Classifications

• • 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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