A widespread family of viral sponge proteins reveals specific inhibition of nucleotide signals in anti-phage defense

Abstract

Cyclic oligonucleotide-based antiviral signaling systems (CBASS) are bacterial anti-phage defense operons that use nucleotide signals to control immune activation. Here we biochemically screen 57 diverse E. coli and Bacillus phages for the ability to disrupt CBASS immunity and discover anti-CBASS 4 (Acb4) from the Bacillus phage SPO1 as the founding member of a large family of >1,300 immune evasion proteins. A 2.1 Å crystal structure of Acb4 in complex with 3'3'-cGAMP reveals a tetrameric assembly that functions as a sponge to sequester CBASS signals and inhibit immune activation. We demonstrate Acb4 alone is sufficient to disrupt CBASS activation in vitro and enable immune evasion in vivo. Analyzing phages that infect diverse bacteria, we explain how Acb4 selectively targets nucleotide signals in host defense and avoids disruption of cellular homeostasis. Together, our results reveal principles of immune evasion protein evolution and explain a major mechanism phages use to inhibit host immunity.

Figure 1.. Discovery of SPO1 Acb4 as a 3′3′-cGAMP binding protein

(A) Schematic of biochemical screen for discovery of 3′3′-cGAMP binding activity in phage-infected lysates using electrophoretic mobility shift assay (EMSA). Representative EMSA depicting 3′3′-cGAMP binding activity following infection with SPO1 or SPO1-like phages (SPOL1–SPOL5). Data are representative of at least n = 3 independent experiments. Phages used in the screen are listed in Table S1, and primary data are shown in Figure S1C. (B) EMSA analysis depicting the time course of 3′3′-cGAMP binding activity in SPO1-infected lysates. SPO1 infection experiments were performed at 37°C and data are representative of n = 2 independent experiments. (C) Representative EMSA analysis demonstrating enrichment of candidate Acb4 genes by activity-guided fractionation. SPO1-infected lysates were fractionated using hydrophobic interaction chromatography followed by S200 size-exclusion chromatography. Fractions enriched in 3′3′-cGAMP binding activity were analyzed by mass spectrometry (MS) to identify candidate SPO1 proteins. For additional details on purification scheme, see Figure S2A. (D) MS analysis of SPO1 proteins detected in a fraction with peak 3′3′-cGAMP binding activity following biochemical fractionation. Abundance was determined by normalizing the sum intensity of each SPO1 protein to the total number of detected peptides. Complete list of SPO1 proteins identified in the selected active fraction are shown in Figure S2B. (E) Comparison of candidate Acb4 proteins identified independently by thermal proteome profiling (TPP), or biochemical fractionation followed by MS. For a detailed list of proteins identified by each approach, see Figure S2B. (F) Volcano plot depicting SPO1 proteins that undergo a shift in thermal stability upon treatment with 100 μM 3′3′-cGAMP. Data are representative of n = 5 independent replicates for control and 3′3′-cGAMP-treated samples. (G) EMSA analysis of 3′3′-cGAMP binding with recombinant Acb4 produced in Escherichia coli. Data are representative of at least n = 3 independent experiments. For details on recombinant Acb4 expression and purification, refer to Figures S2C and S2D. (H) Schematic illustrating genes neighboring SPO1 acb4.

Figure 2.. Acb4 subverts host CBASS immunity

(A) DNA cleavage analysis of uncut plasmid DNA (pGEM9Z) incubated with purified Burkholderia pseudomallei Cap5 and 3′3′-cGAMP pre-treated with a titration (500 nM–5 μM) of recombinant Acb4. Data are representative of at least n = 3 independent experiments. (B) Summary of 3′3′-cGAMP binding and degradation properties in wildtype phage T4 and engineered phage T4 variants. (C) Representative plaque assay performed using Escherichia coli BL21 cells harboring active or inactive forms of a CBASS operon from Yersinia aleksiciae and challenged with T4 phages in Figure 2B. Data are representative of n = 3 independent experiments. (D) Representative plaque assay performed using Escherichia coli BL21 cells harboring active or inactive forms of a CBASS operon from Citrobacter portucalensis / Escherichia coli and challenged with T4 phages in Figure 2B. Data are representative of n = 3 independent experiments.

Figure 3.. Acb4 forms a tetrameric assembly that sequesters four molecules of 3′3′-cGAMP

(A) Co-crystal structure of Acb4 from Bacillus phage SPO1 in complex with 3′3′-cGAMP. (B) Views of Acb4 dimers in complex with 3′3′-cGAMP and cartoon schematic illustrating mechanism of Acb4 tetramer assembly. (C) Surface electrostatic view of Acb4 binding pocket and polder omit map of 3′3′-cGAMP contoured at 2.9 σ. (D) EMSA analysis of Acb4 3′3′-cGAMP complex formation. A titration of recombinant Acb4 was incubated with 20 nM α³²P-radiolabeled 3′3′-cGAMP, and reactions were analyzed by nondenaturing polyacrylamide gel electrophoresis. Data are representative of at least n = 3 independent experiments. (E) Quantification of EMSA analysis to determine Acb4 3′3′-cGAMP affinity. The fraction bound (bound intensity / total intensity) was calculated for each protein concentration and fit to a single binding model. Data represent the mean ± SD from n = 2 independent experiments.

Figure 4.. Structural basis of Acb4 nucleotide immune signal recognition

(A) Overview of the Acb4 ligand binding site with individual Acb4 protomers contacting 3′3′-cGAMP colored in light pink, magenta, and gray, respectively. Subscript denotes protomer chain of Acb4 tetramer. (B) Detailed view of Acb4 residues that interact the adenine base (top left), guanine base (top right), and phosphodiester linkages (bottom left) of 3′3′-cGAMP. Green dashed lines represent hydrogen bonding interactions and subscript denotes protomer chain of Acb4 tetramer. (C) Schematic highlighting key Acb4 residues that form contacts with 3′3′-cGAMP. Green dashed lines represent hydrogen bonds and grey dashed lines represent hydrophobic interactions. (D) Quantification of 3′3′-cGAMP binding with recombinant Acb4 proteins harboring point mutations in binding pocket residues. Data represent the mean ± SD from n = 2 independent experiments. For primary EMSA data, see Figure S6A. (E) Quantification of binding affinity of Acb4 with the cyclic dinucleotides 3′3′-cGAMP, 3′2′-cGAMP, 2′3′-cGAMP, 3′3′-cUA, 2′3′-cUA, 3′3′-cAA, 3′3′-cGG, and 3′3′-cUU calculated from EMSA analysis. Data represent the mean ± SD from n = 2 independent experiments. For primary EMSA data, refer to Figure S6C.

Figure 5.. SPO1 Acb4 is the founding member of a widespread family of viral sponges

(A) Phylogenetic analysis of 1,331 Acb4 homologs. SPO1 Acb4 is denoted with a yellow star. Red circles with interior numbering represent Acb4 family proteins selected for bioinformatic and biochemical analysis. For additional information, refer to Figure S4 and Table S3. (B) Predicted structural models of Acb4 homologs generated using AlphaFold3 and colored by predicted local distance difference test (pLDDT). (C) Summary of purified Acb4 homologs and their binding properties. (D) Electrophoretic mobility shift assay to determine binding of SPO1 Acb4 and purified homologs against 3′3′-cGAMP (top gel) and 3′3′-cAA (bottom gel). Proteins were present at 5 μM and incubated with 20 nM ³²P-labeled 3′3′-cGAMP or 3′3′-cAA, and bound complexes were visualized by nondenaturing polyacrylamide gel electrophoresis. Data are representative of at least n = 3 independent experiments. (E) DALI Z-scores for the top 100 hits for SPO1 Acb4 monomer when searched against all entries in the Protein Data Bank (PDB). (F) Topology map diagrams comparing Bacillus phage SPO1 Acb4 monomer with dsRNA-binding domain containing region of Thermotoga maritima ribonuclease III (PDB: 1O0W). Rectangles denote α-helices and arrows denote β-strands. (G) Structural comparison of SPO1 Acb4 with Vibrio cholerae ribosome hibernation factor (PDB: 4HEI) and Thermotoga maritima ribonuclease III (PDB: 1O0W). SPO1 Acb4 is colored in light pink and bacterial proteins are colored in teal.

Figure 6.. Summary model of CBASS immune evasion by Acb4

(A) Conceptual model of Acb4-mediated inhibition of CBASS anti-phage defense. During phage infection, Acb4 inhibits CBASS signaling by sequestering nucleotide immune signals while avoiding disruption of signals required for normal cellular homeostasis.