RESUMEN
During viral infection, cells can deploy immune strategies that deprive viruses of molecules essential for their replication. Here, we report a family of immune effectors in bacteria that, upon phage infection, degrade cellular adenosine triphosphate (ATP) and deoxyadenosine triphosphate (dATP) by cleaving the N-glycosidic bond between the adenine and sugar moieties. These ATP nucleosidase effectors are widely distributed within multiple bacterial defense systems, including cyclic oligonucleotide-based antiviral signaling systems (CBASS), prokaryotic argonautes, and nucleotide-binding leucine-rich repeat (NLR)-like proteins, and we show that ATP and dATP degradation during infection halts phage propagation. By analyzing homologs of the immune ATP nucleosidase domain, we discover and characterize Detocs, a family of bacterial defense systems with a two-component phosphotransfer-signaling architecture. The immune ATP nucleosidase domain is also encoded within diverse eukaryotic proteins with immune-like architectures, and we show biochemically that eukaryotic homologs preserve the ATP nucleosidase activity. Our findings suggest that ATP and dATP degradation is a cell-autonomous innate immune strategy conserved across the tree of life.
Asunto(s)
Virosis , Humanos , Células Eucariotas , Células Procariotas , Adenosina Trifosfato , N-Glicosil HidrolasasRESUMEN
Over the past few years, numerous anti-phage defense systems have been discovered in bacteria. Although the mechanism of defense for some of these systems is understood, a major unanswered question is how these systems sense phage infection. To systematically address this question, we isolated 177 phage mutants that escape 15 different defense systems. In many cases, these escaper phages were mutated in the gene sensed by the defense system, enabling us to map the phage determinants that confer sensitivity to bacterial immunity. Our data identify specificity determinants of diverse retron systems and reveal phage-encoded triggers for multiple abortive infection systems. We find general themes in phage sensing and demonstrate that mechanistically diverse systems have converged to sense either the core replication machinery of the phage, phage structural components, or host takeover mechanisms. Combining our data with previous findings, we formulate key principles on how bacterial immune systems sense phage invaders.
Asunto(s)
Bacterias , Bacteriófagos , Bacterias/genética , Bacterias/virología , Bacteriófagos/genética , Sistemas CRISPR-Cas , Proteínas Virales/metabolismo , Mutación , Fenómenos Fisiológicos BacterianosRESUMEN
RADAR is a two-protein bacterial defense system that was reported to defend against phage by "editing" messenger RNA. Here, we determine cryo-EM structures of the RADAR defense complex, revealing RdrA as a heptameric, two-layered AAA+ ATPase and RdrB as a dodecameric, hollow complex with twelve surface-exposed deaminase active sites. RdrA and RdrB join to form a giant assembly up to 10 MDa, with RdrA docked as a funnel over the RdrB active site. Surprisingly, our structures reveal an RdrB active site that targets mononucleotides. We show that RdrB catalyzes ATP-to-ITP conversion in vitro and induces the massive accumulation of inosine mononucleotides during phage infection in vivo, limiting phage replication. Our results define ATP mononucleotide deamination as a determinant of RADAR immunity and reveal supramolecular assembly of a nucleotide-modifying machine as a mechanism of anti-phage defense.
Asunto(s)
Bacteriófagos , Bacteriófagos/metabolismo , Microscopía por Crioelectrón/métodos , ATPasas Asociadas con Actividades Celulares Diversas , Adenosina Trifosfato , Adenosina Desaminasa/metabolismoRESUMEN
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.
Asunto(s)
Bacteriófagos , NAD , NAD/metabolismo , Proteínas Reguladoras de Información Silente de Saccharomyces cerevisiae/genética , Proteínas Reguladoras de Información Silente de Saccharomyces cerevisiae/metabolismo , Sirtuina 2/genética , Bacteriófagos/genética , Bacteriófagos/metabolismo , NAD+ NucleosidasaRESUMEN
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.
Asunto(s)
Bacteriófagos , Bacteriófagos/genética , Bacterias/genética , Genoma Microbiano , Genómica , Sistema InmunológicoRESUMEN
DNA viruses and retroviruses consume large quantities of deoxynucleotides (dNTPs) when replicating. The human antiviral factor SAMHD1 takes advantage of this vulnerability in the viral lifecycle, and inhibits viral replication by degrading dNTPs into their constituent deoxynucleosides and inorganic phosphate. Here, we report that bacteria use a similar strategy to defend against bacteriophage infection. We identify a family of defensive bacterial deoxycytidine triphosphate (dCTP) deaminase proteins that convert dCTP into deoxyuracil nucleotides in response to phage infection. We also identify a family of phage resistance genes that encode deoxyguanosine triphosphatase (dGTPase) enzymes, which degrade dGTP into phosphate-free deoxyguanosine and are distant homologues of human SAMHD1. Our results suggest that bacterial defensive proteins deplete specific deoxynucleotides (either dCTP or dGTP) from the nucleotide pool during phage infection, thus starving the phage of an essential DNA building block and halting its replication. Our study shows that manipulation of the dNTP pool is a potent antiviral strategy shared by both prokaryotes and eukaryotes.
Asunto(s)
Bacteriófagos , Antivirales , Bacterias , Bacteriófagos/genética , Desoxiguanosina , Humanos , Proteína 1 que Contiene Dominios SAM y HDRESUMEN
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.
Asunto(s)
Antivirales/metabolismo , Proteínas Arqueales/metabolismo , Proteínas Bacterianas/metabolismo , Bacteriófago T7/inmunología , Evolución Molecular , Células Procariotas/metabolismo , Proteínas/metabolismo , Antivirales/inmunología , Proteínas Arqueales/química , Bacterias/inmunología , Bacterias/metabolismo , Bacterias/virología , Proteínas Bacterianas/química , Bacteriófago T7/enzimología , Bacteriófago T7/fisiología , ADN Polimerasa Dirigida por ADN/metabolismo , Humanos , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH , Células Procariotas/inmunología , Células Procariotas/virología , Proteínas/química , Proteínas/genética , Ribonucleótidos/biosíntesis , Ribonucleótidos/química , Ribonucleótidos/metabolismo , Transcripción Genética/efectos de los fármacosRESUMEN
Cyclic-oligonucleotide-based anti-phage signalling systems (CBASS) are a family of defence systems against bacteriophages (hereafter phages) that share ancestry with the cGAS-STING innate immune pathway in animals. CBASS systems are composed of an oligonucleotide cyclase, which generates signalling cyclic oligonucleotides in response to phage infection, and an effector that is activated by the cyclic oligonucleotides and promotes cell death. Cell death occurs before phage replication is completed, therefore preventing the spread of phages to nearby cells. Here, we analysed 38,000 bacterial and archaeal genomes and identified more than 5,000 CBASS systems, which have diverse architectures with multiple signalling molecules, effectors and ancillary genes. We propose a classification system for CBASS that groups systems according to their operon organization, signalling molecules and effector function. Four major CBASS types were identified, sharing at least six effector subtypes that promote cell death by membrane impairment, DNA degradation or other means. We observed evidence of extensive gain and loss of CBASS systems, as well as shuffling of effector genes between systems. We expect that our classification and nomenclature scheme will guide future research in the developing CBASS field.
Asunto(s)
Bacterias/inmunología , Bacterias/virología , Proteínas Bacterianas/inmunología , Bacteriófagos/fisiología , Oligonucleótidos/inmunología , Bacterias/clasificación , Bacterias/genética , Proteínas Bacterianas/genética , Bacteriófagos/genética , Genoma Bacteriano , Inmunidad Innata , Oligonucleótidos/genética , Filogenia , Transducción de SeñalRESUMEN
The cyclic GMP-AMP synthase (cGAS)-STING pathway is a central component of the cell-autonomous innate immune system in animals1,2. The cGAS protein is a sensor of cytosolic viral DNA and, upon sensing DNA, it produces a cyclic GMP-AMP (cGAMP) signalling molecule that binds to the STING protein and activates the immune response3-5. The production of cGAMP has also been detected in bacteria6, and has been shown, in Vibrio cholerae, to activate a phospholipase that degrades the inner bacterial membrane7. However, the biological role of cGAMP signalling in bacteria remains unknown. Here we show that cGAMP signalling is part of an antiphage defence system that is common in bacteria. This system is composed of a four-gene operon that encodes the bacterial cGAS and the associated phospholipase, as well as two enzymes with the eukaryotic-like domains E1, E2 and JAB. We show that this operon confers resistance against a wide variety of phages. Phage infection triggers the production of cGAMP, which-in turn-activates the phospholipase, leading to a loss of membrane integrity and to cell death before completion of phage reproduction. Diverged versions of this system appear in more than 10% of prokaryotic genomes, and we show that variants with effectors other than phospholipase also protect against phage infection. Our results suggest that the eukaryotic cGAS-STING antiviral pathway has ancient evolutionary roots that stem from microbial defences against phages.
Asunto(s)
Bacterias/virología , Nucleótidos Cíclicos/metabolismo , Transducción de Señal , Bacterias/inmunología , Bacterias/metabolismo , Proteínas Bacterianas/metabolismo , Bacteriófagos/fisiología , Nucleótidos Cíclicos/inmunología , Replicación ViralRESUMEN
The arms race between bacteria and phages led to the development of sophisticated antiphage defense systems, including CRISPR-Cas and restriction-modification systems. Evidence suggests that known and unknown defense systems are located in "defense islands" in microbial genomes. Here, we comprehensively characterized the bacterial defensive arsenal by examining gene families that are clustered next to known defense genes in prokaryotic genomes. Candidate defense systems were systematically engineered and validated in model bacteria for their antiphage activities. We report nine previously unknown antiphage systems and one antiplasmid system that are widespread in microbes and strongly protect against foreign invaders. These include systems that adopted components of the bacterial flagella and condensin complexes. Our data also suggest a common, ancient ancestry of innate immunity components shared between animals, plants, and bacteria.
Asunto(s)
Bacillus subtilis/inmunología , Bacillus subtilis/virología , Bacteriófagos/inmunología , Bacteriófagos/patogenicidad , Escherichia coli/inmunología , Escherichia coli/virología , Genes Bacterianos/fisiología , Bacillus subtilis/genética , Escherichia coli/genética , Genoma Bacteriano , Familia de MultigenesRESUMEN
The larval pool of coral reef fish has a crucial role in the dynamics of adult fish populations. However, large-scale species-level monitoring of species-rich larval pools has been technically impractical. Here, we use high-throughput metabarcoding to study larval ecology in the Gulf of Aqaba, a region that is inhabited by >500 reef fish species. We analysed 9,933 larvae from 383 samples that were stratified over sites, depth and time. Metagenomic DNA extracted from pooled larvae was matched to a mitochondrial cytochrome c oxidase subunit I barcode database compiled for 77% of known fish species within this region. This yielded species-level reconstruction of the larval community, allowing robust estimation of larval spatio-temporal distributions. We found significant correlations between species abundance in the larval pool and in local adult assemblages, suggesting a major role for larval supply in determining local adult densities. We documented larval flux of species whose adults were never documented in the region, suggesting environmental filtering as the reason for the absence of these species. Larvae of several deep-sea fishes were found in shallow waters, supporting their dispersal over shallow bathymetries, potentially allowing Lessepsian migration into the Mediterranean Sea. Our method is applicable to any larval community and could assist coral reef conservation and fishery management efforts.
Asunto(s)
Distribución Animal , Peces/fisiología , Metagenoma , Animales , Arrecifes de Coral , Complejo IV de Transporte de Electrones/análisis , Proteínas de Peces/análisis , Peces/crecimiento & desarrollo , Israel , Larva/crecimiento & desarrollo , Larva/fisiología , Proteínas Mitocondriales/análisis , Océanos y Mares , Densidad de Población , Análisis Espacio-TemporalRESUMEN
The evolutionary pressure imposed by phage predation on bacteria and archaea has resulted in the development of effective anti-phage defence mechanisms, including restriction-modification and CRISPR-Cas systems. Here, we report on a new defence system, DISARM (defence island system associated with restriction-modification), which is widespread in bacteria and archaea. DISARM is composed of five genes, including a DNA methylase and four other genes annotated as a helicase domain, a phospholipase D (PLD) domain, a DUF1998 domain and a gene of unknown function. Engineering the Bacillus paralicheniformis 9945a DISARM system into Bacillus subtilis has rendered the engineered bacteria protected against phages from all three major families of tailed double-stranded DNA phages. Using a series of gene deletions, we show that four of the five genes are essential for DISARM-mediated defence, with the fifth (PLD) being redundant for defence against some of the phages. We further show that DISARM restricts incoming phage DNA and that the B. paralicheniformis DISARM methylase modifies host CCWGG motifs as a marker of self DNA akin to restriction-modification systems. Our results suggest that DISARM is a new type of multi-gene restriction-modification module, expanding the arsenal of defence systems known to be at the disposal of prokaryotes against their viruses.
Asunto(s)
Bacillus subtilis/genética , Bacillus subtilis/virología , Proteínas Bacterianas/metabolismo , Bacteriófagos/fisiología , Enzimas de Restricción-Modificación del ADN/genética , Familia de Multigenes/genética , Proteínas Bacterianas/genética , Bacteriófagos/clasificación , Bacteriófagos/crecimiento & desarrollo , Clonación Molecular , Biología Computacional , Genoma Bacteriano/genética , Islas Genómicas , Metiltransferasas/genética , Modelos Genéticos , Eliminación de Secuencia , Replicación ViralRESUMEN
Temperate viruses can become dormant in their host cells, a process called lysogeny. In every infection, such viruses decide between the lytic and the lysogenic cycles, that is, whether to replicate and lyse their host or to lysogenize and keep the host viable. Here we show that viruses (phages) of the SPbeta group use a small-molecule communication system to coordinate lysis-lysogeny decisions. During infection of its Bacillus host cell, the phage produces a six amino-acids-long communication peptide that is released into the medium. In subsequent infections, progeny phages measure the concentration of this peptide and lysogenize if the concentration is sufficiently high. We found that different phages encode different versions of the communication peptide, demonstrating a phage-specific peptide communication code for lysogeny decisions. We term this communication system the 'arbitrium' system, and further show that it is encoded by three phage genes: aimP, which produces the peptide; aimR, the intracellular peptide receptor; and aimX, a negative regulator of lysogeny. The arbitrium system enables a descendant phage to 'communicate' with its predecessors, that is, to estimate the amount of recent previous infections and hence decide whether to employ the lytic or lysogenic cycle.
Asunto(s)
Bacteriólisis , Bacteriófagos/fisiología , Lisogenia , Secuencia de Aminoácidos , Bacillus/citología , Bacillus/virología , Bacteriólisis/efectos de los fármacos , Bacteriófagos/efectos de los fármacos , Medios de Cultivo Condicionados/química , Medios de Cultivo Condicionados/farmacología , ADN Viral/metabolismo , Lisogenia/efectos de los fármacos , Modelos Biológicos , Péptidos/química , Péptidos/metabolismo , Péptidos/farmacología , Multimerización de Proteína , Transcripción Genética/efectos de los fármacos , Proteínas Virales/química , Proteínas Virales/metabolismo , Proteínas Virales/farmacologíaRESUMEN
Addition of pyrolyzed biomass ("biochar") to soils has commonly been shown to increase crop yields and alleviate plant stresses associated with drought and exposure to toxic materials. Here we investigate the ability of biochar (at two dosages: 5 and 50 t ha(-1)) to mitigate salt-induced stress, simulating road salt additions in a factorial glasshouse experiment involving the broadleaved herbaceous plants Abutilon theophrasti and Prunella vulgaris. Salt additions of 30 g m(-2) NaCl to unamended soils resulted in high mortality rates for both species. Biochar (Fagus grandifolia sawdust pyrolyzed at 378 °C), when applied at 50 t ha(-1) as a top dressing, completely alleviated salt-induced mortality in A. theophrasti and prolonged survival of P. vulgaris. Surviving A. theophrasti plants that received both 50 t ha(-1) biochar and salt addition treatments showed growth rates and physiological performance similar to plants without salt addition. Biochar treatments alone also substantially increased biomass of P. vulgaris, with a â¼50% increase relative to untreated controls at both biochar dosages. Biochar did not significantly affect photosynthetic carbon gain (Amax), water use efficiency, or chlorophyll fluorescence (Fv/Fm) in either species. Our results indicate that biochar can ameliorate salt stress effects on plants through salt sorption, suggesting novel applications of biochar to mitigate effects of salinization in agricultural, urban, and contaminated soils.
Asunto(s)
Carbón Orgánico/metabolismo , Contaminación Ambiental/prevención & control , Restauración y Remediación Ambiental/métodos , Malvaceae/efectos de los fármacos , Prunella/efectos de los fármacos , Cloruro de Sodio/toxicidad , Contaminantes del Suelo/toxicidad , Carbón Orgánico/administración & dosificación , Malvaceae/crecimiento & desarrollo , Malvaceae/fisiología , Ontario , Fotosíntesis/efectos de los fármacos , Prunella/crecimiento & desarrollo , Prunella/fisiología , Distribución Aleatoria , Estrés FisiológicoRESUMEN
The use of DNA barcodes, short DNA sequences from a standardized region of the genome, has recently been proposed as a tool to facilitate species identification and discovery. Here we show that second internal transcribed spacer of nuclear ribosomal DNA (rDNA-ITS2) barcodes effectively discriminate among 16 species of spider mites (Acari: Tetranychidae) from Israel. The barcode sequences of each species were unambiguously distinguishable from all other species and formed distinct, nonoverlapping monophyletic groups in the maximum-parsimony tree. Sequence divergences were generally much greater between species than within them. Using a 0.02 (2%) threshold for species diagnosis in our data set, 14 out of 16 species recognized by morphological criteria would be accurately identified. The only exceptions involved the low divergence, 0.011-0.015 (1.1-1.5%), between Tetranychus urticae and Tetranychus turkestani, where speciation may have occurred only recently. Still, these species had fixed alternative rDNA-ITS2 variants, with five diagnostic nucleotide substitutions. As a result, we tentatively conclude that rDNA-ITS2 sequence barcodes may serve as an effective tool for the identification of spider mite species and can be applicable as a diagnostic tool for quarantine and other pest management activities and decision-making. We predict that our work, together with similar efforts, will provide in the future the platform for a uniform, accurate, practical and easy-to-use method of spider mite species identification.