9 technology as a eukaryotic antiviral defense.

Construction and expression of Dermatophagoides pteronyssinus group 1 major allergen T cell fusion epitope peptide vaccine vector based on the MHC II pathway / Construcción y expresión de vector de vacuna de péptido epítopo de fusión de células t de alérgeno principal del grupo 1 dermatophagoides pteronyssinus basado en la vía MHC II

Backgound and aims: Dermatophagoides peteronyssinus is one of the important house dust mites responsible for allergic asthma that can be tentatively managed by specific immunotherapy. The present study was to construct a vector encoding T-cell epitopes of major allergen group 1 of Dermatophagoides pteronyssinus as a vaccine delivered by MHC class II pathway. Methods: the nucleotide sequences of the 3 target genes were synthesized, including TAT, IhC and the recombinant fragment of Der p 1 encoding 3 T-cell epitopes. After amplification of the 3 target fragments by PCR and digestion with corresponding restriction endonucleases, the recombinant gene TAT-IhC-Der p 1-3T was ligated using T4 DNA ligase and inserted into the prokaryotic expression vector pET28a(+) to construct the recombinant plasmid pET- 28a(+)-TAT-IhC-Der p 1-3T, which was confirmed by digestion with restriction endonucleases and sequencing. The recombinant vector was transformed into E. coli strain BL21 (DE3) and induced with IPTG, and the induced protein TAT-IhC-Der p1-3T was detected by SDS-PAGE. After purification, the recombinant protein was confirmed by Western blotting and its allergenicity tested using IgE-binding assay. Results: the recombinant plasmid pET-28a-TAT-IhCDer p1-3T was successfully constructed as confirmed by restriction endonuclease digestion and sequencing, and the expression of the recombinant protein TAT-IhC-Der p1-3T was induced in E. coli. Western blotting verified successfull purification of the target protein, which showed a stronger IgE-binding ability than Der p1. Conclusion: we successfully constructed the recombinant expression vector pET-28a-TAT-IhC-Der p1-3T expressing a T-cell epitope vaccine delivered by MHC II pathway with strong IgE-binding ability, which provides a basis for further study on specific immunotherapy via MHC class II pathway (AU)

Antecedentes y objetivo: el Dermatophagoides peteronyssinus es uno de los principales ácaros del polvo doméstico responsables del asma alérgica que se pueden administrar provisionalmente para una inmunoterapia específica. El presente estudio busca construir un vector que codifique epítopos de células T del grupo de alérgenos principal, el Grupo 1 de Dermatophagoides pteronyssinus como una vacuna suministrada mediante la vía MHC de clase II. Métodos: se sintetizaron las secuencias de nucleótidos de los 3 genes objetivo, incluyendo TAT, IhC y el fragmento recombinante de Der p 1 encargado de codificar 3 epítopos de célula T. Después de la amplificación de los 3 fragmentos objetivo por PCR y digestión con endonucleasas de restricción correspondientes, el gen recombinante TAT-IhC-Der p 1-3T se ligó usando T4 DNA ligasa y se insertó en el vector de expresión procariota pET28a (+) para construir el plásmido recombinante pET 28a (+)-TAT-IHC-Der p 1-3T, que se confirmó por digestión con endonucleasas de restricción y secuenciación. El vector recombinante se transformó en E. coli cepa BL21 (DE3) y se indujo con IPTG, y la proteína inducida TATIHC-Der p1-3T se detectó mediante SDS-PAGE. Después de la purificación, la proteina recombinante se confirmó por análisis de inmunotransferencia (Western blot) y se probó su alergenicidad usando el ensayo de unión a IgE. Resultados: el plásmido recombinante pET-28a-TATIHCDer p1-3T se construyó con éxito, se confirmó por digestión con endonucleasas de restricción y la secuenciación y la expresión de la proteína recombinante TAT-IHCDer p1-3T fue inducida en E. coli. Purificación con éxito verificada mediante Western blot de la proteína objetivo, que mostró una capacidad de unión a IgE más fuerte que Der p1. Conclusión: hemos construido con éxito el vector de expresión recombinante pET-28a-TAT-IHC-Der p1-3T que expresa una vacuna de epítopo de células T administrada por vía MHC II con fuerte capacidad de unión a IgE. Este trabajo proporciona una base para seguir estudiando la inmunoterapia específica mediante la vía MHC de clase II (AU)

CRISPR-Cas immunity in prokaryotes.

Prokaryotic organisms are threatened by a large array of viruses and have developed numerous defence strategies. Among these, only clustered, regularly interspaced short palindromic repeat (CRISPR)-Cas systems provide adaptive immunity against foreign elements. Upon viral injection, a small sequence of the viral genome, known as a spacer, is integrated into the CRISPR locus to immunize the host cell. Spacers are transcribed into small RNA guides that direct the cleavage of the viral DNA by Cas nucleases. Immunization through spacer acquisition enables a unique form of evolution whereby a population not only rapidly acquires resistance to its predators but also passes this resistance mechanism vertically to its progeny.