Diverse enzymatic activities mediate antiviral immunity in prokaryotes

Abstract

Bacteria and archaea are frequently attacked by viruses and other mobile genetic elements and rely on dedicated antiviral defense systems, such as restriction endonucleases and CRISPR, to survive. The enormous diversity of viruses suggests that more types of defense systems exist than are currently known. By systematic defense gene prediction and heterologous reconstitution, here we discover 29 widespread antiviral gene cassettes, collectively present in 32% of all sequenced bacterial and archaeal genomes, that mediate protection against specific bacteriophages. These systems incorporate enzymatic activities not previously implicated in antiviral defense, including RNA editing and retron satellite DNA synthesis. In addition, we computationally predict a diverse set of other putative defense genes that remain to be characterized. These results highlight an immense array of molecular functions that microbes use against viruses.

Fig. 1:

Domain-independent prediction of putative antiviral defense systems. (A) Computational pipeline to identify uncharacterized putative defense systems across all sequenced bacterial and archaeal genomes. Defense systems were predicted on the basis of analysis of amino acid sequences, independent of domain annotations. (B) Histograms of defense association frequencies before filtering and after neighborhood context–based filtering (minimum 50 homologs). Seeds to the right of the dashed line (0.1) were selected for further analysis. TA, toxin-antitoxin. (C) Pie chart of the domain diversity among predicted defense genes, based on additional analysis using HHpred against pfam domains.

Fig. 2:

Candidate defense systems exhibit antiviral activity in a heterologous system. (A) Experimental validation pipeline using phage plaque assays on E. coli heterologously expressing a cloned candidate defense system. (B) Example plaques and (C) zones of lysis for six candidate defense systems. (D) Anti-phage activity across a panel of 12 coliphages with dsDNA, ssDNA, or ssRNA genomes (mean of two replicates). The bar graph shows the abundance of each system in sequenced bacterial and archaeal genomes. Domains: RT, reverse transcriptase; TIR, Toll/interleukin-1 receptor homology domain; TOPRIM, topoisomerase-primase domain; QueC, 7-cyano-7-deazaguanine synthase-like domain; SIR2, sirtuin; membrane, transmembrane helix; DUF, domain of unknown function. Proposed gene names: DRT, defense-associated reverse transcriptase; RADAR, phage restriction by an adenosine deaminase acting on RNA; AVAST, antiviral ATPase/NTPase of the STAND superfamily; dsr, defense-associated sirtuin; tmn, transmembrane NTPase; qat, QueC-like associated with ATPase and TatD DNase; hhe, HEPN, helicase, and Vsr endonuclease; mza, MutL, Z1, and AIPR; upx, uncharacterized (P)D-(D/E)-XK defense protein; ppl, polymerase/histidinol phosphatase-like; HerA, helicase; MBL, metallo β-lactamase.

Fig. 3:

RADAR mediates RNA editing in response to phage infection. (A) Examples of genomic loci containing three subtypes of RADAR (standalone, Csx27-associated, and SLATT-associated). (B) Essentiality of the core RADAR genes rdrAB and the accessory gene rdrD against phages T2 and T5. D215A, Asp²¹⁵→Ala; H168A, His¹⁶⁸→Ala; H170A, His¹⁷⁰→Ala; WT, wild type. (C) Representative RNA sequencing (RNAseq) reads from E. coli expressing either RADAR or an empty vector control. (D) Expression of phage T2 RNA relative to total host RNA in E. coli containing RADAR. Each dot represents a phage gene. Cells were infected at a MOI of 2. The p value was determined by a Wilcoxon signed-rank test. (E) Representative editing sites in the host and phage transcriptomes, with corresponding predicted RNA secondary structures. (F) Growth kinetics of RADAR-containing E. coli in comparison with an empty vector control under varying MOI by phage T2.

Fig. 4:

Diverse families of RTs mediate antiviral defense. (A) Examples of genomic loci containing two RT-based defense systems (DRT type 1 and type 3), with two representative subtypes shown for each system. (B) Essential components of non-retron RTs (left panel) and retrons (right panel). TM, transmembrane; ncRNA, noncoding RNA; msr/msd: genes encoding msRNA and msDNA, respectively; a2, retron 5’ inverted repeat. (C) Effect of defense RTs on the expression of phage T2 genes in E. coli infected at an MOI of 2. (D) RNAseq reads mapping to the DRT type 3 system. (E) Predicted secondary structure of the highly expressed noncoding RNA identified in (D).

Fig. 5:

Domain architectures and mutational analysis of additional defense systems. Graphics show domains identified by using HHpred, and stars indicate locations of active site mutations. Bar graphs (n = 4 replicates per bar) show either log₁₀ fold change of efficiency of plating (for phages T2, P1, and λ) or fold change in the area of the zone of lysis (for phages T7 and φV-1) relative to the empty vector control. MBL, metallo β-lactamase; SIR2, sirtuin; HerA, helicase; QueC, 7-cyano-7-deazaguanine synthase-like domain; Vsr, very short patch repair endonuclease; TatD, DNase; vWA, von Willebrand factor type A; Prot phos, serine/threonine protein phosphatase; PHP, polymerase/histidinol phosphatase; MTase, methyltransferase; PLD, phospholipase D; DUF, domain of unknown function.