EP4673163A2 - Proteins that inhibit cyclic-oligonucleotide-based anti-phage signaling system (cbass) - Google Patents

Proteins that inhibit cyclic-oligonucleotide-based anti-phage signaling system (cbass)

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Publication number
EP4673163A2
EP4673163A2 EP24764423.0A EP24764423A EP4673163A2 EP 4673163 A2 EP4673163 A2 EP 4673163A2 EP 24764423 A EP24764423 A EP 24764423A EP 4673163 A2 EP4673163 A2 EP 4673163A2
Authority
EP
European Patent Office
Prior art keywords
cbass
acb2
protein
phage
cyclic
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP24764423.0A
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German (de)
French (fr)
Inventor
Joseph BONDY-DENOMY
Erin HUITING
Yue FENG
Xueli CAO
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Beijing University of Chemical Technology
University of California
University of California Berkeley
University of California San Diego UCSD
Original Assignee
Beijing University of Chemical Technology
University of California
University of California Berkeley
University of California San Diego UCSD
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Application filed by Beijing University of Chemical Technology, University of California, University of California Berkeley, University of California San Diego UCSD filed Critical Beijing University of Chemical Technology
Publication of EP4673163A2 publication Critical patent/EP4673163A2/en
Pending legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1241—Nucleotidyltransferases (2.7.7)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/21—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Pseudomonadaceae (F)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
    • C12Y207/07—Nucleotidyltransferases (2.7.7)

Definitions

  • a fundamental strategy of eukaryotic anti-viral immunity involves the cyclic GMP- AMP synthase (cGAS) enzyme, which synthesizes 2’,3’-cGAMP and activates a STING effector to prevent viral replication.
  • cGAS cyclic GMP- AMP synthase
  • Diverse bacteria contain cGAS-like enzymes that produce cyclic oligonucleotides and induce anti-phage activity, known as cyclic- oligonucleotide-based anti-phage signaling system (CBASS).
  • CBASS cyclic- oligonucleotide-based anti-phage signaling system
  • dsDNA double-stranded DNA
  • cGAS cyclic GMP-AMP synthase
  • the activated cGAS enzyme produces 2’,3’-cyclic GMP-AMP (2’,3’-cGAMP) dinucleotides that bind to the STING effector protein and induces a type I interferon response 5,6 .
  • CD-NTases cGAS/DncV- like nucleotidyltransferases
  • CD-NTases and effectors comprise the core CBASS genes (Type I CBASS), and additional ‘signature’ CD-NTase- associated proteins (Cap) have been identified in Type II and III CBASS that regulate CD- NTase activity 10,13–16 .
  • Phage infection introduces nucleic acids and numerous foreign proteins into the bacterial cell.
  • molecules that cause a phage to be sensitive, or resistant, to a given anti-phage immune system are largely unknown.
  • a recent study discovered the first family of phage-encoded anti-CBASS phosphodiesterase enzymes (Acb1), which cleave cyclic oligonucleotides 17 similarly to poxin enzymes encoded by eukaryotic viruses 18 .
  • Pseudomonas aeruginosa is a human opportunistic pathogen that encodes a diversity of CBASS operons and is a generalist microbe that survives in many niches.
  • P. aeruginosa also has a diverse phage population and is a leading candidate for phage therapy, but our limited understanding of anti-phage immunity is a barrier for basic biology and phage therapeutic development.
  • the disclosure features a method of killing bacteria, the method comprising: contacting an anti-cyclic-oligonucleotide-based anti-phage signaling system (CBASS) protein to one or more cyclic oligonucleotides, wherein anti-CBASS protein is substantially (e.g., at least 60%, 70%, 80%, 90%, 95%, 99%) identical to a sequence of any one of SEQ ID NOS:1-3, thereby inhibiting the CBASS in the bacteria.
  • the cyclic oligonucleotides are present in bacteria and the contacting occurs inside bacterial cells.
  • the cyclic oligonucleotides are present in mammalian cells and the contacting occurs inside mammalian cells. In some embodiments, the mammalian cells are infected with bacteria. [0010] In some embodiments, the cyclic oligonucleotides are extracellular and the contacting occurs outside a cell. [0011] The contacting can occur in vitro. [0012] In some embodiments, the contacting can occur ex vivo. In certain embodiments, the contacting occurs within a population of cells comprising bacterial cells and eukaryotic cells (e.g., mammalian cells (e.g., human cells)).
  • eukaryotic cells e.g., mammalian cells (e.g., human cells
  • the population of cells is introduced into a mammal after the introducing and contacting.
  • the contacting comprises introducing the anti-CBASS protein into the cell.
  • the introducing comprises introducing an expression cassette comprising a nucleic acid encoding the anti-CBASS protein and a promoter operably linked to the nucleic acid. The promoter can be inducible.
  • the introducing comprises administering an engineered bacteriophage comprising an anti-CBASS protein that is substantially (e.g., at least 60%, 70%, 80%, 90%, 95%, 99%) identical to a sequence of any one of SEQ ID NOS:1-3 to the cell.
  • the cyclic oligonucleotide is present in the cell prior to the introducing. In other embodiments, the cyclic oligonucleotide is introduced to the cell when or after the anti-CBASS protein is introduced to the cell. [0016] In some embodiments, the cells are introduced into a mammal after the introducing and contacting in the methods. [0017] In some embodiments, the cell is an eukaryotic cell (e.g., a mammalian cell; e.g., a human cell). In some embodiments, the cell is a prokaryotic cell.
  • the disclosure provides a method of treating a bacterial infection in a subject, comprising administering to the subject anti-CBASS protein substantially (e.g., at least 60%, 70%, 80%, 90%, 95%, 99%) identical to a sequence of any one of SEQ ID NOS:1-3, or an engineered bacteriophage comprising thereof, wherein the anti-CBASS protein binds to one or more cyclic oligonucleotides.
  • the anti-CBASS protein binds to the cyclic oligonucleotides present inside cells.
  • the anti-CBASS protein binds to the cyclic oligonucleotides present inside bacterial cells.
  • the anti-CBASS protein binds to the cyclic oligonucleotides present inside mammalian cells (e.g., mammalian cells that are infected with bacteria). [0020] In some embodiments of the method, the anti-CBASS protein binds to the cyclic oligonucleotides present outside of cells. [0021] In some embodiments of the methods described herein, the the cyclic oligonucleotide is a cyclic dinucleotide or a cyclic trinucleotide.
  • the cyclic oligonucleotide is selected from the group consist of 3’,3’cUU, 3’,3’cAA, 3’,3’cGAMP, 3’,3’cUG, 3’,3’cUA, 3’,3’,3’-cAAA, 3’,3’,3’-cAAG, 2’,3’cGAMP, and 3’,2’cGAMP.
  • the disclosure provides an expression cassette comprising a nucleic acid encoding an anti-CBASS protein and a promoter operably linked to the nucleic acid, wherein the anti-CBASS protein comprises a sequence that is substantially (e.g., at least 60%, 70%, 80%, 90%, 95%, 99%) identical to a sequence of any one of SEQ ID NOS:1-3.
  • the promoter is heterologous to the nucleic acid encoding the anti- CBASS protein.
  • the promoter is inducible.
  • the nucleic acid is DNA or RNA.
  • the disclosure also provides a vector comprising the expression cassette described herein.
  • the vector can be a viral vector.
  • the disclosure also provides an engineered bacteriophage comprising the expression cassette described herein.
  • the disclosure also provides a pharmaceutical composition comprising an anti- CBASS protein or a polynucleotide comprising a nucleic acid encoding an anti-CBASS protein, wherein the anti-CBASS protein comprises a sequence that is substantially (e.g., at least 60%, 70%, 80%, 90%, 95%, 99%) identical to a sequence of any one of SEQ ID NOS:1- 3, or the engineered bacteriophage described herein.
  • the disclosure also provides an engineered bacteriophage comprising a nucleic acid encoding an anti-CBASS protein, wherein the anti-CBASS protein comprises a sequence that is substantially (e.g., at least 60%, 70%, 80%, 90%, 95%, 99%) identical to a sequence of any one of SEQ ID NOS:1-3.
  • FIGS.1A-1F P. aeruginosa BWHPSA011 (Pa011) CBASS-based immunity protects against PaMx41 infection.
  • A The presence of different anti-phage immune systems in P. aeruginosa strains that were used in this study.
  • FIGS.2A-2F Phage-encoded acb2 is necessary for replication in the presence of CBASS.
  • PaMx41-like ⁇ acb2 phages have the acb2 gene substituted with the type VI-A anti-CRISPR gene (acrVIA1) as part of the knockout procedure, and JBD67 ⁇ acb2 phages have the acb2 gene removed from its genome.
  • Genes with known protein functions are indicated with names, and genes with hypothetical proteins are indicated with “orf”.
  • Acb2 percent amino acid identity is shown in (D).
  • FIGS.3A-3E Acb2 antagonizes CBASS activity by sequestering the 3’,3’-cGAMP signaling molecule.
  • A CapV enzyme activity in the presence of the indicated cyclic dinucleotides and resorufin butyrate, which is a phospholipase substrate that emits fluorescence when hydrolyzed. The enzyme activity rate was measured by the accumulation rate of fluorescence units (FU) per second.
  • the concentration of 3’,3’-cGAMP ranged from 0.025 to 0.8 ⁇ M (0.025, 0.05, 0.1, 0.2, 0.4, 0.8 ⁇ M), and the other cyclic-dinucleotides were added at 0.8 ⁇ M.
  • (C) Isothermal titration calorimetry (ITC) assays to test binding of cyclic- dinucleotides to Acb2. Representative binding curves and binding affinities are shown. The K D values are mean ⁇ s.d. (n 3). Raw data for these curves are shown in FIGS.10A-10K.
  • D CapV activity assay to test the effects of Acb2 on 3’,3’-cGAMP. The concentration of 3’,3’-cGAMP was 0.8 ⁇ M and Acb2 ranged from 0.25 to 8 ⁇ M (0.25, 0.5, 2, 8 ⁇ M).
  • FIGS.4A-4I Structure of Acb2 reveals hexamer bound to three molecules of 3’,3’- cGAMP.
  • A Overall structure of the Acb2 hexamer. Two views are shown.
  • FIGS.5A-5D CBASS escape phages have mutations in the major capsid gene.
  • A Plaque assays were performed with the indicated control/WT and escape phages spotted in 10-fold serial dilutions on lawns of bacteria expressing CBASS+ (left) or lacking CBASS- (right); clearings represent phage replication.
  • B Schematic of major capsid genes with corresponding missense mutations and associated CBASS Escape (ESC) phages.
  • C Schematic of in vivo homologous recombination of parental phages with homology-directed repair (HDR) template 1 (encoding I121S or I121T capsid mutations) or template 2 (S330P capsid mutation) and resultant engineered/recombinant phages.
  • HDR homology-directed repair
  • FIG.6 Table showing anti-CBASS proteins sequestered a spectrum of cyclic di- and trinucleotides.
  • FIGS.7A-7H Diversity of CBASS in Pseudomonas aeruginosa strains and CBASS-dependent and -independent phage targeting.
  • A Percentage of P. aeruginosa genomes that encode a CBASS operon and
  • B percentage of effector genes in each CBASS type. Data are shown for P.
  • C CBASS type/operon.
  • Core genes include cGAS/DncV-like nucleotidyltransferase (CD-NTase) and effector genes.
  • CD-NTase cGAS/DncV-like nucleotidyltransferase
  • effector genes Known and predicted (*) cyclic nucleotides are denoted next to the CD-NTase gene (Whiteley et al.2019).
  • Signature genes are denoted as CD-NTase-associated proteins (Cap).
  • FIGS.8A-8F Acb2 protects phage and reduces 3’,3’-cGAMP molecules in CBASS-containing cells.
  • A Schematic of in vivo phage infection and cGAMP detection: (1) Pa011 cells with catalytically dead CapV S48A strain overexpressing a wildtype version of an anti-CBASS gene (Acb2 WT; inhibited CBASS) or mutant version (Acb2 K26A; uninhibited or active CBASS).
  • C Plaque assays were performed with PaMx33, 35, 41, and 43 WT phages, as well as an evolved PaMx41 CBASS escape (ESC) phage, spotted in 10-fold serial dilutions on a lawn of Pa011 WT [CBASS+] or ⁇ CBASS [CBASS-] over-expressing the indicated genes; black arrowhead highlights increase in PaMx41 WT phage titer.
  • D Plaque assays were performed with the indicated phages spotted in 10-fold serial dilutions on a lawn of Pa011 WT, ⁇ CBASS, or WT over-expressing acb2.
  • E Plaque assays with indicated phages on a lawn of P. aeruginosa cells (PAO1) with a chromosomally integrated Pa011 CBASS operon (Pa CBASS ), or empty vector (Pa EV ), and overexpressing acb2.
  • Pa CBASS chromosomally integrated Pa011 CBASS operon
  • Pa EV empty vector
  • Black arrowhead highlights CBASS-dependent change in phage titer.
  • FIGS.9A and 9B Acb2 is found in a broad diversity of phages and bacteria.
  • FIGS.10A-10K Acb2 does not bind CBASS proteins, but does bind 3’,3’-cGAMP, 2’,3’-cGAMP, and c-di-AMP.
  • A -(D) Gel filtration profile of incubated Acb2 with Cap2- CdnA complex (A), Cap2 (B), CdnA (C) or CapV (D) (Superdex-200 increase 10/300 GL, GE Healthcare).
  • E ITC assays to test binding of 3’,3’-cGAMP to Acb2 WT.
  • F ITC assays to test binding of 2’,3’-cGAMP to Acb2.
  • FIGS.11A-11K Structures of apo and di-nucleotide bound Acb2.
  • AUC Analytical Ultracentrifugation
  • B Structure of the Acb2 hexamer.
  • C Structure of the Acb2 monomer.
  • D-E Two types of dimer of protomers as shown in (B) are shown.
  • F -(H), The ability of Acb2 to bind 2’,3’- cGAMP/c-di-AMP/c-di-GMP was analyzed by HPLC.2’,3’-cGAMP/c-di-AMP/c-di-GMP standards were used as a control. The remaining cyclic dinucleotides after incubation with Acb2 were tested.
  • FIGS.12A-12G Acb2 binds to the predicted CdnE cyclic dinucleotide products and protects phages against Type I-A and Type I-B CBASS immunity that encode CdnE cyclase.
  • ITC Isothermal titration calorimetry
  • cUU, cUA, and cUG represent 3’,3’-cyclic-di-UMP, 3’,3’-cyclic-UMP-AMP, and 3’,3’-cyclic-UMP-GMP, respectively.
  • Representative binding curves and binding affinities are shown.
  • Raw data for these curves are shown in (B- D).
  • E Native PAGE showed the binding of Acb2 to cyclic dinucleotides.
  • F P. aeruginosa ATCC 33351 and JD332 CBASS operons with predicted cyclic dinucleotides (Whiteley et al. 2019).
  • FIGS.13A-13E PaMx41 phage remains sensitive to CBASS immunity in the presence of major capsid escape allele expression.
  • A Plaque assays were performed with PaMx41 ⁇ acb2 phage, harboring a wildtype (WT) capsid, spotted in 10-fold serial dilutions on a lawn of Pa011 WT [CBASS+] or ⁇ CBASS [CBASS-] over-expressing the indicated genes; clearings represent phage replication.
  • B Plaque assays were performed with PaMx41 ⁇ acb2 CBASS Escaper phage 7, harboring a mutant (I121T) capsid, spotted in 10- fold serial dilutions on a lawn of Pa011 WT or ⁇ CBASS.
  • (C) Alphafold2 prediction of the PaMx41 ⁇ acb2 (blue) and JDB18 (green) major capsid protein monomer structures overlaid using PyMOL (RMSD: 4.194). Red spheres represent amino acid residues that are mutated and are labeled with the corresponding a.a. change.
  • (D) Alphafold2 prediction of the PaMx41 ⁇ acb2 and (E) JDB18 capsid hexamer structures based on the experimentally solved E. coli T4 phage capsid structure (PDB: 6UZC). Spheres represent the a.a. residues that are mutated.
  • FIGS.14A-14F Acb2 from phage PaMx33 binds cyclic trinucleotides and 3’, 2’- cGAMP.
  • B Overall structure of Acb2 complexed with 3’,2’-cGAMP, which are indicated by arrows.
  • FIGS.15A-15I Acb2 binds to cyclic trinucleotides with binding sites different from those of cyclic dinucleotides.
  • A ITC assays to test the binding of cAAG and cA 3 to PaMx33-Acb2, and binding of cA 3 to PaMx33-Acb2 mutants. Representative binding curves and binding affinities are shown.
  • the two mutants R67A and T74A in the panel represent their binding to cA 3 .
  • FIGS.16A-16C Acb2 binds to cyclic trinucleotides and dinucleotides simultaneously.
  • A Overall structure of Acb2 complexed with cA3 and 3’,3’-cGAMP. cA3 and 3’,3’-cGAMP are shown as blue and light gray sticks. Two views are shown.
  • FIGS.17A-17H The binding spectra are different among Acb2 homologs.
  • A Sequence alignment among Acb2 homologs. Residues that are >80 % conserved, >60 % conserved and >40% conserved are shaded in dark purple, light purple, and light grey, respectively. Residues involved in binding of cyclic CDNs and CTNs are marked with green and blue triangles, respectively.
  • (B) ITC assays to test binding of cyclic oligonucleotides to JBD67-Acb2. Representative binding curves and binding affinities are shown. The KD values are mean ⁇ s.d. (n 3).
  • (C) ITC assays to test binding of cyclic oligonucleotides to T4-Acb2. Representative binding curves and binding affinities are shown. The KD values are mean ⁇ s.d. (n 3).
  • FIGS.18A-18E Acb2 antagonizes tri- and di-nucleotide based CBASS immunity.
  • A Pseudomonas aeruginosa BWHPSA011 (Pa011) Type II-A CBASS and ATCC 27853 (Pa278) Type III-C CBASS operons.
  • B Pseudomonas aeruginosa PaMx33 and JBD67 phages acb2 gene annotated with residues essential for CDN (3’,3’-cGAMP) binding and CTN (cA3) binding.
  • C Effect of PaMx33 Acb2 or its mutants on cA3-activated NucC effector protein function.
  • the concentration of NucC, cA 3 , Acb2 and proteinase K is 10 nM, 5 nM, 50 nM and 1 ⁇ M, respectively.
  • N denotes nicked plasmid
  • SC denotes closed-circular supercoiled plasmid
  • cut denotes fully digested DNA.
  • D Plaque assays with JBD67 ⁇ acb2 phage spotted in 10-fold serial dilutions on PAO1 strains harboring an empty vector (E.V.) plasmid or JBD67 Acb2 variants.
  • the PAO1 strains either contain no CBASS operon (-CBASS), a chromosomally integrated Pa011 CBASS operon (PAO1 Pa011 ), or a chromosomally integrated Pa278 CBASS operon (PAO1 Pa278 ).
  • CBASS CBASS operon
  • PAO1 Pa011 a chromosomally integrated Pa011 CBASS operon
  • PAO1 Pa278 chromosomally integrated Pa278 CBASS operon
  • Basal expression of the Pa011 CBASS operon and 0.3mM IPTG-inducible expression of the Pa278 CBASS operon is sufficient for phage targeting. Black arrowheads highlight significant CBASS- dependent reductions in phage titer.
  • anti-CBASS protein refers to a protein that can bind to one or more cyclic oligonycleotides to inhibit bacterial cyclic-oligonucleotide-based anti-phage signaling system (CBASS). In some embodiments, the anti-CBASS protein binds to cyclic oligonucleotides inside cells (e.g., inside bacterial and/or mammalian cells).
  • the anti-CBASS protein binds to cyclic oligonucleotides outside of cells, i.e., the anti-CBASS protein binds to extracellular cyclic oligonucleotides.
  • nucleic acid or “polynucleotide” refers to deoxyribonucleic acids (DNA) or ribonucleic acids (RNA) and polymers thereof in either single- or double-stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogs of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides.
  • nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences as well as the sequence explicitly indicated.
  • degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed- base and/or deoxyinosine residues (Batzer et al., Nucleic Acid Res.19:5081 (1991); Ohtsuka et al., J. Biol. Chem.260:2605-2608 (1985); and Rossolini et al., Mol. Cell.
  • a “promoter” is defined as an array of nucleic acid control sequences that direct transcription of a nucleic acid.
  • a promoter includes necessary nucleic acid sequences near the start site of transcription, such as, in the case of a polymerase II type promoter, a TATA element.
  • a promoter also optionally includes distal enhancer or repressor elements, which can be located as much as several thousand base pairs from the start site of transcription.
  • the promoter can be a heterologous promoter.
  • An “expression cassette” is a nucleic acid construct, generated recombinantly or synthetically, with a series of specified nucleic acid elements that permit transcription of a particular polynucleotide sequence in a host cell.
  • An expression cassette may be part of a plasmid, viral genome, or nucleic acid fragment.
  • an expression cassette includes a polynucleotide to be transcribed, operably linked to a promoter.
  • the promoter can be a heterologous promoter.
  • a “heterologous promoter” refers to a promoter that would not be so operably linked to the same polynucleotide as found in a product of nature (e.g., in a wild-type organism).
  • the term “heterologous” refers to a protein or nucleic acid in a cell or an organism, or being introduced into a cell or an organism, where the protein or nucleic acid originates from a foreign species compared to the cell or the organism, or originates from the same species but is modified from its original form.
  • a promoter when a promoter is said to be operably linked to a heterologous coding sequence, it means that the coding sequence is derived from one species whereas the promoter sequence is derived from another, different species; or, if both are derived from the same species, the coding sequence is not naturally associated with the promoter (e.g., is a genetically engineered coding sequence).
  • the terms “polypeptide,” “peptide,” and “protein” are used interchangeably herein to refer to a polymer of amino acid residues. All three terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymers.
  • the terms encompass amino acid chains of any length, including full-length proteins, wherein the amino acid residues are linked by covalent peptide bonds.
  • the term “conservatively modified variants” applies to both amino acid and nucleic acid sequences. With respect to particular nucleic acid sequences, “conservatively modified variants” refers to those nucleic acids that encode identical or essentially identical amino acid sequences, or where the nucleic acid does not encode an amino acid sequence, to essentially identical sequences. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For instance, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine.
  • nucleic acid variations are “silent variations,” which are one species of conservatively modified variations. Every nucleic acid sequence herein that encodes a polypeptide also describes every possible silent variation of the nucleic acid.
  • AUG which is ordinarily the only codon for methionine
  • TGG which is ordinarily the only codon for tryptophan
  • each silent variation of a nucleic acid that encodes a polypeptide is implicit in each described sequence.
  • amino acid sequences one of skill will recognize that individual substitutions, deletions or additions to a nucleic acid, peptide, polypeptide, or protein sequence which alters, adds or deletes a single amino acid or a small percentage of amino acids in the encoded sequence is a “conservatively modified variant” where the alteration results in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. Such conservatively modified variants are in addition to and do not exclude polymorphic variants, interspecies homologs, and alleles of the invention.
  • conservatively modified variants of Cas9 or sgRNA can have an increased stability, assembly, or activity as described herein.
  • the following eight groups each contain amino acids that are conservative substitutions for one another: 1) Alanine (A), Glycine (G); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W); 7) Serine (S), Threonine (T); and 8) Cysteine (C), Methionine (M) (see, e.g., Creighton, Proteins, W.
  • Two sequences that are “substantially identical” have at least 60% identity, preferably 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity, when compared and aligned for maximum correspondence over a comparison window, or designated region as measured using a sequence comparison algorithm or by manual alignment and visual inspection where a specific region is not designated.
  • this definition also refers to the complement of a test sequence.
  • the identity exists over a region that is at least about 50 amino acids or nucleotides in length, or more preferably over a region that is 75-100 amino acids or nucleotides in length.
  • sequence comparison algorithm typically one sequence acts as a reference sequence, to which test sequences are compared.
  • test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated.
  • the sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters.
  • a “comparison window”, as used herein, includes reference to a segment of any one of the number of contiguous positions selected from the group consisting of from 20 to 600, usually about 50 to about 200, more usually about 100 to about 150 in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned.
  • An algorithm for determining percent sequence identity and sequence similarity is the BLAST 2.0 algorithm, which are described in Altschul et al., (1990) J. Mol. Biol.215: 403-410.
  • HSPs high scoring sequence pairs
  • Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always ⁇ 0).
  • M forward score for a pair of matching residues; always >0
  • N penalty score for mismatching residues; always ⁇ 0.
  • a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative- scoring residue alignments; or the end of either sequence is reached.
  • the BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment.
  • the BLASTP program uses as defaults a word size (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989)).
  • the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin & Altschul, Proc. Nat’l. Acad. Sci. USA 90:5873-5787 (1993)).
  • BLAST algorithm One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance.
  • P(N) the smallest sum probability
  • a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.2, more preferably less than about 0.01, and most preferably less than about 0.001.
  • the present disclosure describes proteins that can bind to multiple cyclic oligonucleotides, for example, in a bacterial host, as a way to inhibit host immunity (such as cyclic-oligonucleotide-based anti-phage signaling system (CBASS) in bacteria).
  • CBASS cyclic-oligonucleotide-based anti-phage signaling system
  • anti-CBASS proteins can be used, for example, to engineer bacteriophages that are particularly effective in treating bacterial infections.
  • the anti-CBASS proteins can also bind to cyclic oligonucleotides outside of a cell.
  • An anti-CBASS protein described herein can have a sequence that is substantially (e.g., at least 60%, 70%, 80%, 90%, 95%, or 99%) identical to a sequence of SEQ ID NO:1 below: MDNQHKKIKGYRDLSQEEIDMMNRVKELGSQFEKLIQDVSDHLRGQYNASLHNRD EITRIANAEPGRWLAIGKTDIQTGMMAIIRAIAQPDSF (SEQ ID NO:1).
  • An anti-CBASS protein can also have a sequence that is substantially (e.g., at least 60%, 70%, 80%, 90%, 95%, or 99%) identical to a sequence of SEQ ID NO:2 below: MIEDIKGYKPHTEEKIGKVNAIKDAEVRLGLIFDALYDEFWEALDNCEDCEFAKNYA ESLDQLTIAKTKLKEASMWACRAVFQPEEKY (SEQ ID NO:2).
  • An anti-CBASS protein can also have a sequence that is substantially (e.g., at least 60%, 70%, 80%, 90%, 95%, or 99%) identical to a sequence of SEQ ID NO:3 below: MDNQHRKIAGYRELTQDDIDLMNRVKAVGAELLALQAALAGRLSTDLEVKQAAAK ASKLAPEHESSPECVELRRFLAAEPLRWAAIAKTDIQTGVMALVRAIAQPEGC (SEQ ID NO:3).
  • an anti-CBASS protein can further include other amino acid sequences or other chemical moieties (e.g., detectable labels) at the amino terminus, carboxyl terminus, or both.
  • Additional amino acid sequences can include, but are not limited to, tags, detectable markers, or nuclear localization signal sequences.
  • an anti-CBASS protein described herein is demonstrated to bind to a number of cyclic oligonucleotides, e.g., 3’,3’cUU, 3’,3’cAA, 3’,3’cGAMP, 3’,3’cUG, 3’,3’cUA, 3’,3’,3’-cAAA, 3’,3’,3’-cAAG, 2’,3’cGAMP, and 3’,2’cGAMP.
  • an anti-CBASS having a sequence that is at least substantially (e.g., at least 60%, 70%, 80%, 90%, 95%, or 99%) identical to, or comprises the sequence of SEQ ID NO:1 can bind to cyclic oligonucleotides such as 3’,3’cUU, 3’,3’cAA, 3’,3’cGAMP, 3’,3’cUG, 3’,3’cUA, 3’,3’,3’-cAAA, 3’,3’,3’-cAAG, 2’,3’cGAMP, and 3’,2’cGAMP.
  • cyclic oligonucleotides such as 3’,3’cUU, 3’,3’cAA, 3’,3’cGAMP, 3’,3’cUG, 3’,3’cUA, 3’,3’,3’-cAAA, 3’,3’,3’-cAAG, 2’,3’cGAMP, and 3’,2
  • an anti-CBASS having a sequence that is at least substantially (e.g., at least 60%, 70%, 80%, 90%, 95%, or 99%) identical to, or comprises the sequence of SEQ ID NO:2, can bind to cyclic oligonucleotides such as 3’,3’cUU, 3’,3’cAA, 3’,3’cGAMP, 3’,3’cUG, 3’,3’cUA, 2’,3’cGAMP, and 3’,2’cGAMP.
  • the disclosure also includes an engineered bacteriophage comprising an anti- CBASS protein described herein (e.g., SEQ ID NO:1, 2, or 3).
  • the engineered bacteriophage can contain an expression cassette comprising a nucleic acid (e.g., DNA or RNA) encoding an anti-CBASS protein (e.g., SEQ ID NO:1, 2, or 3) and a promoter operably linked to the nucleic acid, wherein the anti-CBASS protein comprises a sequence that is substantially (e.g., at least 60%, 70%, 80%, 90%, 95%, 99%) identical to a sequence of any one of SEQ ID NOS:1-3.
  • the promoter can be heterologous to the nucleic acid encoding the anti-CBASS protein.
  • the promoter can also be an inducible promoter.
  • the disclosure also includes a pharmaceutical composition comprising an anti-CBASS protein described herein (e.g., SEQ ID NO:1, 2, or 3) or an engineered bacteriophage comprising an anti-CBASS protein described herein (e.g., SEQ ID NO:1, 2, or 3).
  • the anti-CBASs proteins described herein can be be generated by any method.
  • the protein can be purified from naturally-occurring sources, synthesized, or more typically can be made by recombinant production in a cell engineered to produce the protein.
  • Exemplary expression systems include various bacterial, yeast, insect, and mammalian expression systems.
  • an expression cassette comprising a nucleic acid (e.g., DNA or RNA) encoding an anti-CBASS protein (e.g., SEQ ID NO:1, 2, or 3) and a promoter operably linked to the nucleic acid, wherein the anti-CBASS protein comprises a sequence that is substantially (e.g., at least 60%, 70%, 80%, 90%, 95%, 99%) identical to a sequence of any one of SEQ ID NOS:1-3.
  • the promoter can be heterologous to the nucleic acid encoding the anti-CBASS protein.
  • the promoter can also be an inducible promoter.
  • the disclosure also includes a vector (e.g., a viral vector) comprising the expression cassette as described herein.
  • a vector e.g., a viral vector
  • the anti-CBASS proteins as described herein can be fused to one or more fusion partners and/or heterologous amino acids to form a fusion protein.
  • Fusion partner sequences can include, but are not limited to, amino acid tags, non-L (e.g., D-) amino acids or other amino acid mimetics to extend in vivo half-life and/or protease resistance, targeting sequences or other sequences.
  • functional variants or modified forms of the anti-CBASS proteins include fusion proteins of an anti-CBASS protein and one or more fusion domains.
  • Exemplary fusion domains include, but are not limited to, polyhistidine, Glu-Glu, glutathione S transferase (GST), thioredoxin, protein A, protein G, an immunoglobulin heavy chain constant region (Fc), maltose binding protein (MBP), and/or human serum albumin (HSA).
  • a fusion domain or a fragment thereof may be selected so as to confer a desired property.
  • some fusion domains are particularly useful for isolation of the fusion proteins by affinity chromatography.
  • relevant matrices for affinity chromatography such as glutathione-, amylase-, and nickel- or cobalt-conjugated resins are used.
  • fusion domain may be selected so as to facilitate detection of the anti-CBASS proteins.
  • detection domains include the various fluorescent proteins (e.g., GFP) as well as “epitope tags,” which are usually short peptide sequences for which a specific antibody is available.
  • Epitope tags for which specific monoclonal antibodies are readily available include FLAG, influenza virus haemagglutinin (HA), and c-myc tags.
  • the fusion domains have a protease cleavage site, such as for Factor Xa or Thrombin, which allows the relevant protease to partially digest the fusion proteins and thereby liberate the recombinant proteins therefrom. The liberated proteins can then be isolated from the fusion domain by subsequent chromatographic separation.
  • an anti-CBASS protein is fused with a domain that stabilizes the anti-CBASS protein in vivo (a “stabilizer” domain).
  • stabilizing is meant anything that increases serum half-life, regardless of whether this is because of decreased destruction, decreased clearance by the kidney, or other pharmacokinetic effect.
  • Fusions with the Fc portion of an immunoglobulin are known to confer desirable pharmacokinetic properties on a wide range of proteins. See, e.g., US Patent Publication No.2014/056879. Likewise, fusions to human serum albumin can confer desirable properties. Other types of fusion domains that may be selected include multimerizing (e.g., dimerizing, tetramerizing) domains and functional domains (that confer an additional biological function, as desired). Fusions may be constructed such that the heterologous peptide is fused at the amino and/or carboxyl terminus of an anti-CBASS protein. [0072] In some embodiments, the anti-CBASS proteins as described herein comprise at least one non-naturally encoded amino acid.
  • an anti-CBASS protein comprises 1, 2, 3, 4, or more unnatural amino acids.
  • Methods of making and introducing a non-naturally-occurring amino acid into a protein are known. See, e.g., U.S. Pat. Nos. 7,083,970; and 7,524,647.
  • the general principles for the production of orthogonal translation systems that are suitable for making proteins that comprise one or more desired unnatural amino acid are known in the art, as are the general methods for producing orthogonal translation systems. For example, see International Publication Numbers WO 2002/086075, WO 2002/085923, WO 2004/094593, and WO 2005/007624.
  • a non-naturally encoded amino acid is typically any structure having any substituent side chain other than one used in the twenty natural amino acids. Because non- naturally encoded amino acids typically differ from the natural amino acids only in the structure of the side chain, the non-naturally encoded amino acids form amide bonds with other amino acids, including but not limited to, natural or non-naturally encoded, in the same manner in which they are formed in naturally occurring polypeptides. However, the non-naturally encoded amino acids have side chain groups that distinguish them from the natural amino acids.
  • R optionally comprises an alkyl-, aryl-, acyl-, keto-, azido-, hydroxyl-, hydrazine, cyano-, halo-, hydrazide, alkenyl, alkynl, ether, thiol, seleno-, sulfonyl- , borate, boronate, phospho, phosphono, phosphine, heterocyclic, enone, imine, aldehyde, ester, thioacid, hydroxylamine, amino group, or the like or any combination thereof.
  • amino acids comprising a photoactivatable cross-linker include, but are not limited to, amino acids comprising a photoactivatable cross-linker, spin-labeled amino acids, fluorescent amino acids, metal binding amino acids, metal-containing amino acids, radioactive amino acids, amino acids with novel functional groups, amino acids that covalently or noncovalently interact with other molecules, photocaged and/or photoisomerizable amino acids, amino acids comprising biotin or a biotin analog, glycosylated amino acids such as a sugar substituted serine, other carbohydrate modified amino acids, keto-containing amino acids, amino acids comprising polyethylene glycol or polyether, heavy atom substituted amino acids, chemically cleavable and/or photocleavable amino acids, amino acids with an elongated side chains as compared to natural amino acids, including but not limited to, polyethers or long chain hydrocarbons, including but not limited to, greater than about 5 or greater than about 10 carbons, carbon-linked sugar-containing amino acids, redox-active amino acids
  • PEGylation or incorporation of long-chain polyethylene glycol polymers PEG
  • Introduction of PEG or long chain polymers of PEG increases the effective molecular weight of the present polypeptides, for example, to prevent rapid filtration into the urine.
  • a Lysine residue in a protein can be conjugated to PEG directly or through a linker.
  • Such linker can be, for example, a Glu residue or an acyl residue containing a thiol functional group for linkage to the appropriately modified PEG chain.
  • An alternative method for introducing a PEG chain is to first introduce a Cys residue at the C-terminus or at solvent exposed residues such as replacements for Arg or Lys residues. This Cys residue is then site-specifically attached to a PEG chain containing, for example, a maleimide function.
  • Methods for incorporating PEG or long chain polymers of PEG can include, for example, those described in Veronese, F. M., et al., Drug Disc. Today 10: 1451-8 (2005); Greenwald, R. B., et al., Adv. Drug Deliv.
  • p-azidophenylalanine can be incorporated into the present polypeptides and then reacted with a PEG polymer having an acetylene moiety in the presence of a reducing agent and copper ions to facilitate an organic reaction known as “Huisgen [3+2]cycloaddition.”
  • specific mutations of anti-CBASS proteins can be made to alter the glycosylation of the protein, if needed. Such mutations may be selected to introduce or eliminate one or more glycosylation sites, including but not limited to, O-linked or N- linked glycosylation sites as recognized by eukaryotic expression systems.
  • the present inventors have discovered a protein that can bind to a number of cyclic oligonucleotides in order to inhibit CBASS and evade bacterial immune system.
  • the disclosure also provides methods of killing bacteria by contacting an anti- CBASS protein to one or more cyclic oligonucleotides.
  • the anti- CBASS protein binds to cyclic oligonucleotides that are present inside bacterial cells.
  • the anti-CBASS protein binds to cyclic oligonucleotides that are present inside mammalian cells.
  • the anti-CBASS protein binds to cyclic oligonucleotides that are present inside mammalian cells that have been infected with bacteria. In some embodiments, the anti-CBASS protein binds to cyclic oligonucleotides that are present outside of cells, i.e., the anti-CBASS protein binds to extracellular cyclic oligonucleotides.
  • the anti-CBASS protein can have a sequence that is substantially (e.g., at least 60%, 70%, 80%, 90%, 95%, 99%) identical to a sequence of any one of SEQ ID NOS:1- 3.
  • the anti-CBASS protein has a sequence that is 95%, 97%, 99%, or 100% identical to the sequence of SEQ ID NO:1. In some embodiments, the anti-CBASS protein has a sequence that is 95%, 97%, 99%, or 100% identical to the sequence of SEQ ID NO:2. In some embodiments, the anti-CBASS protein has a sequence that is 95%, 97%, 99%, or 100% identical to the sequence of SEQ ID NO:3.
  • contacting the anti-CBASS protein to one or more cyclic oligonucleotides can occur in a cell, such as an eukaryotic cell (e.g., an eukaryotic cell (e.g., a mammalian cell) infected with bacteria).
  • the cell can be a human cell (e.g., a human cell infected with bacteria).
  • the cell can be a prokaryotic cell (e.g., a bacterial cell).
  • the contacting can occur in vitro, in vivo, or ex vivo.
  • contacting the anti-CBASS protein to one or more cyclic oligonucleotides can comprise introducing the anti-CBASS protein into the cell.
  • the anti-CBASS protein can be introduced into the cell as an isolated protein or as a polynucleotide or expression cassette comprising a nucleic acid encoding an anti-CBASS protein described herein in which the expression of the anti-CBASS protein can be induced inside the cell.
  • the anti-CBASS protein can be introduced into the cell by a bacteriophage comprising the anti-CBASs protein.
  • an anti-CBASS protein described herein can be introduced into the cell by an expression cassette comprising a nucleic acid encoding the anti-CBASS protein and a promoter operably linked to the nucleic acid.
  • the promoter can be an inducible promoter.
  • an anti-CBASS protein described herein can be introduced into the cell by administering an engineered bacteriophage comprising an anti-CBASS protein described herein.
  • the cyclic oligonucleotides are already present inside the cell prior to the anti-CBASS protein is introduced into the cell.
  • the cyclic oligonucleotides can additionally be introduced into the cell before, during, or after the the anti-CBASS protein is introduced to the cell.
  • the methods of killing bacteria as described herein can also be performed ex vivo, in which cells infected by bacteria from a subject can be isolated from the subject, and the anti-CBASS protein can be introduced into the cells to kill the bacteria. The isolated cells containing the anti-CBASS protein can then be introduced back into the subject.
  • the disclosure also provides methods of treating a bacterial infection in a subject by administering to the subject an anti-CBASS protein substantially (e.g., at least 60%, 70%, 80%, 90%, 95%, 99%) identical to a sequence of any one of SEQ ID NOS:1-3, or an engineered bacteriophage comprising thereof, wherein the anti-CBASS protein binds to one or more cyclic oligonucleotides in the bacteria.
  • an anti-CBASS protein substantially (e.g., at least 60%, 70%, 80%, 90%, 95%, 99%) identical to a sequence of any one of SEQ ID NOS:1-3, or an engineered bacteriophage comprising thereof, wherein the anti-CBASS protein binds to one or more cyclic oligonucleotides in the bacteria.
  • bacterial infections include, but are not limited to, infections caused by bacteria in the genus Pseudomonas (e.g., Pseudomonas aeruginosa), infections caused by bacteria in the genus Acinetobacter (e.g., Acinetobacter baumanii), infections caused by bacteria in the genus Klebsiella, infections caused by bacteria in the genus Yersinia, infections caused by bacteria in the genus Enterobacter, infections caused by bacteria in the genus Streptococcus (e.g., Streptococcus pyogenes), infections caused by bacteria in the genus Escherichia (e.g., Escherichia coli), infections caused by bacteria in the genus Vibrio (e.g., Vibrio cholerae), and infections caused by bacteria in the genus Salmonella (e.g., Salmonella typhi).
  • Pseudomonas e.g., Pseudom
  • an anti-CBASS protein e.g., SEQ ID NO:1, 2, or 3
  • a cyclic oligonucleotide that can be a cyclic dinucleotide or a cyclic trinucleotide.
  • an anti-CBASS protein (e.g., SEQ ID NO:1, 2, or 3) can bind to a cyclic oligonucleotide selected from the group consist of 3’,3’cUU, 3’,3’cAA, 3’,3’cGAMP, 3’,3’cUG, 3’,3’cUA, 3’,3’,3’-cAAA, 3’,3’,3’-cAAG, 2’,3’cGAMP, and 3’,2’cGAMP.
  • a cyclic oligonucleotide selected from the group consist of 3’,3’cUU, 3’,3’cAA, 3’,3’cGAMP, 3’,3’cUG, 3’,3’cUA, 3’,3’,3’-cAAA, 3’,3’,3’-cAAG, 2’,3’cGAMP, and 3’,2’cGAMP.
  • an anti-CBASS protein having a sequence that is at least substantially (e.g., at least 60%, 70%, 80%, 90%, 95%, or 99%) identical to, or comprises the sequence of SEQ ID NO:1 can be used in methods of killing bacteria and/or treating a bacterial infection as described herein by binding to cyclic oligonucleotides such as 3’,3’cUU, 3’,3’cAA, 3’,3’cGAMP, 3’,3’cUG, 3’,3’cUA, 3’,3’,3’-cAAA, 3’,3’,3’-cAAG, 2’,3’cGAMP, and 3’,2’cGAMP.
  • cyclic oligonucleotides such as 3’,3’cUU, 3’,3’cAA, 3’,3’cGAMP, 3’,3’cUG, 3’,3’cUA, 3’,3’,3’-cAAA, 3’,3’,
  • an anti-CBASS protein having a sequence that is at least substantially (e.g., at least 60%, 70%, 80%, 90%, 95%, or 99%) identical to, or comprises the sequence of SEQ ID NO:2, can be used in methods of killing bacteria and/or treating a bacterial infection as described herein by binding to cyclic oligonucleotides such as 3’,3’cUU, 3’,3’cAA, 3’,3’cGAMP, 3’,3’cUG, 3’,3’cUA, 2’,3’cGAMP, and 3’,2’cGAMP.
  • cyclic oligonucleotides such as 3’,3’cUU, 3’,3’cAA, 3’,3’cGAMP, 3’,3’cUG, 3’,3’cUA, 2’,3’cGAMP, and 3’,2’cGAMP.
  • aeruginosa strain that harbors Type II-A CBASS (3’,3’-cGAMP producing CD-NTase; CdnA) with a phospholipase (CapV) effector that limits phage replication by >10,000-fold.
  • CBASS anti-phage immunity has been shown to function naturally without heterologous overexpression.
  • Acb2 a widespread phage protein that forms a hexamer complex with three 3’,3-cGAMP molecules, acting as a “sponge” to reduce the available molecules to activate the phospholipase effector.
  • Acb2 binds to multiple other cyclic dinucleotides, including 3’,3’-c-di-UMP, 3’,3’-cUA, and 3’,3’-cUG, and is necessary for optimal phage replication in the presence of Type I or II CBASS that are predicted to encode the aforementioned cyclic dinucleotides.
  • Phages with acb2 deleted were unable to replicate in the lytic cycle or during exit from lysogeny in the presence of CBASS.
  • mutations in the major capsid gene enabled phages to escape CBASS. This work provides direct evidence of phage inhibition and evasion of CBASS, demonstrating a robust arms race between the two.
  • CBASS loci were deleted from the genome of four strains possessing representatives of the common CBASS types (Type I-A, II-A, II-C, and III-C; FIG.7C) using a CRISPR-Cas3 tool 19 .
  • these strains also encode numerous other anti-phage immune systems (FIG.1A). Therefore, due to the multitude of immune systems, we screened the CBASS mutants against a diverse panel of ⁇ 70 phages, which spanned 23 different genomic families and four different morphologies (Myoviridae, Siphoviridae, Podoviridae, and Inoviridae 20 ). A single P.
  • capV, cdnA, and cap2 mutations abolished anti-phage activity whereas the cap3 mutation did not (FIG.7H).
  • PaMx41 infection generated low levels of 3’,3’-cGAMP ( ⁇ 8 nM; FIGS.8A and 8B) whereas the molecule was nearly undetectable in CdnA mutant strain ( ⁇ 0.5 nM; L.O.D.0.24 nM).
  • PaMx41-like phages encode a CBASS antagonist
  • CBASS CBASS-like phages encode a CBASS antagonist
  • FIG.1D How CBASS detects and targets phage is currently unknown. Therefore, to identify phage genes required for successful CBASS activity, we isolated PaMx41 mutants that resist Pa011 CBASS-based immunity. With a frequency of 3.7 x 10 -5 (FIG.1D), 10 independent PaMx41 CBASS “escape” phages were isolated that replicate well on Pa011 WT (FIG.1B).
  • Whole genome sequencing revealed one mutation in all CBASS escape phages: a no-stop extension mutation (X37Q) in orf24 (FIG.1E).
  • the naturally CBASS resistant phages, PaMx33, PaMx35, and PaMx43 share >96% nucleotide identity across the genome and naturally encode the 94 a.a. version with >98% a.a. identity.
  • the PaMx41 gp24 variants were overexpressed in Pa011 WT and ⁇ CBASS cells and then plaque assays were performed.
  • Acb2 has conserved function in a broadly distributed temperate phage family [0093] Homology searches with Acb2 revealed that it is encoded in a striking number of tailed phages, including those infecting Pseudomonas, Vibrio, Acinetobacter, Salmonella, Serratia, Erwinia, and Escherichia sp. (T2 and T4 phages; gene: vs.4), among others (FIG. 9A). We observed no other examples of the truncated Acb2 variant encoded in PaMx41 WT phage.
  • acb2 is commonly encoded by P. aeruginosa B3- like temperate phages (e.g. JBD67), which are unrelated to the PaMx41-like lytic phages. Since JBD67 phage does not replicate on the Pa011 strains, we integrated the Type II-A CBASS operon with its native promoter into the chromosome of a P. aeruginosa strain (POA1) that is sensitive to this phage and naturally lacks CBASS.
  • P. aeruginosa strain POA1
  • JBD67 ⁇ acb2 and JBD18 phages were reduced by >5 orders of magnitude in the presence of CBASS.
  • Expression of acb2 derived from either JBD67 or PaMx41-orf24 X37Q on a plasmid fully restored the titer of the PaMx41 ⁇ acb2 phage, and only partially restored the titer of the JBD67 ⁇ acb2 and JBD18 phages (FIG.2C, FIG.8E).
  • JBD67 and JBD18 more strongly activate CBASS, and consequently, Acb2 expression in trans becomes partially overwhelmed.
  • CapV phospholipase activity in vitro and confirmed that it is only activated by 3 ⁇ ,3 ⁇ -cGAMP in a concentration-dependent manner, but not by 2 ⁇ ,3 ⁇ -cGAMP, c-di-AMP, or c-di-GMP (FIG.3A).
  • CapV activity was abrogated (FIG.3A), suggesting that Acb2 may directly bind to the 3 ⁇ ,3 ⁇ -cGAMP molecule.
  • a native gel assay showed a significant shift of the purified Acb2 protein upon adding 3 ⁇ ,3 ⁇ -cGAMP (FIG.3B).
  • Each protomer mainly interacts with two adjacent protomers (FIG.11B), allowing the six protomers to interlock into a compact assembly.
  • An Acb2 protomer consists of one short N- terminal helix and two long anti-parallel helices, with a kink in the long helix at the C- terminus (FIG.11C).
  • the ligand-bound structure showed that one Acb2 hexamer binds three 3 ⁇ ,3 ⁇ -cGAMP molecules (FIG.4B).
  • Each cGAMP binding pocket is formed by two Acb2 protomers that interact in a head-to-head manner, and is mainly composed of N- and C- terminal helices/loops from each protomer (FIG.11D).
  • cGAMP is stabilized through hydrophobic interactions by several residues from both protomers, such as L14, M22, M81, I84 and P90 (FIG.4F). Consistent with these analyses, Acb2 Y11A and K26A mutants were inactive in plaque assays (FIG.4I) and the Acb2 K26A mutant expressed in the Pa011 WT strain lost its ability to sequester 3’,3’-cGAMP in vivo (FIGS.8A and 8B).
  • the PaMx41 WT or mutant major capsid gene with its native promoter was cloned and expressed in Pa011 WT and ⁇ CBASS cells.
  • the WT major capsid we did not observe any CBASS-dependent cellular toxicity, indicating that capsid monomer is not sufficient to activate CBASS.
  • FIG.13A CBASS-dependent targeting of resistant phage
  • FIG.13B expression of the mutant major capsid gene did not induce escape of CBASS sensitive phage
  • CBASS escape phages were also isolated from JBD67 ⁇ acb2 and JBD18 phages on the Pa CBASS strain (FIG.5A). Strikingly, whole genome sequencing revealed missense point mutations in the genes encoding their major capsid proteins [orf32 in JBD67 (NCBI: YP_009625956) and orf35 in JBD18 (NCBI: AFR52188); FIG.5B, Table S2].
  • the PaMx41 major capsid protein shares no significant amino acid identity with the JBD67 and JBD18 major capsid proteins, yet the mutations all converge on the same protein.
  • FIG.13C When modeling the location of the mutations on the predicted major capsid monomer structures, we did not observe overlap between the distinct phages (FIG.13C). However, modeling of the predicted major capsid hexamer structures indicated that the mutations lie on the protein-protein interface within or between hexamers (FIGS.13D and 13E), suggesting that a higher ordered capsid structure or process may be implicated in CBASS immunity.
  • some of the observed capsid mutant genotypes i.e.
  • aeruginosa BWHPSA011 (Pa011) strain harbors a cGAS-like enzyme (CdnA) that produces 3’,3’-cGAMP dinucleotides in response to PaMx41 phage infection, which activates the phospholipase (CapV) effector.
  • Phages related to PaMx41 (or an escape mutant derived from PaMx41) produce an anti-CBASS protein (Acb2) that is expressed as a middle gene in the phage replication cycle 24 and sequesters 3’,3’-cGAMP.
  • Acb2 likely accumulates prior to the production of the dinucleotide given that previous work demonstrated 3’,3’-cGAMP levels increase later in the E.
  • a Thoeris anti-defense (Tad1) protein was found to specifically bind to and sequester gcADPR molecules 25 .
  • the first anti-CBASS gene identified (Acb1) is another class of phage enzymes, similar to Apyc1, that harbors a phosphodiesterase fold that specifically binds and cleaves cyclic nucleotides 17 .
  • This is a common inhibitory mechanism of the eukaryotic cGAS-STING signaling system and is utilized by poxviruses 18 and a host pyrophosphatase/phosphodiesterase protein 26 , which enzymatically cleaves and depletes 2’,3’-cGAMP.
  • Acb2 binds to and sequesters bacterial 3’,3’-cGAMP, c-di-AMP, 3’,3’-c-di-UMP, 3’,3’-cUA, 3’,3’-cUG, and human 2’,3’- cGAMP.
  • the structure and mechanism of Acb2 were independently confirmed with a homolog from E. coli phage T4 in a recent preprint 16 .
  • the cGAMP “sponge” mechanism is an entirely new inhibitory strategy.
  • capsid mutants that escape CBASS mirrors the recently identified major capsid mutations in E. coli phage T5, which enables escape from Pycsar (pyrimidine cyclase system for anti-phage resistance) 28 .
  • Pycsar pyrimidine cyclase system for anti-phage resistance
  • phage capsid protein which is one of many phage structural proteins mutated to evade targeting of anti-phage bacterial immune systems 31 . Additionally, binding to other phage structural proteins directly activate Avs (anti- viral STAND NTPases) 32 and DSR (defense-associated sirtuins) systems 33 .
  • Avs anti- viral STAND NTPases
  • DSR defense-associated sirtuins
  • the major capsid gene mutations identified in our study are enriched at the capsid protein interface, suggesting that a higher ordered capsid structure or process, rather than a capsid monomer, may be implicated in CBASS immunity. However, the mechanism behind the mutant phage capsid that enables CBASS evasion is unknown and remains an area of future investigation.
  • Plating was performed on LB solid agar with 10 mM MgSO4 when performing phage infections, and when indicated, gentamicin (50 ⁇ g ml -1 for P. aeruginosa and 15 ⁇ g ml -1 for E. coli) was used to maintain the pHERD30T plasmid. Gene expression was induced by the addition of L-arabinose (0.01% final for BWHPSA011 bacterial genes and 0.1% for phage genes, unless otherwise specified). [0106] The E. coli BL21 (DE3) strain was used for recombinant protein overexpression and grown in Lysogeny broth (LB) medium.
  • LB Lysogeny broth
  • tBLASTn was used to query the amino acid sequence of eight known CD-NTases (CdnA-H) against sequenced Pseudomonas aeruginosa genomes contained in the NCBI 36 and IMG 37 databases as well as our sequenced UCSF clinical isolates. Proteins with >25% amino acid sequence identity to a validated CD-NTase were accepted as “hits” 7 , leading to the identification of >300 CBASS operons in 252 distinct P. aeruginosa strains. The P.
  • aeruginosa BWHPSA011 (Pa011) strain contains a Type II-A CBASS operon in contig 12 (NCBI Genome ID: NZ_AXQR000000012.1) ranging from 1250439-1254679bp, with CapV (phospholipase effector, NCBI Gene ID: Q024_30602), CdnA (cyclase, Q024_30601), Cap2 (E1/E2, Q024_30600), and Cap3 (JAB, intergenic region 1250439-1250912bp).
  • Identification of anti-phage immune systems [0108] DefenseFinder was used to systematically identify all known anti-phage bacterial immune system operons in P.
  • aeruginosa strains 34,38 were used to construct the table in FIG.1A.
  • Episomal gene expression [0109] The shuttle vector that replicates in P. aeruginosa and E. coli, pHERD30T 39 was used for cloning and episomal expression of genes in P. aeruginosa BWHPSA011 (Pa011) or PAO1 strains. This vector has an arabinose-inducible promoter and a selectable gentamicin marker. Vector was digested with SacI and PstI restriction enzymes and purified.
  • Inserts were amplified by PCR using bacterial overnight culture or phage lysate as the DNA template, and joined into the pHERD30T vector at the SacI-PstI restriction enzyme cut sites by Hi-Fi DNA Gibson Assembly (NEB) following the manufacturer’s protocol.
  • the resulting plasmids were transformed into E. coli DH5 ⁇ . All plasmid constructs were verified by sequencing using primers that annealed to sites outside the multiple cloning site.
  • P. aeruginosa cells were electroporated with the pHERD30T constructs and selected on gentamicin.
  • integrants were screened by colony PCR with primers PTn7R and PglmS-down, and then verified by sequencing using primers that anneal to sites outside the attTn7 site. Electrocompetent cell preparations, transformations, integrations, selections, plasmid curing, and FLP-recombinase-mediated marker excision with pFLP were performed as described previously 40 . Chromosomal mutants of P. aeruginosa BWHPSA011 [0111] The allelic exchange vector that replicates in P. aeruginosa and E. coli, pMQ30 42 was used for generating the chromosomal CBASS knockout and CBASS mutant genes in P.
  • aeruginosa BWHPSA011 (Pa011). Vector was digested with HindIII and BamHI restriction enzymes and purified.
  • CBASS knockout strain homology arms >500bp up- and downstream of CBASS operon were amplified by PCR using Pa011 overnight culture as the template DNA.
  • CBASS gene mutant strains homology arms >500bp up- and downstream of CBASS gene catalytic residue(s), with the appropriate mutant nucleotides, were amplified by PCR using Pa011 overnight culture as the template DNA.
  • aeruginosa PAO1 WT which naturally lacks CBASS.150 ⁇ l of overnight cultures of PAO1 were infected with 10 ⁇ l of low titer phage lysate (>10 4-7 pfu/ml) and then mixed with 3 ml of 0.7% top agar 10 mM MgSO 4 for plating on the LB solid agar. After incubating at 37°C overnight, individual phage plaques were picked from top agar and resuspended in 200 ⁇ l SM phage buffer. For high titer lysates, the purified phage was further amplified on LB solid agar plates with PAO1 WT.
  • Plaque assays [0113] Plaque assays were conducted at 37°C with solid LB agar plates.150 ⁇ l of overnight bacterial culture was mixed with top agar and plated.
  • Lysogen construction with JDB67 phage [0114] Lysogens were constructed by spotting serial dilutions of JDB67 WT or JBD67 ⁇ acb2 phage lysates on the engineered P. aeruginosa PAO1 strain that harbors BWHPSA011 CBASS in the chromosome (Pa CBASS ), or a mini-Tn7 E.V.
  • aeruginosa strain BWHPSA011 (Pa011) were infected with 10 ⁇ l of high titer phage lysate (>10 9 pfu/ml) and then plated on LB solid agar. After incubating at 37°C overnight, 10 individual phage plaques were picked from top agar and resuspended in 200 ⁇ l SM phage buffer. Phage lysates were purified for three rounds using the CBASS expressing strain. Three PaMx41 WT control phages were picked, purified, and propagated in parallel by infecting the Pa011 ⁇ CBASS strain.
  • Genomic DNA from phage lysates was extracted using a modified SDS/Proteinase K method. Briefly, 200 ⁇ L high titer phage lysate (>10 9 pfu/ml) was mixed with an equal volume of lysis buffer (10 mM Tris, 10 mM EDTA, 100 ⁇ g/mL proteinase K, 100 ⁇ g/mL RNaseA, 0.5% SDS) and incubated at 37°C for 30 min, and then 55°C for 30 min.
  • lysis buffer 10 mM Tris, 10 mM EDTA, 100 ⁇ g/mL proteinase K, 100 ⁇ g/mL RNaseA, 0.5% SDS
  • Preps were further purified using the DNA Clean & Concentrator Kit (Zymo Research). DNA was quantified using the Qubit 4.0 Fluorometer (Life Technologies).20-100 ng genomic DNA was used to prepare WGS libraries using the Illumina DNA Prep Kit (formerly known as Illumina Nextera Flex Kit) using a modified protocol that utilized 5x reduced quantities of tagmentation reagents per prep, except for the bead washing step with Tagment Wash Buffer (TWB), where the recommended 100 ⁇ L of TWB was used. Subsequent on-bead PCR indexing-amplification of tagmented DNA was performed using 2x Phusion Master Mix (NEB) and custom-ordered indexing primers (IDT) matching the sequences from the Illumina Nextera Index Kit.
  • NEB 2x Phusion Master Mix
  • IDT custom-ordered indexing primers
  • Each 50 ⁇ L reaction was split in two tubes, amplified for 9 and 12 cycles respectively.
  • Libraries were further purified by agarose gel electrophoresis; DNA was excised around the ⁇ 400 bp size range and purified using the Zymoclean Gel DNA Recovery Kit (Zymo Research). Libraries were quantified by Qubit and the 9-cycle reaction was used unless the yield was too low for sequencing, in which case the 12-cycle reaction was used. Libraries were pooled in equimolar ratios and sequenced with Illumina MiSeq v3 reagents (150 cycles, Read 1; 8 cycles, Index 1; 8 cycles, Index 2). WGS data were demultiplexed either on-instrument or using a custom demultiplexing Python script (written by Dr.
  • homology arms of >500bp up- and downstream of PaMx41 acb2 were amplified by PCR using Pamx41 WT phage genomic DNA as the template.
  • the acrVIA1 gene was amplified from plasmid pAM383 48 , a gift from Luciano Marraffini, The Rockefeller University. PCR products were purified and assembled as a recombineering substrate and then inserted into the NheI site of the pHERD30T vector. The resulting plasmids were electroporated into P. aeruginosa PAO1 cells.
  • PAO1 strains carrying the recombination plasmid were grown in LB media supplemented with gentamicin.150 ⁇ l of overnight cultures were infected with 10 ⁇ l of high titer phage lysate (>10 9 pfu/ml; PaMx33 WT, PaMx35 WT, PaMx43 WT, PaMx41 ESC or PaMx41 WT) and then plated on LB solid agar. After incubating at 37°C overnight, SM phage buffer was added to the entire lawn and whole cell lysate collected.
  • the resulting phage lysate containing both WT and recombinant phages were tittered on PAO1 strains with a chromosomally integrated Type VI-A CRISPR-Cas13a system, and the most efficiently targeting crRNA guide (specific to orf11; guide #5) was used to screen for recombinants.
  • PAO1 strains carrying the Cas13a system and crRNA of choice were grown overnight in LB media supplemented with gentamicin.150 ⁇ l of overnight cultures were infected with 10 ⁇ l of low titer phage lysate (10 4-7 pfu/ml), and then plated onto LB solid agar containing 0.3% arabinose and 1 mM isopropyl ⁇ -d-1-thiogalactopyranoside (IPTG). After incubating at 37°C overnight, individual phage plaques were picked from top agar and resuspended in 200 ⁇ l SM phage buffer.
  • IPTG isopropyl ⁇ -d-1-thiogalactopyranoside
  • Phage lysates were purified for three rounds using the Cas13a counter-selection strain (guide #5), and further propagated on a complementary Cas13a counter-selection strain (guide #4), to select against Cas13a escaper phages.
  • PCR was performed with the appropriate pairs of primers amplifying the region outside of the homology arms, an internal region of acrVIA1, and acb2.
  • Homologous recombination-mediated mutation of phage gene [0119] Construction of template plasmids for homologous recombination consisted of homology arms >500bp up- and downstream of the mutation of interest encoded in PaMx41 orf11.
  • the homology arms were amplified by PCR using PaMx41 ⁇ acb2 escapers phage genomic DNA as the template, and PaMx41 WT phage genomic DNA as the control template.
  • Template 1 primers were designed to symmetrically flank the PaMx41 orf11 mutations I121S and I121T, and template 2 primers were designed to symmetrically flank mutation I327T and S330P.
  • PCR products were purified and assembled as a recombineering substrate and then inserted into the SacI-PstI site of the pHERD30T vector. The resulting plasmids were electroporated into P. aeruginosa BWHPSA011 (Pa011) ⁇ CBASS cells.
  • Pa011 strains carrying the recombination plasmid were grown in LB media supplemented with gentamicin.150 ⁇ l of overnight cultures were infected with 10 ⁇ l of high titer phage lysate (>10 9 pfu/ml; PaMx41 ⁇ acb2) and then plated on LB solid agar. After incubating at 37°C overnight, SM phage buffer was added to the entire lawn and whole cell lysate collected. The resulting phage lysate containing both WT and recombinant phages were screened on a lawn of Pa011 WT cells harboring an active CBASS system.
  • Cas3 (Type I-C)-specific guides targeting JBD67 acb2 were cloned into a pHERD30T-derived vector containing modified I-C repeats as previously described 19 .
  • the guides electroporated into P. aeruginosa PAO1 strains with a chromosomally integrated Type I-C helicase attenuated Cas3 system.
  • JBD67 WT phage lysate was tittered on the PAO1 strains and the efficiently targeting crRNA guide (specific to acb2; guide #3) was identified.
  • PAO1 strains carrying the Type I-C CRISPR-Cas system with a helicase attenuated Cas3 enzyme and crRNA targeting phage JBD67 acb2 were grown overnight in LB media supplemented with gentamicin.150 ⁇ l of overnight cultures were infected with 10 ⁇ l of high titer phage lysate (>10 9 pfu/ml; JBD67) and plated on LB agar plates containing gentamicin, 0.1% arabinose, and 1 mM isopropyl ⁇ -d-1-thiogalactopyranoside (IPTG).
  • IPTG isopropyl ⁇ -d-1-thiogalactopyranoside
  • SM phage buffer was added to the entire lawn and whole cell lysate collected.
  • the resulting phage lysate containing both WT and acb2 knockout phages were grown on a complementary Cas3 counter-selection strain (guide #4) to select against Cas3 escaper phages.150 ⁇ l of overnight cultures were infected with 10 ⁇ l of low titer phage lysate (10 4-7 pfu/ml; JBD67 WT) and then plated on LB solid agar containing 0.1% arabinose and 1 mM IPTG.
  • phage plaques were picked from top agar and replica-plated onto LB solid agar with PAO1 EV and PAO1 CBASS strains. JDB67 plaque sizes that were reduced on the PAO1 CBASS plate compared to the positive control (JBD18 WT phage) were identified as potential CBASS sensitive phages. Corresponding plaques on the PAO1 EV plate were picked and resuspended in 200 ⁇ l SM phage buffer. To determine whether the phages harbored deletions in acb2, PCR was performed with the appropriate pairs of primers amplifying a ⁇ 1kb region outside of acb2.
  • Intracellular 3’,3’-cGAMP measurements [0121] Cell lysates were prepared similarly to previous methods 8 , in which P. aeruginosa BWHPA011 (Pa011) cells harboring a catalytically dead capV gene (CapV S48A ) were used and then transformed with a pHERD30T vector expressing acb2 WT or K26A. Cells were taken from overnight culture, diluted 1:100 in 150ml LB medium with G50 and 0.1% arabinose (flask size 500ml), and then grown at 37°C (190 r.p.m.) until reaching an OD600nm of 0.3-0.4.
  • the resuspended pellet was supplemented with 1 ⁇ l hen-lysozyme (Sigma-Aldrich), vortexed briefly, and incubated at 25°C for 10 min.
  • the resuspended cells were then mixed with Lysing Matrix B (MP) beads and cells were disrupted mechanically using Mini-Beadbeater 16 Biospec Products (1 cycle of 2:30, 3,450 oscillations/m, at 4 °C). Cell lysates were then centrifuged at 17,500 g for 10 min at 4°C.
  • MP Lysing Matrix B
  • one 50 ml cell lysate and subsequent supernatant was (i) loaded onto a 3kDa filter (Amicon Ultra-0.5 centrifugal filter unit; Merk) and the corresponding 50 ml cell lysate and subsequent supernatant was (ii) subjected to phenol-chloroform/chloroform nucleotide extraction (Rouillon et al., 2019).
  • the unit was centrifuged at 16,000 g for 45 min at 4 °C and flow-through (containing small molecules less than 3kDa) was used as the sample for 3’,3’-cGAMP measurements.
  • 600 ⁇ l of supernatant was added to 600 ⁇ l of phenol-chloroform, vortexed for 30 sec, and then centrifuged at 17,500 g for 45 min at 4 °C.
  • the top aqueous layer was carefully transferred into another eppendorf tube and 600 ⁇ l of chloroform was added, vortexed for 30 sec, and then centrifuged at 17,500 g for 10 min at 4 °C.
  • the top aqueous layer was added to the 3kD filter, centrifuged at 16,000 g for 45 min at 4 °C, and flow-through collected.
  • aeruginosa PaMx41 (orf11) and JBD18 (orf35) phage capsid proteins were generated using AlphaFold2 53 and aligned using the PyMol “super” function to the different chains of the E. coli T4 phage capsid structure (PDB: 6UZC).
  • PDB PyMol “super” function to the different chains of the E. coli T4 phage capsid structure
  • the Acb2 mutants were generated by two-step PCR and were subcloned, overexpressed and purified in the same way as wild-type protein.
  • the proteins were expressed in E. coli strain BL21 (DE3) and induced by 0.2 mM isopropyl- ⁇ -D-thiogalactopyranoside (IPTG) when the cell density reached an OD 600nm of 0.8. After growth at 18°C for 12 h, the cells were harvested, re-suspended in lysis buffer (50 mM Tris–HCl pH 8.0, 300 mM NaCl, 10 mM imidazole and 1 mM PMSF) and lysed by sonication.
  • lysis buffer 50 mM Tris–HCl pH 8.0, 300 mM NaCl, 10 mM imidazole and 1 mM PMSF
  • the cell lysate was centrifuged at 20,000 g for 50 min at 4°C to remove cell debris.
  • the supernatant was applied onto a self-packaged Ni-affinity column (2 mL Ni-NTA, Genscript) and contaminant proteins were removed with wash buffer (50 mM Tris pH 8.0, 300 mM NaCl, 30 mM imidazole).
  • wash buffer 50 mM Tris pH 8.0, 300 mM NaCl, 30 mM imidazole.
  • the fusion protein was then digested with Ulp1 at 18°C for 2 h, and then the Acb2 protein was eluted with wash buffer.
  • the eluant of Acb2 was concentrated and further purified using a Superdex-200 increase 10/300 GL (GE Healthcare) column equilibrated with a buffer containing 10 mM Tris-HCl pH 8.0, 200 mM NaCl and 5 mM DTT.
  • the purified protein was analyzed by SDS-PAGE. The fractions containing the target protein were pooled and concentrated.
  • the CdnA, Cap2 and CdnA-Cap2 complex were purified as His-tagged proteins, which were eluted with elution buffer (50 mM Tris pH 8.0, 300 mM NaCl, 300 mM imidazole) after removing contaminant proteins with wash buffer.
  • CapV The cells expressing CapV were resuspended with lysis buffer containing 50 mM phosphate buffer pH 7.4, 300 mM NaCl, 10% glycerol (v/v).
  • the CapV proteins bound to Ni-NTA beads were washed with a buffer containing 50 mM phosphate buffer pH 7.4, 300 mM NaCl, 10% glycerol (v/v), 30 mM imidazole and then eluted with the 50 mM phosphate buffer (pH 7.4), 300 mM NaCl, 10% glycerol (v/v), 300 mM imidazole.
  • the eluant of CapV was concentrated and further purified using a Superdex-200 increase 10/300 GL (GE Healthcare) column equilibrated with a reaction buffer containing 50 mM phosphate buffer (pH 7.4), 300 mM NaCl, 10% glycerol (v/v).
  • the purified protein was analyzed as described above. Crystallization, data collection and structural determination [0126]
  • the Acb2 protein was concentrated to 24 mg/mL in 10 mM Tris-HCl pH 8.0, 200 mM NaCl and 5 mM DTT. Crystals were grown using the hanging-drop vapor diffusion method.
  • Crystals of Acb2 were grown at 18°C by mixing an equal volume of the protein (24 mg/mL) with reservoir solution containing 0.2 M Sodium bromide, 0.1 M Bis-Tris propane pH 6.5, 10% Ethylene glycol and 20% v/v PEG 3350. Crystals of Acb2 in complex with 3’,3’-cGAMP or c-di-AMP were grown under the same reservoir solution. Prior to crystallization, 3’,3’-cGAMP or c-di-AMP were mixed with the protein at a molar ratio of 0.8:1. The crystals appeared overnight and grew to full size in about two to three days. The crystals were cryoprotected in the reservoir solution containing 20% glycerol before its transferring to liquid nitrogen.
  • Isothermal titration calorimetry binding assay [0128] The dissociation constants of binding reactions of Acb2 or Acb2 mutants with the 3’,3’-cGAMP/2’,3’-cGAMP/c-di-GMP/c-di-AMP/3’,3’-c-di-UMP/3’,3’-c-UMP-AMP/3’,3’- c-UMP-GMP were determined by isothermal titration calorimetry (ITC) using a MicroCal ITC200 calorimeter.
  • ITC isothermal titration calorimetry
  • Both proteins and cyclic dinucleotides were desalted into the working buffer (20 mM HEPES pH 7.5 and 200 mM NaCl). The titration was carried out with 19 successive injections of 2 ⁇ L cyclic dinucleotides at the 0.4 mM concentration, spaced 120 s apart, into the sample cell containing the Acb2 or Acb2 mutants with a concentration of 0.1 mM by 700 rpm at 25°C.
  • the Origin software was used for baseline correction, integration, and curve fitting to a single site binding model. Fluorogenic biochemical assay for CapV activity [0129] The enzymatic reaction velocity was measured as previously described 8 .
  • the esterase activity of the 6 ⁇ His-tagged CapV was probed with the fluorogenic substrate resorufin butyrate.
  • the CapV protein was diluted in 50 mM sodium phosphate pH 7.4, 300 mM NaCl, 10% (v/v) glycerol to a final concentration of 1.77 ⁇ M.
  • the purified 6 ⁇ His-tagged CapV was added to the reaction solution containing 3’,3’-cGAMP to a final assay volume of 50 ⁇ L, and fluorescence was measured in a 96-well plate (Corning 96-well half area black non-treated plate with a flat bottom). Plates were read once every 30 s for 20 min at 37°C using a EnSpire Multimode Plate Reader (PerkinElmer) with excitation and emission wavelengths of 550 and 591 nm, respectively. To determine the function of Acb2, 32 ⁇ M Acb2 and 0.8 ⁇ M 3’,3’-cGAMP were pre-incubated at 18°C, and the subsequent detection method was as described above.
  • Proteinase K was subsequently added to the reaction system at a final concentration of 0.25 mg/mL and the reaction was performed at 58°C for 1 h. Reaction products were transferred to Amicon Ultra-15 Centrifugal Filter Unit 3 kDa and centrifuged at 4°C, 4,000 g. The products obtained by filtration were further filtered with a 0.22 ⁇ m filter and subsequently used for HPLC experiments.
  • the HPLC analysis was performed on an Agilent 1200 system with a ZORBAX Bonus-RP column (4.6 ⁇ 150 mm). A mixture of acetonitrile (2%) and 0.1% tri ⁇ uoroacetic acid solution in water (98%) were used as mobile phase with 0.8 mL/min.
  • Example 2 This example shows that anti-CBASS protein sequested a number of cyclic di- and trinucleotides.
  • Two methods were used to assess cyclic oligonucleotide binding: isothermal titration calorimietry (ITC) or native polyacrylamide gel electrophoresis (native-PAGE) to see the protein shift when incubated with the indicated nucleotide.
  • ITC isothermal titration calorimietry
  • native-PAGE native polyacrylamide gel electrophoresis
  • coli T4 (SEQ ID NO:2), a mix of ITC and native-PAGE were used.
  • JBD67 Acb2 SEQ ID NO:3
  • native-PAGE was used to confirm similar binding spectrum as that observed with PaMx33 (FIG.6).
  • Data on proteins Apyc1, Acb1, and Tad1 in FIG.6 were taken from the literature.
  • Example 3 Acb2 sequesters diverse cyclic dinucleotides and is active in human cells. [0136] To understand the selectivity of the newly identified Acb2 protein fold, we comprehensively tested an array of cyclic oligonucleotides that Acb2 may bind to.
  • CDN molecules are bound by the N-terminal domains of the two interacting Acb2 protomers, each from one Acb2 dimer (FIG.14D).
  • the stacking from Y11 residue and salt bridges from K26 residue of both protomers further stabilize this interaction (FIGS.14C and 14D).
  • the structure of Acb2 complexed with another cGAMP isomer, 2’,3’-cGAMP, solved at 2.24 ⁇ resolution further confirmed this mode of binding (FIGS.14D and 14E).
  • Their base groups are mainly stabilized by the ⁇ - ⁇ stacking from the Y11 residue (FIGS.14C and 14D).
  • cA 3 and cAAG are major products of the CD-NTase enzymes involved in CBASS whereas cA 4 is only a minor product of a single CD-NTase.
  • cA 6 has not been identified as a product of any known CD-NTases. However, all three cyclic oligoadenylates are known products involved in Type III CRISPR-Cas anti-phage immunity.
  • a native gel assay showed that the Acb2 protein does not shift upon adding cA 4 or cA 6 molecules.
  • both native gel and ITC assays showed that Acb2 does not bind to cUMP, cCMP or cAMP.
  • the two CTN molecules bind at the two ends of the channel, blocking the channel from two opposite sides (FIG.15E).
  • the binding modes of CDNs and CTNs within Acb2 can be described as follows: Each of the two protomers that together bind a CDN is involved in binding to one out of the two CTNs, respectively. Correspondingly, each of the three protomers that together bind a CTN is involved in binding to one out of the three CDNs, respectively. [0139]
  • the CTN is bound mainly through its three phosphate groups, each of which is coordinated by R67 of one protomer and T74 of another protomer through hydrogen bonds (FIGS.15F-15G).
  • the CTN is also stabilized by hydrophobic interactions from R67, A70, and I71 from each of the three protomers (FIG.15G).
  • the Acb2 T74A mutant displayed a significantly decreased binding affinity to cA3 (K D of ⁇ 291 nM), and the Acb2 R67A mutant abolished Acb2 binding of cA 3 in vitro (FIG. 15A).
  • the binding sites of the CTNs and CDNs in Acb2 are independent of each other, we tested the binding of 3’,3’-cGAMP with the T74A or R67A Acb2 mutant proteins.
  • a native gel assay showed similar shifts of the two Acb2 mutants as WT Acb2 upon adding 3’,3’-cGAMP (FIG.15H), suggesting that the binding to 3’,3’-cGAMP is not affected by the two mutations.
  • the native gel results showed a significant shift of Y11A and K26A mutant proteins upon adding cA 3 (FIG.15I). Taken together, these data collectively show that one Acb2 hexamer binds two CTNs through two pockets independent of those that bind CDNs.
  • cA 3 is bound within its SAVED (SMODS-associated) domain, which is a fusion of two CARF (CRISPR-associated Rossman fold) domains derived from Type III CRISPR-Cas system (PDB code: 6WAN).
  • SAVED SMODS-associated domain
  • CARF CRISPR-associated Rossman fold domains derived from Type III CRISPR-Cas system
  • RECON adopts a TIM barrel fold with eight parallel ⁇ strands surrounded by eight crossover ⁇ -helixes and cAAG is bound in a deep crevice at the top of the ⁇ barrel (PDB code: 6M7K).
  • the conformation of cA 3 within Acb2 is also different from those within NucC, Cap4, and RECON complex structures.
  • cA3 in both NucC and Cap4 are almost in an overall planar conformation, and two adenine bases of cAAG within RECON are nearly in the same plane as the phosphodiester ring and the third guanine base is extended out.
  • each base of cA3 forms a ⁇ 46.8 degree angle with the phosphate plane in Acb2.
  • the structure of Acb2 complexed with cA3 reveals a novel CTN-binding fold. Cyclic nucleotide binding spectra are different among Acb2 homologs.
  • aeruginosa phage JBD67 (44.4% a.a. identity), in which both R67 and T74 residues are conserved, alongside Serratia phage CHI14 (23.5% a.a. identity) and Escherichia phage T4 (24.2% a.a. identity), in which only the R67 (Serratia phage) or T74 (Escherichia phage) residue is conserved.
  • ITC analyses showed that JBD67- Acb2 directly binds to 3’,3’-cGAMP with a KD of ⁇ 99 nM and cA3 with a KD of ⁇ 3.5 nM (FIG.17B), both of which are comparable to those of PaMx33-Acb2.
  • Native gel assays also suggest that JBD67-Acb2 binds to the same spectrum of cyclic nucleotides as PaMx33-Acb2. ITC analyses showed that T4-Acb2 directly binds to 3’,3’-cGAMP with a KD of ⁇ 84.4 nM, consistent with previous work, but does not bind to cA3 (FIG.17C). Native gel assays also suggest that T4-Acb2 binds the same spectrum of CDNs as PaMx33-Acb2, but not to the CTNs cA 3 and cAAG.
  • CHI14-Acb2 displayed the same binding spectrum to all cyclic oligonucleotides as T4-Acb2 (FIG.17F-17G).
  • T4-Acb2 The outcomes of binding experiments are summarized, along with a comparison to the enzyme Acb1 (FIG. 17H).
  • Acb2 homologs bind to many CTNs and CDNs used in cGAS-based immunity with certain homologs having a more limited spectrum.
  • Acb2 antagonizes Type III-C CBASS immunity.
  • phage-encoded Acb2 can antagonize Type III-C CBASS immunity that uses a cA3 signaling molecule to activate the endonuclease (NucC) effector protein.
  • NucC is a cyclic nucleotide-activated effector in both CBASS and Type III CRISPR-Cas systems, which non- specifically degrades DNA and limits phage replication.
  • JBD67 WT phage was reduced by 5 orders of magnitude (FIG.18D-18E), whereas JBD67 WT phage was reduced by 1-2 orders of magnitude.
  • Plasmid-based expression of WT Acb2 or R82A and T89A Acb2 (cyclic trinucleotide binding mutants) partially rescued phage titer, while Y11A and K26A Acb2 (CDN binding mutants) did not (FIG.18D).
  • the partial targeting of JBD67 WT phage i.e.
  • CBASS immunity functions via the activation of a cGAS-like enzyme to catalyze the synthesis of a cyclic oligonucleotide signaling molecule.
  • cGAS-like enzyme to catalyze the synthesis of a cyclic oligonucleotide signaling molecule.
  • two phage proteins have been discovered to antagonize the CBASS immunity: Acb1 and Acb2.
  • Acb1 uses an inhibitory mechanism common to the eukaryotic cGAS-STING signaling system, 25 that is, enzymatically cleaving and depleting an array of CDNs and CTNs.15
  • Acb2 acts as a “sponge” and sequesters 3’,3’-cGAMP 16, 17 as well as a variety of other CBASS CDN signaling molecules.16
  • a sponging mechanism was also reported for inhibitors of the anti- phage system Thoeris, including Tad126 and Tad2, 27 that sequester gcADPR signaling molecules.
  • STING is a direct innate immune sensor of cyclic di- GMP. Nature 478, 515–518.10.1038/nature10429. 7.
  • RNA targeting with CRISPR-Cas13a facilitates bacteriophage genome engineering. bioRxiv, 2022.02.14.480438.10.1101/2022.02.14.480438. 22. Athukoralage, J.S., McMahon, S.A., Zhang, C., Grüschow, S., Graham, S., Krupovic, M., Whitaker, R.J., Gloster, T.M., and White, M.F.
  • MacSyFinder a program to mine genomes for molecular systems with an application to CRISPR-Cas systems.
  • 39. Qiu, D., Damron, F.H., Mima, T., Schweizer, H.P., and Yu, H.D. (2008).
  • mini-Tn7 insertion in bacteria with single attTn7 sites example Pseudomonas aeruginosa. Nat. Protoc.1, 153–161. 10.1038/nprot.2006.24. 41. Choi, K.-H., Mima, T., Casart, Y., Rholl, D., Kumar, A., Beacham, I.R., and Schweizer, H.P. (2008). Genetic tools for select-agent-compliant manipulation of Burkholderia pseudomallei. Appl. Environ. Microbiol.74, 1064–1075.10.1128/AEM.02430- 07. 42.
  • the Pseudomonas aeruginosa generalized transducing phage phiPA3 is a new member of the phiKZ-like group of “jumbo” phages, and infects model laboratory strains and clinical isolates from cystic fibrosis patients. Microbiology 157, 859–867.10.1099/mic.0.044701-0. 64.
  • Bondy-Denomy J., Pawluk, A., Maxwell, K.L., and Davidson, A.R. (2013). Bacteriophage genes that inactivate the CRISPR/Cas bacterial immune system. Nature 493, 429–432.10.1038/nature11723. 68. Bondy-Denomy, J., Qian, J., Westra, E.R., Buckling, A., Guttman, D.S., Davidson, A.R., and Maxwell, K.L. (2016). Prophages mediate defense against phage infection through diverse mechanisms. ISME J.10, 2854–2866.10.1038/ismej.2016.79. 69.
  • RDA Representational Difference Analysis

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Abstract

Anti-CBASS proteins and methods of their use are provided.

Description

PATENT Attorney Docket No. 081906-1429035-260910PC Client Ref. No. SF2023-155 PROTEINS THAT INHIBIT CYCLIC-OLIGONUCLEOTIDE-BASED ANTI-PHAGE SIGNALING SYSTEM (CBASS) CROSS-REFERENCE TO RELATED APPLICATION [0001] This application claims priority to U.S. Provisional Application No.63/448,609, filed February 27, 2023, the disclosure of which is hereby incorporated by reference in its entirety for all purposes. STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT [0002] This invention was made with government support under contract no. R21 AI168811 awarded by the National Institute of Health (NIH). The government has certain rights in the invention. BACKGROUND OF THE INVENTION [0003] A fundamental strategy of eukaryotic anti-viral immunity involves the cyclic GMP- AMP synthase (cGAS) enzyme, which synthesizes 2’,3’-cGAMP and activates a STING effector to prevent viral replication. Diverse bacteria contain cGAS-like enzymes that produce cyclic oligonucleotides and induce anti-phage activity, known as cyclic- oligonucleotide-based anti-phage signaling system (CBASS). However, this activity has only been demonstrated through heterologous expression. Whether bacteria harboring CBASS antagonize and co-evolve with phages is unknown. [0004] Sensing specific macromolecules produced or possessed by viruses is an conserved strategy of anti-viral immunity across all kingdoms of life1,2. In mammalian cells, viral double-stranded DNA (dsDNA) is bound by cyclic GMP-AMP synthase (cGAS) in the cytoplasm3,4. The activated cGAS enzyme produces 2’,3’-cyclic GMP-AMP (2’,3’-cGAMP) dinucleotides that bind to the STING effector protein and induces a type I interferon response5,6. Recently, thousands of cGAS-like enzymes named CD-NTases (cGAS/DncV- like nucleotidyltransferases) were identified across the entire bacterial domain and then biochemically characterized, revealing at least 8 enzymatic clades and 10 known cyclic oligonucleotides7. These enzymes are activated during phage infection through an unknown mechanism and produce cyclic oligonucleotides, like 3’,3’-cGAMP, which activate a downstream effector8–12. This strategy of bacterial immunity was coined cyclic- oligonucleotide-based anti-phage signaling system (CBASS)8. CD-NTases and effectors comprise the core CBASS genes (Type I CBASS), and additional ‘signature’ CD-NTase- associated proteins (Cap) have been identified in Type II and III CBASS that regulate CD- NTase activity10,13–16. [0005] Phage infection introduces nucleic acids and numerous foreign proteins into the bacterial cell. However, molecules that cause a phage to be sensitive, or resistant, to a given anti-phage immune system are largely unknown. A recent study discovered the first family of phage-encoded anti-CBASS phosphodiesterase enzymes (Acb1), which cleave cyclic oligonucleotides17 similarly to poxin enzymes encoded by eukaryotic viruses18. Investigating the co-evolution of phages and CBASS in a host with endogenous CBASS function will inform how cGAS-based immunity functions in nature and is the main goal of this study. [0006] Pseudomonas aeruginosa is a human opportunistic pathogen that encodes a diversity of CBASS operons and is a generalist microbe that survives in many niches. P. aeruginosa also has a diverse phage population and is a leading candidate for phage therapy, but our limited understanding of anti-phage immunity is a barrier for basic biology and phage therapeutic development. BRIEF SUMMARY OF THE INVENTION [0007] In one aspect, the disclosure features a method of killing bacteria, the method comprising: contacting an anti-cyclic-oligonucleotide-based anti-phage signaling system (CBASS) protein to one or more cyclic oligonucleotides, wherein anti-CBASS protein is substantially (e.g., at least 60%, 70%, 80%, 90%, 95%, 99%) identical to a sequence of any one of SEQ ID NOS:1-3, thereby inhibiting the CBASS in the bacteria. [0008] In some embodiments, the cyclic oligonucleotides are present in bacteria and the contacting occurs inside bacterial cells. [0009] In some embodiments, the cyclic oligonucleotides are present in mammalian cells and the contacting occurs inside mammalian cells. In some embodiments, the mammalian cells are infected with bacteria. [0010] In some embodiments, the cyclic oligonucleotides are extracellular and the contacting occurs outside a cell. [0011] The contacting can occur in vitro. [0012] In some embodiments, the contacting can occur ex vivo. In certain embodiments, the contacting occurs within a population of cells comprising bacterial cells and eukaryotic cells (e.g., mammalian cells (e.g., human cells)). In some embodiments, the population of cells is introduced into a mammal after the introducing and contacting. [0013] In some embodiments, the contacting comprises introducing the anti-CBASS protein into the cell. In some embodiments, the introducing comprises introducing an expression cassette comprising a nucleic acid encoding the anti-CBASS protein and a promoter operably linked to the nucleic acid. The promoter can be inducible. [0014] In some embodiments, the introducing comprises administering an engineered bacteriophage comprising an anti-CBASS protein that is substantially (e.g., at least 60%, 70%, 80%, 90%, 95%, 99%) identical to a sequence of any one of SEQ ID NOS:1-3 to the cell. [0015] In some embodiments, the cyclic oligonucleotide is present in the cell prior to the introducing. In other embodiments, the cyclic oligonucleotide is introduced to the cell when or after the anti-CBASS protein is introduced to the cell. [0016] In some embodiments, the cells are introduced into a mammal after the introducing and contacting in the methods. [0017] In some embodiments, the cell is an eukaryotic cell (e.g., a mammalian cell; e.g., a human cell). In some embodiments, the cell is a prokaryotic cell. [0018] In another aspect, the disclosure provides a method of treating a bacterial infection in a subject, comprising administering to the subject anti-CBASS protein substantially (e.g., at least 60%, 70%, 80%, 90%, 95%, 99%) identical to a sequence of any one of SEQ ID NOS:1-3, or an engineered bacteriophage comprising thereof, wherein the anti-CBASS protein binds to one or more cyclic oligonucleotides. [0019] In some embodiments, the anti-CBASS protein binds to the cyclic oligonucleotides present inside cells. In some embodiments, the anti-CBASS protein binds to the cyclic oligonucleotides present inside bacterial cells. In some embodiments, the anti-CBASS protein binds to the cyclic oligonucleotides present inside mammalian cells (e.g., mammalian cells that are infected with bacteria). [0020] In some embodiments of the method, the anti-CBASS protein binds to the cyclic oligonucleotides present outside of cells. [0021] In some embodiments of the methods described herein, the the cyclic oligonucleotide is a cyclic dinucleotide or a cyclic trinucleotide. In some embodiments, the cyclic oligonucleotide is selected from the group consist of 3’,3’cUU, 3’,3’cAA, 3’,3’cGAMP, 3’,3’cUG, 3’,3’cUA, 3’,3’,3’-cAAA, 3’,3’,3’-cAAG, 2’,3’cGAMP, and 3’,2’cGAMP. [0022] In another aspect, the disclosure provides an expression cassette comprising a nucleic acid encoding an anti-CBASS protein and a promoter operably linked to the nucleic acid, wherein the anti-CBASS protein comprises a sequence that is substantially (e.g., at least 60%, 70%, 80%, 90%, 95%, 99%) identical to a sequence of any one of SEQ ID NOS:1-3. In some embodiments, the promoter is heterologous to the nucleic acid encoding the anti- CBASS protein. In certain embodiments, the promoter is inducible. In certain embodiments the nucleic acid is DNA or RNA. [0023] The disclosure also provides a vector comprising the expression cassette described herein. The vector can be a viral vector. [0024] The disclosure also provides an engineered bacteriophage comprising the expression cassette described herein. [0025] The disclosure also provides a pharmaceutical composition comprising an anti- CBASS protein or a polynucleotide comprising a nucleic acid encoding an anti-CBASS protein, wherein the anti-CBASS protein comprises a sequence that is substantially (e.g., at least 60%, 70%, 80%, 90%, 95%, 99%) identical to a sequence of any one of SEQ ID NOS:1- 3, or the engineered bacteriophage described herein. [0026] The disclosure also provides an engineered bacteriophage comprising a nucleic acid encoding an anti-CBASS protein, wherein the anti-CBASS protein comprises a sequence that is substantially (e.g., at least 60%, 70%, 80%, 90%, 95%, 99%) identical to a sequence of any one of SEQ ID NOS:1-3. BRIEF DESCRIPTION OF THE DRAWINGS [0027] FIGS.1A-1F: P. aeruginosa BWHPSA011 (Pa011) CBASS-based immunity protects against PaMx41 infection. (A) The presence of different anti-phage immune systems in P. aeruginosa strains that were used in this study. Some systems are present in the genome twice as indicated by the darker shade of gray and number (2). An asterisk (*) indicates the CBASS operon was effectively deleted from the bacterial genome. (B) Pa011 CBASS operon. (C) Plaque assays with PaMx41-like phages and an evolved PaMx41 CBASS Escaper (ESC) phage spotted in 10-fold serial dilutions on a lawn of Pa011 WT [CBASS+] or ¨CBASS [CBASS-]; clearings represent phage replication. Black arrowhead highlights the reduction in PaMx41 WT phage titer. See also FIGS.7A-7H. (D) Efficiency of plating was quantified as plaque-forming units (PFU) per ml on Pa011 WT divided by PFU/ml on the ¨CBASS (n=3). Data are mean + s.d. Non-parametric ANOVA test yielded a P value of <0.0001. (E) Schematic of PaMx41 WT and CBASS escape phage genomes with the no-stop extension mutation. The bold underline indicates mutation of thymine (T) to cytosine (C), resulting in a stop codon (TAG, *) to glutamine (CAG, Q) substitution (in red). (F) Plaque assays with PaMx41 WT phage on a lawn of Pa011 WT or ¨CBASS over-expressing the indicated orf24 variants. See also FIGS.8A-8F. [0028] FIGS.2A-2F: Phage-encoded acb2 is necessary for replication in the presence of CBASS. (A, C, E) Heat maps representing the order of magnitude change in phage titer, where phage titer is quantified by comparing the number of spots (with plaques, or clearing if plaques were not visible) on the CBASS+ strain divided by the CBASS- strain. Plaque assays used for these quantifications can been seen in FIGS.8A-8F (n=3). (B, D) Comparison of the acb2 locus across phage genomes. PaMx41-like ¨acb2 phages have the acb2 gene substituted with the type VI-A anti-CRISPR gene (acrVIA1) as part of the knockout procedure, and JBD67¨acb2 phages have the acb2 gene removed from its genome. Genes with known protein functions are indicated with names, and genes with hypothetical proteins are indicated with “orf”. Acb2 percent amino acid identity is shown in (D). (F) Plaque assays assessing the titer induced prophages spotted on a lawn of PaEV or PaCBASS. Black arrowhead highlights reduction in JBD67¨acb2 phage titer. See also FIGS.8A-8F. [0029] FIGS.3A-3E: Acb2 antagonizes CBASS activity by sequestering the 3’,3’-cGAMP signaling molecule. (A) CapV enzyme activity in the presence of the indicated cyclic dinucleotides and resorufin butyrate, which is a phospholipase substrate that emits fluorescence when hydrolyzed. The enzyme activity rate was measured by the accumulation rate of fluorescence units (FU) per second. The concentration of 3’,3’-cGAMP ranged from 0.025 to 0.8 ^M (0.025, 0.05, 0.1, 0.2, 0.4, 0.8 ^M), and the other cyclic-dinucleotides were added at 0.8 ^M. To test the effects of Acb2 to bind and release 3’,3’-cGAMP, Acb2 (32 ^M) was incubated with 3’,3’-cGAMP (0.8 ^M) for 10 minutes and then Proteinase K (0.065 mg/mL) was added to extract the nucleotides from the Acb2 protein. Filtered nucleotides products were used for the CapV activity assay. Data are mean ± s.d. (n=3). (B) Native PAGE showed the binding of Acb2 to cyclic dinucleotides. (C) Isothermal titration calorimetry (ITC) assays to test binding of cyclic- dinucleotides to Acb2. Representative binding curves and binding affinities are shown. The KD values are mean ± s.d. (n=3). Raw data for these curves are shown in FIGS.10A-10K. (D) CapV activity assay to test the effects of Acb2 on 3’,3’-cGAMP. The concentration of 3’,3’-cGAMP was 0.8 ^M and Acb2 ranged from 0.25 to 8 ^M (0.25, 0.5, 2, 8 ^M). The 3’,3’-cGAMP was pre-incubated with Acb2 for 1, 10, 30 minutes, respectively. Data are mean ± s.d. (n=3). (E) The ability of Acb2 to bind and release 3’,3’-cGAMP when treated with proteinase K was analyzed by HPLC.3’,3’- cGAMP standard was used as a control. The remaining 3’,3’-cGAMP after incubation with Acb2 were tested. [0030] FIGS.4A-4I: Structure of Acb2 reveals hexamer bound to three molecules of 3’,3’- cGAMP. (A) Overall structure of the Acb2 hexamer. Two views are shown. (B) Overall structure of the Acb2 hexamer bound to three molecules of 3’,3’-cGAMP.3’,3’-cGAMP molecules are colored in slate. (C) 2Fo-Fc electron density of 3’,3’-cGAMP in the binding pocket contoured at 1 ı. (D) Structural comparison between an Acb2 dimer in its apo (colored in gray) and 3’,3’-cGAMP-bound form (colored in yellow and light magenta). The loops which move upon 3’,3’-cGAMP binding are marked with black circles. (E) Structural alignment between apo and 3’,3’-cGAMP-bound Acb2, which are colored as in (B). Y11 from the two structures are highlighted as sticks. Red dashed lines represent poplar interactions. (F) Binding between Acb2 or Acb2 mutants and 3’,3’-cGAMP. Red dashed lines represent polar interactions. (G) ITC assays to test the binding of 3’,3’-cGAMP to Acb2 mutants. Representative binding curves and binding affinities are shown. The KD values are mean ± s.d. (n=3). Raw data for these curves are shown in FIGS.10A-10K. (H) CapV activity assay to test the effects of Acb2 mutations. The concentration of 3’,3’-cGAMP was 0.8 ^M and Acb2 or its mutants was 32 ^M. The 3’,3’-cGAMP was pre-incubated with Acb2 or its mutants for 10 minutes. Bar graph represents average of three technical replicates. Data are mean ± s.d. (n=3). (I) Plaque assays on a lawn of Pa011 WT or ¨CBASS overexpressing empty vector (E.V.) or the indicated PaMx41 Acb2 mutants; clearings represent phage replication. [0031] FIGS.5A-5D: CBASS escape phages have mutations in the major capsid gene. (A) Plaque assays were performed with the indicated control/WT and escape phages spotted in 10-fold serial dilutions on lawns of bacteria expressing CBASS+ (left) or lacking CBASS- (right); clearings represent phage replication. (B) Schematic of major capsid genes with corresponding missense mutations and associated CBASS Escape (ESC) phages. (C) Schematic of in vivo homologous recombination of parental phages with homology-directed repair (HDR) template 1 (encoding I121S or I121T capsid mutations) or template 2 (S330P capsid mutation) and resultant engineered/recombinant phages. (D) Plaque assays with recombinant phages possessing major capsid mutations, or WT capsid controls, spotted on lawns of Pa011 WT or ¨CBASS. [0032] FIG.6: Table showing anti-CBASS proteins sequestered a spectrum of cyclic di- and trinucleotides. [0033] FIGS.7A-7H: Diversity of CBASS in Pseudomonas aeruginosa strains and CBASS-dependent and -independent phage targeting. (A) Percentage of P. aeruginosa genomes that encode a CBASS operon and (B) percentage of effector genes in each CBASS type. Data are shown for P. aeruginosa in the IMG database (n = 758); some genomes may encode more than one CBASS type/operon. (C) The presence of different CBASS types in P. aeruginosa strains according to their operon composition of core and signature genes. Core genes include cGAS/DncV-like nucleotidyltransferase (CD-NTase) and effector genes. Known and predicted (*) cyclic nucleotides are denoted next to the CD-NTase gene (Whiteley et al.2019). Signature genes are denoted as CD-NTase-associated proteins (Cap). (D) Heat map representing the order of magnitude reduction in phage titer on a CBASS- encoding (Pa011 WT Native CBASS Host or PaCBASS Heterologous CBASS Host; CBASS+) strain normalized to a strain lacking CBASS (Pa011 ¨CBASS or PaEV; CBASS-).46 (out of 64 total) phages infected one or both strains and are represented in the heat map. (E) Plaque assays with indicated phages spotted in 10-fold serial dilutions on a lawn of Pa011 WT or ¨CBASS, or (F) on a lawn of PaCBASS or PaEV, to highlight CBASS-dependent targeting and CBASS-independent targeting (PB-1 and Ab22 phages). Plaque assays were used to quantify the order of magnitude reduction in phage titer by comparing the number of spots (with plaques, or clearing if plaques were not visible) on the CBASS+ strain divided by the CBASS- strain (n=3). (G) Plaque assays with the indicated phages spotted in 10-fold serial dilutions on a lawn of Pa011 WT or ¨CBASS, and Pa011 ¨CBASS over-expressing the CBASS operon or empty vector (E.V.). (H) Plaque assays on a lawn of Pa011 chromosomal mutants of each CBASS gene [capVS48A (phospholipase), cdnAD87A/D89A (cyclase), cap2C450A/C453A (E1/E2), and cap3E38A (JAB)]. For all plaque assays, clearings represent phage replication and black arrowheads highlight the reduction of PaMx41 WT phage titer. [0034] FIGS.8A-8F: Acb2 protects phage and reduces 3’,3’-cGAMP molecules in CBASS-containing cells. (A) Schematic of in vivo phage infection and cGAMP detection: (1) Pa011 cells with catalytically dead CapVS48A strain overexpressing a wildtype version of an anti-CBASS gene (Acb2 WT; inhibited CBASS) or mutant version (Acb2 K26A; uninhibited or active CBASS). These cells are infected with PaMx41 phage that lacks Acb2 (PaMx41¨acb2) at an MOI of ~5 for 60 minutes to provide the phage time to proceed through its replication cycle (Cruz-Plancarte et al., 2016). (2) Cell lysates are processed without (-) or with phenol-chloroform/chloroform nucleotide extraction. (3) Resultant lysates are filtered to collect cyclic oligonucleotides and then (4) 3’,3’-cGAMP is specifically measured by an ELISA. (B) 3’,3’-cGAMP detected in each respective condition performed in biological triplicate. Data are mean ± s.d. (C) Plaque assays were performed with PaMx33, 35, 41, and 43 WT phages, as well as an evolved PaMx41 CBASS escape (ESC) phage, spotted in 10-fold serial dilutions on a lawn of Pa011 WT [CBASS+] or ¨CBASS [CBASS-] over-expressing the indicated genes; black arrowhead highlights increase in PaMx41 WT phage titer. (D) Plaque assays were performed with the indicated phages spotted in 10-fold serial dilutions on a lawn of Pa011 WT, ¨CBASS, or WT over-expressing acb2. (E) Plaque assays with indicated phages on a lawn of P. aeruginosa cells (PAO1) with a chromosomally integrated Pa011 CBASS operon (PaCBASS), or empty vector (PaEV), and overexpressing acb2. Black arrowhead highlights CBASS-dependent change in phage titer. (F) Plaque assays with the indicated phages spotted in 10-fold serial dilutions on a lawn of PaEV [CBASS-], PaEV (JBD67 WT) lysogen, or PaEV (JBD67¨acb2) lysogen, or PaCBASS [CBASS+], PaCBASS (JBD67 WT) lysogen, or PaCBASS (JBD67¨acb2) lysogen. For all plaque assays, clearings represent phage replication. [0035] FIGS.9A and 9B: Acb2 is found in a broad diversity of phages and bacteria. Phylogenetic tree of acb2 across the genomes of 239 tailed phages (Caudovirales) following two iterations of PSI-BLAST. Phages families that are colored are the most frequently identified. Phages relevant to this study are bolded. (B) Multiple sequence alignment of Acb2, PaMx33, PaMx43, JDB67 Acb2, and other homologous proteins via MAFFT alignment in Jalview. All proteins except T2 and T4 phage homologs were acquired by standard BLASTp, while T2 and T4 were observed on a second round of a PSI- BLAST. Colors indicate an identical amino acid. Black boxes highlight residues Y11 and K26, which are necessary for Acb2 activity. [0036] FIGS.10A-10K: Acb2 does not bind CBASS proteins, but does bind 3’,3’-cGAMP, 2’,3’-cGAMP, and c-di-AMP. (A)-(D) Gel filtration profile of incubated Acb2 with Cap2- CdnA complex (A), Cap2 (B), CdnA (C) or CapV (D) (Superdex-200 increase 10/300 GL, GE Healthcare). (E) ITC assays to test binding of 3’,3’-cGAMP to Acb2 WT. (F) ITC assays to test binding of 2’,3’-cGAMP to Acb2. (G) ITC assays to test binding of c-di-AMP to Acb2 WT. (H) ITC assay to test binding of c-di-GMP to Acb2. (I), (J) ITC assay to test binding of 3’,3’-cGAMP to Acb2 Y11A and K26A, respectively. (K) Acb2 and its mutants were incubated with 3’,3’ƍ-cGAMP at indicated concentrations. Then the samples were subjected to native PAGE. [0037] FIGS.11A-11K: Structures of apo and di-nucleotide bound Acb2. (A) Analytical Ultracentrifugation (AUC) analysis of Acb2 and its complex with 3’,3’-cGAMP. (B) Structure of the Acb2 hexamer. (C) Structure of the Acb2 monomer. (D-E) Two types of dimer of protomers as shown in (B) are shown. (F)-(H), The ability of Acb2 to bind 2’,3’- cGAMP/c-di-AMP/c-di-GMP was analyzed by HPLC.2’,3’-cGAMP/c-di-AMP/c-di-GMP standards were used as a control. The remaining cyclic dinucleotides after incubation with Acb2 were tested. (I) Overall structure of Acb2 bound with c-di-AMP. (J)-(K) Structural comparison between an Acb2 dimer in c-di-AMP-bound form (colored in pink) and 3’,3’- cGAMP-bound form (colored in slate). [0038] FIGS.12A-12G: Acb2 binds to the predicted CdnE cyclic dinucleotide products and protects phages against Type I-A and Type I-B CBASS immunity that encode CdnE cyclase. (A) Isothermal titration calorimetry (ITC) assays to test binding of cyclic dinucleotides to Acb2. cUU, cUA, and cUG represent 3’,3’-cyclic-di-UMP, 3’,3’-cyclic-UMP-AMP, and 3’,3’-cyclic-UMP-GMP, respectively. Representative binding curves and binding affinities are shown. The KD values are mean ± s.d. (n=3). Raw data for these curves are shown in (B- D). (E) Native PAGE showed the binding of Acb2 to cyclic dinucleotides. (F) P. aeruginosa ATCC 33351 and JD332 CBASS operons with predicted cyclic dinucleotides (Whiteley et al. 2019). (G) Plaque assays with the indicated phages spotted in 10-fold serial dilutions on a lawn of P. aeruginosa cells (PAO1), which naturally lacks CBASS, over-expressing a CBASS operon or empty vector (E.V.); clearings represent phage replication. Black arrowhead highlights change in phage titer specific to phages lacking acb2. [0039] FIGS.13A-13E: PaMx41 phage remains sensitive to CBASS immunity in the presence of major capsid escape allele expression. (A) Plaque assays were performed with PaMx41¨acb2 phage, harboring a wildtype (WT) capsid, spotted in 10-fold serial dilutions on a lawn of Pa011 WT [CBASS+] or ¨CBASS [CBASS-] over-expressing the indicated genes; clearings represent phage replication. (B) Plaque assays were performed with PaMx41¨acb2 CBASS Escaper phage 7, harboring a mutant (I121T) capsid, spotted in 10- fold serial dilutions on a lawn of Pa011 WT or ¨CBASS. (C) Alphafold2 prediction of the PaMx41¨acb2 (blue) and JDB18 (green) major capsid protein monomer structures overlaid using PyMOL (RMSD: 4.194). Red spheres represent amino acid residues that are mutated and are labeled with the corresponding a.a. change. (D) Alphafold2 prediction of the PaMx41¨acb2 and (E) JDB18 capsid hexamer structures based on the experimentally solved E. coli T4 phage capsid structure (PDB: 6UZC). Spheres represent the a.a. residues that are mutated. Black and gray colors are indicative of mutations within a capsid monomer and highlight the inter- and intra-protein localization of the mutations. [0040] FIGS.14A-14F: Acb2 from phage PaMx33 binds cyclic trinucleotides and 3’, 2’- cGAMP. (A) ITC assays to test binding of cyclic nucleotides to PaMx33-Acb2. Representative binding curves and binding affinities are shown. The KD values are mean ± s.d. (n=3). (B) Overall structure of Acb2 complexed with 3’,2’-cGAMP, which are indicated by arrows. (C) Detailed binding between Acb2 and 3’,2’-cGAMP. Residues involved in 3’,2’-cGAMP binding are shown as sticks. Red dashed lines represent polar interactions. (D) Structural alignment among 3’,2’-cGAMP, 2’,3’-cGAMP, 3’,3’-cGAMP and c-di-AMP bound Acb2. Surface representation overlaid to cartoon representation, highlighting the binding pocket of CDNs. (E) Electrostatic surface model showing the binding pocket of CDNs. The CDNs are colored as in D. (F) 293T-Dual cells were transfected with hSTING and Acb2 or its mutants, then treated with 2’,3’-cGAMP. STING activation was read as luciferase signal controlled by an interferon promoter. A western blot is shown probing the expression of STING and Acb2. [0041] FIGS.15A-15I: Acb2 binds to cyclic trinucleotides with binding sites different from those of cyclic dinucleotides. (A) ITC assays to test the binding of cAAG and cA3 to PaMx33-Acb2, and binding of cA3 to PaMx33-Acb2 mutants. Representative binding curves and binding affinities are shown. The KD values are mean ± s.d (n = 3). The two mutants R67A and T74A in the panel represent their binding to cA3. (B) The ability of PaMx33-Acb2 to bind and release cA3 when treated with proteinase K was analyzed by HPLC. cA3 standard was used as a control. The remaining cA3 after incubation with PaMx33-Acb2 was tested. (C) Overall structure of Acb2 complexed with cAAG, which are shown as sticks. Two views are shown. (D) Electrostatic surface of Acb2 overlaid on the cartoon model shows the channel in the center of the Acb2 hexamer. (E) Electrostatic surface of Acb2 bound with cAAG. Two views are shown. (F) A closer view of the binding pocket shown in the left panel of D. (G) Detailed binding between Acb2 and cAAG. Residues involved in cAAG binding are shown as sticks. Red dashed lines represent polar interactions. (H-I) Native PAGE showed the binding of PaMx33 Acb2 mutants to cyclic oligonucleotides. [0042] FIGS.16A-16C: Acb2 binds to cyclic trinucleotides and dinucleotides simultaneously. (A) Overall structure of Acb2 complexed with cA3 and 3’,3’-cGAMP. cA3 and 3’,3’-cGAMP are shown as blue and light gray sticks. Two views are shown. (B) 2Fo-Fc electron density of cA3 and 3’,3’-cGAMP within an Acb2 dimer contoured at 1 ı. (C) Distribution of the small molecules within the Acb2 hexamer. The nucleotides are shown as they are in the right panel of (A). [0043] FIGS.17A-17H: The binding spectra are different among Acb2 homologs. (A) Sequence alignment among Acb2 homologs. Residues that are >80 % conserved, >60 % conserved and >40% conserved are shaded in dark purple, light purple, and light grey, respectively. Residues involved in binding of cyclic CDNs and CTNs are marked with green and blue triangles, respectively. (B) ITC assays to test binding of cyclic oligonucleotides to JBD67-Acb2. Representative binding curves and binding affinities are shown. The KD values are mean ± s.d. (n=3). (C) ITC assays to test binding of cyclic oligonucleotides to T4-Acb2. Representative binding curves and binding affinities are shown. The KD values are mean ± s.d. (n=3). (D) Structural alignment between PaMx33-Acb2 and T4-Acb2 at one monomer. Residues with potential steric clash with cA3 in T4-Acb2 and the corresponding residues in PaMx33-Acb2 are shown in sticks. (E) The same alignment shown in (D), highlighting the different relative angels formed by the three helices lining the binding pocket of cA3. (F-G) ITC assays to test binding of cyclic nucleotides to CHI14-Acb2. Representative binding curves and binding affinities are shown. The KD values are mean ± s.d. (n=3). (H) Summary of the binding results of Acb2 homologs and Acb1. [0044] FIGS.18A-18E: Acb2 antagonizes tri- and di-nucleotide based CBASS immunity. (A) Pseudomonas aeruginosa BWHPSA011 (Pa011) Type II-A CBASS and ATCC 27853 (Pa278) Type III-C CBASS operons. (B) Pseudomonas aeruginosa PaMx33 and JBD67 phages acb2 gene annotated with residues essential for CDN (3’,3’-cGAMP) binding and CTN (cA3) binding. (C) Effect of PaMx33 Acb2 or its mutants on cA3-activated NucC effector protein function. After treatment with proteinase K, the released cA3 also showed the ability to activate the nuclease activity of NucC. The concentration of NucC, cA3, Acb2 and proteinase K is 10 nM, 5 nM, 50 nM and 1 μM, respectively. N denotes nicked plasmid, SC denotes closed-circular supercoiled plasmid, and cut denotes fully digested DNA. (D) Plaque assays with JBD67¨acb2 phage spotted in 10-fold serial dilutions on PAO1 strains harboring an empty vector (E.V.) plasmid or JBD67 Acb2 variants. The PAO1 strains either contain no CBASS operon (-CBASS), a chromosomally integrated Pa011 CBASS operon (PAO1Pa011), or a chromosomally integrated Pa278 CBASS operon (PAO1Pa278). These plaque assays were used to quantify the order of magnitude change in phage titer by comparing the number of spots (with plaques, or clearings if plaques were not visible) on the PAO1Pa011 or PAO1Pa278 CBASS-expressing strains divided by the PAO1 (-CBASS) strain (n=3). Basal expression of the Pa011 CBASS operon and 0.3mM IPTG-inducible expression of the Pa278 CBASS operon is sufficient for phage targeting. Black arrowheads highlight significant CBASS- dependent reductions in phage titer. (E) Plaque assays with JBD67 phages spotted in 10-fold serial dilutions on PAO1 strains with and without CBASS. Pa278 CBASS was expressed from the pHERD30T (p30T) plasmid, while Pa011 CBASS was expressed from the chromosome. These plaque assays were used to quantify the order of magnitude change in phage titer (n=3). Basal expression (i.e. no arabinose added) of the Pa278 CBASS operon is sufficient for phage targeting. Black arrowheads highlight significant CBASS-dependent reductions in phage titer. DEFINITIONS [0045] The term “anti-CBASS protein” refers to a protein that can bind to one or more cyclic oligonycleotides to inhibit bacterial cyclic-oligonucleotide-based anti-phage signaling system (CBASS). In some embodiments, the anti-CBASS protein binds to cyclic oligonucleotides inside cells (e.g., inside bacterial and/or mammalian cells). In some embodiments, the anti-CBASS protein binds to cyclic oligonucleotides outside of cells, i.e., the anti-CBASS protein binds to extracellular cyclic oligonucleotides. [0046] The term “nucleic acid” or “polynucleotide” refers to deoxyribonucleic acids (DNA) or ribonucleic acids (RNA) and polymers thereof in either single- or double-stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogs of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed- base and/or deoxyinosine residues (Batzer et al., Nucleic Acid Res.19:5081 (1991); Ohtsuka et al., J. Biol. Chem.260:2605-2608 (1985); and Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)). [0047] A "promoter" is defined as an array of nucleic acid control sequences that direct transcription of a nucleic acid. As used herein, a promoter includes necessary nucleic acid sequences near the start site of transcription, such as, in the case of a polymerase II type promoter, a TATA element. A promoter also optionally includes distal enhancer or repressor elements, which can be located as much as several thousand base pairs from the start site of transcription. The promoter can be a heterologous promoter. [0048] An “expression cassette” is a nucleic acid construct, generated recombinantly or synthetically, with a series of specified nucleic acid elements that permit transcription of a particular polynucleotide sequence in a host cell. An expression cassette may be part of a plasmid, viral genome, or nucleic acid fragment. Typically, an expression cassette includes a polynucleotide to be transcribed, operably linked to a promoter. The promoter can be a heterologous promoter. In the context of promoters operably linked to a polynucleotide, a “heterologous promoter” refers to a promoter that would not be so operably linked to the same polynucleotide as found in a product of nature (e.g., in a wild-type organism). [0049] As used herein, the term "heterologous" refers to a protein or nucleic acid in a cell or an organism, or being introduced into a cell or an organism, where the protein or nucleic acid originates from a foreign species compared to the cell or the organism, or originates from the same species but is modified from its original form. For example, when a promoter is said to be operably linked to a heterologous coding sequence, it means that the coding sequence is derived from one species whereas the promoter sequence is derived from another, different species; or, if both are derived from the same species, the coding sequence is not naturally associated with the promoter (e.g., is a genetically engineered coding sequence). [0050] The terms “polypeptide,” “peptide,” and “protein” are used interchangeably herein to refer to a polymer of amino acid residues. All three terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymers. As used herein, the terms encompass amino acid chains of any length, including full-length proteins, wherein the amino acid residues are linked by covalent peptide bonds. [0051] The term “conservatively modified variants” applies to both amino acid and nucleic acid sequences. With respect to particular nucleic acid sequences, “conservatively modified variants” refers to those nucleic acids that encode identical or essentially identical amino acid sequences, or where the nucleic acid does not encode an amino acid sequence, to essentially identical sequences. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For instance, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, at every position where an alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide. Such nucleic acid variations are “silent variations,” which are one species of conservatively modified variations. Every nucleic acid sequence herein that encodes a polypeptide also describes every possible silent variation of the nucleic acid. One of skill will recognize that each codon in a nucleic acid (except AUG, which is ordinarily the only codon for methionine, and TGG, which is ordinarily the only codon for tryptophan) can be modified to yield a functionally identical molecule. Accordingly, each silent variation of a nucleic acid that encodes a polypeptide is implicit in each described sequence. [0052] As to amino acid sequences, one of skill will recognize that individual substitutions, deletions or additions to a nucleic acid, peptide, polypeptide, or protein sequence which alters, adds or deletes a single amino acid or a small percentage of amino acids in the encoded sequence is a “conservatively modified variant” where the alteration results in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. Such conservatively modified variants are in addition to and do not exclude polymorphic variants, interspecies homologs, and alleles of the invention. In some cases, conservatively modified variants of Cas9 or sgRNA can have an increased stability, assembly, or activity as described herein. [0053] The following eight groups each contain amino acids that are conservative substitutions for one another: 1) Alanine (A), Glycine (G); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W); 7) Serine (S), Threonine (T); and 8) Cysteine (C), Methionine (M) (see, e.g., Creighton, Proteins, W. H. Freeman and Co., N. Y. (1984)). [0054] Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, may be referred to by their commonly accepted single-letter codes. [0055] As used in herein, the terms “identical” or percent “identity,” in the context of describing two or more polynucleotide or amino acid sequences, refer to two or more sequences or specified subsequences that are the same. Two sequences that are “substantially identical” have at least 60% identity, preferably 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity, when compared and aligned for maximum correspondence over a comparison window, or designated region as measured using a sequence comparison algorithm or by manual alignment and visual inspection where a specific region is not designated. With regard to polynucleotide sequences, this definition also refers to the complement of a test sequence. With regard to amino acid sequences, in some cases, the identity exists over a region that is at least about 50 amino acids or nucleotides in length, or more preferably over a region that is 75-100 amino acids or nucleotides in length. [0056] For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters. For sequence comparison of nucleic acids and proteins, the BLAST 2.0 algorithm and the default parameters discussed below are used. [0057] A “comparison window”, as used herein, includes reference to a segment of any one of the number of contiguous positions selected from the group consisting of from 20 to 600, usually about 50 to about 200, more usually about 100 to about 150 in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned. [0058] An algorithm for determining percent sequence identity and sequence similarity is the BLAST 2.0 algorithm, which are described in Altschul et al., (1990) J. Mol. Biol.215: 403-410. Software for performing BLAST analyses is publicly available at the National Center for Biotechnology Information website. The algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al., supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always <0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative- scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a word size (W) of 28, an expectation (E) of 10, M=1, N=-2, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a word size (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989)). [0059] The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin & Altschul, Proc. Nat’l. Acad. Sci. USA 90:5873-5787 (1993)). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.2, more preferably less than about 0.01, and most preferably less than about 0.001. DETAILED DESCRIPTION OF THE INVENTION Introduction [0060] The present disclosure describes proteins that can bind to multiple cyclic oligonucleotides, for example, in a bacterial host, as a way to inhibit host immunity (such as cyclic-oligonucleotide-based anti-phage signaling system (CBASS) in bacteria). Such proteins, referred to as “anti-CBASS proteins” herein, can be used, for example, to engineer bacteriophages that are particularly effective in treating bacterial infections. In some embodiments, the anti-CBASS proteins can also bind to cyclic oligonucleotides outside of a cell. Anti-CBASS Protein [0061] An anti-CBASS protein described herein can have a sequence that is substantially (e.g., at least 60%, 70%, 80%, 90%, 95%, or 99%) identical to a sequence of SEQ ID NO:1 below: MDNQHKKIKGYRDLSQEEIDMMNRVKELGSQFEKLIQDVSDHLRGQYNASLHNRD EITRIANAEPGRWLAIGKTDIQTGMMAIIRAIAQPDSF (SEQ ID NO:1). [0062] An anti-CBASS protein can also have a sequence that is substantially (e.g., at least 60%, 70%, 80%, 90%, 95%, or 99%) identical to a sequence of SEQ ID NO:2 below: MIEDIKGYKPHTEEKIGKVNAIKDAEVRLGLIFDALYDEFWEALDNCEDCEFAKNYA ESLDQLTIAKTKLKEASMWACRAVFQPEEKY (SEQ ID NO:2). [0063] An anti-CBASS protein can also have a sequence that is substantially (e.g., at least 60%, 70%, 80%, 90%, 95%, or 99%) identical to a sequence of SEQ ID NO:3 below: MDNQHRKIAGYRELTQDDIDLMNRVKAVGAELLALQAALAGRLSTDLEVKQAAAK ASKLAPEHESSPECVELRRFLAAEPLRWAAIAKTDIQTGVMALVRAIAQPEGC (SEQ ID NO:3). [0064] In some embodiments, an anti-CBASS protein can further include other amino acid sequences or other chemical moieties (e.g., detectable labels) at the amino terminus, carboxyl terminus, or both. Additional amino acid sequences can include, but are not limited to, tags, detectable markers, or nuclear localization signal sequences. [0065] As described in the examples, an anti-CBASS protein described herein is demonstrated to bind to a number of cyclic oligonucleotides, e.g., 3’,3’cUU, 3’,3’cAA, 3’,3’cGAMP, 3’,3’cUG, 3’,3’cUA, 3’,3’,3’-cAAA, 3’,3’,3’-cAAG, 2’,3’cGAMP, and 3’,2’cGAMP. [0066] In particular embodiments, an anti-CBASS having a sequence that is at least substantially (e.g., at least 60%, 70%, 80%, 90%, 95%, or 99%) identical to, or comprises the sequence of SEQ ID NO:1, can bind to cyclic oligonucleotides such as 3’,3’cUU, 3’,3’cAA, 3’,3’cGAMP, 3’,3’cUG, 3’,3’cUA, 3’,3’,3’-cAAA, 3’,3’,3’-cAAG, 2’,3’cGAMP, and 3’,2’cGAMP. [0067] In particular embodiments, an anti-CBASS having a sequence that is at least substantially (e.g., at least 60%, 70%, 80%, 90%, 95%, or 99%) identical to, or comprises the sequence of SEQ ID NO:2, can bind to cyclic oligonucleotides such as 3’,3’cUU, 3’,3’cAA, 3’,3’cGAMP, 3’,3’cUG, 3’,3’cUA, 2’,3’cGAMP, and 3’,2’cGAMP. [0068] The disclosure also includes an engineered bacteriophage comprising an anti- CBASS protein described herein (e.g., SEQ ID NO:1, 2, or 3). For example, the engineered bacteriophage can contain an expression cassette comprising a nucleic acid (e.g., DNA or RNA) encoding an anti-CBASS protein (e.g., SEQ ID NO:1, 2, or 3) and a promoter operably linked to the nucleic acid, wherein the anti-CBASS protein comprises a sequence that is substantially (e.g., at least 60%, 70%, 80%, 90%, 95%, 99%) identical to a sequence of any one of SEQ ID NOS:1-3. The promoter can be heterologous to the nucleic acid encoding the anti-CBASS protein. The promoter can also be an inducible promoter. The disclosure also includes a pharmaceutical composition comprising an anti-CBASS protein described herein (e.g., SEQ ID NO:1, 2, or 3) or an engineered bacteriophage comprising an anti-CBASS protein described herein (e.g., SEQ ID NO:1, 2, or 3). [0069] The anti-CBASs proteins described herein can be be generated by any method. For example, in some embodiments the protein can be purified from naturally-occurring sources, synthesized, or more typically can be made by recombinant production in a cell engineered to produce the protein. Exemplary expression systems include various bacterial, yeast, insect, and mammalian expression systems. [0070] Also provided herein is an expression cassette comprising a nucleic acid (e.g., DNA or RNA) encoding an anti-CBASS protein (e.g., SEQ ID NO:1, 2, or 3) and a promoter operably linked to the nucleic acid, wherein the anti-CBASS protein comprises a sequence that is substantially (e.g., at least 60%, 70%, 80%, 90%, 95%, 99%) identical to a sequence of any one of SEQ ID NOS:1-3. The promoter can be heterologous to the nucleic acid encoding the anti-CBASS protein. The promoter can also be an inducible promoter. The disclosure also includes a vector (e.g., a viral vector) comprising the expression cassette as described herein. [0071] The anti-CBASS proteins as described herein can be fused to one or more fusion partners and/or heterologous amino acids to form a fusion protein. Fusion partner sequences can include, but are not limited to, amino acid tags, non-L (e.g., D-) amino acids or other amino acid mimetics to extend in vivo half-life and/or protease resistance, targeting sequences or other sequences. In some embodiments, functional variants or modified forms of the anti-CBASS proteins include fusion proteins of an anti-CBASS protein and one or more fusion domains. Exemplary fusion domains include, but are not limited to, polyhistidine, Glu-Glu, glutathione S transferase (GST), thioredoxin, protein A, protein G, an immunoglobulin heavy chain constant region (Fc), maltose binding protein (MBP), and/or human serum albumin (HSA). A fusion domain or a fragment thereof may be selected so as to confer a desired property. For example, some fusion domains are particularly useful for isolation of the fusion proteins by affinity chromatography. For the purpose of affinity purification, relevant matrices for affinity chromatography, such as glutathione-, amylase-, and nickel- or cobalt-conjugated resins are used. Many of such matrices are available in “kit” form, such as the Pharmacia GST purification system and the QLAexpress™ system (Qiagen) useful with (HIS6) fusion partners. As another example, a fusion domain may be selected so as to facilitate detection of the anti-CBASS proteins. Examples of such detection domains include the various fluorescent proteins (e.g., GFP) as well as “epitope tags,” which are usually short peptide sequences for which a specific antibody is available. Epitope tags for which specific monoclonal antibodies are readily available include FLAG, influenza virus haemagglutinin (HA), and c-myc tags. In some cases, the fusion domains have a protease cleavage site, such as for Factor Xa or Thrombin, which allows the relevant protease to partially digest the fusion proteins and thereby liberate the recombinant proteins therefrom. The liberated proteins can then be isolated from the fusion domain by subsequent chromatographic separation. In certain embodiments, an anti-CBASS protein is fused with a domain that stabilizes the anti-CBASS protein in vivo (a “stabilizer” domain). By “stabilizing” is meant anything that increases serum half-life, regardless of whether this is because of decreased destruction, decreased clearance by the kidney, or other pharmacokinetic effect. Fusions with the Fc portion of an immunoglobulin are known to confer desirable pharmacokinetic properties on a wide range of proteins. See, e.g., US Patent Publication No.2014/056879. Likewise, fusions to human serum albumin can confer desirable properties. Other types of fusion domains that may be selected include multimerizing (e.g., dimerizing, tetramerizing) domains and functional domains (that confer an additional biological function, as desired). Fusions may be constructed such that the heterologous peptide is fused at the amino and/or carboxyl terminus of an anti-CBASS protein. [0072] In some embodiments, the anti-CBASS proteins as described herein comprise at least one non-naturally encoded amino acid. In some embodiments, an anti-CBASS protein comprises 1, 2, 3, 4, or more unnatural amino acids. Methods of making and introducing a non-naturally-occurring amino acid into a protein are known. See, e.g., U.S. Pat. Nos. 7,083,970; and 7,524,647. The general principles for the production of orthogonal translation systems that are suitable for making proteins that comprise one or more desired unnatural amino acid are known in the art, as are the general methods for producing orthogonal translation systems. For example, see International Publication Numbers WO 2002/086075, WO 2002/085923, WO 2004/094593, and WO 2005/007624. For discussion of orthogonal translation systems that incorporate unnatural amino acids, and methods for their production and use, see also, Wang and Schultz, (2005) “Expanding the Genetic Code.” Angewandte Chemie Int Ed 44: 34-66; Xie and Schultz, (2005) “An Expanding Genetic Code.” Methods 36: 227-238; Xie and Schultz, (2005) “Adding Amino Acids to the Genetic Repertoire.” Curr Opinion in Chemical Biology 9: 548-554; and Wang, et al., (2006) “Expanding the Genetic Code.” Annu Rev Biophys Biomol Struct 35: 225-249; and International Publication No. W02006/034332. Additional details are found in U.S. Pat. Nos.7,045,337; 7,083,970; 7,199,222; and 7,217,809. [0073] A non-naturally encoded amino acid is typically any structure having any substituent side chain other than one used in the twenty natural amino acids. Because non- naturally encoded amino acids typically differ from the natural amino acids only in the structure of the side chain, the non-naturally encoded amino acids form amide bonds with other amino acids, including but not limited to, natural or non-naturally encoded, in the same manner in which they are formed in naturally occurring polypeptides. However, the non- naturally encoded amino acids have side chain groups that distinguish them from the natural amino acids. For example, R optionally comprises an alkyl-, aryl-, acyl-, keto-, azido-, hydroxyl-, hydrazine, cyano-, halo-, hydrazide, alkenyl, alkynl, ether, thiol, seleno-, sulfonyl- , borate, boronate, phospho, phosphono, phosphine, heterocyclic, enone, imine, aldehyde, ester, thioacid, hydroxylamine, amino group, or the like or any combination thereof. Other non-naturally occurring amino acids of interest that may be suitable for use include, but are not limited to, amino acids comprising a photoactivatable cross-linker, spin-labeled amino acids, fluorescent amino acids, metal binding amino acids, metal-containing amino acids, radioactive amino acids, amino acids with novel functional groups, amino acids that covalently or noncovalently interact with other molecules, photocaged and/or photoisomerizable amino acids, amino acids comprising biotin or a biotin analog, glycosylated amino acids such as a sugar substituted serine, other carbohydrate modified amino acids, keto-containing amino acids, amino acids comprising polyethylene glycol or polyether, heavy atom substituted amino acids, chemically cleavable and/or photocleavable amino acids, amino acids with an elongated side chains as compared to natural amino acids, including but not limited to, polyethers or long chain hydrocarbons, including but not limited to, greater than about 5 or greater than about 10 carbons, carbon-linked sugar-containing amino acids, redox-active amino acids, amino thioacid containing amino acids, and amino acids comprising one or more toxic moiety. [0074] Another type of modification that can optionally be introduced into the anti-CBASS proteins (e.g., within the polypeptide chain or at either the N- or C-terminus), e.g., to extend in vivo half-life, is PEGylation or incorporation of long-chain polyethylene glycol polymers (PEG). Introduction of PEG or long chain polymers of PEG increases the effective molecular weight of the present polypeptides, for example, to prevent rapid filtration into the urine. In some embodiments, a Lysine residue in a protein can be conjugated to PEG directly or through a linker. Such linker can be, for example, a Glu residue or an acyl residue containing a thiol functional group for linkage to the appropriately modified PEG chain. An alternative method for introducing a PEG chain is to first introduce a Cys residue at the C-terminus or at solvent exposed residues such as replacements for Arg or Lys residues. This Cys residue is then site-specifically attached to a PEG chain containing, for example, a maleimide function. Methods for incorporating PEG or long chain polymers of PEG can include, for example, those described in Veronese, F. M., et al., Drug Disc. Today 10: 1451-8 (2005); Greenwald, R. B., et al., Adv. Drug Deliv. Rev.55: 217-50 (2003); Roberts, M. J., et al., Adv. Drug Deliv. Rev., 54: 459-76 (2002)), the contents of which are incorporated herein by reference. [0075] Another alternative approach for incorporating PEG or PEG polymers through incorporation of non-natural amino acids (e.g., as described above) can be performed with the present anti-CBASS proteins. This approach utilizes an evolved tRNA/tRNA synthetase pair and is coded in the expression plasmid by the amber suppressor codon (Deiters, A, et al. (2004). Bio-org. Med. Chem. Lett.14, 5743-5). For example, p-azidophenylalanine can be incorporated into the present polypeptides and then reacted with a PEG polymer having an acetylene moiety in the presence of a reducing agent and copper ions to facilitate an organic reaction known as “Huisgen [3+2]cycloaddition.” [0076] In certain embodiments, specific mutations of anti-CBASS proteins can be made to alter the glycosylation of the protein, if needed. Such mutations may be selected to introduce or eliminate one or more glycosylation sites, including but not limited to, O-linked or N- linked glycosylation sites as recognized by eukaryotic expression systems. Methods [0077] As discussed herein, the present inventors have discovered a protein that can bind to a number of cyclic oligonucleotides in order to inhibit CBASS and evade bacterial immune system. The disclosure also provides methods of killing bacteria by contacting an anti- CBASS protein to one or more cyclic oligonucleotides. In some embodiments, the anti- CBASS protein binds to cyclic oligonucleotides that are present inside bacterial cells. In some embodiments, the anti-CBASS protein binds to cyclic oligonucleotides that are present inside mammalian cells. In some embodiments, the anti-CBASS protein binds to cyclic oligonucleotides that are present inside mammalian cells that have been infected with bacteria. In some embodiments, the anti-CBASS protein binds to cyclic oligonucleotides that are present outside of cells, i.e., the anti-CBASS protein binds to extracellular cyclic oligonucleotides. The anti-CBASS protein can have a sequence that is substantially (e.g., at least 60%, 70%, 80%, 90%, 95%, 99%) identical to a sequence of any one of SEQ ID NOS:1- 3. In some embodiments, the anti-CBASS protein has a sequence that is 95%, 97%, 99%, or 100% identical to the sequence of SEQ ID NO:1. In some embodiments, the anti-CBASS protein has a sequence that is 95%, 97%, 99%, or 100% identical to the sequence of SEQ ID NO:2. In some embodiments, the anti-CBASS protein has a sequence that is 95%, 97%, 99%, or 100% identical to the sequence of SEQ ID NO:3. [0078] In some embodiments, contacting the anti-CBASS protein to one or more cyclic oligonucleotides can occur in a cell, such as an eukaryotic cell (e.g., an eukaryotic cell (e.g., a mammalian cell) infected with bacteria). In certain embodiments, the cell can be a human cell (e.g., a human cell infected with bacteria). In other embodiments, the cell can be a prokaryotic cell (e.g., a bacterial cell). In some embodiments, the contacting can occur in vitro, in vivo, or ex vivo. [0079] In some embodiments, contacting the anti-CBASS protein to one or more cyclic oligonucleotides can comprise introducing the anti-CBASS protein into the cell. In certain embodiments, the anti-CBASS protein can be introduced into the cell as an isolated protein or as a polynucleotide or expression cassette comprising a nucleic acid encoding an anti-CBASS protein described herein in which the expression of the anti-CBASS protein can be induced inside the cell. In other embodiments, the anti-CBASS protein can be introduced into the cell by a bacteriophage comprising the anti-CBASs protein. [0080] In some embodiments, an anti-CBASS protein described herein can be introduced into the cell by an expression cassette comprising a nucleic acid encoding the anti-CBASS protein and a promoter operably linked to the nucleic acid. The promoter can be an inducible promoter. [0081] In some embodiments, an anti-CBASS protein described herein can be introduced into the cell by administering an engineered bacteriophage comprising an anti-CBASS protein described herein. [0082] In some embodiments, the cyclic oligonucleotides are already present inside the cell prior to the anti-CBASS protein is introduced into the cell. In other embodiments, the cyclic oligonucleotides can additionally be introduced into the cell before, during, or after the the anti-CBASS protein is introduced to the cell. [0083] The methods of killing bacteria as described herein can also be performed ex vivo, in which cells infected by bacteria from a subject can be isolated from the subject, and the anti-CBASS protein can be introduced into the cells to kill the bacteria. The isolated cells containing the anti-CBASS protein can then be introduced back into the subject. [0084] The disclosure also provides methods of treating a bacterial infection in a subject by administering to the subject an anti-CBASS protein substantially (e.g., at least 60%, 70%, 80%, 90%, 95%, 99%) identical to a sequence of any one of SEQ ID NOS:1-3, or an engineered bacteriophage comprising thereof, wherein the anti-CBASS protein binds to one or more cyclic oligonucleotides in the bacteria. Examples of bacterial infections include, but are not limited to, infections caused by bacteria in the genus Pseudomonas (e.g., Pseudomonas aeruginosa), infections caused by bacteria in the genus Acinetobacter (e.g., Acinetobacter baumanii), infections caused by bacteria in the genus Klebsiella, infections caused by bacteria in the genus Yersinia, infections caused by bacteria in the genus Enterobacter, infections caused by bacteria in the genus Streptococcus (e.g., Streptococcus pyogenes), infections caused by bacteria in the genus Escherichia (e.g., Escherichia coli), infections caused by bacteria in the genus Vibrio (e.g., Vibrio cholerae), and infections caused by bacteria in the genus Salmonella (e.g., Salmonella typhi). [0085] In the methods of killing bacteria and/or treating a bacterial infection as described herein, an anti-CBASS protein (e.g., SEQ ID NO:1, 2, or 3) can bind to a cyclic oligonucleotide that can be a cyclic dinucleotide or a cyclic trinucleotide. In some embodiments, an anti-CBASS protein (e.g., SEQ ID NO:1, 2, or 3) can bind to a cyclic oligonucleotide selected from the group consist of 3’,3’cUU, 3’,3’cAA, 3’,3’cGAMP, 3’,3’cUG, 3’,3’cUA, 3’,3’,3’-cAAA, 3’,3’,3’-cAAG, 2’,3’cGAMP, and 3’,2’cGAMP. [0086] In particular embodiments, an anti-CBASS protein having a sequence that is at least substantially (e.g., at least 60%, 70%, 80%, 90%, 95%, or 99%) identical to, or comprises the sequence of SEQ ID NO:1, can be used in methods of killing bacteria and/or treating a bacterial infection as described herein by binding to cyclic oligonucleotides such as 3’,3’cUU, 3’,3’cAA, 3’,3’cGAMP, 3’,3’cUG, 3’,3’cUA, 3’,3’,3’-cAAA, 3’,3’,3’-cAAG, 2’,3’cGAMP, and 3’,2’cGAMP. [0087] In particular embodiments, an anti-CBASS protein having a sequence that is at least substantially (e.g., at least 60%, 70%, 80%, 90%, 95%, or 99%) identical to, or comprises the sequence of SEQ ID NO:2, can be used in methods of killing bacteria and/or treating a bacterial infection as described herein by binding to cyclic oligonucleotides such as 3’,3’cUU, 3’,3’cAA, 3’,3’cGAMP, 3’,3’cUG, 3’,3’cUA, 2’,3’cGAMP, and 3’,2’cGAMP. EXAMPLES Example 1 [0088] We identified a P. aeruginosa strain that harbors Type II-A CBASS (3’,3’-cGAMP producing CD-NTase; CdnA) with a phospholipase (CapV) effector that limits phage replication by >10,000-fold. This is the first instance where CBASS anti-phage immunity has been shown to function naturally without heterologous overexpression. We next identified a widespread phage protein (Acb2) that forms a hexamer complex with three 3’,3-cGAMP molecules, acting as a “sponge” to reduce the available molecules to activate the phospholipase effector. In addition, Acb2 binds to multiple other cyclic dinucleotides, including 3’,3’-c-di-UMP, 3’,3’-cUA, and 3’,3’-cUG, and is necessary for optimal phage replication in the presence of Type I or II CBASS that are predicted to encode the aforementioned cyclic dinucleotides. Phages with acb2 deleted were unable to replicate in the lytic cycle or during exit from lysogeny in the presence of CBASS. However, mutations in the major capsid gene enabled phages to escape CBASS. This work provides direct evidence of phage inhibition and evasion of CBASS, demonstrating a robust arms race between the two. Results Endogenous anti-phage CBASS function in Pseudomonas aeruginosa [0089] Previous analyses13, coupled with our own bioinformatics, revealed that 252 distinct P. aeruginosa strains have >300 CBASS operons (FIGS.7A and 7B). These systems span Type I-III and use numerous effector proteins and cyclic oligonucleotides7. The diverse CBASS types in P. aeruginosa suggest that it is important for host fitness and that it may be well suited to study phage-CBASS interactions. To identify naturally functional CBASS immunity in P. aeruginosa, CBASS loci were deleted from the genome of four strains possessing representatives of the common CBASS types (Type I-A, II-A, II-C, and III-C; FIG.7C) using a CRISPR-Cas3 tool19. Notably, these strains also encode numerous other anti-phage immune systems (FIG.1A). Therefore, due to the multitude of immune systems, we screened the CBASS mutants against a diverse panel of ~70 phages, which spanned 23 different genomic families and four different morphologies (Myoviridae, Siphoviridae, Podoviridae, and Inoviridae20). A single P. aeruginosa strain (BWHPSA011; Pa011) was identified with CBASS-dependent anti-phage activity (FIGS.7D and 7E). [0090] Deletion of the Pa011 Type II-A CBASS operon (¨CBASS, FIG.1B) resulted in >4 orders of magnitude increase in titer of the dsDNA podophage PaMx41 (FIGS.1C and 1D). Phage protection was restored when all four CBASS genes (capV (phospholipase effector), cdnA (CD-NTase), cap2 (E1/E2 ubiquitin-ligase-like domains), and cap3 (JAB de- ubiquitinating enzyme-like domain)) were complemented on a plasmid (FIG.7G). To determine which genes are necessary for CBASS anti-phage activity, chromosomal mutants known to disrupt catalytic activity8,15 were generated: capVS48A, cdnAD87A/D89A, cap2C450A/C453A, and cap3E38A. capV, cdnA, and cap2 mutations abolished anti-phage activity whereas the cap3 mutation did not (FIG.7H). Furthermore, using an ELISA, we observed that PaMx41 infection generated low levels of 3’,3’-cGAMP (~8 nM; FIGS.8A and 8B) whereas the molecule was nearly undetectable in CdnA mutant strain (~0.5 nM; L.O.D.0.24 nM). These data demonstrate that Pa011 CBASS-based immunity is naturally active, significantly limits phage replication, and requires CapV, CdnA, and Cap2 enzyme activities for phage targeting. PaMx41-like phages encode a CBASS antagonist [0091] How CBASS detects and targets phage is currently unknown. Therefore, to identify phage genes required for successful CBASS activity, we isolated PaMx41 mutants that resist Pa011 CBASS-based immunity. With a frequency of 3.7 x 10-5 (FIG.1D), 10 independent PaMx41 CBASS “escape” phages were isolated that replicate well on Pa011 WT (FIG.1B). Whole genome sequencing revealed one mutation in all CBASS escape phages: a no-stop extension mutation (X37Q) in orf24 (FIG.1E). X37Q lengthens gp24 from a 37 amino acid (a.a.) protein to 94 a.a. Interestingly, the naturally CBASS resistant phages, PaMx33, PaMx35, and PaMx43, share >96% nucleotide identity across the genome and naturally encode the 94 a.a. version with >98% a.a. identity. [0092] To determine whether the short gp24 activates CBASS or the long gp24 antagonizes it, the PaMx41 gp24 variants were overexpressed in Pa011 WT and ¨CBASS cells and then plaque assays were performed. In the presence of CBASS, the long gp24 increased the titer of the PaMx41 WT phage by >4 orders of magnitude while the truncated gp24 versions had no effect (FIG.1F). In contrast, the PaMx41 escaper phage and PaMx33, PaMx35, and PaMx43 phages exhibited high titer on all strains (FIG.8C), demonstrating that the short gene is not a dominant CBASS activator. We next deleted orf24 from all of the resistant PaMx41-like phages using a Cas13a selection tool because these phages were surprisingly resistant to all tested DNA-targeting CRISPR-Cas systems21. In Pa011 WT cells, the titer of the ¨orf24 phages was reduced 2-4 orders of magnitude; however, expression of the long gp24 in trans rescued the phages (FIGS.2A and 2B, FIG.8D). Taken together, these results indicate that the long gp24, or Acb2 (anti-cbass 2; PaMx33 NCBI: ANA48877) hereafter, inhibits Pa011 CBASS immunity and is necessary for phage replication in the presence of CBASS. Acb2 has conserved function in a broadly distributed temperate phage family [0093] Homology searches with Acb2 revealed that it is encoded in a striking number of tailed phages, including those infecting Pseudomonas, Vibrio, Acinetobacter, Salmonella, Serratia, Erwinia, and Escherichia sp. (T2 and T4 phages; gene: vs.4), among others (FIG. 9A). We observed no other examples of the truncated Acb2 variant encoded in PaMx41 WT phage. A multi-sequence alignment with diverse homologs revealed highly conserved N- and C-termini with a middle region of varied length and sequence (FIG.9B), but no molecular function could be predicted. Furthermore, acb2 is commonly encoded by P. aeruginosa B3- like temperate phages (e.g. JBD67), which are unrelated to the PaMx41-like lytic phages. Since JBD67 phage does not replicate on the Pa011 strains, we integrated the Type II-A CBASS operon with its native promoter into the chromosome of a P. aeruginosa strain (POA1) that is sensitive to this phage and naturally lacks CBASS. This engineered strain (PaCBASS) was active and reduced PaMx41 WT phage titer by 3-4 orders of magnitude (FIGS. 7D and 7F). By contrast, JBD67 exhibited resistance to the PaCBASS strain while a related phage that naturally lacks the acb2 gene, JBD18, was robustly inhibited (FIGS.2C and 2D, FIG.8E). Deletion of acb2 from JBD67 using a helicase attenuated Cascade-Cas3 system (see Methods) sensitized it to CBASS immunity (FIGS.2C and 2D, FIG.8E). The titer of JBD67¨acb2 and JBD18 phages were reduced by >5 orders of magnitude in the presence of CBASS. Expression of acb2 derived from either JBD67 or PaMx41-orf24X37Q on a plasmid fully restored the titer of the PaMx41¨acb2 phage, and only partially restored the titer of the JBD67¨acb2 and JBD18 phages (FIG.2C, FIG.8E). We hypothesize that JBD67 and JBD18 more strongly activate CBASS, and consequently, Acb2 expression in trans becomes partially overwhelmed. Together, these results collectively demonstrate that acb2 retains its anti-CBASS function across distinct phage families. [0094] Given that JBD67 is a temperate phage, we investigated whether acb2 is active during lysogeny as a prophage and therefore inhibits a co-encoded CBASS system. Lysogens were constructed with JBD67 in the PaCBASS strain, where CBASS targets the super-infecting phage D3. JBD67 and D3 are from hetero-immune groups, so there is no super-infection exclusion between these phages. Interestingly, the degree to which CBASS targets D3 was not impacted by the presence of acb2 in the JBD67 prophage (FIG.2E, FIG.8F). This suggests that acb2 is not expressed during lysogeny and allows functional CBASS immunity despite a prophage-encoded inhibitor. However, upon exit from lysogeny, CBASS significantly blocks JBD67¨acb2 prophage induction, reducing the induced titer by ~5-orders of magnitude whereas JBD67 WT prophage induction was unaffected (FIG.8F). These collective findings demonstrate that acb2 is active during lysogenic induction, but not lysogenic maintenance, and importantly shows for the first time that CBASS can dramatically limit prophage induction. Acb2 sequesters 3’,3’-cGAMP to inhibit CBASS [0095] To determine the mechanism of Acb2, we purified Acb2 from the naturally CBASS-resistant PaMx33 phage and each CBASS protein to test direct CBASS antagonism. First, we tested binding and found that Acb2 did not bind to purified CapV, CdnA, Cap2, nor the CdnA-Cap2 complex, which was purified as described previously (Ledvina et al., 2022; FIGS.10A-10D). Next, we reconstituted CapV phospholipase activity in vitro and confirmed that it is only activated by 3ƍ,3ƍ-cGAMP in a concentration-dependent manner, but not by 2ƍ,3ƍ-cGAMP, c-di-AMP, or c-di-GMP (FIG.3A). When Acb2 was preincubated with 3ƍ,3ƍ- cGAMP, CapV activity was abrogated (FIG.3A), suggesting that Acb2 may directly bind to the 3ƍ,3ƍ-cGAMP molecule. A native gel assay showed a significant shift of the purified Acb2 protein upon adding 3ƍ,3ƍ-cGAMP (FIG.3B). Isothermal calorimetry (ITC) experiments further verified that Acb2 directly binds to 3ƍ,3ƍ-cGAMP with a KD of ~87 nM (FIG.3C, FIG. 10E). Together, these data suggest that Acb2 binding of 3ƍ,3ƍ-cGAMP antagonizes CBASS by reducing available signaling molecules. [0096] Recent studies have reported that eukaryotic18 and prokaryotic17,22 viruses express proteins that directly degrade cyclic oligonucleotides. Therefore, to determine whether Acb2 is an enzyme that cleaves 3’,3’-cGAMP molecules, we performed the CapV phospholipase activity assay using a series of Acb2 concentrations and Acb2-3ƍ,3ƍ-cGAMP incubation times. Inhibition of CapV activity was concentration-dependent, but not time-dependent (FIG.3D), suggesting that Acb2 is a protein that “sponges” and sequesters 3’,3’-cGAMP rather than degrade it. High-performance liquid chromatography (HPLC) clearly showed that incubation of Acb2 depletes detectable 3ƍ,3ƍ-cGAMP, and following proteolysis of Acb2, the molecule is released back into the buffer (FIG.3E). Filtration of the unbound 3’,3’-cGAMP enabled CapV to regain its enzymatic activity, demonstrating that the molecule is still active (FIG. 3A). Consistent with these results, overexpression of Acb2 in phage-infected Pa011 WT cells reduced detectable 3’,3’-cGAMP levels, but following phenol-chloroform/chloroform nucleotide extraction, the molecule was released and levels increased ~4-fold (FIGS.8A and 8B). Altogether, these results demonstrate that Acb2 antagonizes Type II-A CBASS immunity via binding and sequestering the 3’3’-cGAMP dinucleotide, which prevents phospholipase effector activation. Crystal structure of Acb2 and its complex with 3ƍ,3ƍ-cGAMP [0097] To further understand how Acb2 interacts with 3’,3’-cGAMP, we determined the crystal structures of apo Acb2 and its complex with the signaling molecule (Table S1). Interestingly, Acb2 folds as a homo-hexamer in its apo (FIG.4A) and 3’,3’-cGAMP bound form (FIG.4B). Analytical ultracentrifugation (AUC) assay also indicated that Acb2 is a hexamer in its apo and 3’,3’-cGAMP-bound forms in solution (FIGS.11A-11E). Each protomer mainly interacts with two adjacent protomers (FIG.11B), allowing the six protomers to interlock into a compact assembly. An Acb2 protomer consists of one short N- terminal helix and two long anti-parallel helices, with a kink in the long helix at the C- terminus (FIG.11C). The ligand-bound structure showed that one Acb2 hexamer binds three 3ƍ,3ƍ-cGAMP molecules (FIG.4B). Each cGAMP binding pocket is formed by two Acb2 protomers that interact in a head-to-head manner, and is mainly composed of N- and C- terminal helices/loops from each protomer (FIG.11D). This is consistent with the Acb2 multiple sequence alignment, which revealed highly conserved N- and C-termini (FIG.9B). Searches using the Dali (distance-matrix alignment) server did not return entries with the same protein fold as Acb223, indicating that Acb2 adopts an overall novel cyclic dinucleotide binding fold. In support of these findings, a recent preprint 16 demonstrated that the Acb2 homolog from E. coli phage T4 (gene vs.4) tightly binds to 3’,3’-cGAMP (KD of ~30 nM) and adopts a hexameric structure similar to the one reported here. [0098] Acb2 binding of a cyclic dinucleotide ligand only causes a slight movement of the loop linking Į1 and Į2 (named loop L12) towards the ligand (FIG.4D), but several other residues display rotations and movements upon ligand binding. Among them, the most dramatic movement happens in Y11 from both protomers, which rotates and forms ʌ-ʌ interactions with the purine bases of cGAMP, as well as hydrogen bonds with the phosphate group of cGAMP (FIG.4E). In addition, K26 in Į2 of both protomers forms a salt bridge with the phosphate group of the cGAMP, possibly stabilizing the molecule together with Y11 (FIG.4F). Additionally, cGAMP is stabilized through hydrophobic interactions by several residues from both protomers, such as L14, M22, M81, I84 and P90 (FIG.4F). Consistent with these analyses, Acb2 Y11A and K26A mutants were inactive in plaque assays (FIG.4I) and the Acb2 K26A mutant expressed in the Pa011 WT strain lost its ability to sequester 3’,3’-cGAMP in vivo (FIGS.8A and 8B). Furthermore, Acb2 Y11A and K26A mutants abolished Acb2 binding of 3’,3’-cGAMP in vitro (FIG.4G, FIGS.10I-10K) and abrogated its ability to reduce CapV activity (FIG.4H). These data collectively show that Acb2 proteins form a complex with cyclic dinucleotides and subsequently reduces downstream CBASS activation. Acb2 sequesters variety of cyclic dinucleotides [0099] The binding mode of 3ƍ,3ƍ-cGAMP within Acb2 implied that other cyclic dinucleotides could also be sequestered by this protein. Indeed, native gels and ITC experiments demonstrated that Acb2 also binds to 2ƍ,3ƍ-cGAMP and c-di-AMP with high affinity (KD of ~103 and 66 nM, respectively), but not c-di-GMP (FIGS.3B and 3C, FIGS. 10F and 10G). HPLC assays confirmed that Acb2 could also sequester c-di-AMP and 2ƍ,3ƍ- cGAMP, and showed a very weak sequestering effect on c-di-GMP (FIGS.11F-11H). The Acb2-c-di-AMP structure was also solved (Table S1) and c-di-AMP showed a similar binding mode as 3ƍ,3ƍ-cGAMP (FIGS 11I-11K). Additional native gels and ITC experiments showed that Acb2 can bind to 3’,3’-c-di-UMP (cUU) and 3’,3’-c-UMP-AMP (cUA) with high affinity (KD of ~99 and 97 nM, respectively), and binds to 3’,3’-c-UMP-GMP (cUG) with lower affinity (KD of ~524 nM) (FIGS.12A-12E). Next, we tested whether phages expressing acb2 could inhibit CBASS subtypes in vivo that harbor a CdnE cyclase, which are predicted to generate cUU, cUA, and/or cUG7. In our strain collection, we identified that P. aeruginosa strains ATCC 33351 and JD332 encode Type I-A and Type I-B CBASS operons, respectively, with a CdnE cyclase (FIG.12F). When expressed in a P. aeruginosa strain (PAO1 WT) that naturally lacks CBASS, the titer of phages lacking acb2 was reduced 1-3 orders of magnitude – most notably by JD332 Type I-B CBASS – and the isogenic phages containing acb2 remained resistant to CBASS (FIG.12G). Together, these in vitro and in vivo results suggest that Acb2 can broadly inhibit CBASS types that utilize cyclic dinucleotides with uracil and adenine bases. Phages escape CBASS via mutations in the capsid gene [0100] The function and mechanism of acb2 demonstrates that CBASS places strong evolutionary pressure on phage. Therefore, we sought to determine whether any alternative mechanisms to evade CBASS exist. We again attempted to identify mutant escape phages, but this time, we used phage lacking acb2 (PaMx41¨acb2). Phages were first exposed directly to CBASS by plating on Pa011 WT, which yielded no escape plaques. However, the invisible phage population on the plate was collected, amplified in the Pa011 ¨CBASS strain, and ultimately escape phages were isolated under CBASS selection (FIG.5A). Whole genome sequencing revealed that all seven escape phages had one of four different missense point mutations in the major capsid gene (orf11, NCBI ID: YP_010088887.1; FIG.5B, Table S2). To confirm the causality of the capsid mutations for CBASS escape, we used plasmid- based recombination to introduce de novo mutations I121A, I121T, and S330P into a naive PaMx41¨acb2 phage and confirmed that these mutations induced CBASS escape (FIGS.5C and 5D). To test whether the major capsid is sufficient to trigger CBASS, the PaMx41 WT or mutant major capsid gene with its native promoter was cloned and expressed in Pa011 WT and ¨CBASS cells. In the presence of the WT major capsid, we did not observe any CBASS- dependent cellular toxicity, indicating that capsid monomer is not sufficient to activate CBASS. We also did not observe CBASS-dependent targeting of resistant phage (FIG.13A). Likewise, expression of the mutant major capsid gene did not induce escape of CBASS sensitive phage (FIG.13B). We anticipate that the plasmid-encoded major capsid gene does not express well enough to sufficiently displace the phage-expressed major capsid gene in a manner that impacts the CBASS phenotype. [0101] CBASS escape phages were also isolated from JBD67¨acb2 and JBD18 phages on the PaCBASS strain (FIG.5A). Strikingly, whole genome sequencing revealed missense point mutations in the genes encoding their major capsid proteins [orf32 in JBD67 (NCBI: YP_009625956) and orf35 in JBD18 (NCBI: AFR52188); FIG.5B, Table S2]. The PaMx41 major capsid protein shares no significant amino acid identity with the JBD67 and JBD18 major capsid proteins, yet the mutations all converge on the same protein. When modeling the location of the mutations on the predicted major capsid monomer structures, we did not observe overlap between the distinct phages (FIG.13C). However, modeling of the predicted major capsid hexamer structures indicated that the mutations lie on the protein-protein interface within or between hexamers (FIGS.13D and 13E), suggesting that a higher ordered capsid structure or process may be implicated in CBASS immunity. Interestingly, some of the observed capsid mutant genotypes (i.e. I121S/T from PaMx41, N191H from JBD67, and N275D from JBD18) are common natural alleles in the capsids of other phages, suggesting that these genotypes are fit. Follow-up studies are needed to determine the mechanistic connection between the major capsid protein and CBASS. Discussion [0102] The discovery and characterization of CBASS marked an exciting connection between prokaryotic and eukaryotic immunity7,8. However, many questions remain in this nascent field regarding phage-host evolution, as well as CBASS regulation and activation mechanisms, that can be best addressed through utilization of endogenous CBASS model systems. We found that the P. aeruginosa BWHPSA011 (Pa011) strain harbors a cGAS-like enzyme (CdnA) that produces 3’,3’-cGAMP dinucleotides in response to PaMx41 phage infection, which activates the phospholipase (CapV) effector. Phages related to PaMx41 (or an escape mutant derived from PaMx41) produce an anti-CBASS protein (Acb2) that is expressed as a middle gene in the phage replication cycle24 and sequesters 3’,3’-cGAMP. Acb2 likely accumulates prior to the production of the dinucleotide given that previous work demonstrated 3’,3’-cGAMP levels increase later in the E. coli phage P1 replication cycle8. Furthermore, when studying the temperate phage JBD67 with its acb2 gene removed, we found that CBASS limits prophage induction and lytic phage replication, which had not been previously noted. [0103] The cyclic dinucleotide “sponge” protein discovered in this study, Acb2, joins an expanding repertoire of phage-encoded antagonists that directly act on signaling molecules involved in bacterial anti-phage immunity. Previous examples include the anti-CRISPR (AcrIII1) protein that cleaves c-A4 molecules22, and the anti-Pyscar (Apyc1) protein that cleaves a spectrum of cyclic nucleotides17. In parallel, a Thoeris anti-defense (Tad1) protein was found to specifically bind to and sequester gcADPR molecules25. Additionally, the first anti-CBASS gene identified (Acb1) is another class of phage enzymes, similar to Apyc1, that harbors a phosphodiesterase fold that specifically binds and cleaves cyclic nucleotides17. This is a common inhibitory mechanism of the eukaryotic cGAS-STING signaling system and is utilized by poxviruses18 and a host pyrophosphatase/phosphodiesterase protein26, which enzymatically cleaves and depletes 2’,3’-cGAMP. By contrast, Acb2 binds to and sequesters bacterial 3’,3’-cGAMP, c-di-AMP, 3’,3’-c-di-UMP, 3’,3’-cUA, 3’,3’-cUG, and human 2’,3’- cGAMP. The structure and mechanism of Acb2 were independently confirmed with a homolog from E. coli phage T4 in a recent preprint16. Despite the numerous different mechanisms employed by viruses to inhibit the eukaryotic cGAS-STING signaling system27, the cGAMP “sponge” mechanism is an entirely new inhibitory strategy. [0104] The identification of capsid mutants that escape CBASS mirrors the recently identified major capsid mutations in E. coli phage T5, which enables escape from Pycsar (pyrimidine cyclase system for anti-phage resistance)28. This study also noted that expression of the T5 WT major capsid protein alone did not induce Pyscar-mediated toxicity, which we similarly observed, nor was direct binding observed between the T5 major capsid protein and any of the Pyscar proteins28, suggesting a more complex or indirect activation mechanism. However, abortive immune systems can be activated through direct binding to a capsid monomer, such as the CapRelSJ46 Toxin-Antitoxin system29 and the Lit protease30. These mechanistically diverse systems all converge on the phage capsid protein, which is one of many phage structural proteins mutated to evade targeting of anti-phage bacterial immune systems31. Additionally, binding to other phage structural proteins directly activate Avs (anti- viral STAND NTPases)32 and DSR (defense-associated sirtuins) systems33. The major capsid gene mutations identified in our study are enriched at the capsid protein interface, suggesting that a higher ordered capsid structure or process, rather than a capsid monomer, may be implicated in CBASS immunity. However, the mechanism behind the mutant phage capsid that enables CBASS evasion is unknown and remains an area of future investigation. Altogether, we present direct evidence of CBASS as an anti-phage bacterial immune system, uncover distinct paths taken by phage to inhibit or evade immunity, and expand our understanding of viral evasion strategies of cGAS-based immunity. Experimental Model and Subject Details Bacterial strains and phages [0105] The bacterial strains and phages used in this study are listed in the Key Resources Table. The P. aeruginosa strains (ATCC 33351, JD332, BWHPSA011, BWH058, ATCC 27853, PAO1) and E. coli strains (DH5^ and SM10) were grown in Lysogeny broth (LB) medium at 37°C both with aeration at 225 r.p.m. Plating was performed on LB solid agar with 10 mM MgSO4 when performing phage infections, and when indicated, gentamicin (50 μg ml-1 for P. aeruginosa and 15 μg ml-1 for E. coli) was used to maintain the pHERD30T plasmid. Gene expression was induced by the addition of L-arabinose (0.01% final for BWHPSA011 bacterial genes and 0.1% for phage genes, unless otherwise specified). [0106] The E. coli BL21 (DE3) strain was used for recombinant protein overexpression and grown in Lysogeny broth (LB) medium. The cells were grown at 37^ until OD600nm reached 0.8 and then induced at 18°C for 12 h. Method Details Identification of CBASS operons [0107] tBLASTn was used to query the amino acid sequence of eight known CD-NTases (CdnA-H) against sequenced Pseudomonas aeruginosa genomes contained in the NCBI36 and IMG37 databases as well as our sequenced UCSF clinical isolates. Proteins with >25% amino acid sequence identity to a validated CD-NTase were accepted as “hits”7, leading to the identification of >300 CBASS operons in 252 distinct P. aeruginosa strains. The P. aeruginosa BWHPSA011 (Pa011) strain contains a Type II-A CBASS operon in contig 12 (NCBI Genome ID: NZ_AXQR000000012.1) ranging from 1250439-1254679bp, with CapV (phospholipase effector, NCBI Gene ID: Q024_30602), CdnA (cyclase, Q024_30601), Cap2 (E1/E2, Q024_30600), and Cap3 (JAB, intergenic region 1250439-1250912bp). Identification of anti-phage immune systems [0108] DefenseFinder was used to systematically identify all known anti-phage bacterial immune system operons in P. aeruginosa strains34,38, and the output was used to construct the table in FIG.1A. Episomal gene expression [0109] The shuttle vector that replicates in P. aeruginosa and E. coli, pHERD30T39 was used for cloning and episomal expression of genes in P. aeruginosa BWHPSA011 (Pa011) or PAO1 strains. This vector has an arabinose-inducible promoter and a selectable gentamicin marker. Vector was digested with SacI and PstI restriction enzymes and purified. Inserts were amplified by PCR using bacterial overnight culture or phage lysate as the DNA template, and joined into the pHERD30T vector at the SacI-PstI restriction enzyme cut sites by Hi-Fi DNA Gibson Assembly (NEB) following the manufacturer’s protocol. The resulting plasmids were transformed into E. coli DH5^. All plasmid constructs were verified by sequencing using primers that annealed to sites outside the multiple cloning site. P. aeruginosa cells were electroporated with the pHERD30T constructs and selected on gentamicin. Chromosomal CBASS integration [0110] For chromosomal insertion of the Pa011 CBASS operon, the integrating vector pUC18-mini-Tn7T-LAC40 and the transposase expressing helper plasmid pTNS341 were used to insert the BWHPSA011 CBASS operon at the Tn7 locus in P. aeruginosa PAO1 strain (PaCBASS), or an pUC18-mini-Tn7T-LAC empty vector (E.V.) control strain (PaEV). The vector was linearized using around-the-world PCR, treated with DpnI, and then purified. Two overlapping inserts encompassing the CBASS operon were amplified by PCR using Pa011 overnight culture as the DNA template, and joined into the pUC18-mini-Tn7T-LAC vector a the SacI-PstI restriction enzyme cut sites by Hi-Fi DNA Gibson Assembly (NEB) following the manufacturer’s protocol. The resulting plasmids were used to transform E. coli DH5^. All plasmid constructs were verified by sequencing using primers that annealed to sites outside the multiple cloning site. P. aeruginosa PAO1 cells were electroporated with pUC18-mini- Tn7T-LAC and pTNS3 and selected for on gentamicin. Potential integrants were screened by colony PCR with primers PTn7R and PglmS-down, and then verified by sequencing using primers that anneal to sites outside the attTn7 site. Electrocompetent cell preparations, transformations, integrations, selections, plasmid curing, and FLP-recombinase-mediated marker excision with pFLP were performed as described previously40. Chromosomal mutants of P. aeruginosa BWHPSA011 [0111] The allelic exchange vector that replicates in P. aeruginosa and E. coli, pMQ3042 was used for generating the chromosomal CBASS knockout and CBASS mutant genes in P. aeruginosa BWHPSA011 (Pa011). Vector was digested with HindIII and BamHI restriction enzymes and purified. For the CBASS knockout strain, homology arms >500bp up- and downstream of CBASS operon were amplified by PCR using Pa011 overnight culture as the template DNA. For the CBASS gene mutant strains, homology arms >500bp up- and downstream of CBASS gene catalytic residue(s), with the appropriate mutant nucleotides, were amplified by PCR using Pa011 overnight culture as the template DNA. Previously identified catalytic residues in Escherichia coli TW11681 (NZ_AELD01000000)8 were used to aid the identification of the catalytic residues in Pseudomonas aeruginosa BWHPSA011. Multiple Sequence Comparison by Log-Expectation (MUSCLE, 43 and NCBI Multiple Sequence Alignment Viewer (MSA) were subsequently used to validate conserved catalytic residues between P. aeruginosa BWHPSA011, Vibrio cholerae El Tor N16961 (NC_002505.1), and E.coli TW11681. The inserts were joined into the pMQ30 vector at the HindIII-BamHI restriction enzyme cut sites by Hi-Fi DNA Gibson Assembly (NEB) following the manufacturer’s protocol. The resulting plasmids were transformed into E. coli DH5^. All plasmid constructs were verified by sequencing using primers that annealed to sites outside the multiple cloning site. E. coli SM10 cells were electroporated with pMQ30 constructs and selected for on gentamicin. E. coli SM10 harboring the pMQ30 construct were mated with Pa011 to transfer the plasmid and enable allelic exchange. Potential mutant Pa011 strains were subjected to a phenotype cross streak screen with PaMx41-like phages and then verified by sequencing using primers that anneal to sites outside of the homology arms. Electrocompetent cell preparations, transformations, selections, and plasmid curing were performed as described previously44. Phage growth [0112] All phages were grown at 37°C with solid LB agar plates containing 20 ml of bottom agar containing 10 mM MgSO4 and any necessary inducers or antibiotics. Phages were initially grown on the permissible host P. aeruginosa PAO1 WT, which naturally lacks CBASS.150 μl of overnight cultures of PAO1 were infected with 10 μl of low titer phage lysate (>104-7 pfu/ml) and then mixed with 3 ml of 0.7% top agar 10 mM MgSO4 for plating on the LB solid agar. After incubating at 37°C overnight, individual phage plaques were picked from top agar and resuspended in 200 ^l SM phage buffer. For high titer lysates, the purified phage was further amplified on LB solid agar plates with PAO1 WT. After incubating 37°C overnight, SM phage buffer was added until the solid agar lawn was completely covered and then incubated for 5-10 minutes at room temperature. The whole cell lysate was collected and a 1% volume of chloroform was added, and then left to shake gently on an orbital shaker at room temperature for 15 min followed by centrifugation at maximum g for 3 min to remove cell debris. The supernatant phage lysate was stored at 4°C for downstream assays. Plaque assays [0113] Plaque assays were conducted at 37°C with solid LB agar plates.150 μl of overnight bacterial culture was mixed with top agar and plated. Phage lysates were diluted 10-fold then 2 μl spots were applied to the top agar after it had been poured and solidified. Lysogen construction with JDB67 phage [0114] Lysogens were constructed by spotting serial dilutions of JDB67 WT or JBD67¨acb2 phage lysates on the engineered P. aeruginosa PAO1 strain that harbors BWHPSA011 CBASS in the chromosome (PaCBASS), or a mini-Tn7 E.V. control (PaEV) strain, and streaking out the bacteria (that is, putative lysogens) from the inside of the clearing resulting from a clutter of plaques onto a solid LB agar plate. Colonies were then screened using a cross streak test to confirm resistance to the phage used to lysogenize the strain. The putative lysogens were grown in liquid culture, and the presence of spontaneously produced phage in the supernatant that could plaque on the PAO1 wildtype strain confirmed lysogeny. Isolation of CBASS phage escapers [0115] For identifying PaMx41 WT phage escapers of CBASS, 150 μl of overnight cultures of the P. aeruginosa strain BWHPSA011 (Pa011) were infected with 10 μl of high titer phage lysate (>109 pfu/ml) and then plated on LB solid agar. After incubating at 37°C overnight, 10 individual phage plaques were picked from top agar and resuspended in 200 ^l SM phage buffer. Phage lysates were purified for three rounds using the CBASS expressing strain. Three PaMx41 WT control phages were picked, purified, and propagated in parallel by infecting the Pa011 ¨CBASS strain. To validate the phage identity, PCR and Sanger sequencing were performed on nucleotide sequences unique to PaMx41. [0116] To identify PaMx41 ¨acb2, JBD67 ¨acb2, and JBD18 WT phage escapers of CBASS, 150 μl of overnight cultures of the CBASS expressing strains (Pa011 WT or PaCBASS) were infected with 10 μl of high titer phage lysate (>109 pfu/ml) and plated on LB solid agar. After incubating at 37°C overnight, no obvious plaques were observed. SM phage buffer was added to the entire lawn and whole cell lysate collected. Next, to propagate the mutant escaper phage population, 150 μl of overnight cultures of the P. aeruginosa strains lacking CBASS (Pa011 ¨CBASS or PaEV) were infected with 10 μl of the phage lysates and plated on LB solid agar. After incubating at 37°C overnight, SM phage buffer was added to the entire lawn and whole cell lysate collected. Lastly, to isolate individual escaper plaques, 150 μl of overnight cultures of the CBASS expressing strains (Pa011 WT or PaCBASS) were infected with 10 μl of the previously collected phage lysate and plated on LB solid agar After incubating at 37°C overnight, at least four individual phage plaques were picked from top agar and resuspended in 200 ^l SM phage buffer. Phage lysates were purified for three rounds using the CBASS expressing strain. At least two control or WT phages were picked, purified, and propagated in parallel by infecting the Pa011 ¨CBASS or PaEV strains. To validate the phage identity, PCR and Sanger sequencing were performed on nucleotide sequences unique to each phage. Whole genome sequencing (WGS) and analysis [0117] Genomic DNA from phage lysates was extracted using a modified SDS/Proteinase K method. Briefly, 200 ^L high titer phage lysate (>109 pfu/ml) was mixed with an equal volume of lysis buffer (10 mM Tris, 10 mM EDTA, 100 ^g/mL proteinase K, 100 ^g/mL RNaseA, 0.5% SDS) and incubated at 37°C for 30 min, and then 55°C for 30 min. Preps were further purified using the DNA Clean & Concentrator Kit (Zymo Research). DNA was quantified using the Qubit 4.0 Fluorometer (Life Technologies).20-100 ng genomic DNA was used to prepare WGS libraries using the Illumina DNA Prep Kit (formerly known as Illumina Nextera Flex Kit) using a modified protocol that utilized 5x reduced quantities of tagmentation reagents per prep, except for the bead washing step with Tagment Wash Buffer (TWB), where the recommended 100 ^L of TWB was used. Subsequent on-bead PCR indexing-amplification of tagmented DNA was performed using 2x Phusion Master Mix (NEB) and custom-ordered indexing primers (IDT) matching the sequences from the Illumina Nextera Index Kit. Each 50 ^L reaction was split in two tubes, amplified for 9 and 12 cycles respectively. Libraries were further purified by agarose gel electrophoresis; DNA was excised around the ~400 bp size range and purified using the Zymoclean Gel DNA Recovery Kit (Zymo Research). Libraries were quantified by Qubit and the 9-cycle reaction was used unless the yield was too low for sequencing, in which case the 12-cycle reaction was used. Libraries were pooled in equimolar ratios and sequenced with Illumina MiSeq v3 reagents (150 cycles, Read 1; 8 cycles, Index 1; 8 cycles, Index 2). WGS data were demultiplexed either on-instrument or using a custom demultiplexing Python script (written by Dr. Nimit Jain), and trimmed using cutadapt (v 3.445) to remove Nextera adapters. Trimmed reads were mapped using Bowtie 2.0 (--very-sensitive-local alignments46) and alignments were visualized using IGV (v 2.9.447). Variants were detected using the SeqDiff program . CRISPR-Cas13a phage gene editing [0118] Construction of template plasmids for homologous recombination and selection of engineered phages via the CRISPR-Cas13a system were performed as described previously21. Specifically, homology arms of >500bp up- and downstream of PaMx41 acb2 were amplified by PCR using Pamx41 WT phage genomic DNA as the template. The acrVIA1 gene was amplified from plasmid pAM38348, a gift from Luciano Marraffini, The Rockefeller University. PCR products were purified and assembled as a recombineering substrate and then inserted into the NheI site of the pHERD30T vector. The resulting plasmids were electroporated into P. aeruginosa PAO1 cells. PAO1 strains carrying the recombination plasmid were grown in LB media supplemented with gentamicin.150 μl of overnight cultures were infected with 10 μl of high titer phage lysate (>109 pfu/ml; PaMx33 WT, PaMx35 WT, PaMx43 WT, PaMx41 ESC or PaMx41 WT) and then plated on LB solid agar. After incubating at 37°C overnight, SM phage buffer was added to the entire lawn and whole cell lysate collected. The resulting phage lysate containing both WT and recombinant phages were tittered on PAO1 strains with a chromosomally integrated Type VI-A CRISPR-Cas13a system, and the most efficiently targeting crRNA guide (specific to orf11; guide #5) was used to screen for recombinants. PAO1 strains carrying the Cas13a system and crRNA of choice were grown overnight in LB media supplemented with gentamicin.150 μl of overnight cultures were infected with 10 μl of low titer phage lysate (104-7 pfu/ml), and then plated onto LB solid agar containing 0.3% arabinose and 1 mM isopropyl ȕ-d-1-thiogalactopyranoside (IPTG). After incubating at 37°C overnight, individual phage plaques were picked from top agar and resuspended in 200 ^l SM phage buffer. Phage lysates were purified for three rounds using the Cas13a counter-selection strain (guide #5), and further propagated on a complementary Cas13a counter-selection strain (guide #4), to select against Cas13a escaper phages. To determine whether the phages were recombinants, PCR was performed with the appropriate pairs of primers amplifying the region outside of the homology arms, an internal region of acrVIA1, and acb2. Homologous recombination-mediated mutation of phage gene [0119] Construction of template plasmids for homologous recombination consisted of homology arms >500bp up- and downstream of the mutation of interest encoded in PaMx41 orf11. The homology arms were amplified by PCR using PaMx41¨acb2 escapers phage genomic DNA as the template, and PaMx41 WT phage genomic DNA as the control template. Template 1 primers were designed to symmetrically flank the PaMx41 orf11 mutations I121S and I121T, and template 2 primers were designed to symmetrically flank mutation I327T and S330P. PCR products were purified and assembled as a recombineering substrate and then inserted into the SacI-PstI site of the pHERD30T vector. The resulting plasmids were electroporated into P. aeruginosa BWHPSA011 (Pa011) ¨CBASS cells. Pa011 strains carrying the recombination plasmid were grown in LB media supplemented with gentamicin.150 μl of overnight cultures were infected with 10 μl of high titer phage lysate (>109 pfu/ml; PaMx41¨acb2) and then plated on LB solid agar. After incubating at 37°C overnight, SM phage buffer was added to the entire lawn and whole cell lysate collected. The resulting phage lysate containing both WT and recombinant phages were screened on a lawn of Pa011 WT cells harboring an active CBASS system. Specifically, 150 μl of overnight Pa011 WT cultures were infected with 10 μl of low titer phage lysate (104-7 pfu/ml), and then plated onto LB solid agar. After incubating at 37°C overnight, individual phage plaques were picked from top agar and resuspended in 200 ^l SM phage buffer. Phage lysates were purified for three rounds using the Pa011 WT strain. To confirm whether the phages were recombinants, PCR was performed with the appropriate pairs of primers amplifying the region outside of the homology arms and subject to Sanger Sequencing. Helicase attenuated Cas3 removal of phage genes [0120] Cas3 (Type I-C)-specific guides targeting JBD67 acb2 were cloned into a pHERD30T-derived vector containing modified I-C repeats as previously described19. The guides electroporated into P. aeruginosa PAO1 strains with a chromosomally integrated Type I-C helicase attenuated Cas3 system. JBD67 WT phage lysate was tittered on the PAO1 strains and the efficiently targeting crRNA guide (specific to acb2; guide #3) was identified. PAO1 strains carrying the Type I-C CRISPR-Cas system with a helicase attenuated Cas3 enzyme and crRNA targeting phage JBD67 acb2 were grown overnight in LB media supplemented with gentamicin.150 μl of overnight cultures were infected with 10 μl of high titer phage lysate (>109 pfu/ml; JBD67) and plated on LB agar plates containing gentamicin, 0.1% arabinose, and 1 mM isopropyl ȕ-d-1-thiogalactopyranoside (IPTG). After incubating at 37°C overnight, SM phage buffer was added to the entire lawn and whole cell lysate collected. The resulting phage lysate containing both WT and acb2 knockout phages were grown on a complementary Cas3 counter-selection strain (guide #4) to select against Cas3 escaper phages.150 μl of overnight cultures were infected with 10 μl of low titer phage lysate (104-7 pfu/ml; JBD67 WT) and then plated on LB solid agar containing 0.1% arabinose and 1 mM IPTG. After incubating at 37°C overnight, individual phage plaques were picked from top agar and replica-plated onto LB solid agar with PAO1EV and PAO1CBASS strains. JDB67 plaque sizes that were reduced on the PAO1CBASS plate compared to the positive control (JBD18 WT phage) were identified as potential CBASS sensitive phages. Corresponding plaques on the PAO1EV plate were picked and resuspended in 200 ^l SM phage buffer. To determine whether the phages harbored deletions in acb2, PCR was performed with the appropriate pairs of primers amplifying a ~1kb region outside of acb2. Intracellular 3’,3’-cGAMP measurements [0121] Cell lysates were prepared similarly to previous methods8, in which P. aeruginosa BWHPA011 (Pa011) cells harboring a catalytically dead capV gene (CapVS48A) were used and then transformed with a pHERD30T vector expressing acb2 WT or K26A. Cells were taken from overnight culture, diluted 1:100 in 150ml LB medium with G50 and 0.1% arabinose (flask size 500ml), and then grown at 37°C (190 r.p.m.) until reaching an OD600nm of 0.3-0.4. From the culture, 100 ml was aliquoted and 10 mM MgSO4 added. The cells were then infected with PaMx41¨acb2 to obtain an MOI of ~5 and ensure at least one or more phages were infecting each bacterial cell. After 60 minutes following infection, the culture was separated into two 50 ml samples and centrifuged at 7,500 g for 10 mins at 4°C. Following centrifugation, supernatant was removed and pellets were kept on ice until resuspended in 600 μl of phosphate buffer (50 mM sodium phosphate (pH 7.4), 300 mM NaCl, 10% (v/v) glycerol). The resuspended pellet was supplemented with 1 μl hen-lysozyme (Sigma-Aldrich), vortexed briefly, and incubated at 25°C for 10 min. The resuspended cells were then mixed with Lysing Matrix B (MP) beads and cells were disrupted mechanically using Mini-Beadbeater 16 Biospec Products (1 cycle of 2:30, 3,450 oscillations/m, at 4 °C). Cell lysates were then centrifuged at 17,500 g for 10 min at 4°C. For each condition, one 50 ml cell lysate and subsequent supernatant was (i) loaded onto a 3kDa filter (Amicon Ultra-0.5 centrifugal filter unit; Merk) and the corresponding 50 ml cell lysate and subsequent supernatant was (ii) subjected to phenol-chloroform/chloroform nucleotide extraction (Rouillon et al., 2019). For the filtration step, the unit was centrifuged at 16,000 g for 45 min at 4 °C and flow-through (containing small molecules less than 3kDa) was used as the sample for 3’,3’-cGAMP measurements. For the nucleotide extraction step, 600 μl of supernatant was added to 600 μl of phenol-chloroform, vortexed for 30 sec, and then centrifuged at 17,500 g for 45 min at 4 °C. The top aqueous layer was carefully transferred into another eppendorf tube and 600 μl of chloroform was added, vortexed for 30 sec, and then centrifuged at 17,500 g for 10 min at 4 °C. The top aqueous layer was added to the 3kD filter, centrifuged at 16,000 g for 45 min at 4 °C, and flow-through collected. Each flow-through sample was run in technical triplicate on a 3’,3’-cGAMP ELISA Kit (Arbor Assays) and standards were prepared in the same phosphate buffer.3’,3’-cGAMP concentrations were calculated using a sigmoidal standard curve via GraphPad Prism (v 9.4.1). Phylogenetic analysis [0122] Phylogenetic reconstructions were conducted similar to previous work in our lab49. Homologs of Acb2 were acquired through 3 iterations of psiBLASTp search the non- redundant protein database. Hits with >70% coverage and an E value <0.0005 were included in the generation of the position specific scoring matrix (PSSM). High confidence homologs (>70% coverage, E value < 0.0005) represented in unique species of bacteria were then aligned using NCBI COBALT50 using default settings and a phylogeny was generated in Cobalt using the fastest minimum evolution method51 employing a maximum sequence difference of 0.85 and Grishin distance to calculate the tree. The resulting phylogeny was then displayed as a phylogenetic tree using iTOL: Interactive Tree of Life52. Computational modeling of phage capsid [0123] Models of P. aeruginosa PaMx41 (orf11) and JBD18 (orf35) phage capsid proteins were generated using AlphaFold253 and aligned using the PyMol “super” function to the different chains of the E. coli T4 phage capsid structure (PDB: 6UZC). Protein expression and purification [0124] The Acb2, CapV, CdnA and Cap2 genes were synthesized by GenScript. The full- length Acb2 gene was amplified by PCR and cloned into a modified pET28a vector in which the expressed Acb2 protein contains a His-SUMO tag. The Acb2 mutants were generated by two-step PCR and were subcloned, overexpressed and purified in the same way as wild-type protein. The proteins were expressed in E. coli strain BL21 (DE3) and induced by 0.2 mM isopropyl-ȕ-D-thiogalactopyranoside (IPTG) when the cell density reached an OD600nm of 0.8. After growth at 18°C for 12 h, the cells were harvested, re-suspended in lysis buffer (50 mM Tris–HCl pH 8.0, 300 mM NaCl, 10 mM imidazole and 1 mM PMSF) and lysed by sonication. The cell lysate was centrifuged at 20,000 g for 50 min at 4°C to remove cell debris. The supernatant was applied onto a self-packaged Ni-affinity column (2 mL Ni-NTA, Genscript) and contaminant proteins were removed with wash buffer (50 mM Tris pH 8.0, 300 mM NaCl, 30 mM imidazole). The fusion protein was then digested with Ulp1 at 18°C for 2 h, and then the Acb2 protein was eluted with wash buffer. The eluant of Acb2 was concentrated and further purified using a Superdex-200 increase 10/300 GL (GE Healthcare) column equilibrated with a buffer containing 10 mM Tris-HCl pH 8.0, 200 mM NaCl and 5 mM DTT. The purified protein was analyzed by SDS-PAGE. The fractions containing the target protein were pooled and concentrated. [0125] The CdnA, Cap2 and CdnA-Cap2 complex were purified as His-tagged proteins, which were eluted with elution buffer (50 mM Tris pH 8.0, 300 mM NaCl, 300 mM imidazole) after removing contaminant proteins with wash buffer. The cells expressing CapV were resuspended with lysis buffer containing 50 mM phosphate buffer pH 7.4, 300 mM NaCl, 10% glycerol (v/v). The CapV proteins bound to Ni-NTA beads were washed with a buffer containing 50 mM phosphate buffer pH 7.4, 300 mM NaCl, 10% glycerol (v/v), 30 mM imidazole and then eluted with the 50 mM phosphate buffer (pH 7.4), 300 mM NaCl, 10% glycerol (v/v), 300 mM imidazole. The eluant of CapV was concentrated and further purified using a Superdex-200 increase 10/300 GL (GE Healthcare) column equilibrated with a reaction buffer containing 50 mM phosphate buffer (pH 7.4), 300 mM NaCl, 10% glycerol (v/v). The purified protein was analyzed as described above. Crystallization, data collection and structural determination [0126] The Acb2 protein was concentrated to 24 mg/mL in 10 mM Tris-HCl pH 8.0, 200 mM NaCl and 5 mM DTT. Crystals were grown using the hanging-drop vapor diffusion method. Crystals of Acb2 were grown at 18°C by mixing an equal volume of the protein (24 mg/mL) with reservoir solution containing 0.2 M Sodium bromide, 0.1 M Bis-Tris propane pH 6.5, 10% Ethylene glycol and 20% v/v PEG 3350. Crystals of Acb2 in complex with 3’,3’-cGAMP or c-di-AMP were grown under the same reservoir solution. Prior to crystallization, 3’,3’-cGAMP or c-di-AMP were mixed with the protein at a molar ratio of 0.8:1. The crystals appeared overnight and grew to full size in about two to three days. The crystals were cryoprotected in the reservoir solution containing 20% glycerol before its transferring to liquid nitrogen. [0127] All the data were collected at the X-ray crystallography facility at Tsinghua University (XtaLAB Synergy Custom FRX and a hybrid photon counting detector HyPix- 6000, Rigaku, Japan) and SSRF beamlines BL02U1 and BL19U1, integrated and scaled using the HKL2000 package54. The initial model of Acb2 was obtained through modeling using AlphaFold253. The structures of Acb2 and its complex with ligands were solved through molecular replacement and refined manually using COOT55. The structure was further refined with PHENIX56 using non-crystallographic symmetry and stereochemistry information as restraints. The final structure was obtained through several rounds of refinement. Data collection and structure refinement statistics are summarized in Table S1. Isothermal titration calorimetry binding assay [0128] The dissociation constants of binding reactions of Acb2 or Acb2 mutants with the 3’,3’-cGAMP/2’,3’-cGAMP/c-di-GMP/c-di-AMP/3’,3’-c-di-UMP/3’,3’-c-UMP-AMP/3’,3’- c-UMP-GMP were determined by isothermal titration calorimetry (ITC) using a MicroCal ITC200 calorimeter. Both proteins and cyclic dinucleotides were desalted into the working buffer (20 mM HEPES pH 7.5 and 200 mM NaCl). The titration was carried out with 19 successive injections of 2 μL cyclic dinucleotides at the 0.4 mM concentration, spaced 120 s apart, into the sample cell containing the Acb2 or Acb2 mutants with a concentration of 0.1 mM by 700 rpm at 25°C. The Origin software was used for baseline correction, integration, and curve fitting to a single site binding model. Fluorogenic biochemical assay for CapV activity [0129] The enzymatic reaction velocity was measured as previously described8. Briefly, the esterase activity of the 6×His-tagged CapV was probed with the fluorogenic substrate resorufin butyrate. The CapV protein was diluted in 50 mM sodium phosphate pH 7.4, 300 mM NaCl, 10% (v/v) glycerol to a final concentration of 1.77 ^M. To determine the enzymatic activity of CapV activated by 3’,3’-cGAMP, increasing concentrations ranging from 0.025 to 0.8 ^M of 3’,3’-cGAMP was added to DMSO solubilized resorufin butyrate (stock of 20 mM mixed with 50 mM sodium phosphate pH 7.4, 300 mM NaCl, 10% v/v glycerol reaching a final concentration of 100 ^M). Subsequently, the purified 6×His-tagged CapV was added to the reaction solution containing 3’,3’-cGAMP to a final assay volume of 50 ^L, and fluorescence was measured in a 96-well plate (Corning 96-well half area black non-treated plate with a flat bottom). Plates were read once every 30 s for 20 min at 37°C using a EnSpire Multimode Plate Reader (PerkinElmer) with excitation and emission wavelengths of 550 and 591 nm, respectively. To determine the function of Acb2, 32 ^M Acb2 and 0.8 ^M 3’,3’-cGAMP were pre-incubated at 18°C, and the subsequent detection method was as described above. To examine whether the released molecule from Acb2 is able to activate CapV, 0.8 ^M 3’,3’-cGAMP was incubated with 32 ^M Acb2 for 10 min at 18°C. Proteinase K was subsequently added to the reaction system at a final concentration of 0.065 mg/mL and the reaction was performed at 58°C for 3 h. Reaction products were transferred to Amicon Ultra-4 Centrifugal Filter Unit 3 kDa and centrifuged at 4°C, 4,000 g. Filtered products were used for CapV activity assay. Gel filtration assay [0130] The Acb2, CapV, CdnA, Cap2 and the CdnA-Cap2 complex purified as described above were subjected to gel filtration analysis (Superdex-200 increase 10/300 GL, GE Healthcare). The Acb2 was incubated with CapV, CdnA, Cap2 or the CdnA-Cap2 complex at a molar ratio of 5:1 overnight on ice before the gel filtration analysis in buffer containing 10 mM Tris–HCl pH 8.0, 200 mM NaCl, and 5 mM DTT. The assays were performed with a flow rate of 0.5 mL/min and an injection volume of 1 mL for each run. Samples from relevant fractions were subjected to SDS-PAGE and visualized by Coomassie blue staining. Analytical ultracentrifugation [0131] Proteins were extensively dialyzed against AUC buffer (10 mM Tris pH 8.0, 200 mM NaCl). Sedimentation velocity studies were performed in a Beckman XL-A analytical ultracentrifuge at 20°C and 35,000 rpm. The absorbance at 280 nm was collected every 4 min for a total of 200 scans. These values were used to fit the data to the Lamm equation in SEDFIT software using the continuous c(s) distribution model. Graphs were prepared using Origin software. Native-PAGE assay [0132] Acb2 was pre-incubated with cyclic dinucleotides for 10 min at 18°C, where Acb2 was 14.3 ^M and the concentrations of cyclic dinucleotides ranged from 1.8 to 7.2 ^M (1.8, 3.6, 7.2 ^M). Products of the reaction were analyzed using 5% native polyacrylamide gels and visualized by Coomassie blue staining. High-performance liquid chromatography (HPLC) [0133] 40 ^M Acb2 was pre-incubated with 4 ^M 3’,3’-cGAMP for 10 min at 18°C. Proteinase K was subsequently added to the reaction system at a final concentration of 0.25 mg/mL and the reaction was performed at 58°C for 1 h. Reaction products were transferred to Amicon Ultra-15 Centrifugal Filter Unit 3 kDa and centrifuged at 4°C, 4,000 g. The products obtained by filtration were further filtered with a 0.22 ^m filter and subsequently used for HPLC experiments. The HPLC analysis was performed on an Agilent 1200 system with a ZORBAX Bonus-RP column (4.6 × 150 mm). A mixture of acetonitrile (2%) and 0.1% triÀuoroacetic acid solution in water (98%) were used as mobile phase with 0.8 mL/min. The compounds were detected at 254 nm Example 2 [0134] This example shows that anti-CBASS protein sequested a number of cyclic di- and trinucleotides. [0135] Two methods were used to assess cyclic oligonucleotide binding: isothermal titration calorimietry (ITC) or native polyacrylamide gel electrophoresis (native-PAGE) to see the protein shift when incubated with the indicated nucleotide. For PaMx33 (SEQ ID NO:1), ITC and native-PAGE were performed with each indicated oligonucleotide and binding was observed for the boxes indicated by (FIG.6). For Acb2 from E. coli T4 (SEQ ID NO:2), a mix of ITC and native-PAGE were used. For JBD67 Acb2 (SEQ ID NO:3), native-PAGE was used to confirm similar binding spectrum as that observed with PaMx33 (FIG.6). Data on proteins Apyc1, Acb1, and Tad1 in FIG.6 were taken from the literature. Example 3 Acb2 sequesters diverse cyclic dinucleotides and is active in human cells. [0136] To understand the selectivity of the newly identified Acb2 protein fold, we comprehensively tested an array of cyclic oligonucleotides that Acb2 may bind to. Previous work revealed that Acb2 binds to 3’,3’-cGAMP, 2’,3’-cGAMP and 3’,3’-cUU/UA/UG/AA, but not 3’,3’-cGG. In this current study, we first tested Acb2 binding of 3’,2’-cGAMP, which was recently identified as a signaling molecule for both CBASS and cGAS-like enzymes in eukaryotes. A native gel assay showed a significant shift of the Acb2 protein upon adding 3’,2’-cGAMP, and isothermal calorimetry (ITC) experiments verified that Acb2 binds to 3’,2’-cGAMP with a KD of ~297.7 nM (FIG.14A). Next, we solved the structure of Acb2 complexed with 3’,2’-cGAMP (2.33 Å, FIG.14B), in which 3’,2’-cGAMP binds in the same binding pocket and shows a similar binding mode as 3’,3’-cGAMP and c-di-AMP (FIGS. 14C-14E). Specifically, these CDN molecules are bound by the N-terminal domains of the two interacting Acb2 protomers, each from one Acb2 dimer (FIG.14D). The stacking from Y11 residue and salt bridges from K26 residue of both protomers further stabilize this interaction (FIGS.14C and 14D). The structure of Acb2 complexed with another cGAMP isomer, 2’,3’-cGAMP, solved at 2.24 Å resolution further confirmed this mode of binding (FIGS.14D and 14E). Interestingly, multiple CDNs are tolerated in the binding pocket. Their base groups are mainly stabilized by the ʌ-ʌ stacking from the Y11 residue (FIGS.14C and 14D). Therefore, all the three tested base groups (adenine, guanine, and uridine) are tolerated, albeit with different affinities. For the phosphate-ribose backbone, interestingly, 3’,3’-, 2’,3’- and 3’,2’-cGAMP linkages are all tolerated by Acb2. Detailed analysis further shows that for each linkage, its phosphate group can be stabilized by polar interactions from both K26 and Y11 (FIG.14D), while the other interacting Acb2 residues might vary a little. However, the cavity in Acb2 is large enough for all the three linkages (FIG.14E). Since 3’,2’-cGAMP and 2’,3’-cGAMP are ligands used in eukaryotic cGAS-STING immunity, we also tested whether Acb2 can antagonize cGAS-STING signaling pathway in human cells. The results showed that upon expression of WT Acb2, interferon (IFN) signaling mediated by 2’,3’-cGAMP is significantly reduced while Y11A and K26A Acb2 mutants were less active (FIG.14F). Consistent with these data, native gel assays showed that Y11A and K26A mutations abrogated 2’,3’-cGAMP binding (data not shown). Taken together, the data demonstrates that Acb2 harbors a binding pocket that is well suited for many CDNs. Acb2 sequesters cyclic trinucleotides with higher affinity than cyclic dinucleotides. [0137] Based on the binding pocket of Acb2, it was hypothesized that Acb2 may not bind cyclic mononucleotides or oligonucleotides, such as cA3, cA4 or cA6, due to potential steric clash caused by the nucleotides. Of note, cA3 and cAAG are major products of the CD-NTase enzymes involved in CBASS whereas cA4 is only a minor product of a single CD-NTase. cA6 has not been identified as a product of any known CD-NTases. However, all three cyclic oligoadenylates are known products involved in Type III CRISPR-Cas anti-phage immunity. A native gel assay showed that the Acb2 protein does not shift upon adding cA4 or cA6 molecules. Furthermore, both native gel and ITC assays showed that Acb2 does not bind to cUMP, cCMP or cAMP. However, the native gel assay revealed a significant shift of the Acb2 protein upon adding cA3 or cAAG. ITC experiments revealed that Acb2 binds to cA3 and cAAG with a KD of ~1.5 and ~2.7 nM (FIG.15A), respectively, which is more than an order of magnitude stronger than Acb2 binding to 3’,3’-cGAMP (KD of ~87 nM). To determine whether Acb2 sequesters or cleaves the cyclic trinucleotide molecules, high- performance liquid chromatography (HPLC) revealed that incubating Acb2 with cA3 depletes any detectable molecules, and following proteolysis of Acb2, cA3 is released back into the buffer unmodified (FIG.15B). Collectively, these results demonstrate that Acb2 binds to and sequesters cyclic trinucleotides commonly used in CBASS immunity with a significantly higher affinity than CDNs. Acb2 binds cyclic trinucleotides and dinucleotides with different binding sites. [0138] The binding of CTNs was unexpected because the Acb2 binding pocket appears well suited for only CDNs. To understand how Acb2 interacts with CTNs, we determined the crystal structures of Acb2 in complex with cA3 (2.26 Å) or cAAG (2.10 Å). Surprisingly, the structures showed that one Acb2 hexamer binds two CTNs with two distinct binding pockets that are far from the three pockets for the CDN binding (FIG.15C). An Acb2 hexamer can be viewed as a trimer of dimers, which forms a channel in the center of the hexamer (FIG.15D). Interestingly, each binding pocket of the CTNs is formed by three Acb2 protomers, each from one different Acb2 dimer, in a three-fold symmetry. The two CTN molecules bind at the two ends of the channel, blocking the channel from two opposite sides (FIG.15E). The binding modes of CDNs and CTNs within Acb2 can be described as follows: Each of the two protomers that together bind a CDN is involved in binding to one out of the two CTNs, respectively. Correspondingly, each of the three protomers that together bind a CTN is involved in binding to one out of the three CDNs, respectively. [0139] The CTN is bound mainly through its three phosphate groups, each of which is coordinated by R67 of one protomer and T74 of another protomer through hydrogen bonds (FIGS.15F-15G). Moreover, the CTN is also stabilized by hydrophobic interactions from R67, A70, and I71 from each of the three protomers (FIG.15G). Consistent with this analysis, the Acb2 T74A mutant displayed a significantly decreased binding affinity to cA3 (K D of ~291 nM), and the Acb2 R67A mutant abolished Acb2 binding of cA 3 in vitro (FIG. 15A). To confirm that the binding sites of the CTNs and CDNs in Acb2 are independent of each other, we tested the binding of 3’,3’-cGAMP with the T74A or R67A Acb2 mutant proteins. A native gel assay showed similar shifts of the two Acb2 mutants as WT Acb2 upon adding 3’,3’-cGAMP (FIG.15H), suggesting that the binding to 3’,3’-cGAMP is not affected by the two mutations. In turn, we tested the binding of cA3 with Y11A and K26A Acb2 mutants, which lose their binding to 3’,3’-cGAMP. The native gel results showed a significant shift of Y11A and K26A mutant proteins upon adding cA3 (FIG.15I). Taken together, these data collectively show that one Acb2 hexamer binds two CTNs through two pockets independent of those that bind CDNs. [0140] Structural alignment between apo Acb2 and its complexes with CTNs showed that the binding of CTNs does not induce a conformational change of Acb2, with a root mean square deviation (RMSD) of 0.224 and 0.261 Å (CĮ atoms) for Acb2-cA3 and Acb2-cAAG compared to the apo Acb2, respectively. Therefore, we co-crystallized Acb2 with both cA3 and 3’,3’-cGAMP and then solved its crystal structure at a resolution of 2.76 Å (Table 1). The structure clearly showed that Acb2 binds to two cA3 and three 3’,3’-cGAMP molecules simultaneously (FIGS.3A-3C). Structural alignment between Acb2-cA3-3’,3’-cGAMP and apo Acb2 also showed little conformational changes with an RMSD of 0.298 Å for CĮ atoms. Acb2 binds to cA3 with a novel fold. [0141] Dali search did not return entries of experimentally determined proteins with the same fold as Acb2 nor did Foldseek searches of computationally predicted proteins, suggesting that both the CDN and CTN-binding folds are novel. Foldseek also did not reveal any similar structures encoded by viruses that infect eukaryotes. Several experimentally determined proteins have been reported to bind CTNs, including the CBASS effector proteins NucC and Cap4 that directly bind cA3, as well as the human CDN sensor RECON that directly binds cAAG. Compared to the cA3 binding pocket in Acb2, those in NucC, Cap4, and RECON are significantly different. In NucC, one cA3 molecule is bound in a three-fold symmetric allosteric pocket at the ‘‘bottom’’ of the protein trimer, mainly formed by an extended hairpin loop from each protomer. Additionally, each adenine base is stabilized by hydrogen bonds and ʌ stacking interactions in NucC (PDB code: 6Q1H). In Cap4, cA3 is bound within its SAVED (SMODS-associated) domain, which is a fusion of two CARF (CRISPR-associated Rossman fold) domains derived from Type III CRISPR-Cas system (PDB code: 6WAN). RECON adopts a TIM barrel fold with eight parallel ȕ strands surrounded by eight crossover Į-helixes and cAAG is bound in a deep crevice at the top of the ȕ barrel (PDB code: 6M7K). Moreover, the conformation of cA3 within Acb2 is also different from those within NucC, Cap4, and RECON complex structures. Specifically, cA3 in both NucC and Cap4 are almost in an overall planar conformation, and two adenine bases of cAAG within RECON are nearly in the same plane as the phosphodiester ring and the third guanine base is extended out. However, each base of cA3 forms a ~46.8 degree angle with the phosphate plane in Acb2. Together, the structure of Acb2 complexed with cA3 reveals a novel CTN-binding fold. Cyclic nucleotide binding spectra are different among Acb2 homologs. [0142] To determine the conservation of each binding site across the Acb2 family, PSI- BLAST was used to identify 2,242 total homologs. From these, clustering of the Acb2 homologs revealed 878 unique and non-redundant proteins (see Methods for details; Table S1). Multi-sequence alignments and phylogenetic analyses of these unique Acb2 homologs revealed that both binding sites are predicted to be intact in most homologs (78%). However, some homologs have a mutation in a residue homologous to R67 or T74 that are essential for CTN binding (19%), and very few proteins had mutated Y11 or K26 sites that are essential for CDN binding (3%; FiG.17A). We therefore assessed the binding spectrum of representative Acb2 homologs to determine their cyclic oligonucleotide binding preferences. We chose Acb2 homologs from P. aeruginosa phage JBD67 (44.4% a.a. identity), in which both R67 and T74 residues are conserved, alongside Serratia phage CHI14 (23.5% a.a. identity) and Escherichia phage T4 (24.2% a.a. identity), in which only the R67 (Serratia phage) or T74 (Escherichia phage) residue is conserved. ITC analyses showed that JBD67- Acb2 directly binds to 3’,3’-cGAMP with a KD of ~99 nM and cA3 with a KD of ~3.5 nM (FIG.17B), both of which are comparable to those of PaMx33-Acb2. Native gel assays also suggest that JBD67-Acb2 binds to the same spectrum of cyclic nucleotides as PaMx33-Acb2. ITC analyses showed that T4-Acb2 directly binds to 3’,3’-cGAMP with a KD of ~84.4 nM, consistent with previous work, but does not bind to cA3 (FIG.17C). Native gel assays also suggest that T4-Acb2 binds the same spectrum of CDNs as PaMx33-Acb2, but not to the CTNs cA3 and cAAG. Next, we mutated D61 of T4-Acb2 to Arginine to see whether it can endow T4-Acb2 with the binding activity of cA3 because T4-Acb2 already has T68 residue in the place of T74 of PaMx33-Acb2 that is essential for cA3 binding. However, based on ITC assays, we observed that the D61R mutant of T4-Acb2 was still unable to bind cA3 (FIG. 17C). Interestingly, structural alignment between T4-Acb2 and PaMx33-Acb2 complexed with cA3 showed that the helix lining the cA3 binding pocket of PaMx33-Acb2 has a kink at E64, which enlarges the pocket to accommodate the base groups of cA3 (FIG.17D). However, the corresponding helix of T4-Acb2 does not kink here, so the Y37, A57, L60, D61, and T64 residues of T4-Acb2 may undergo steric clashing and prevent cA3 binding (FIG.17D). More importantly, the relative angles among the three helices lining the binding pocket are also different between PaMx33-Acb2 and T4-Acb2, resulting in a smaller binding pocket in T4-Acb2 (FIG.17E). Together, these observations may explain the inability of T4- Acb2 binding to CTNs. Lastly, ITC analyses showed that CHI14-Acb2 directly binds to 3’,3’-cGAMP with a KD of ~62.4 nM, but also does not bind to cA3 (FIG.17F). Of note, native gel assays showed almost no shift of the CHI14-Acb2 protein upon adding any cyclic oligonucleotides, including 3’,3’-cGAMP, suggesting that native gel assay is not suitable for studying the binding spectrum of CHI14-Acb2. Using ITC, CHI14-Acb2 displayed the same binding spectrum to all cyclic oligonucleotides as T4-Acb2 (FIG.17F-17G). The outcomes of binding experiments are summarized, along with a comparison to the enzyme Acb1 (FIG. 17H). In summary, Acb2 homologs bind to many CTNs and CDNs used in cGAS-based immunity with certain homologs having a more limited spectrum. Acb2 antagonizes Type III-C CBASS immunity. [0143] Since Acb2 displays high affinity binding to cyclic trinucleotides, it was tested whether phage-encoded Acb2 can antagonize Type III-C CBASS immunity that uses a cA3 signaling molecule to activate the endonuclease (NucC) effector protein. NucC is a cyclic nucleotide-activated effector in both CBASS and Type III CRISPR-Cas systems, which non- specifically degrades DNA and limits phage replication. First, we established an in vitro NucC activity assay using purified NucC from the P. aeruginosa strain ATCC 27853 (Pa278) and Acb2 from P. aeruginosa phage PaMx33 (FIGS.18A-18B). While cA3 activates the DNA cleavage activity of NucC, WT Acb2 significantly decreased NucC activity (FIG.18C). Moreover, following proteolysis of WT Acb2, the released cA3 molecule again activated NucC activity (FIG.18C; last two lanes). The R67A and T74A Acb2 mutant proteins, which lost or exhibited decreased cA3 binding, displayed minimal inhibition of NucC activity. However, the Y11A and K26A Acb2 mutants, whose CDN binding pockets are disrupted, inhibited NucC activity similarly to WT Acb2 (FIG.18C). These results demonstrate that Acb2 antagonizes Type III-C CBASS immunity in vitro through sequestering the cA3 molecule. [0144] To determine whether Acb2 can inhibit this same cA3-based CBASS system in vivo, we performed phage infection assays with plasmid or phage-encoded Acb2 and the Pa278 Type III-C CBASS operon (FIG.18B). Phages naturally expressing acb2 were unable to replicate on the native P. aeruginosa ATCC 27853 strain, so the Pa278 system was chromosomally integrated into PAO1 that is a phage sensitive strain and naturally lacks CBASS. In the presence Pa278 Type III-C CBASS, the titer of JBD67 phage lacking acb2 (JBD67¨acb2) was reduced by 3 orders of magnitude compared to its replication in the absence of CBASS (FIGS.18D-E). Plasmid-based expression of WT Acb2 or Y11A and K26A Acb2 (CDN binding mutants) fully rescued phage titer, while R82A and T89A (cyclic trinucleotide binding mutants) did not (FIG.18D). In the presence of the Pa011 Type II-A CBASS (cGAMP producing), the titer of JBD67¨acb2 phage was reduced by 5 orders of magnitude (FIG.18D-18E), whereas JBD67 WT phage was reduced by 1-2 orders of magnitude. Plasmid-based expression of WT Acb2 or R82A and T89A Acb2 (cyclic trinucleotide binding mutants) partially rescued phage titer, while Y11A and K26A Acb2 (CDN binding mutants) did not (FIG.18D). The partial targeting of JBD67 WT phage (i.e. naturally encoding Acb2) was fully reversed by plasmid-based expression of WT Acb2 or the cyclic trinucleotide binding mutants, but not the CDN binding mutants. We additionally introduced a K26A mutation into acb2 within the genome of JBD67 WT phage. This rendered mutant phage completely sensitive to cells expressing the cGAMP-based system but maintained resistance against the cA3 system (FIG.18E). These findings demonstrate Acb2 protects phage from Type III-C (cA3-producing) and Type II-A (3’,3’-cGAMP-producing) CBASS using different interfaces, further highlighting the versatility of the Acb2 protein. Discussion [0145] Following phage infection, CBASS immunity functions via the activation of a cGAS-like enzyme to catalyze the synthesis of a cyclic oligonucleotide signaling molecule. To date, two phage proteins have been discovered to antagonize the CBASS immunity: Acb1 and Acb2. Acb1 uses an inhibitory mechanism common to the eukaryotic cGAS-STING signaling system, 25 that is, enzymatically cleaving and depleting an array of CDNs and CTNs.15 In contrast, we, alongside another independent group, reported that Acb2 acts as a “sponge” and sequesters 3’,3’-cGAMP 16, 17 as well as a variety of other CBASS CDN signaling molecules.16 A sponging mechanism was also reported for inhibitors of the anti- phage system Thoeris, including Tad126 and Tad2, 27 that sequester gcADPR signaling molecules. Here, we extend the CDN binding spectrum of Acb2 to include 3’,2’-cGAMP, a signaling molecule not cleaved by Acb1, but recently implicated in both CBASS and cGAS like signaling in eukaryotes.9, 22, 28 The ability of Acb2 to function in human cells against 2’,3’-cGAMP reinforces the flexibility of the “sponging” mechanism (i.e. no need to bind to cGAS, STING, or host proteins), and the remarkable cross-kingdom conservation of this cyclic-oligonucleotide-based immune system. The activity of Acb2 in mammalian cells also implies the possibility that pathogenic bacteria with prophage-encoded Acb1, Acb2, or other undiscovered cGAMP interactors could use them to dampen the human immune response during intracellular infection. Similar cross-kingdom interactions have been previously reported with bacterial c-di-GMP 3 and c-di-AMP produced by Listeria monocytogenes, 29 serving as ligands for human STING. While we could not identify proteins with similar structures to Acb2 in eukaryotic viruses, we speculate that novel cGAMP sponges await discovery in eukaryotes and their viruses. Taken together, the work suggests that discoveries with human health implications remain to be made at the interface of cyclic oligonucleotide inhibitors and cGAS-STING immunity. [0146] Acb2 binds to a wider spectrum of cyclic oligonucleotides compared to the enzyme Acb1, including 3’,3’-cUU, 3’,3’-cUG, and 3’,2’-cGAMP, while also sequestering CTNs with a stronger binding affinity. 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Claims

WHAT IS CLAIMED IS: 1. A method of killing bacteria, the method comprising, contacting an anti-cyclic-oligonucleotide-based anti-phage signaling system (CBASS) protein to one or more cyclic oligonucleotides, wherein anti-CBASS protein is substantially (e.g., at least 60%, 70%, 80%, 90%, 95%, 99%) identical to a sequence of any one of SEQ ID NOS:1-3, thereby inhibiting the CBASS in the bacteria.
2. The method of claim 1, wherein the cyclic oligonucleotides are present in bacteria and the contacting occurs inside bacterial cells.
3. The method of claim 1, wherein the cyclic oligonucleotides are present in mammalian cells and the contacting occurs inside mammalian cells.
4. The method of claim 3, wherein the mammalian cells are infected with bacteria.
5. The method of any one of claims 1 to 4, wherein the contacting comprises introducing the anti-CBASS protein into the cell.
6. The method of claim 5, wherein the introducing comprises introducing an expression cassette comprising a nucleic acid encoding the anti-CBASS protein and a promoter operably linked to the nucleic acid.
7. The method of claim 6, wherein the promoter is inducible.
8. The method of any one of claims 5 to 7, wherein the introducing comprises administering an engineered bacteriophage comprising an anti-CBASS protein that is substantially (e.g., at least 60%, 70%, 80%, 90%, 95%, 99%) identical to a sequence of any one of SEQ ID NOS:1-3 to the cell.
9. The method of any one of claims 5 to 8, wherein the cyclic oligonucleotide is present in the cell prior to the introducing.
10. The method of any one of claims 5 to 8, wherein the cyclic oligonucleotide is introduced to the cell when or after the anti-CBASS protein is introduced to the cell.
11. The method of claim 1, wherein the cyclic oligonucleotides are extracellular and the contacting occurs outside a cell.
12. The method of any one of claims 1 to 11, wherein the contacting occurs in vitro.
13. The method of any one of claims 1 to 11, wherein the contacting occurs ex vivo.
14. The method of claim 13, wherein the contacting occurs within a population of cells comprising bacterial cells and eukaryotic cells.
15. The method of claim 14, wherein the eukaryotic cells are mammalian cells.
16. The method of claim 15, wherein the mammalian cells are human cells.
17. The method of any one of claims 13 to 16, wherein the cells are introduced into a mammal after the introducing and contacting.
18. A method of treating a bacterial infection in a subject, comprising administering to the subject anti-CBASS protein substantially (e.g., at least 60%, 70%, 80%, 90%, 95%, 99%) identical to a sequence of any one of SEQ ID NOS:1-3, or an engineered bacteriophage comprising thereof, wherein the anti-CBASS protein binds to one or more cyclic oligonucleotides.
19. The method of claim 18, wherein the anti-CBASS protein binds to the cyclic oligonucleotides present inside cells.
20. The method of claim 19, wherein the anti-CBASS protein binds to the cyclic oligonucleotides present inside bacterial cells.
21. The method of claim 19, wherein the anti-CBASS protein binds to the cyclic oligonucleotides present inside mammalian cells.
22. The method of claim 21, wherein the mammalian cells are infected with bacteria.
23. The method of claim 18, wherein the anti-CBASS protein binds to the cyclic oligonucleotides present outside of cells.
24. An expression cassette comprising a nucleic acid encoding an anti- CBASS protein and a promoter operably linked to the nucleic acid, wherein the anti-CBASS protein comprises a sequence that is substantially (e.g., at least 60%, 70%, 80%, 90%, 95%, 99%) identical to a sequence of any one of SEQ ID NOS:1-3.
25. The expression cassette of claim 24, wherein the promoter is heterologous to the nucleic acid encoding the anti-CBASS protein.
26. The expression cassette of claim 24 or 25, wherein the promoter is inducible.
27. The expression cassette of any one of claims 24 to 26, wherein the nucleic acid is DNA or RNA.
28. A vector comprising the expression cassette of any one of claims 24 to 27.
29. The vector of claim 28, wherein the vector is a viral vector.
30. An engineered bacteriophage comprising the expression cassette of any one of claims 24 to 27.
31. A pharmaceutical composition comprising an anti-CBASS protein or a polynucleotide comprising a nucleic acid encoding an anti-CBASS protein, wherein the anti- CBASS protein comprises a sequence that is substantially (e.g., at least 60%, 70%, 80%, 90%, 95%, 99%) identical to a sequence of any one of SEQ ID NOS:1-3, or the engineered bacteriophage of claim 30.
32. An engineered bacteriophage comprising a nucleic acid encoding an anti-CBASS protein, wherein the anti-CBASS protein comprises a sequence that is substantially (e.g., at least 60%, 70%, 80%, 90%, 95%, 99%) identical to a sequence of any one of SEQ ID NOS:1-3.
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