RESUMO
Sterile alpha and TIR motif-containing 1 (SARM1) is a protein involved in programmed death of injured axons. Following axon injury or a drug-induced insult, the TIR domain of SARM1 degrades the essential molecule nicotinamide adenine dinucleotide (NAD+), leading to a form of axonal death called Wallerian degeneration. Degradation of NAD+ by SARM1 is essential for the Wallerian degeneration process, but accumulating evidence suggest that other activities of SARM1, beyond the mere degradation of NAD+, may be necessary for programmed axonal death. In this study we show that the TIR domains of both human and fruit fly SARM1 produce 1''-2' and 1''-3' glycocyclic ADP-ribose (gcADPR) molecules as minor products. As previously reported, we observed that SARM1 TIR domains mostly convert NAD+ to ADPR (for human SARM1) or cADPR (in the case of SARM1 from Drosophila melanogaster). However, we now show that human and Drosophila SARM1 additionally convert ~0.1-0.5% of NAD+ into gcADPR molecules. We find that SARM1 TIR domains produce gcADPR molecules both when purified in vitro and when expressed in bacterial cells. Given that gcADPR is a second messenger involved in programmed cell death in bacteria and likely in plants, we propose that gcADPR may play a role in SARM1-induced programmed axonal death in animals.
Assuntos
NAD , Degeneração Walleriana , Animais , Humanos , Degeneração Walleriana/metabolismo , Degeneração Walleriana/patologia , NAD/metabolismo , Drosophila melanogaster/metabolismo , Axônios/metabolismo , Bactérias/metabolismo , Adenosina Difosfato Ribose/metabolismo , Proteínas do Domínio Armadillo/genética , Proteínas do Domínio Armadillo/metabolismo , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismoRESUMO
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.
Assuntos
Bacteriófagos , Bacteriófagos/metabolismo , Microscopia Crioeletrônica/métodos , ATPases Associadas a Diversas Atividades Celulares , Trifosfato de Adenosina , Adenosina Desaminase/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 Toll/interleukin-1 receptor (TIR) domain is a canonical component of animal and plant immune systems1,2. In plants, intracellular pathogen sensing by immune receptors triggers their TIR domains to generate a molecule that is a variant of cyclic ADP-ribose3,4. This molecule is hypothesized to mediate plant cell death through a pathway that has yet to be resolved5. TIR domains have also been shown to be involved in a bacterial anti-phage defence system called Thoeris6, but the mechanism of Thoeris defence remained unknown. Here we show that phage infection triggers Thoeris TIR-domain proteins to produce an isomer of cyclic ADP-ribose. This molecular signal activates a second protein, ThsA, which then depletes the cell of the essential molecule nicotinamide adenine dinucleotide (NAD) and leads to abortive infection and cell death. We also show that, similar to eukaryotic innate immune systems, bacterial TIR-domain proteins determine the immunological specificity to the invading pathogen. Our results describe an antiviral signalling pathway in bacteria, and suggest that the generation of intracellular signalling molecules is an ancient immunological function of TIR domains that is conserved in both plant and bacterial immunity.
Assuntos
Bacillus/imunologia , Bacillus/virologia , Proteínas de Bactérias/química , Proteínas de Bactérias/imunologia , Bacteriófagos/imunologia , Receptores de Interleucina-1/química , Transdução de Sinais/imunologia , Receptores Toll-Like/química , ADP-Ribose Cíclica/análogos & derivados , ADP-Ribose Cíclica/metabolismo , Evolução Molecular , Modelos Moleculares , NAD/metabolismo , Domínios Proteicos , Especificidade por Substrato/imunologiaRESUMO
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
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.
Assuntos
Bactérias/virologia , Nucleotídeos Cíclicos/metabolismo , Transdução de Sinais , Bactérias/imunologia , Bactérias/metabolismo , Proteínas de Bactérias/metabolismo , Bacteriófagos/fisiologia , Nucleotídeos Cíclicos/imunologia , Replicação ViralRESUMO
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.
Assuntos
Bacteriólise , Bacteriófagos/fisiologia , Lisogenia , Sequência de Aminoácidos , Bacillus/citologia , Bacillus/virologia , Bacteriólise/efeitos dos fármacos , Bacteriófagos/efeitos dos fármacos , Meios de Cultivo Condicionados/química , Meios de Cultivo Condicionados/farmacologia , DNA Viral/metabolismo , Lisogenia/efeitos dos fármacos , Modelos Biológicos , Peptídeos/química , Peptídeos/metabolismo , Peptídeos/farmacologia , Multimerização Proteica , Transcrição Gênica/efeitos dos fármacos , Proteínas Virais/química , Proteínas Virais/metabolismo , Proteínas Virais/farmacologiaRESUMO
Inteins are protein-splicing domains present in many proteins. They self-catalyze their excision from the host protein, ligating their former flanks by a peptide bond. The C-terminal residue of inteins is typically an asparagine (Asn). Cyclization of this residue to succinimide causes the final detachment of inteins from their hosts. We studied protein-splicing activity of two inteins with atypical C-terminal residues. One having a C-terminal glutamine (Gln), isolated from Chilo iridescent virus (CIV), and another unique intein, first reported here, with a C-terminal aspartate, isolated from Carboxydothermus hydrogenoformans (Chy). Protein-splicing activity was examined in the wild-type inteins and in several mutants with N- and C-terminal amino acid substitutions. We demonstrate that both wild-type inteins can protein splice, probably by new variations of the typical protein-splicing mechanism. Substituting the atypical C-terminal residue to the typical Asn retained protein-splicing only in the CIV intein. All diverse C-terminal substitutions in the Chy intein (Asp(345) to Asn, Gln, Glu, and Ala) abolished protein-splicing and generated N- and C-terminal cleavage. The observed C-terminal cleavage in the Chy intein ending with Ala cannot be explained by cyclization of this residue. We present and discuss several new models for reactions in the protein-splicing pathway.