Cryo-EM structure of the RADAR supramolecular anti-phage defense complex

Abstract

SummaryRADAR is a two-protein bacterial defense system which 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 a RdrB active site that targets mononucleotides, not RNA. We show that RdrB catalyzes ATP-to-ITP conversion in vitro and induces the 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 novel mechanism of anti-phage defense.

Toxin/Antitoxin Systems Induce Persistence and Work in Concert with Restriction/Modification Systems to Inhibit Phage

“…Corroborating our findings, many phage-inhibition systems reduce energy metabolites like ATP (RADAR 11 and Detocs 36 ) and NAD + (Thoeris 23 ), and the reduction of energy metabolites like ATP consistently induces persistence 8,21 . Furthermore, reduction of ATP by toxin/antitoxin systems leads to persistence 6 .…”

“…For example, after transcription shutoff by T4 phage activates the Hok/Sok toxin/antitoxin system, Hok damages the cell membrane to cease cell metabolism to halt phage propagation, in the first link of a toxin/antitoxin system to phage inhibition (1996) 32 . Similarly, phage attack activates various toxins to reduce cell metabolism including those of the CBASS (phospholipases, nucleases, and pore-forming proteins 22 ), RADAR (RdrB converts ATP to ITP 11 ), and Thoeris (ThsA degrades NAD +23 ) phage inhibition systems.…”

Structural basis of Gabija anti-phage defense and viral immune evasion

“…Our study defines the structural basis of Gabija supramolecular complex formation and explains how phages block DNA cleavage to defeat this form of host immunity. Similar to supramolecular complexes in CRISPR 32 , CBASS 33,34 , and RADAR immunity 35,36 , the ~500 kDa GajAB complex extends an emerging theme in anti-phage defense where protein subunits assemble into large machines to resist phage infection. These results parallel human innate immunity, where key effectors in inflammasome, Toll-like receptor, RIG-I-like receptor, and cGAS-STING signaling pathways also oligomerize into large assemblies to block viral replication 37,38 .…”

Ribosome inactivation by Escherichia coli GTPase RsgA inhibits T4 phage

“…Since control of protein production is critical for phages, many antiphage systems reduce energy compounds in the cell, which inhibits protein translation and 10.3389/fmicb.2023.1242163 phage propagation. For example, NAD + is depleted by the Bacillus cereus Thoeris anti-phage system (Leavitt et al, 2022), and ATP is depleted by the E. coli RADAR anti-phage system (Duncan-Lowey et al, 2022) and by the GhoT/GhoS toxin/antitoxin system as the toxin GhoT damages the membrane (Cheng et al, 2014). In addition, as the most prevalent phage inhibition system, most toxin/antitoxin systems reduce translation; for example, by degrading mRNA via RNases.…”