RESUMO
It was recently shown that bacteria use, apart from CRISPR-Cas and restriction systems, a considerable diversity of phage resistance systems1-4, but it is largely unknown how phages cope with this multilayered bacterial immunity. Here we analysed groups of closely related Bacillus phages that showed differential sensitivity to bacterial defence systems, and discovered four distinct families of anti-defence proteins that inhibit the Gabija, Thoeris and Hachiman systems. We show that these proteins Gad1, Gad2, Tad2 and Had1 efficiently cancel the defensive activity when co-expressed with the respective defence system or introduced into phage genomes. Homologues of these anti-defence proteins are found in hundreds of phages that infect taxonomically diverse bacterial species. We show that the anti-Gabija protein Gad1 blocks the ability of the Gabija defence complex to cleave phage-derived DNA. Our data further reveal that the anti-Thoeris protein Tad2 is a 'sponge' that sequesters the immune signalling molecules produced by Thoeris TIR-domain proteins in response to phage infection. Our results demonstrate that phages encode an arsenal of anti-defence proteins that can disable a variety of bacterial defence mechanisms.
Assuntos
Fagos Bacilares , Bactérias , Proteínas Virais , Fagos Bacilares/classificação , Fagos Bacilares/genética , Fagos Bacilares/imunologia , Fagos Bacilares/metabolismo , Bactérias/classificação , Bactérias/genética , Bactérias/imunologia , Bactérias/virologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , DNA Viral/genética , DNA Viral/metabolismo , Proteínas Virais/genética , Proteínas Virais/metabolismoRESUMO
Bacteria encode hundreds of diverse defence systems that protect them from viral infection and inhibit phage propagation1-5. Gabija is one of the most prevalent anti-phage defence systems, occurring in more than 15% of all sequenced bacterial and archaeal genomes1,6,7, but the molecular basis of how Gabija defends cells from viral infection remains poorly understood. Here we use X-ray crystallography and cryo-electron microscopy (cryo-EM) to define how Gabija proteins assemble into a supramolecular complex of around 500 kDa that degrades phage DNA. Gabija protein A (GajA) is a DNA endonuclease that tetramerizes to form the core of the anti-phage defence complex. Two sets of Gabija protein B (GajB) dimers dock at opposite sides of the complex and create a 4:4 GajA-GajB assembly (hereafter, GajAB) that is essential for phage resistance in vivo. We show that a phage-encoded protein, Gabija anti-defence 1 (Gad1), directly binds to the Gabija GajAB complex and inactivates defence. A cryo-EM structure of the virally inhibited state shows that Gad1 forms an octameric web that encases the GajAB complex and inhibits DNA recognition and cleavage. Our results reveal the structural basis of assembly of the Gabija anti-phage defence complex and define a unique mechanism of viral immune evasion.
Assuntos
Bactérias , Proteínas de Bactérias , Bacteriófagos , Evasão da Resposta Imune , Multimerização Proteica , Bactérias/genética , Bactérias/imunologia , Bactérias/metabolismo , Bactérias/virologia , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/ultraestrutura , Bacteriófagos/genética , Bacteriófagos/imunologia , Bacteriófagos/metabolismo , Microscopia Crioeletrônica , Cristalografia por Raios X , Desoxirribonucleases/química , Desoxirribonucleases/metabolismo , Desoxirribonucleases/ultraestrutura , DNA Viral/química , DNA Viral/metabolismo , DNA Viral/ultraestruturaRESUMO
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.
Assuntos
Viroses , Humanos , Células Eucarióticas , Células Procarióticas , Trifosfato de Adenosina , N-Glicosil HidrolasesRESUMO
Caspase recruitment domains (CARDs) and pyrin domains are important facilitators of inflammasome activity and pyroptosis. Upon pathogen recognition by NLR proteins, CARDs recruit and activate caspases, which, in turn, activate gasdermin pore forming proteins to and induce pyroptotic cell death. Here we show that CARD-like domains are present in defense systems that protect bacteria against phage. The bacterial CARD is essential for protease-mediated activation of certain bacterial gasdermins, which promote cell death once phage infection is recognized. We further show that multiple anti-phage defense systems utilize CARD-like domains to activate a variety of cell death effectors. We find that these systems are triggered by a conserved immune evasion protein that phages use to overcome the bacterial defense system RexAB, demonstrating that phage proteins inhibiting one defense system can activate another. We also detect a phage protein with a predicted CARD-like structure that can inhibit the CARD-containing bacterial gasdermin system. Our results suggest that CARD domains represent an ancient component of innate immune systems conserved from bacteria to humans, and that CARD-dependent activation of gasdermins is conserved in organisms across the tree of life.
RESUMO
The Toll/interleukin-1 receptor (TIR) domain is a key component of immune receptors that identify pathogen invasion in bacteria, plants and animals1-3. In the bacterial antiphage system Thoeris, as well as in plants, recognition of infection stimulates TIR domains to produce an immune signalling molecule whose molecular structure remains elusive. This molecule binds and activates the Thoeris immune effector, which then executes the immune function1. We identified a large family of phage-encoded proteins, denoted here as Thoeris anti-defence 1 (Tad1), that inhibit Thoeris immunity. We found that Tad1 proteins are 'sponges' that bind and sequester the immune signalling molecule produced by TIR-domain proteins, thus decoupling phage sensing from immune effector activation and rendering Thoeris inactive. Tad1 can also efficiently sequester molecules derived from a plant TIR-domain protein, and a high-resolution crystal structure of Tad1 bound to a plant-derived molecule showed a unique chemical structure of 1 ''-2' glycocyclic ADPR (gcADPR). Our data furthermore suggest that Thoeris TIR proteins produce a closely related molecule, 1''-3' gcADPR, which activates ThsA an order of magnitude more efficiently than the plant-derived 1''-2' gcADPR. Our results define the chemical structure of a central immune signalling molecule and show a new mode of action by which pathogens can suppress host immunity.
Assuntos
Bactérias , Bacteriófagos , Domínios Proteicos , Receptores de Interleucina-1 , Transdução de Sinais , Receptores Toll-Like , Proteínas Virais , Bactérias/imunologia , Bactérias/metabolismo , Bactérias/virologia , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/química , Proteínas de Bactérias/imunologia , Proteínas de Bactérias/metabolismo , Proteínas de Plantas/antagonistas & inibidores , Proteínas de Plantas/química , Proteínas de Plantas/imunologia , Proteínas de Plantas/metabolismo , Receptores de Interleucina-1/química , Transdução de Sinais/imunologia , Bacteriófagos/química , Bacteriófagos/imunologia , Bacteriófagos/metabolismo , Proteínas Virais/química , Proteínas Virais/imunologia , Proteínas Virais/metabolismo , Receptores Toll-Like/química , Cristalografia por Raios XRESUMO
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.
Assuntos
Bacteriófagos , Antivirais , Bactérias , Bacteriófagos/genética , Desoxiguanosina , Humanos , Proteína 1 com Domínio SAM e Domínio HDRESUMO
The cyclic oligonucleotide-based antiphage signalling system (CBASS) and the pyrimidine cyclase system for antiphage resistance (Pycsar) are antiphage defence systems in diverse bacteria that use cyclic nucleotide signals to induce cell death and prevent viral propagation1,2. Phages use several strategies to defeat host CRISPR and restriction-modification systems3-10, but no mechanisms are known to evade CBASS and Pycsar immunity. Here we show that phages encode anti-CBASS (Acb) and anti-Pycsar (Apyc) proteins that counteract defence by specifically degrading cyclic nucleotide signals that activate host immunity. Using a biochemical screen of 57 phages in Escherichia coli and Bacillus subtilis, we discover Acb1 from phage T4 and Apyc1 from phage SBSphiJ as founding members of distinct families of immune evasion proteins. Crystal structures of Acb1 in complex with 3'3'-cyclic GMP-AMP define a mechanism of metal-independent hydrolysis 3' of adenosine bases, enabling broad recognition and degradation of cyclic dinucleotide and trinucleotide CBASS signals. Structures of Apyc1 reveal a metal-dependent cyclic NMP phosphodiesterase that uses relaxed specificity to target Pycsar cyclic pyrimidine mononucleotide signals. We show that Acb1 and Apyc1 block downstream effector activation and protect from CBASS and Pycsar defence in vivo. Active Acb1 and Apyc1 enzymes are conserved in phylogenetically diverse phages, demonstrating that cleavage of host cyclic nucleotide signals is a key strategy of immune evasion in phage biology.
Assuntos
Bacteriófagos , Bactérias/metabolismo , Proteínas de Bactérias/metabolismo , Bacteriófago T4/metabolismo , Bacteriófagos/fisiologia , Sistemas CRISPR-Cas/genética , Endonucleases/metabolismo , Escherichia coli/metabolismo , Nucleotídeos Cíclicos/metabolismo , Oligonucleotídeos , Pirimidinas/metabolismoRESUMO
Gasdermin proteins form large membrane pores in human cells that release immune cytokines and induce lytic cell death. Gasdermin pore formation is triggered by caspase-mediated cleavage during inflammasome signaling and is critical for defense against pathogens and cancer. We discovered gasdermin homologs encoded in bacteria that defended against phages and executed cell death. Structures of bacterial gasdermins revealed a conserved pore-forming domain that was stabilized in the inactive state with a buried lipid modification. Bacterial gasdermins were activated by dedicated caspase-like proteases that catalyzed site-specific cleavage and the removal of an inhibitory C-terminal peptide. Release of autoinhibition induced the assembly of large and heterogeneous pores that disrupted membrane integrity. Thus, pyroptosis is an ancient form of regulated cell death shared between bacteria and animals.
Assuntos
Bactérias/química , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Bacteriófagos/fisiologia , Piroptose , Proteínas Reguladoras de Apoptose/química , Proteínas Reguladoras de Apoptose/metabolismo , Bactérias/metabolismo , Bactérias/virologia , Bradyrhizobium/química , Membrana Celular/metabolismo , Cristalografia por Raios X , Cytophagaceae/química , Modelos Moleculares , Myxococcales/química , Fragmentos de Peptídeos/metabolismo , Peptídeo Hidrolases/metabolismo , Conformação Proteica , Conformação Proteica em alfa-Hélice , Domínios ProteicosRESUMO
The cyclic pyrimidines 3',5'-cyclic cytidine monophosphate (cCMP) and 3',5'-cyclic uridine monophosphate (cUMP) have been reported in multiple organisms and cell types. As opposed to the cyclic nucleotides 3',5'-cyclic adenosine monophosphate (cAMP) and 3',5'-cyclic guanosine monophosphate (cGMP), which are second messenger molecules with well-established regulatory roles across all domains of life, the biological role of cyclic pyrimidines has remained unclear. Here we report that cCMP and cUMP are second messengers functioning in bacterial immunity against viruses. We discovered a family of bacterial pyrimidine cyclase enzymes that specifically synthesize cCMP and cUMP following phage infection and demonstrate that these molecules activate immune effectors that execute an antiviral response. A crystal structure of a uridylate cyclase enzyme from this family explains the molecular mechanism of selectivity for pyrimidines as cyclization substrates. Defense systems encoding pyrimidine cyclases, denoted here Pycsar (pyrimidine cyclase system for antiphage resistance), are widespread in prokaryotes. Our results assign clear biological function to cCMP and cUMP as immunity signaling molecules in bacteria.
Assuntos
Bactérias/imunologia , Bactérias/virologia , Bacteriófagos/fisiologia , CMP Cíclico/metabolismo , Nucleotídeos Cíclicos/metabolismo , Uridina Monofosfato/metabolismo , Sequência de Aminoácidos , Bactérias/genética , Burkholderia/enzimologia , CMP Cíclico/química , Ciclização , Escherichia coli/enzimologia , Modelos Moleculares , Mutação/genética , Nucleotídeos Cíclicos/química , Fósforo-Oxigênio Liases/química , Fósforo-Oxigênio Liases/metabolismo , Pirimidinas/metabolismo , Uridina Monofosfato/químicaRESUMO
Multiple sclerosis (MS) is an immune-mediated disease whose precise etiology is unknown. Several studies found alterations in the microbiome of individuals with MS, but the mechanism by which it may affect MS is poorly understood. Here we analyze the microbiome of 129 individuals with MS and find that they harbor distinct microbial patterns compared with controls. To study the functional consequences of these differences, we measure levels of 1,251 serum metabolites in a subgroup of subjects and unravel a distinct metabolite signature that separates affected individuals from controls nearly perfectly (AUC = 0.97). Individuals with MS are found to be depleted in butyrate-producing bacteria and in bacteria that produce indolelactate, an intermediate in generation of the potent neuroprotective antioxidant indolepropionate, which we found to be lower in their serum. We identify microbial and metabolite candidates that may contribute to MS and should be explored further for their causal role and therapeutic potential.
Assuntos
Butiratos/metabolismo , Metaboloma/fisiologia , Microbiota/fisiologia , Esclerose Múltipla/etiologia , Esclerose Múltipla/microbiologia , Adulto , Bactérias/metabolismo , Bactérias/patogenicidade , Feminino , Microbioma Gastrointestinal/fisiologia , Humanos , MasculinoRESUMO
22q11.2 deletion syndrome (22q11.DS) is a neurogenetic disorder caused by a microdeletion in chromosome 22. Its phenotype includes high rates of psychiatric disorders, immune system abnormalities, and cognitive impairments. We assessed the quality of sleep in 22q11.2DS and its potential link to inflammatory markers and cognitive deficits. Thirty-three 22q11.2DS individuals and 24 healthy controls were studied. Sleep parameters were assessed by the Pittsburgh sleep quality index (PSQI) questionnaire and correlated with serum cytokine levels and cognitive functioning, measured using the Penn computerized neurocognitive battery (CNB). The 22q11.2DS individuals had significantly worse sleep quality scores than the controls, unrelated to the psychiatric or physical comorbidities common to 22q11.2DS. Interleukin 6 levels were correlated with the overall score of the PSQI questionnaire for nonpsychotic 22q11.2DS participants only. Several domains of the CNB were associated with poorer sleep quality, suggesting that cognitive impairments in 22q11.2DS may be at least partially explained by poor sleep quality. Our findings confirm sleep impairments in individuals with 22q11.2DS, which might negatively affect their cognitive functioning, and corroborate a potential role of immunological pathways in the 22q11.2DS neuro-phenotype.