Viral proteins activate PARIS-mediated tRNA degradation and viral tRNAs rescue infection.

Viruses compete with each other for limited cellular resources, and some viruses deliver defense mechanisms that protect the host from competing genetic parasites. PARIS is a defense system, often encoded in viral genomes, that is composed of a 53 kDa ABC ATPase (AriA) and a 35 kDa TOPRIM nuclease (AriB). Here we show that AriA and AriB assemble into a 425 kDa supramolecular immune complex. We use cryo-EM to determine the structure of this complex which explains how six molecules of AriA assemble into a propeller-shaped scaffold that coordinates three subunits of AriB. ATP-dependent detection of foreign proteins triggers the release of AriB, which assembles into a homodimeric nuclease that blocks infection by cleaving the host tRNALys. Phage T5 subverts PARIS immunity through expression of a tRNALys variant that prevents PARIS-mediated cleavage, and thereby restores viral infection. Collectively, these data explain how AriA functions as an ATP-dependent sensor that detects viral proteins and activates the AriB toxin. PARIS is one of an emerging set of immune systems that form macromolecular complexes for the recognition of foreign proteins, rather than foreign nucleic acids.

[Bacterial immunity: Uncovering a new world]. / Immunité bactérienne : à la découverte d'un nouveau monde.

Viruses are parasites that infect all living organisms, and bacteria are no exception. To defend themselves against their viruses (phages), bacteria have developed numerous and sophisticated defense mechanisms, our understanding of which is rapidly growing. In the 2000s, only a handful of mechanisms were known and only two of them seemed to be found in most bacteria. In 2018, a new key method based on genome analysis revealed that there were likely many others. Indeed, over the past five years, more than 150 new mechanisms have been discovered. It is now estimated that there are probably thousands. This remarkable diversity, paralleled with the tremendous viral diversity, is evident both in terms of possible combinations of systems in bacterial genomes and in molecular mechanisms. One of the most surprising observations emerging from the exploration of this diversity is the discovery of striking similarities between certain bacterial defense systems and antiviral systems in humans, as well as plant (and eukaryotes in general) immune systems. Contrary to the previously accepted paradigm, organisms as diverse as fungi, plants, bacteria and humans share certain molecular strategies to fight viral infections, suggesting that an underestimated part of eukaryotic antiviral immunity could have evolved from bacterial antiviral defense systems.

Title: Immunité bactérienne : à la découverte d'un nouveau monde. Abstract: Les virus sont des parasites qui infectent tous les organismes vivants, et les bactéries n'y font pas exception. Pour se défendre contre leurs virus (les bactériophages ou phages), les bactéries se sont dotées d'un éventail de mécanismes élaborés, dont la découverte et la compréhension sont en pleine expansion. Dans les années 2000, seuls quelques systèmes de défense étaient connus et deux semblaient présents chez la plupart des bactéries. En 2018, une nouvelle méthode fondée sur l'analyse des génomes a révélé l'existence potentielle de nombreux autres. Plus de 150 nouveaux systèmes anti-phages ont été découverts au cours des cinq dernières années. On estime maintenant qu'il en existe probablement des milliers. Cette formidable diversité, qui est à mettre en parallèle avec la considérable diversité virale, s'exprime tant en termes de combinaisons de systèmes possibles dans les génomes bactériens que de mécanismes moléculaires. Une des observations les plus surprenantes qui émerge est la découverte de similarités entre certains systèmes de défense bactériens et des mécanismes antiviraux eucaryotes. Contrairement au paradigme jusqu'alors en place, des organismes aussi différents que des champignons, des plantes, des bactéries ou des êtres humains partagent certaines stratégies moléculaires pour combattre des infections virales, suggérant qu'une part sous-estimée de l'immunité antivirale eucaryote a directement évolué à partir des systèmes de défense bactériens.

The highly diverse antiphage defence systems of bacteria.

Bacteria and their viruses have coevolved for billions of years. This ancient and still ongoing arms race has led bacteria to develop a vast antiphage arsenal. The development of high-throughput screening methods expanded our knowledge of defence systems from a handful to more than a hundred systems, unveiling many different molecular mechanisms. These findings reveal that bacterial immunity is much more complex than previously thought. In this Review, we explore recently discovered bacterial antiphage defence systems, with a particular focus on their molecular diversity, and discuss the ecological and evolutionary drivers and implications of the existing diversity of antiphage defence mechanisms.

Synergy and regulation of antiphage systems: toward the existence of a bacterial immune system?

Bacteria encode a vast repertoire of diverse antiphage defense systems. Recent studies revealed that different defense systems are often encoded within the same genome, raising the question of their possible interactions in a cell. Here, we review the known synergies and coregulations of antiphage systems. The emerging complexities suggest a potential existence of an additional level of organization of antiviral defense in prokaryotes. We argue that this organization could be compared with immune systems of animals and plants. We discuss this concept and explore what it could mean in bacteria.

Multiple phage resistance systems inhibit infection via SIR2-dependent NAD+ depletion.

Defence-associated sirtuins (DSRs) comprise a family of proteins that defend bacteria from phage infection via an unknown mechanism. These proteins are common in bacteria and harbour an N-terminal sirtuin (SIR2) domain. In this study we report that DSR proteins degrade nicotinamide adenine dinucleotide (NAD+) during infection, depleting the cell of this essential molecule and aborting phage propagation. Our data show that one of these proteins, DSR2, directly identifies phage tail tube proteins and then becomes an active NADase in Bacillus subtilis. Using a phage mating methodology that promotes genetic exchange between pairs of DSR2-sensitive and DSR2-resistant phages, we further show that some phages express anti-DSR2 proteins that bind and repress DSR2. Finally, we demonstrate that the SIR2 domain serves as an effector NADase in a diverse set of phage defence systems outside the DSR family. Our results establish the general role of SIR2 domains in bacterial immunity against phages.

An expanded arsenal of immune systems that protect bacteria from phages.

Bacterial anti-phage systems are frequently clustered in microbial genomes, forming defense islands. This property enabled the recent discovery of multiple defense systems based on their genomic co-localization with known systems, but the full arsenal of anti-phage mechanisms remains unknown. We report the discovery of 21 defense systems that protect bacteria from phages, based on computational genomic analyses and phage-infection experiments. We identified multiple systems with domains involved in eukaryotic antiviral immunity, including those homologous to the ubiquitin-like ISG15 protein, dynamin-like domains, and SEFIR domains, and show their participation in bacterial defenses. Additional systems include domains predicted to manipulate DNA and RNA molecules, alongside toxin-antitoxin systems shown here to function in anti-phage defense. These systems are widely distributed in microbial genomes, and in some bacteria, they form a considerable fraction of the immune arsenal. Our data substantially expand the inventory of defense systems utilized by bacteria to counteract phage infection.

Systematic and quantitative view of the antiviral arsenal of prokaryotes.

Bacteria and archaea have developed multiple antiviral mechanisms, and genomic evidence indicates that several of these antiviral systems co-occur in the same strain. Here, we introduce DefenseFinder, a tool that automatically detects known antiviral systems in prokaryotic genomes. We use DefenseFinder to analyse 21000 fully sequenced prokaryotic genomes, and find that antiviral strategies vary drastically between phyla, species and strains. Variations in composition of antiviral systems correlate with genome size, viral threat, and lifestyle traits. DefenseFinder will facilitate large-scale genomic analysis of antiviral defense systems and the study of host-virus interactions in prokaryotes.

CRISPR-Cas and restriction-modification team up to achieve long-term immunity.

Bacteria have been shown to harbor a growing arsenal of various defense systems against phages. Maguin et al. have uncovered how two of the most frequent defense systems interact: the clustered regularly interspaced short palindromic repeats-Cas (CRISPR-Cas) system recycles by-products of the restriction-modification (RM) system to increase bacterial defense in the long run.

Phages and their satellites encode hotspots of antiviral systems.

Bacteria carry diverse genetic systems to defend against viral infection, some of which are found within prophages where they inhibit competing viruses. Phage satellites pose additional pressures on phages by hijacking key viral elements to their own benefit. Here, we show that E. coli P2-like phages and their parasitic P4-like satellites carry hotspots of genetic variation containing reservoirs of anti-phage systems. We validate the activity of diverse systems and describe PARIS, an abortive infection system triggered by a phage-encoded anti-restriction protein. Antiviral hotspots participate in inter-viral competition and shape dynamics between the bacterial host, P2-like phages, and P4-like satellites. Notably, the anti-phage activity of satellites can benefit the helper phage during competition with virulent phages, turning a parasitic relationship into a mutualistic one. Anti-phage hotspots are present across distant species and constitute a substantial source of systems that participate in the competition between mobile genetic elements.

Evolutionary and mechanistic diversity of Type I-F CRISPR-associated transposons.

Canonical CRISPR-Cas systems utilize RNA-guided nucleases for targeted cleavage of foreign nucleic acids, whereas some nuclease-deficient CRISPR-Cas complexes have been repurposed to direct the insertion of Tn7-like transposons. Here, we established a bioinformatic and experimental pipeline to comprehensively explore the diversity of Type I-F CRISPR-associated transposons. We report DNA integration for 20 systems and identify a highly active subset that exhibits complete orthogonality in transposon DNA mobilization. We reveal the modular nature of CRISPR-associated transposons by exploring the horizontal acquisition of targeting modules and by characterizing a system that encodes both a programmable, RNA-dependent pathway, and a fixed, RNA-independent pathway. Finally, we analyzed transposon-encoded cargo genes and found the striking presence of anti-phage defense systems, suggesting a role in transmitting innate immunity between bacteria. Collectively, this study substantially advances our biological understanding of CRISPR-associated transposon function and expands the suite of RNA-guided transposases for programmable, large-scale genome engineering.

Engineering gene overlaps to sustain genetic constructs in vivo.

Evolution is often an obstacle to the engineering of stable biological systems due to the selection of mutations inactivating costly gene circuits. Gene overlaps induce important constraints on sequences and their evolution. We show that these constraints can be harnessed to increase the stability of costly genes by purging loss-of-function mutations. We combine computational and synthetic biology approaches to rationally design an overlapping reading frame expressing an essential gene within an existing gene to protect. Our algorithm succeeded in creating overlapping reading frames in 80% of E. coli genes. Experimentally, scoring mutations in both genes of such overlapping construct, we found that a significant fraction of mutations impacting the gene to protect have a deleterious effect on the essential gene. Such an overlap thus protects a costly gene from removal by natural selection by associating the benefit of this removal with a larger or even lethal cost. In our synthetic constructs, the overlap converts many of the possible mutants into evolutionary dead-ends, reducing the evolutionary potential of the system and thus increasing its stability over time.

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.

Bacterial Retrons Function In Anti-Phage Defense.

Retrons are bacterial genetic elements comprised of a reverse transcriptase (RT) and a non-coding RNA (ncRNA). The RT uses the ncRNA as template, generating a chimeric RNA/DNA molecule in which the RNA and DNA components are covalently linked. Although retrons were discovered three decades ago, their function remained unknown. We report that retrons function as anti-phage defense systems. The defensive unit is composed of three components: the RT, the ncRNA, and an effector protein. We examined multiple retron systems and show that they confer defense against a broad range of phages via abortive infection. Focusing on retron Ec48, we show evidence that it "guards" RecBCD, a complex with central anti-phage functions in bacteria. Inhibition of RecBCD by phage proteins activates the retron, leading to abortive infection and cell death. Thus, the Ec48 retron forms a second line of defense that is triggered if the first lines of defense have collapsed.

Methods for the Analysis and Characterization of Defense Mechanisms Against Horizontal Gene Transfer: CRISPR Systems.

CRISPR-Cas systems provide RNA-guided adaptive immunity to the majority of archaea and many bacteria. They are able to capture pieces of invading genetic elements in the form of novel spacers in an array of repeats. These elements can then be used as a memory to destroy incoming DNA through the action of RNA-guided nucleases. This chapter describes general procedures to determine the ability of CRISPR-Cas systems to capture novel sequences and to use them to block phages and horizontal gene transfer. All protocols are performed in Staphylococcus aureus using Type II-A CRISPR-Cas systems. Nonetheless, the protocols provided can be adapted to work with other bacteria and other types of CRISPR-Cas systems.

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.

The pan-immune system of bacteria: antiviral defence as a community resource.

Viruses and their hosts are engaged in a constant arms race leading to the evolution of antiviral defence mechanisms. Recent studies have revealed that the immune arsenal of bacteria against bacteriophages is much more diverse than previously envisioned. These discoveries have led to seemingly contradictory observations: on one hand, individual microorganisms often encode multiple distinct defence systems, some of which are acquired by horizontal gene transfer, alluding to their fitness benefit. On the other hand, defence systems are frequently lost from prokaryotic genomes on short evolutionary time scales, suggesting that they impose a fitness cost. In this Perspective article, we present the 'pan-immune system' model in which we suggest that, although a single strain cannot carry all possible defence systems owing to their burden on fitness, it can employ horizontal gene transfer to access immune defence mechanisms encoded by closely related strains. Thus, the 'effective' immune system is not the one encoded by the genome of a single microorganism but rather by its pan-genome, comprising the sum of all immune systems available for a microorganism to horizontally acquire and use.

A matter of background: DNA repair pathways as a possible cause for the sparse distribution of CRISPR-Cas systems in bacteria.

The absence of CRISPR-Cas systems in more than half of the sequenced bacterial genomes is intriguing, because their role in adaptive immunity and their frequent transfer between species should have made them almost ubiquitous, as is the case in Archaea. Here, we investigate the possibility that the success of CRISPR-Cas acquisition by horizontal gene transfer is affected by the interactions of these systems with the host genetic background and especially with components of double-strand break repair systems (DSB-RS). We first described the distribution of systems specialized in the repair of double-strand breaks in Bacteria: homologous recombination and non-homologous end joining. This allowed us to show that such systems are more often positively or negatively correlated with the frequency of CRISPR-Cas systems than random genes of similar frequency. The detailed analysis of these co-occurrence patterns shows that our method identifies previously known cases of mechanistic interactions between these systems. It also reveals other positive and negative patterns of co-occurrence between DSB-RS and CRISPR-Cas systems. Notably, it shows that the patterns of distribution of CRISPR-Cas systems in Proteobacteria are strongly dependent on the epistatic groups including RecBCD and AddAB. Our results suggest that the genetic background plays an important role in the success of adaptive immunity in different bacterial clades and provide insights to guide further experimental research on the interactions between CRISPR-Cas and DSB-RS. This article is part of a discussion meeting issue 'The ecology and evolution of prokaryotic CRISPR-Cas adaptive immune systems'.

Genetic exchanges are more frequent in bacteria encoding capsules.

Capsules allow bacteria to colonize novel environments, to withstand numerous stresses, and to resist antibiotics. Yet, even though genetic exchanges with other cells should be adaptive under such circumstances, it has been suggested that capsules lower the rates of homologous recombination and horizontal gene transfer. We analysed over one hundred pan-genomes and thousands of bacterial genomes for the evidence of an association between genetic exchanges (or lack thereof) and the presence of a capsule system. We found that bacteria encoding capsules have larger pan-genomes, higher rates of horizontal gene transfer, and higher rates of homologous recombination in their core genomes. Accordingly, genomes encoding capsules have more plasmids, conjugative elements, transposases, prophages, and integrons. Furthermore, capsular loci are frequent in plasmids, and can be found in prophages. These results are valid for Bacteria, independently of their ability to be naturally transformable. Since we have shown previously that capsules are commonly present in nosocomial pathogens, we analysed their co-occurrence with antibiotic resistance genes. Genomes encoding capsules have more antibiotic resistance genes, especially those encoding efflux pumps, and they constitute the majority of the most worrisome nosocomial bacteria. We conclude that bacteria with capsule systems are more genetically diverse and have fast-evolving gene repertoires, which may further contribute to their success in colonizing novel niches such as humans under antibiotic therapy.