Prokaryotic innate immunity through pattern recognition of conserved viral proteins

Many organisms have evolved specialized immune pattern-recognition receptors, including nucleotide-binding oligomerization domain–like receptors (NLRs) of the STAND superfamily that are ubiquitous in plants, animals, and fungi. Although the roles of NLRs in eukaryotic immunity are well established, it is unknown whether prokaryotes use similar defense mechanisms. Here, we show that antiviral STAND (Avs) homologs in bacteria and archaea detect hallmark viral proteins, triggering Avs tetramerization and the activation of diverse N-terminal effector domains, including DNA endonucleases, to abrogate infection. Cryo–electron microscopy reveals that Avs sensor domains recognize conserved folds, active-site residues, and enzyme ligands, allowing a single Avs receptor to detect a wide variety of viruses. These findings extend the paradigm of pattern recognition of pathogen-specific proteins across all three domains of life. Description STAND against viral invaders The innate immune systems of animals, plants, and fungi universally use nucleotide binding oligomerization domain–like receptors (NLRs) of the STAND superfamily to detect molecular patterns common to pathogens. Gao et al. show that NLR-based immune pattern recognition is also prevalent in bacteria and archaea, something that was not known before. In particular, the authors characterized four families of NLR-like genes, finding that they are specific sensors for two highly conserved bacteriophage proteins. Upon binding to the target, these NLRs activate diverse effector domains, including nucleases, to prevent phage propagation. These findings demonstrate that pattern recognition of pathogen-specific proteins is a common mechanism of immunity across all domains of life. —DJ Bacteria and archaea have innate immune receptors that recognize conserved viral proteins and prevent infection. INTRODUCTION Many organisms have evolved specialized immune pattern-recognition receptors, including nucleotide-binding oligomerization domain-like receptors (NLRs) of the STAND superfamily that are ubiquitous in plants, animals, and fungi. NLRs oligomerize upon recognition of pathogen-associated molecular patterns, leading to the activation of an effector domain that mediates an inflammatory or cell death response. Although the roles of NLRs in eukaryotic immunity are well established, it is unknown whether prokaryotes use similar defense mechanisms. RATIONALE We previously identified a set of bacterial and archaeal STAND nucleoside triphosphatases (NTPases), dubbed Avs (antiviral STAND), that protect bacteria from tailed phages through an unknown mechanism. Like eukaryotic NLRs, Avs proteins have a characteristic tripartite domain architecture consisting of a central NTPase, an extended C-terminal sensor, and an N-terminal effector. Here, we investigate the defense mechanism of these Avs proteins. RESULTS Using genetic screens in Escherichia coli, we characterized four Avs families (Avs1 to Avs4) and found that they detect hallmark viral proteins that are expressed during infection. In particular, Avs1 to Avs3 recognize the large terminase subunit, and Avs4 recognizes the portal. These two proteins together make up the conserved DNA packaging machinery of tailed phages. Coexpression of an Avs protein with its cognate target in E. coli resulted in cell death. We assessed the specificity of Avs target recognition with a panel of terminases and portals from 24 tailed phages, spanning nine major families. Notably, a single Avs protein was capable of recognizing a large variety of targets (terminase or portal), with less than 5% sequence identity in some cases. We next reconstituted Avs activity in vitro, focusing on representatives from Salmonella enterica (SeAvs3) and E. coli (EcAvs4), both of which contain N-terminal PD-DExK nuclease effectors. In the presence of their cognate target, SeAvs3 and EcAvs4 mediated degradation of double-stranded DNA. Nuclease activity required the presence of Mg2+ and adenosine triphosphate (ATP); however, the hydrolysis of ATP was not strictly required. Single-stranded DNA and RNA substrates were not cleaved. We determined the cryo–electron microscopy structures of the SeAvs3-terminase and EcAvs4-portal complexes, revealing that both form tetramers in which the C-terminal sensor domain of each Avs subunit binds to a single target protein. Binding is mediated by shape complementarity across an extended interface, consistent with fold recognition. Additionally, SeAvs3 directly recognizes terminase active-site residues and its ATP ligand. Tetramerization of both SeAvs3 and EcAvs4 is mediated by their STAND ATPase domains and allows the N-terminal nucleases to adopt active dimeric configurations. Bioinformatic analysis of Avs proteins across prokaryotic lineages revealed at least 18 distinct types of N-terminal effectors that are modularly swapped between Avs homologs, as well as widespread distribution of avs genes across phyla with extensive horizontal gene transfer. Finally, we also discovered phage-encoded Avs inhibitors, highlighting an extensive arms race between prokaryotes and viruses. CONCLUSION NLR-like defense proteins in bacteria and archaea recognize the conserved folds of hallmark viral proteins and assemble into tetramers that activate diverse N-terminal effectors. The mechanism of these proteins highlights the similarity between the defense strategies of prokaryotes and eukaryotes and extends the paradigm of pattern recognition of pathogen-specific proteins across all three domains of life. Mechanism and structures of NLR-like defense proteins in prokaryotes. (Left) Comparison of the domain architectures of 11 representative NLR-like pattern-recognition receptors across four kingdoms of life. Selected structures of activated complexes are shown as examples. T3SS, type 3 secretion system. (Right) Defense mechanism of Avs proteins in bacteria and archaea (this study). Target binding triggers the formation of Avs tetramers, which activates an N-terminal effector that disrupts the viral life cycle.