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WO2025215636A1 - Polypeptides de système anti-défense et leurs utilisations - Google Patents

Polypeptides de système anti-défense et leurs utilisations

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Publication number
WO2025215636A1
WO2025215636A1 PCT/IL2025/050306 IL2025050306W WO2025215636A1 WO 2025215636 A1 WO2025215636 A1 WO 2025215636A1 IL 2025050306 W IL2025050306 W IL 2025050306W WO 2025215636 A1 WO2025215636 A1 WO 2025215636A1
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Prior art keywords
polypeptide
seq
protein
amino acid
group
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English (en)
Inventor
Rotem Sorek
Azita Leavitt
Gil Amitai
Erez YIRMIYA
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Yeda Research and Development Co Ltd
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Yeda Research and Development Co Ltd
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Publication of WO2025215636A1 publication Critical patent/WO2025215636A1/fr
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    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • 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
    • C07K14/315—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Streptococcus (G), e.g. Enterococci
    • 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
    • C12N9/14—Hydrolases (3)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983—Viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1034—Isolating an individual clone by screening libraries
    • C12N15/1037—Screening libraries presented on the surface of microorganisms, e.g. phage display, E. coli display
    • 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
    • C12N2795/00—Bacteriophages
    • C12N2795/00011—Details
    • C12N2795/10011—Details dsDNA Bacteriophages
    • C12N2795/10021—Viruses as such, e.g. new isolates, mutants or their genomic sequences
    • 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
    • C12N2795/00—Bacteriophages
    • C12N2795/00011—Details
    • C12N2795/10011—Details dsDNA Bacteriophages
    • C12N2795/10022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • 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
    • C12N2795/00—Bacteriophages
    • C12N2795/00011—Details
    • C12N2795/10011—Details dsDNA Bacteriophages
    • C12N2795/10111—Myoviridae
    • C12N2795/10121—Viruses as such, e.g. new isolates, mutants or their genomic sequences
    • 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
    • C12N2795/00—Bacteriophages
    • C12N2795/00011—Details
    • C12N2795/10011—Details dsDNA Bacteriophages
    • C12N2795/10111—Myoviridae
    • C12N2795/10122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/07—Bacillus
    • C12R2001/125—Bacillus subtilis ; Hay bacillus; Grass bacillus
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B40/00—Libraries per se, e.g. arrays, mixtures
    • C40B40/02—Libraries contained in or displayed by microorganisms, e.g. bacteria or animal cells; Libraries contained in or displayed by vectors, e.g. plasmids; Libraries containing only microorganisms or vectors

Definitions

  • the present invention in some embodiments thereof, relates to anti-defense system polypeptides and uses thereof.
  • Harnessing phages and their defense mechanisms as anti-bacterial agents for therapeutic uses has gained much interest over the last decade, especially in light of the substantial rise in the prevalence of bacterial antibiotic resistance, coupled with an inadequate number of new antibiotics (see e.g. Gibb et al. Pharmaceuticals 2021, 14, 634).
  • avirus comprising an exogenous polynucleotide encoding an anti-defense system polypeptide, wherein the anti-defense system polypeptide is selected from the group consisting of:
  • RMSD Root Mean Square Deviation
  • RMSD Root Mean Square Deviation
  • RMSD Root Mean Square Deviation
  • RMSD Root Mean Square Deviation
  • RMSD Root Mean Square Deviation
  • RMSD Root Mean Square Deviation
  • RMSD Root Mean Square Deviation
  • the virus having an increased infectivity to at least one cell as compared to a virus of the same species not comprising the exogenous polynucleotide.
  • a cell comprising an exogenous anti-defense system polypeptide or a polynucleotide encoding same, wherein the anti-defense system polypeptide is selected from the group consisting of:
  • RMSD Root Mean Square Deviation
  • RMSD Root Mean Square Deviation
  • RMSD Root Mean Square Deviation
  • RMSD Root Mean Square Deviation
  • RMSD Root Mean Square Deviation
  • RMSD Root Mean Square Deviation
  • RMSD Root Mean Square Deviation
  • RMSD Root Mean Square Deviation
  • RMSD Root Mean Square Deviation
  • RMSD Root Mean Square Deviation
  • RMSD Root Mean Square Devi
  • the nucleic acid sequence heterologous to the polynucleotide is selected from the group consisting of: a promoter, a recombination element, an element for expression of multiple polynucleotides from a single construct, a transmissible element and a selectable marker.
  • a method of producing the virus comprising introducing into a virus the exogenous polynucleotide encoding the anti-defense system polypeptide, thereby producing the virus.
  • the method comprises introducing into the virus the nucleic acid construct, under conditions which allow integration of the polynucleotide in a genome of the virus.
  • the virus does not endogenously comprise the polynucleotide encoding the anti-defense system polypeptide.
  • a method of infecting a cell comprising contacting the cell with the virus.
  • a method of producing the cell comprising introducing into a cell the anti-defense system polypeptide or the polynucleotide encoding same, thereby producing the cell.
  • a method of impairing ability of a cell to respond to stress comprising introducing into the cell an anti-defense system polypeptide or a polynucleotide encoding same, wherein the antidefense system polypeptide is selected from the group consisting of:
  • RMSD Root Mean Square Deviation
  • RMSD Root Mean Square Deviation
  • RMSD Root Mean Square Deviation
  • RMSD Root Mean Square Deviation
  • RMSD Root Mean Square Deviation
  • the stress comprises a viral infection.
  • the cell is a prokaryotic cell.
  • the virus is a phage.
  • the cell is a eukaryotic cell.
  • the eukaryotic cell is a plant cell.
  • the eukaryotic cell is a human cell.
  • the cell expresses the defense system polypeptide or a homolog thereof.
  • the cell further comprises an exogenous polynucleotide of interest.
  • the method further comprising introducing into the cell an exogenous polynucleotide of interest.
  • the method being effected in-vitro or ex-vivo.
  • the method being effected in-vivo.
  • a method of treating a bacterial infection in a subject in need thereof comprising administering to the subject a therapeutically effective amount of the phage comprising the exogenous polynucleotide encoding the anti-defense system polypeptide, thereby treating the bacterial infection in the subject.
  • the method comprising administering to the subject a therapeutically effective amount of an antibiotic.
  • an article of manufacture comprising as active ingredients the phage comprising the exogenous polynucleotide encoding the anti-defense system polypeptide; and an antibiotic.
  • a method of improving resistance of a plant to biotic stress comprising introducing into the plant an anti-defense system polypeptide or a polynucleotide encoding same, wherein the anti-defense system polypeptide is selected from the group consisting of:
  • RMSD Root Mean Square Deviation
  • RMSD Root Mean Square Deviation
  • RMSD Root Mean Square Deviation
  • RMSD Root Mean Square Deviation
  • RMSD Root Mean Square Deviation
  • RMSD Root Mean Square Deviation
  • RMSD Root Mean Square Deviation
  • a method of treating a disease that can benefit from inhibiting a defense system polypeptide in a subject in need thereof comprising administering to the subject a therapeutically effective amount of an anti-defense system polypeptide or a polynucleotide encoding same, wherein: when the defense system polypeptide is a homolog of the defense system polypeptide ThsB of a Type I Thoeris set forth in SEQ ID NO: 10 endogenously expressed in the subject, the anti-defense system polypeptide is a Tad3 polypeptide, a Tad4 polypeptide, a Tad5 polypeptide and/or a Tad6 polypeptide which binds the homolog of SEQ ID NO: 10; when the defense system polypeptide is a homolog of the defense system polypeptide ThsB of a Type II Thoeris set forth in SEQ ID NO: 228 endogenously expressed in the subject, the anti-defense system polypeptide is a Tad
  • the anti-defense system polypeptide is a Tad8 polypeptide which binds the homolog of SEQ ID NO: 227 endogenously expressed in the subject
  • the anti-defense system polypeptide is an Acb3 polypeptide which binds the homolog of amino acid sequence selected from the group consisting of SEQ ID NO: 229-231 and 233; wherein:
  • the Tad3 polypeptide has a sequence similarity defined by an e- value ⁇ 0.05 to a sequence of a protein having an amino acid sequence selected from the group consisting of SEQ ID NO: 58, 67-76, 245-5265 and 7594-11520 and/or a structural similarity defined by a Root Mean Square Deviation (RMSD) ⁇ 2.0 angstroms to a structure of a protein having an amino acid sequence selected from the group consisting of SEQ ID NO: 58, 67-76, 245-5265 and 7594- 11520;
  • RMSD Root Mean Square Deviation
  • the Tad4 polypeptide has a sequence similarity defined by an e- value ⁇ 0.05 to a sequence of a protein having an amino acid sequence selected from the group consisting of SEQ ID NO: 66, 77-86, 5266-5865 and 11521-11766 and/or a structural similarity defined by a Root Mean Square Deviation (RMSD) ⁇ 2.0 angstroms to a structure of a protein having an amino acid sequence selected from the group consisting of SEQ ID NO: 66, 77-86, 5266-5865 and 11521-11766;
  • RMSD Root Mean Square Deviation
  • the Tad5 polypeptide has a sequence similarity defined by an e- value ⁇ 0.05 to a sequence of a protein having an amino acid sequence selected from the group consisting of SEQ ID NO: 59, 87-96, 5866-5961 and 11767-11849 and/or a structural similarity defined by a Root Mean Square Deviation (RMSD) ⁇ 2.0 angstroms to a structure of a protein having an amino acid sequence selected from the group consisting of SEQ ID NO: 59, 87-96, 5866-5961 and 11767-11849;
  • RMSD Root Mean Square Deviation
  • the Tad6 polypeptide has a sequence similarity defined by an e-value ⁇ 0.05 to a sequence of a protein having an amino acid sequence selected from the group consisting of SEQ ID NO: 65, 97-106, 5962-6369 and 11850-11888 and/or a structural similarity defined by a Root Mean Square Deviation (RMSD) ⁇ 2.0 angstroms to a structure of a protein having an amino acid sequence selected from the group consisting of SEQ ID NO: 65, 97-106, 5962-6369 and 11850-11888;
  • RMSD Root Mean Square Deviation
  • the Tad7 polypeptide has a sequence similarity defined by an e-value ⁇ 0.05 to a sequence of a protein having an amino acid sequence selected from the group consisting of SEQ ID NO: 239, 6370-6461 and 11889-11918 and/or a structural similarity defined by a Root Mean Square Deviation (RMSD) ⁇ 2.0 angstroms to a structure of a protein having an amino acid sequence selected from the group consisting of SEQ ID NO: 239, 6370-6461 and 11889-11918;
  • RMSD Root Mean Square Deviation
  • the Tad8 polypeptide has a sequence similarity defined by an e-value ⁇ 0.05 to a sequence of a protein having an amino acid sequence selected from the group consisting of SEQ ID NO: 240, 6462-6616 and 11919-12290 and/or a structural similarity defined by a Root Mean Square Deviation (RMSD) ⁇ 2.0 angstroms to a structure of a protein having an amino acid sequence selected from the group consisting of SEQ ID NO: 240, 6462-6616 and 11919-12290; and
  • RMSD Root Mean Square Deviation
  • the Acb3 polypeptide has a sequence similarity defined by an e-value ⁇ 0.05 to a sequence of a protein having an amino acid sequence selected from the group consisting of SEQ ID NO: 244 and 6617-7593 and/or a structural similarity defined by a Root Mean Square Deviation (RMSD) ⁇ 2.0 angstroms to a structure of a protein having an amino acid sequence selected from the group consisting of SEQ ID NO: 244 and 6617-7593, thereby treating the disease in the subject.
  • RMSD Root Mean Square Deviation
  • the disease is selected from the group consisting of autoimmune disease, interferonopathy and a disease associated with neuronal degeneration.
  • the Tad3 polypeptide, Tad4 polypeptide, Tad5 polypeptide, Tad6 polypeptide, Tad7 polypeptide, Tad8 polypeptide and/or Acb3 polypeptide inhibits activity of the defense system polypeptide.
  • the Tad3 polypeptide and/or Tad8 polypeptide is capable of forming a homodimer.
  • the Tad3 polypeptide and/or Tad8 polypeptide binds as a homodimer two monomers of the defense system polypeptide.
  • expression of the Tad7 polypeptide and/or Tad8 polypeptide in a B. subtilis BEST7003 comprising SEQ ID NO: 2 increases sensitivity of the B. subtilis BEST7003 to infection by a B. subtilis phage SBSphiJ.
  • expression of the Acb3 polypeptide in a E. coil MG1655 comprising SEQ ID NO: 4 increases sensitivity of the E. coll MG1655 to infection by an E. coll phage BAS 18.
  • expression of the Acb3 polypeptide in a B. subtilis BEST7003 comprising SEQ ID NO: 3 increases sensitivity of the B. subtilis BEST7003 to infection by a B. subtilis phage SBSphiC.
  • the Tad4 polypeptide binds and/or inhibits activity of plant Brachypodium distachyon (BdTIR) set forth SEQ ID NO: 8.
  • the Tad3 polypeptide and/or Tad4 polypeptide binds and/or inhibits activity of SARM1TIR set forth SEQ ID NO: 6.
  • the Acb3 polypeptide binds and/or inhibits activity of human cGAS of a CBASS set forth SEQ ID NO: 12291.
  • the anti-defense polypeptide comprises the SEQ ID NO.
  • the amino acid sequence of the Tad3 polypeptide comprises the SEQ ID NO: 58; the amino acid sequence of the Tad4 polypeptide comprises the SEQ ID NO: 66; the amino acid sequence of the Tad5 polypeptide comprises the SEQ ID NO: 59; the amino acid sequence of the Tad6 polypeptide comprises the SEQ ID NO: 65; the amino acid sequence of the Tad7 polypeptide comprises the SEQ ID NO: 239; the amino acid sequence of the Tad8 polypeptide comprises the SEQ ID NO: 240; and/or the amino acid sequence of the Acb3 polypeptide comprises the SEQ ID NO: 244.
  • a putative anti-defense system polypeptide comprising:
  • the method comprises in-silico modelling a three dimensional structure of the single viral protein of the cluster prior to the (ii), and proceeding to step (ii) with a viral protein having a predicted structural confidence score above a predetermined threshold.
  • the (ii) when the viral protein is predicted to be a homodimer in the three dimensional structure modelling, the (ii) is effected on a homodimer conformation.
  • the method comprising selecting only viral proteins with an unknown function prior to the (iii).
  • the method comprising selecting only viral proteins having a length ⁇ 200 amino acids prior to the (iii).
  • the method comprising determining an amount of amino acid residues in the selected viral protein having the predicted co-folding confidence score above the predetermined threshold interacting with the defense system polypeptide, to thereby select a viral protein having at least 20 interacting residues with the defense system polypeptide.
  • the method further comprising in-vitro or in-vivo determining a functional activity of the putative anti-defense system polypeptide following the (iii).
  • the sequence similarity is defined by an e-value ⁇ 0.05.
  • the e-value ⁇ 0.001.
  • sequence similarity is defined by an alignment length covering at least 80 % of the aligned viral proteins sequences.
  • the structural similarity is defined by a Root Mean Square Deviation (RMSD) ⁇ 2.0 angstroms.
  • RMSD Root Mean Square Deviation
  • the defense system is a prokaryotic defense system.
  • the defense system is a eukaryotic defense system.
  • the viral proteins are phage proteins.
  • FIGs. 1A-F show structure-guided discovery of protein inhibitors of type I Thoeris.
  • Figure 1A is a schematic representation of clustering the phage protein space and establishing a database of phage clusters representing short proteins of unknown function.
  • Figure IB is a schematic representation of an iterative pipeline for the prediction of interactions between a bacterial immune protein and phage proteins. High-scoring interactions based on a single AlphaFold2-Multimer model are further examined via multiple models.
  • Figure 1C is a graph demonstrating the anti-defense activity of the indicated anti-Thoeris candidates.
  • Figure IF is an SDS-PAGE image demonstrating that the Tad3, Tad4, Tad5 and Tad6 anti-defense proteins bind ThsB. Pulldown of a 6xHis-SUMO2-tagged ThsB protein coexpressed with Tad3, Tad4 Tad5 or Tad6 retrieves the respective anti-Thoeris protein.
  • FIGs. 2A-B demonstrate that the Tad3, Tad4, Tad5 and Tad6 anti-defense proteins bind ThsB.
  • Figure 2A is a graph demonstrating that knock-in of Tad.3 in phage SBSphiJ renders the phage resistant to Thoeris type I. Data represent plaque-forming units per milliliter (PFU/ml) of phages infecting control cells (no defense system) and cells expressing the Thoeris defense system. Shown is the average of three replicates, with individual data points overlaid.
  • Figure 2B shows the predicted complex structures of ThsB bound to each one of the anti-Thoeris proteins Tad3, Tad4, Tad5 and Tad6. ThsB is presented in the same orientation, with the active site frontfacing, in all cases.
  • FIGs. 3A-C shows comparison between in-vitro verified inhibitors to non-verified candidates.
  • Figure 3A shows that homologs of in-vitro verified Thoeris inhibitors are also predicted to bind ThsB when analyzed via AlphaFold2-Multimer, but homologs of most of the in-vitro non-verified candidates are not.
  • 10 homologs ranging in sequence identity between 25 % and 95 % were analyzed by AlphaFold2-Multimer.
  • FIGs. 4A-I show an improved computational pipeline for predicting phage-encoded inhibitors of bacterial immune proteins.
  • Figure 4 A is a schematic representation of the modified computational pipeline which takes into account co-folding of protein homologs.
  • Figure 4B is a graph demonstrating the anti-defense activity of the anti-Thoeris type II candidates referred to herein as “Tad7” and “Tad8”.
  • Data represent plaque-forming units per milliliter (PFU/ml) of phage SBSphiJ infecting control cells (no defense system), cells expressing type II Thoeris system and cells co-expressing type II Thoeris and each of the indicated anti-Thoeris candidates.
  • Figure 4C shows the structure of the AlphaFold2-Multimer predicted complex formed between Tad7 (red) and ThsB (dark green).
  • Figure 4D is a zoom in on the Tad7-ThsB complex, showing a detailed view of how the active site of ThsB is blocked by a loop of Tad7. E99 is the catalytic residue of the type II ThsB.
  • Figure 4E shows the structure of the AlphaFold2-Multimer predicted complex formed by a Tad8 homodimer (shades of red) binding to ThsA (dark green).
  • Figure 4F shows a detailed view of the Macro domain pocket of ThsA blocked by Tad8. Residues in the Macro domain that also interact with His-ADPR are indicated.
  • Figure 4G shows a detailed view of the Macro domain pocket in complex with the His-ADPR immune signal 40 , showing that His-ADPR occupies the same pocket that is blocked by Tad8.
  • Figure 4H is a graph demonstrating the anti-defense activity of the anti-CBASS type III candidate referred to herein as “Acb3”.
  • Data represent plaque-forming units per milliliter (PFU/ml) of phage Bas 18 infecting control cells (no defense system), cells expressing the type III CBASS system from E. coli KTE188, and cells co-expressing type III CBASS and Acb3.
  • Figure 41 shows the structure of the AlphaFold2-Multimer predicted complex formed between Acb3 (red) and the CD-NTase enzyme of the E.
  • FIGs. 5A-E demonstrate that phage-derived anti-defense proteins bind and inhibit human and plant immune proteins.
  • Figure 5A is an SDS-PAGE image showing pulldown of a 6xHis- SUMO2-tagged BdTIR co-expressed with Tad4, demonstrating that these proteins co-elute together.
  • Figure 5B is a graph demonstrating NADase activity of purified ThsA incubated with filtered lysates derived from cells expressing BdTIR alone, BdTIR together with Tad4, or control cells that do not express BdTIR. NADase activity was measured using a nicotinamide 1,N6- ethenoadenine dinucleotide (sNAD) cleavage fluorescence assay.
  • sNAD nicotinamide 1,N6- ethenoadenine dinucleotide
  • Figure 5C is an SDS-PAGE image showing pulldown of a His-tagged TIR domain of the human SARM1 protein (SARMITIR) that was co-expressed with Tad4, demonstrating physical interactions.
  • Figure 5D is a graph demonstrating NAD + levels in filtered lysates derived from cells expressing IISARMITIR, cells co-expressing IISARMITIR and Tad4, or control cells that do not express IISARMITIR. Bars represent the mean of five experiments, with individual data points overlaid.
  • Figure 5E is a graph demonstrating that co-expression of Acb3 with the human cGAS prevents 2'3'-cGAMP production. Lysates were analyzed by LC-MS.
  • FIG. 6 shows multiple sequence alignment of Tad3 anti-Thoeris proteins. Alignment was generated using 10 randomly selected homologous sequences of each anti-Thoeris protein. conserveed residues are colored in purple. Red boxes indicate residues that comprise a loop that blocks the ThsB pocket. Positions in the alignments not occupied in the verified anti-Thoeris protein sequences are not shown.
  • FIGs. 7A-B show multiple sequence alignment of anti-Thoeris proteinsTad4 ( Figure 7 A) and Tad6 ( Figure 7B). Alignment was generated using 10 randomly selected homologous sequences of each anti-Thoeris protein. conserveed residues are colored in purple. Red boxes indicate residues that comprise a loop that blocks the ThsB pocket. Positions in the alignments not occupied in the verified anti-Thoeris protein sequences are not shown.
  • FIG. 8 is a graph demonstrating that substitutions in the loops predicted to block the ThsB pocket impact anti-Thoeris function.
  • Data represents plaque-forming units per ml (PFU/ml) of phage SBSphiJ infecting cells co-expressing the Thoeris system and a WT or mutated anti-Thoeris protein (SEQ ID NOs: 58, 12292 and 12293 for WT and mutated Tad3, SEQ ID NOs: 66, 12294 and 12295 for WT and mutated Tad4 and SEQ ID NOs: 65, 12296 and 12297 for WT and mutated Tad6), as well as control cells expressing no defense system and cells expressing the Thoeris system alone (“type I Thoeris”). Shown is the average of three replicates, with individual datapoints overlaid. Data for type I Thoeris and the WT anti-defense proteins are the same as those presented in Figure 1C.
  • FIG. 9 shows a structural comparison between the ThsB proteins of type I and of type II Thoeris systems.
  • FIG. 10 demonstrates that Type I Thoeris inhibitors Tad3, Tad4 and Tad6 do not inhibit type II Thoeris.
  • Data represents plaque-forming units per ml (PFU/ml) of phage SBSphiJ infecting cells co-expressing the type II Thoeris system and a type I Thoeris inhibitor, as well as control cells expressing no defense system and cells expressing the type II Thoeris system alone (“type II Thoeris”). Shown is the average of three replicates, with individual data points overlaid.
  • FIGs. 11A-B show that Acb3 is predicted to bind diverse CD-NTase enzymes.
  • Figure 11A is an overview of the predicted complex between Acb3 and the CD-NTase of the E. coli KTE188 CBASS (SEQ ID NO: 229).
  • Acb3 surface electrostatics are shown to highlight the hydrophobic patch that covers the CD-NTase ligand binding groove.
  • Figure 11B shows that predicted complexes formed between Acb3 (red) and four bacterial CD-NTase enzymes (green, SEQ ID NOs: 230, 231, 234 and 233).
  • AlphaFold2-Multimer model co-folding confidence scores are indicated above the models.
  • FIG. 12 demonstrates that Acb3 inhibits type I CBASS from B.
  • Data represent plaque-forming units per ml (PFU/ml) of phage SBSphiC infecting cells co-expressing the type I CBASS system and Acb3, as well as control cells expressing no defense system and cells expressing the type I CBASS system alone (“type I CBASS”). Shown is the average of three replicates, with individual datapoints overlaid.
  • FIGs. 13A-D demonstrate that phage-derived anti-defense proteins (red/orange) are predicted to bind eukaryotic immune proteins (green).
  • Predicted complexes formed between BdTIR and Tad4 Figure 13 A
  • the TIR domain of SARM1 and Tad3 Figure 13B
  • the TIR domain of SARM1 and Tad4 Figure 13C
  • cGAS and Acb3 Figure 13D
  • AlphaFold2- Multimer model co-folding confidence scores are indicated above the models.
  • the present invention in some embodiments thereof, relates to anti-defense system polypeptides and uses thereof.
  • the present inventors developed an iterative process that utilizes AlphaFold2 -Multimer, a software that predicts physical interactions between input proteins 37 , combined with a dimension-reduction process and a set of reliability measures aimed to reduce computation loads to thereby systematically screen phage-encoded proteins for inhibitors that physically bind and antagonize bacterial defense system proteins (Examples 1-5 of the Examples section which follows). While AlphaFold2-Multimer notoriously yields false positive predictions, the present inventors showed that false positive rates can be substantially reduced by co-folding sets of protein homologs with the target defense system protein.
  • phage- encoded inhibitors of bacterial Thoeris proteins can also bind and inhibit distantly related human and plant immune TIRs, and that the phage-derived inhibitor of bacterial CBASS can inhibit the activity of human cGAS as well (Example 6 of the Examples section which follows).
  • This system comprises a Cyclic GMP-AMP synthase (cGAS)-like enzyme named cGAS/DncV-like nucleotidyltransferases (CD-NTases).
  • cGAS Cyclic GMP-AMP synthase
  • CD-NTases Cyclic GMP-AMP synthase
  • phage infection these enzymes are activated and produce cyclic oligonucleotides that bind to and activate a downstream effector protein.
  • the activated effector proteins are proposed to induce premature cell death through various mechanisms, including membrane impairment, DNA degradation, NAD+ depletion, etc.
  • CBASS operons are classified on the basis of their architecture, with type I CBASS encoding only a CD-NTase and an effector protein, and a non-Type I CBASS (e.g. types II, III and IV) encoding additional proteins with proposed regulatory roles.
  • type I CBASS encoding only a CD-NTase and an effector protein
  • non-Type I CBASS e.g. types II, III and IV
  • the CBASS is a non-Type I CBASS.
  • Non-limiting examples of non-Type I CBASS include those of Escherichia coli KTE188 (SEQ ID NO: 229), mycobacterium absessus ATCC 19977 (SEQ ID NO: 230), Escherichia coli ECOR31 (SEQ ID NO: 231) and Escherichia coli MOD1-EX1698 (SEQ ID NO: 233).
  • the CBASS is a Type III CBASS.
  • a non-limiting example of a Type III CBASS includes the one of Escherichia coli KTE188 (SEQ ID NO: 229).
  • the CBASS is a Type I CBASS.
  • Type I includes the one of B. cereus VD146 (SEQ ID NO: 234).
  • Cyclic GMP-AMP synthase binds viral DNA and is activated to produce 2’, 3’- cyclic GMP-AMP (2’,3’-cGAMP) dinucleotides, which activate the STING (stimulator of interferon genes) effector protein to initiate a potent interferon response
  • a non-limiting example of such homologs includes the human cGAS (e.g. SEQ ID NO: 12291).
  • anti-defense system polypeptide refers to a polypeptide which expression in a cell disables a defense system polypeptide expressed in the cell, thereby impairing ability of the cell to respond to stress.
  • the phrase “impairing an ability to respond to stress” or “impaired ability to respond to stress” refers to a significant decrease in the ability of the cell to autonomously respond to stress, as compared to a control cell of the same species not expressing the antidefense system polypeptide and under the same conditions, as may be manifested by the cell phenotype e.g. in growth arrest, death (e.g., apoptotic, necrotic), change in gene expression (secretion of cytokines etc.), sensitivity to infection by a pathogen e.g. a virus.
  • death e.g., apoptotic, necrotic
  • change in gene expression secretion of cytokines etc.
  • sensitivity to infection by a pathogen e.g. a virus.
  • the decrease is by at least 5 %, at least 10%, 20 %, 30 %, 40 % or even higher say, 50 %, 60 %, 70 %, 80 %, 90 % or more than 99 %. According to specific embodiments the decrease is by at least 1.5 fold, at least 2 fold, at least 3 fold, at least 5 fold, at least 10 fold, or at least 20 fold.
  • the impaired ability to respond to stress is manifested by increased sensitivity of the cell to infection by at least one virus.
  • increasing sensitivity or “increased infectivity” refers to a significant increase in the cell susceptibility towards a virus, as compared to a cell of the same species not expressing the anti-defense system polypeptide, as may be manifested e.g. in growth arrest, death, integration of the viral nucleic acid sequence into the cell genome, prevention of lysogeny and/or viral genomic replication.
  • the increase is in at least 5 %, at least 10%, 20 %, 30 %, 40 % or even higher say, 50 %, 60 %, 70 %, 80 %, 90 % or more than 100 %.
  • the increase is at least 1.5 fold, at least 2 fold, at least 3 fold, at least 5 fold, at least 10 fold, or at least 20 fold.
  • the lysogenic activity of a virus can be assessed by PCR or DNA sequencing.
  • the DNA replication activity of a virus can be assessed e.g. by DNA sequencing or southern blot analysis.
  • the lytic activity of a virus can be assessed e.g. by optical density, plaque assay or living dye indicators.
  • the lytic activity of a phage can be measured indirectly by following the decrease in optical density of the bacterial cultures owing to lysis. This method involves introduction of phage into a fluid bacterial culture medium.
  • optical density assays can be adapted for use with eukaryotic cells.
  • methods that include redox chemistry employing cell respiration as a universal reporter.
  • cellular respiration reduces a dye (e.g., tetrazolium dye) leading to a color change that can be measured automatically.
  • a virus successfully infects its host cell and replicates, it often disrupts normal cellular processes, including growth and respiration. This viral activity results in reduced cellular respiration, which is reflected by a corresponding decrease in the intensity of the color change.
  • Such colorimetric assays include for example the MTT assay or WST-1 assay that involves the reduction of a tetrazolium salt to a colored formazan product by metabolically active cells.
  • the amount of color produced which can be measured by optical density, correlates with the number of viable cells in the sample.
  • plaque assay Another exemplary method, known as the plaque assay, in which cultured cells are infected with a diluted virus sample. After incubation, the virus causes localized cell death, resulting in clear zones called plaques. These plaques can be counted to determine the virus titer.
  • phages may be introduced into a few milliliters of soft agar along with some bacterial host cells. This soft agar mixture is laid over a hard agar base (seeded-agar overlay). The phage adsorbs onto the host bacterial cells, infect and lyse the cells, and then begin the process anew with other bacterial cells in the vicinity. After 6 - 24 hours, plaques are observable within the lawn of bacterial growth on the plate. Each plaque represents a single infective phage particle in the original sample.
  • Yet another exemplary method is the one-step viral (e.g. phage) growth curve which allows determining the production of progeny virions by cells as a function of time after infection.
  • the assay is based on the fact that cells in the culture are infected simultaneously with a low number of viruses so that no cell can be infected with more than one virus. At various time intervals, samples are removed for a plaque assay allowing quantitative determination of the number of phages present in the medium.
  • the cell is a bacterial cell and the defense system is an anti-phage bacterial defense system.
  • the anti-defense system polypeptide is a polypeptide which expression in an infected bacteria disables an anti-phage bacterial defense system, thereby increasing sensitivity of the bacteria to a phage.
  • the anti-defense system polypeptide directly binds the defense system polypeptide.
  • Assays for testing binding include both in-silico and experimental methods (e.g. flow cytometry, western blow, immunoprecipitation, surface plasmon resonance (e.g. Biacore), biolayer interferometry Blitz® assay, HPLC and functional assays depending on the polypeptide of interest). Such methods are well known in the art, and exemplary assays for specific defense system polypeptides are also described infra and in the Examples section which follows.
  • clustering refers to the process of grouping a set of viral proteins such that the proteins within the same cluster, exhibit greater similarity in sequence and/or structure to each other than to proteins in other groups (clusters). Sequence similarity and structure similarity can be determined by methods which are well known in the art as further described herein. Clustering is a general analytical task rather than a specific algorithm, and it can be accomplished through various algorithms that differ in their definitions of what constitutes a cluster and in the methods they use to identify them. For instance, clustering can involve techniques that emphasize different aspects of similarity, such as sequence alignment or structural motifs. A non-limiting example of a clustering tool includes the one provided by MMseqs2. Specific embodiments suggest that clustering is effected based on sequence similarity, structure similarity or a combination of both.
  • the clustering is based on sequence similarity.
  • the sequence similarity is defined by percentage of sequence identity between the polypeptides or polynucleotides when aligned to each other.
  • percentage of sequence identity is used in reference to polypeptides it is recognized that residue positions which are not identical often differ by conservative amino acid substitutions, where amino acid residues are substituted for other amino acid residues with similar chemical properties (e.g. charge or hydrophobicity) and therefore do not change the functional properties of the molecule.
  • sequences differ in conservative substitutions the percent sequence identity may be adjusted upwards to correct for the conservative nature of the substitution. Means for making this adjustment are well-known to those of skill in the art. Typically this involves scoring a conservative substitution as a partial rather than a full mismatch, thereby increasing the percentage sequence identity.
  • the sequence alignment program is a basic local alignment program, e.g., BLAST.
  • the identity is a global identity, i.e., an identity over the entire sequences aligned and not over portions thereof.
  • the sequence alignment program is Mmseqs2 (which can do both local alignment or global alignment).
  • sequence similarity is defined by an alignment length covering at least 50 %, at least 60 %, at least 70 %, at least 80 % or at least 90 % of the aligned sequences.
  • sequence similarity is defined by an alignment length covering at least 80 % of the aligned sequences.
  • sequence similarity is defined by at least 20 % identity between the aligned sequences.
  • the at least 20 % identity comprises at least 25 %, at least 30 %, at least 35%, at least 40 %, at least 45 %, at least 50 %, at least 60 %, at least 65 %, at least 70 %, at least 75 %, at least 80 %, at least 85 %, at least 90 %, at least 91 %, at least 92 %, at least 93 %, at least 94 %, at least 95 %, at least 96 %, at least 97 %, at least 98 %, or at least 99 % or 100 % identity.
  • sequence similarity is defined by an e- value.
  • e-value refers to a parameter that describes the number of hits one can expect to see by chance in a sequence alignment search to the recited sequence using a database of a particular size. It decreases exponentially as the Score (S) of the match increases. Essentially, the e-value describes the random background noise. An e-value of 1 assigned to a hit can be interpreted as meaning that in a database of the current size one might expect to see 1 match with a similar score simply by chance.
  • the e-value represents a statistically significant similarity between the sequences.
  • the e-value is ⁇ 0.05. According to specific embodiments, the e-value is ⁇ 0.01, ⁇ 0.001 or ⁇ 0.0001.
  • the clustering is based on structural similarity.
  • structural similarity refers to the extent to which the three-dimensional (3D) shapes of two or more proteins resemble each other. This similarity can be assessed by comparing the spatial arrangement of the protein's secondary structure (such as alpha-helices, beta-sheets), the overall folding pattern (tertiary structure) and/or quaternary structure. According to specific embodiments, the structure similarity is determined using computational tools that align protein structures and calculate the degree of similarity, such as root-mean-square deviation (RMSD) of atomic positions or other structural alignment metrics.
  • RMSD root-mean-square deviation
  • Commercial computations tools for computing RMSD are known in the art and include for example Dali, PDBeFOLD, PyMOL, FoldSeek.
  • RMSD >5 angstroms indicates a low structural similarity
  • 2 angstroms ⁇ RMSD ⁇ 2 angstroms indicates a moderate similarity
  • RMSD ⁇ 2.0 angstroms indicates high similarity
  • the structure similarity is defined by a Root Mean Square Deviation (RMSD) ⁇ 2.0 angstroms.
  • RMSD Root Mean Square Deviation
  • the structure similarity is defined by a Root Mean Square Deviation (RMSD) ⁇ 1.5 angstroms.
  • RMSD Root Mean Square Deviation
  • the structure similarity is defined by a Root Mean Square Deviation (RMSD) ⁇ 1 angstroms.
  • RMSD Root Mean Square Deviation
  • the method comprises in-silico modeling an interaction between a single viral protein of a cluster and the defense system polypeptide of interest.
  • Selection of the single viral protein of the cluster may be effected either randomly of systematically.
  • the clustering is effected such that there is a representative sequence (“center” of the cluster) that is significantly similar in sequence and/or structure to each member in the cluster.
  • the selected viral protein is the one in the center of the cluster (in other words, the closes member to all the sequences in the cluster).
  • Modeling of an interaction between a viral protein and a defense system polypeptide refers to in-silico predicting formation of a protein complex comprising both the viral protein and the defense system polypeptide. Such prediction can be effected by any method known in the art, such as, but not limited to AlphaFold-Multimer. According to specific embodiments, the method comprises proceeding to the following step only with a viral protein having a predicted co-folding confidence score with the defense system polypeptide above a predetermined threshold.
  • the phrase “predicted co-folding confidence score above a predetermined threshold” refers to a score that signifies a high level of confidence that the model’s predicted co-folding of the viral protein and the defense system closely represents a true complex formation.
  • the predetermined threshold comprises a predicted co-folding confidence score > 0.5 in a confidence scale of 0 to 1.
  • the predetermined threshold comprises a predicted co-folding confidence score > 0.6, > 0.7, > 0.8 or > 0.9 in a confidence scale of 0 to 1.
  • the predetermined threshold comprises a predicted co-folding confidence score > 0.8 in a confidence scale of 0 to 1.
  • the co-folding confidence score comprises the average co-folding scores of all rounds.
  • the present inventors show in the Examples section which follows that modeling of an interaction between a single viral protein of a cluster and the defense system polypeptide yield false positive results, and that these rates are substantially reduced by modelling sets of homologs of the viral protein being members of the same cluster with the target immune protein.
  • the method comprises in-silico modeling an interaction between at least one additional viral protein of the cluster of the selected protein.
  • predetermined number of viral proteins having predicted cofolding confidence scores refer to a number of viral proteins that signifies a high level of confidence that viral proteins of the selected cluster are indeed anti-defense system polypeptides.
  • the predetermined number comprises at least 50 %, at least 60 %, at least 70 %, at least 80 %, at least 90 % or more of the modelled viral proteins of the cluster.
  • the predetermined number comprises the average co-folding score of all the modelled viral proteins of the cluster.
  • the method further comprises in-silico modelling a three dimensional structure of the viral protein prior to modeling its interaction with the defense system polypeptide.
  • the method comprises proceeding to the following step only with a viral protein having a predicted structural confidence score above a predetermined threshold.
  • the viral protein when predicted to be a homodimer modelling its interaction with the defense system polypeptide is effected on a homodimer conformation.
  • predicted structural confidence score above a predetermined threshold refers to a score that signifies a high level of confidence that the model’s predicted structure closely represents the true structure.
  • the predetermined threshold comprises a predicted structural confidence score > 50 in a confidence scale of 0 to 100.
  • the predetermined threshold comprises a predicted structural confidence score > 60, > 70, > 80 or > 90 in a confidence scale of 0 to 100.
  • the predetermined threshold comprises a predicted structural confidence score > 80 in a confidence scale of 0 to 100.
  • the method described above can have additional steps imposing sequence, structure or function limitations aiming to reduce computational load and/or improve the method. According to specific embodiments, such steps can be inserted prior or following any of the recited steps hereinabove.
  • the method comprises omitting identical viral proteins from the dataset.
  • the method comprises selecting only a viral protein(s) with an unknown function.
  • viral proteins do not have a previous annotation in publicly available databases.
  • the method comprises filtering out proteins that are not biased towards viruses.
  • proteins with a higher number of homologs detected in a general metagenomic database (e.g. MGnify database) than the viral protein database used may be considered as not biased towards viruses.
  • the method comprises selecting only a viral protein(s) having a length ⁇ 1000 amino acids, ⁇ 800 amino acids, ⁇ 600 amino acids, ⁇ 500 amino acids, ⁇ 400 amino acids, ⁇ 300 amino acids, or ⁇ 200 amino acids.
  • the method comprises selecting only a viral protein(s) having a length ⁇ 200 amino acids.
  • the method comprises determining an amount of amino acid residues in said selected viral protein having said predicted co-folding confidence score above said predetermined threshold interacting with said defense system polypeptide system polypeptide.
  • Amino acid residues of the viral protein may form various interactions with the defense system polypeptides, including but not limited to hydrogen (H)-bonds, 7t-7t stacking, 7t-Cation interaction, ionic bonds (salt bridges), 7t-H Bonds, van der Waals and disulphide bonds. Methods of determining such bonds are known in the art such as, but not limited to the RING version 4 server and the Biopython PDBparser
  • the method comprises selecting a viral protein having at least 20, at least 25, at least 30, at least 35 or at least 40 interacting residues with said defense system polypeptide
  • the method comprises screening for additional proteins having sequence and/or structure similarity to the selected putative anti-defense system polypeptide (e.g. in other databases or with less constringent parameters), to thereby identify additional protein(s) being a putative anti-defense system polypeptide.
  • the method comprises experimentally determining in-vitro, ex-vivo or in-vivo the functional activity of the putative anti-defense system polypeptide.
  • the method may further comprises experimentally testing binding of the putative anti-defense system polypeptide to the defense system polypeptide or testing the inhibitory activity of the putative anti-defense system polypeptide on the defense system polypeptide.
  • Such methods are well known in the art and depend on the specific function of the target defense system polypeptide. Exemplary non limiting assays for specific defense system polypeptides are also described hereinabove and below and in the Examples section which follows.
  • the method further comprises synthesizing the proteins by any techniques known to those skilled in the art of peptide synthesis, for example but not limited to recombinant DNA techniques or solid phase peptide synthesis.
  • the viral protein with the highest co-folding confidence score with the defense system polypeptide is the one selected (e.g. for experimental verification).
  • phage-encoded inhibitors of bacterial defense systems can also inhibit their eukaryotic homologs (Example 6 of the Examples section which follows).
  • specific embodiments of the present invention contemplates that the selected anti-defense system polypeptide may be used to inhibit a homolog of the defense system polypeptide used in the identification method.
  • a homolog of a defense system refers to a defense system protein(s) of a different species (orthologs, such as in prokaryotes and eukaryotes), of a different gene within the same organism (paralogs) or a synthetic version thereof that exhibits similarities in sequence, structure and/or function.
  • the homolog may comprise a deletion, insertion, or substitution variant, including an amino acid substitution in the sequence of the defense system polypeptide.
  • anti-defense system polypeptides identified by the methods described herein may further be used, according to specific embodiments, in various research, biotechnological, agricultural and clinical applications associated with the defense system polypeptide or a homolog thereof.
  • the present inventors discovered several anti-defense system polypeptides. Specifically, six families of Thoeris inhibitors (referred to herein as “Tad3”, “Tad4”, “Tad5”, “Tad6”, “Tad7” and “Tad8”) and one family of CBASS inhibitors (referred to herein as “Acb3”).
  • the anti-defense system polypeptide is a Tad3 polypeptide.
  • a Tad3 polypeptide disables the anti -phage bacterial system Thoeris type I.
  • RMSD Root Mean Square Deviation
  • the Tad3 polypeptide has an e-value ⁇ 0.05 to a sequence of a protein having an amino acid sequence selected from the group consisting of SEQ ID NO: 58, 67-76, 245-5265 and 7594-11520, each possibility represents a separate embodiment of the claimed invention.
  • the Tad3 polypeptide of some embodiments has a sequence similarity defined by an e- value ⁇ 0.05 to SEQ ID NO: 58 and/or a structural similarity defined by a RMSD ⁇ 2.0 angstroms to the structure of SEQ ID NO: 58.
  • the Tad3 polypeptide comprises a serine residue at position 48 corresponding to SEQ ID NO: 58.
  • the Tad3 polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 58, 67-76, 245-5265 and 7594- 11520, each possibility represents a separate embodiment of the claimed invention.
  • the Tad3 polypeptide comprises SEQ ID NO: 58.
  • the Tad3 polypeptide consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 58, 67-76, 245-5265 and 7594- 11520, each possibility represents a separate embodiment of the claimed invention. According to specific embodiments, the Tad3 polypeptide consists of SEQ ID NO: 58.
  • the Tad3 polypeptide binds the defense system polypeptide ThsB of a Type I Thoeris set forth in SEQ ID NO: 10.
  • Determining binding to a defense system polypeptide can be effected by any method known in the art.
  • a defense system polypeptide e.g. ThsB of a Type I Thoeris set forth in SEQ ID NO: 10.
  • Non-limiting examples are described hereinabove and in the Examples section which follows, and include e.g. in-silico prediction interaction models, immunoprecipitation/pull-down followed by SDS-PAGE or SEC-MALS.
  • the Tad3 polypeptide binds the active site of ThsB (and specifically forms a loop that penetrates the active site).
  • the Tad3 polypeptide forms a hydrogen bond with the catalytic E85 residue of ThsB set forth in SEQ ID NO: 10.
  • the Tad3 polypeptide is capable of forming a homodimer.
  • the Tad3 polypeptide binds as a homodimer two monomers of ThsB.
  • the Tad3 polypeptide inhibits activity of the defense system polypeptide ThsB of a Type I Thoeris set forth in SEQ ID NO: 10.
  • Determining inhibition of activity of a defense system polypeptide can be effected by any method known in the art. Non-limiting examples are described hereinabove and in the Examples section which follows. For example, one can measure the ability of filtered cell lysates derived from Thoeris-infected cells to activate the Thoeris effector ThsA (e.g. the NADase activity of the Thoeris effector ThsA). Additionally or alternatively, one can measure phage infectivity of a bacteria expressing the defense system by e.g. a plaque assay or liquid culture infection. For example, inhibition can be determined by increased sensitivity of B.
  • ThsB set forth in SEQ ID NO: 10
  • subtilis BEST7003 expressing an exogenous Thoeris type I and the anti-defense system polypeptide to infection by the SBSphiJ phage.
  • expression of the Tad3 polypeptide in a B. subtilis BEST7003 comprising SEQ ID NO: 1 increases sensitivity of B. subtilis BEST7003 to infection by a B. subtilis phage SBSphiJ.
  • inhibition can be determined by increased sensitivity of B. subtilis BEST7003 expressing an exogenous Thoeris type I to infection by a SBSphiJ phage genetically modified to express the anti-defense system polypeptide.
  • the Tad3 polypeptide binds a homolog of the defense system polypeptide ThsB of a Type I Thoeris set forth in SEQ ID NO: 10.
  • the Tad3 polypeptide inhibits activity of a homolog of the defense system polypeptide ThsB of a Type I Thoeris set forth in SEQ ID NO: 10.
  • the homolog is SARM1TIR set forth SEQ ID NO: 6.
  • Methods of determining binding to and activity of SARM1TIR are known in the art and also described in the examples section which follows, and include e.g. computational modeling and experimental (e.g. immunoprecipitation, NADase activity, axonal cell death in response to neuronal injury etc.).
  • the anti-defense system polypeptide is a Tad4 polypeptide.
  • a Tad4 polypeptide disables the anti -phage bacterial system Thoeris type I.
  • a “Tad4 polypeptide having a sequence similarity defined by an e-value ⁇ 0.05 to a sequence of a protein having an amino acid sequence selected from the group consisting of SEQ ID NO: 66, 77-86, 5266-5865 and 11521-11766 and/or a structural similarity defined by a Root Mean Square Deviation (RMSD) ⁇ 2.0 angstroms to a structure of a protein having an amino acid sequence selected from the group consisting of SEQ ID NO: 66, 77- 86, 5266-5865 and 11521-11766, wherein the polypeptide binds the defense system polypeptide ThsB of a Type I Thoeris set forth in SEQ ID NO: 10 and/or a homolog thereof’ is also referred to herein as a “Tad4 polypeptide”.
  • the Tad4 polypeptide has an e-value ⁇ 0.05 to a sequence of a protein having an amino acid sequence selected from the group consisting of SEQ ID NO: 66, 77-86, 5266-5865 and 11521-11766, each possibility represents a separate embodiment of the claimed invention.
  • the Tad4 polypeptide has a RMSD ⁇ 2.0 angstroms to a structure of a protein having an amino acid sequence selected from the group consisting of SEQ ID NO: 66, 77-86, 5266-5865 and 11521-11766, each possibility represents a separate embodiment of the claimed invention.
  • the Tad4 polypeptide of some embodiments has a sequence similarity defined by an e- value ⁇ 0.05 to SEQ ID NO: 66 and/or a structural similarity defined by a RMSD ⁇ 2.0 angstroms to the structure of SEQ ID NO: 66.
  • the Tad4 polypeptide comprises a threonine residue at position 69 corresponding to SEQ ID NO: 66.
  • the Tad4 polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 66, 77-86, 5266-5865 and 11521- 11766, each possibility represents a separate embodiment of the claimed invention.
  • the Tad4 polypeptide comprises SEQ ID NO: 66.
  • the Tad4 polypeptide consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 66, 77-86, 5266-5865 and 11521- 11766, each possibility represents a separate embodiment of the claimed invention.
  • the Tad4 polypeptide consists of SEQ ID NO: 66.
  • the Tad4 polypeptide binds the defense system polypeptide ThsB of a Type I Thoeris set forth in SEQ ID NO: 10.
  • the Tad4 polypeptide binds the active site of ThsB (and specifically forms a loop that penetrates the active site).
  • the Tad4 polypeptide forms a hydrogen bond with the catalytic E85 residue of ThsB set forth in SEQ ID NO: 10.
  • the Tad4 polypeptide inhibits activity of the defense system polypeptide ThsB of a Type I Thoeris set forth in SEQ ID NO: 10.
  • expression of the Tad4 polypeptide in a B. subtilis BEST7003 comprising SEQ ID NO: 1 increases sensitivity of B. subtilis BEST7003 to infection by a B. subtilis phage SBSphiJ.
  • the Tad4 polypeptide binds a homolog of the defense system polypeptide ThsB of a Type I Thoeris set forth in SEQ ID NO: 10.
  • the Tad4 polypeptide inhibits activity of a homolog of the defense system polypeptide ThsB of a Type I Thoeris set forth in SEQ ID NO: 10.
  • the homolog is plant Brachypodium distachyon (BdTIR) set forth SEQ ID NO: 8.
  • BdTIR plant Brachypodium distachyon
  • Methods of determining binding to and activity of BdTIR are known in the art and also described in the examples section which follows, and include e.g. computational modeling and experimental (e.g. immunoprecipitation, NADase activity).
  • RMSD Root Mean Square Deviation
  • the Tad5 polypeptide has an e-value ⁇ 0.05 to a sequence of a protein having an amino acid sequence selected from the group consisting of SEQ ID NO: 59, 87-96, 5866-5961 and 11767-11849, each possibility represents a separate embodiment of the claimed invention.
  • the Tad5 polypeptide has a RMSD ⁇ 2.0 angstroms to a structure of a protein having an amino acid sequence selected from the group consisting of SEQ ID NO: 59, 87-96, 5866-5961 and 11767-11849, each possibility represents a separate embodiment of the claimed invention.
  • the Tad5 polypeptide of some embodiments has a sequence similarity defined by an e- value ⁇ 0.05 to SEQ ID NO: 59 and/or a structural similarity defined by a RMSD ⁇ 2.0 angstroms to the structure of SEQ ID NO: 59.
  • the Tad5 polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 59, 87-96, 5866-5961 and 11767- 11849, each possibility represents a separate embodiment of the claimed invention.
  • the Tad5 polypeptide comprises SEQ ID NO: 59.
  • the Tad5 polypeptide consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 59, 87-96, 5866-5961 and 11767- 11849, each possibility represents a separate embodiment of the claimed invention.
  • the Tad5 polypeptide consists of SEQ ID NO: 59.
  • the Tad5 polypeptide binds the defense system polypeptide ThsB of a Type I Thoeris set forth in SEQ ID NO: 10.
  • the Tad5 polypeptide binds the active site of ThsB (and specifically forms a loop that penetrates the active site). According to specific embodiments, the Tad5 polypeptide forms a hydrogen bond with the catalytic E85 residue of ThsB set forth in SEQ ID NO: 10.
  • the Tad5 polypeptide inhibits activity of the defense system polypeptide ThsB of a Type I Thoeris set forth in SEQ ID NO: 10.
  • expression of the Tad5 polypeptide in a B. subtilis BEST7003 comprising SEQ ID NO: 1 increases sensitivity of B. subtilis BEST7003 to infection by a B. subtilis phage SBSphiJ.
  • the anti-defense system polypeptide is a Tad6 polypeptide.
  • a Tad6 polypeptide disables the anti -phage bacterial system Thoeris type I.
  • RMSD Root Mean Square Deviation
  • the Tad6 polypeptide has an e-value ⁇ 0.05 to a sequence of a protein having an amino acid sequence selected from the group consisting of SEQ ID NO: 65, 97-106, 5962-6369 and 11850-11888, each possibility represents a separate embodiment of the claimed invention.
  • the Tad6 polypeptide has a RMSD ⁇ 2.0 angstroms to a structure of a protein having an amino acid sequence selected from the group consisting of SEQ ID NO: 65, 97-106, 5962-6369 and 11850-11888, each possibility represents a separate embodiment of the claimed invention.
  • the Tad6 polypeptide of some embodiments has a sequence similarity defined by an e- value ⁇ 0.05 to SEQ ID NO: 65 and/or a structural similarity defined by a RMSD ⁇ 2.0 angstroms to the structure of SEQ ID NO: 65.
  • the Tad6 polypeptide comprises an arginine residue at position 92 corresponding to SEQ ID NO: 65.
  • the Tad6 polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 65, 97-106, 5962-6369 and 11850- 11888, each possibility represents a separate embodiment of the claimed invention.
  • the Tad6 polypeptide comprises SEQ ID NO: 65.
  • the Tad6 polypeptide consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 65, 97-106, 5962-6369 and 11850- 11888, each possibility represents a separate embodiment of the claimed invention.
  • the Tad6 polypeptide consists of SEQ ID NO: 65.
  • the Tad6 polypeptide binds the defense system polypeptide ThsB of a Type I Thoeris set forth in SEQ ID NO: 10.
  • the Tad6 polypeptide binds the active site of ThsB (and specifically forms a loop that penetrates the active site).
  • the Tad6 polypeptide forms a hydrogen bond with the catalytic E85 residue of ThsB set forth in SEQ ID NO: 10.
  • the Tad6 polypeptide inhibits activity of the defense system polypeptide ThsB of a Type I Thoeris set forth in SEQ ID NO: 10.
  • expression of the Tad6 polypeptide in a B. subtilis BEST7003 comprising SEQ ID NO: 1 increases sensitivity of B. subtilis BEST7003 to infection by a B. subtilis phage SBSphiJ.
  • the anti-defense system polypeptide is a Tad7 polypeptide.
  • a Tad7 polypeptide disables the anti -phage bacterial system Thoeris type II.
  • RMSD Root Mean Square Deviation
  • the Tad7 polypeptide has an e-value ⁇ 0.05 to a sequence of a protein having an amino acid sequence selected from the group consisting of SEQ ID NO: 239, 6370-6461 and 11889-11918, each possibility represents a separate embodiment of the claimed invention.
  • the Tad7 polypeptide has a RMSD ⁇ 2.0 angstroms to a structure of a protein having an amino acid sequence selected from the group consisting of SEQ ID NO: 239, 6370-6461 and 11889-11918, each possibility represents a separate embodiment of the claimed invention.
  • the Tad7 polypeptide of some embodiments has a sequence similarity defined by an e- value ⁇ 0.05 to SEQ ID NO: 239 and/or a structural similarity defined by a RMSD ⁇ 2.0 angstroms to the structure of SEQ ID NO: 239.
  • the Tad7 polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 239, 6370-6461 and 11889-11918, each possibility represents a separate embodiment of the claimed invention.
  • the Tad7 polypeptide comprises SEQ ID NO: 239.
  • the Tad7 polypeptide consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 239, 6370-6461 and 11889-11918, each possibility represents a separate embodiment of the claimed invention.
  • the Tad7 polypeptide consists of SEQ ID NO: 239.
  • the Tad7 polypeptide binds the defense system polypeptide ThsB of a Type II Thoeris set forth in SEQ ID NO: 228.
  • the Tad7 polypeptide binds the active site of ThsB (and specifically forms a loop that penetrates the active site).
  • the Tad7 polypeptide forms a bond with the catalytic E99 residue of ThsB set forth in SEQ ID NO: 228.
  • the Tad7 polypeptide inhibits activity of the defense system polypeptide ThsB of a Type I Thoeris set forth in SEQ ID NO: 228.
  • expression of the Tad7 polypeptide in a B. subtilis BEST7003 comprising SEQ ID NO: 2 increases sensitivity of said B. subtilis BEST7003 to infection by a B. subtilis phage SBSphiJ.
  • the anti-defense system polypeptide is a Tad8 polypeptide.
  • a Tad8 polypeptide disables the anti -phage bacterial system Thoeris type II.
  • the Tad8 polypeptide has an e-value ⁇ 0.05 to a sequence of a protein having an amino acid sequence selected from the group consisting of SEQ ID NO: 240, 6462-6616 and 11919-12290, each possibility represents a separate embodiment of the claimed invention.
  • the Tad8 polypeptide has a RMSD ⁇ 2.0 angstroms toa structure of a protein having an amino acid sequence selected from the group consisting of SEQ ID NO: 240, 6462-6616 and 11919-12290, each possibility represents a separate embodiment of the claimed invention.
  • the Tad8 polypeptide of some embodiments has a sequence similarity defined by an e- value ⁇ 0.05 to SEQ ID NO: 240 and/or a structural similarity defined by a RMSD ⁇ 2.0 angstroms to the structure of SEQ ID NO: 240.
  • the Tad8 polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 240, 6462-6616 and 11919-12290, each possibility represents a separate embodiment of the claimed invention.
  • the Tad8 polypeptide comprises SEQ ID NO: 240.
  • the Tad8 polypeptide consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 240, 6462-6616 and 11919-12290, each possibility represents a separate embodiment of the claimed invention.
  • the Tad8 polypeptide consists of SEQ ID NO: 240.
  • the Tad8 polypeptide binds the defense system polypeptide ThsA of a Type II Thoeris set forth in SEQ ID NO: 227.
  • the Tad8 polypeptide binds the active site of ThsA (and specifically forms a loop that penetrates the Macro domain of ThsA).
  • the Tad8 polypeptide forms a bond with a residue of the Macro domain pocked of ThsA selected from the group consisting of A202, R240, Q135 and Y277 of ThsA set forth in SEQ ID NO: 227. According to specific embodiments, the Tad8 polypeptide is capable of forming a homodimer.
  • the Tad8 polypeptide binds as a homodimer two monomers of ThsA.
  • the Tad8 polypeptide inhibits activity of the defense system polypeptide ThsA of a Type I Thoeris set forth in SEQ ID NO: 227.
  • expression of the Tad8 polypeptide in a B. subtilis BEST7003 comprising SEQ ID NO: 2 increases sensitivity of said B. subtilis BEST7003 to infection by a B. subtilis phage SBSphiJ.
  • the anti-defense system polypeptide is an Acb3 polypeptide.
  • An Acb3 polypeptide disables the anti-phage bacterial system CBASS (e.g. CBASS Type III).
  • the Acb3 polypeptide has an e-value ⁇ 0.05 to a sequence of a protein having an amino acid sequence selected from the group consisting of SEQ ID NO: 244 and 6617-7593, each possibility represents a separate embodiment of the claimed invention.
  • the Acb3 polypeptide has a RMSD ⁇ 2.0 angstroms to a structure of a protein having an amino acid sequence selected from the group consisting of SEQ ID NO: 244 and 6617-7593, each possibility represents a separate embodiment of the claimed invention.
  • the Acb3 polypeptide of some embodiments has a sequence similarity defined by an e- value ⁇ 0.05 to SEQ ID NO: 244 and/or a structural similarity defined by a RMSD ⁇ 2.0 angstroms to the structure of SEQ ID NO: 244.
  • the Acb3 polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 244 and 6617-7593, each possibility represents a separate embodiment of the claimed invention.
  • the Acb3 polypeptide comprises SEQ ID NO: 244.
  • the Acb3 polypeptide consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 244 and 6617-7593, each possibility represents a separate embodiment of the claimed invention.
  • the Acb3 polypeptide consists of SEQ ID NO: 244.
  • the Acb3 polypeptide binds the bacterial defense system polypeptide cGAS of CBASS set forth in amino acid sequence selected from the group consisting of SEQ ID NO: 229-231 and 233.
  • the Acb3 polypeptide binds the active site of cGAS.
  • the Acb3 polypeptide forms a bond with the nucleotidyltransferase active site and/or the putative ligand binding domain that is required for CD-NTase activation.
  • the Acb3 polypeptide inhibits activity of the defense system polypeptide cGAS of a CBASS set forth in amino acid sequence selected from the group consisting of SEQ ID NOs: 229-231 and 233.
  • expression of the Acb3 polypeptide in a E. coil MG1655 comprising SEQ ID NO: 4 increases sensitivity of the E. coll MG 1655 to infection by an E. coli phage BAS 18.
  • expression of the Acb3 polypeptide in a B. subtilis BEST7003 comprising SEQ ID NO: 3 increases sensitivity of the B. subtilis BEST7003 to infection by a B. subtilis phage SBSphiC.
  • the Acb3 polypeptide binds a homolog of the defense system polypeptide cGAS of a CBASS set forth in amino acid sequence selected from the group consisting of SEQ ID NOs: 229-231 and 233. According to specific embodiments, the Acb3 polypeptide inhibits activity of a homolog of the defense system polypeptide cGAS of CBASS set forth in amino acid sequence selected from the group consisting of SEQ ID NOs: 229-231 and 233.
  • the homolog is cGAS of a Type I CBASS set forth SEQ ID NO: 234.
  • the homolog is human cGAS of a CBASS set forth SEQ ID NO: 12291.
  • the “Tad3 polypeptide”, “Tad4 polypeptide”, “Tad5 polypeptide”, “Tad6 polypeptide”, “Tad7 polypeptide”, “Tad8 polypeptide” and/or “Acb3 polypeptide” of some embodiments of the invention may comprise a homolog, an ortholog, a deletion, insertion, or substitution variant, including an amino acid substitution of the recited amino acid sequence.
  • the “Tad3 polypeptide”, “Tad4 polypeptide”, “Tad5 polypeptide”, “Tad6 polypeptide”, “Tad7 polypeptide”, “Tad8 polypeptide” and/or “Acb3 polypeptide” comprises the recited amino acid sequence.
  • the “Tad3 polypeptide”, “Tad4 polypeptide”, “Tad5 polypeptide”, “Tad6 polypeptide”, “Tad7 polypeptide”, “Tad8 polypeptide” and/or “Acb3 polypeptide” consists of the recited amino acid sequence.
  • the terms “Tad3 polypeptide”, “Tad4 polypeptide”, “Tad5 polypeptide”, “Tad6 polypeptide”, “Tad7 polypeptide”, “Tad8 polypeptide” and/or “Acb3 polypeptide” refer to a fragment of the amino acid sequence of “Tad3 polypeptide”, “Tad4 polypeptide”, “Tad5 polypeptide”, “Tad6 polypeptide”, “Tad7 polypeptide”, “Tad8 polypeptide” and/or “Acb3 polypeptide”, respectively, provided herein, which maintains the activity as described herein.
  • “Tad3 polypeptide”, “Tad4 polypeptide”, “Tad5 polypeptide”, “Tad6 polypeptide”, “Tad7 polypeptide”, “Tad8 polypeptide” and/or “Acb3 polypeptide” is up to 800, up to 700, up to 600, up to 500, up to 400, up to 300 or up to 200 amino acids long.
  • “Tad3 polypeptide”, “Tad4 polypeptide”, “Tad5 polypeptide”, “Tad6 polypeptide”, “Tad7 polypeptide”, “Tad8 polypeptide” and/or “Acb3 polypeptide” is at least 50, at least 100, at least 150, at least 200, at least 250 amino acids long.
  • embodiments of the present invention also contemplate viruses genetically modified to express these polypeptide, cells comprising these polypeptides, methods of producing same and methods of use thereof.
  • a virus comprising an exogenous polynucleotide encoding an anti-defense system polypeptide, wherein said antidefense system polypeptide is selected from the group consisting of:
  • a genetically modified virus comprising a polynucleotide encoding an anti-defense system polypeptide as described herein.
  • a virus will be characterized by: 1) the nature of the nucleic acids that make up its genome, e.g., DNA, RNA, single-stranded or double- stranded; 2) the nature of its infectivity, e.g., lytic or temperate; and 3) the particular cell species that it infects (and in certain instances the particular subspecies or strain). This is known as “host range”.
  • the viral genome can be ssDNA or dsDNA.
  • the viral genome can be ssRNA or dsRNA.
  • the virus is a lytic virus.
  • lytic virus refers to a virus that infects a host cell and causes that host cell to lyse without incorporating the virus nucleic acids into the host genome.
  • a lytic virus is typically not capable of reproducing using the lysogenic cycle.
  • the virus is temperate (also referred to as lysogenic).
  • emperate virus refers to a virus that is capable of reproducing using both the lysogenic cycle and the lytic cycle. Lysogeny is characterized by integration of the virus nucleic acid into the host cell’s genome or formation of a circular replicon in the cytoplasm.
  • the virus infects eukaryotic cells.
  • Non-limiting examples of such viruses include retroviruses, circoviruses, parvoviruses, papovaviruses, adenoviruses, adeno- associated virus, herpesviruses, iridoviruses, poxviruses, hepadnaviruses, picomaviruses, caliciviruses, togaviruses, flaviviruses, reoviruses, orthomyxoviruses, paramyxoviruses, rhabdoviruses, bunyaviruses, coronaviruses, arenaviruses, filoviruses, lentivirus, baculovirus, vesicular stomatitis virus.
  • viruses encompassed by the present invention include human immunodeficiency virus (HlV)-induced acquired immunodeficiency syndrome (AIDS), influenza, rhinoviral infection, viral meningitis, Epstein-Barr virus (EBV) infection, hepatitis A, B or C virus infection, measles, papilloma virus infection/warts, cytomegalovirus (CMV) infection, Herpes simplex virus infection, yellow fever, Ebola virus infection, rabies, etc.
  • HlV human immunodeficiency virus
  • AIDS human immunodeficiency virus
  • EBV Epstein-Barr virus
  • CMV cytomegalovirus
  • Herpes simplex virus infection yellow fever
  • Ebola virus infection rabies, etc.
  • the virus infects prokaryotic cells.
  • the virus is a phage.
  • phage or “bacteriophage” refers to a virus that selectively infects one or more bacterial species. Many phages are specific to a particular genus or species or strain of bacteria.
  • phages that infect bacteria that are pathogenic to plants and/or animals find particular use.
  • Exemplary phages which fall under the scope of some embodiments of the invention include, but are not limited to, phages that belong to any of the following virus families: Corticoviridae, Cystoviridae, Inoviridae, Leviviridae, Microviridae, Myoviridae, Podoviridae, Siphoviridae, or Tectiviridae.
  • the phage is isolated and/or developed to target a specific bacteria. Isolation and characterization of such phages can be done, for example, as described in Hayman Pharmaceuticals (Basel) (2019) 12(1): 35, DOI: 10.3390/phl2010035 and https://doi(dot)org/10.1016/j. cels.2015.08.013, the contents of which are fully incorporated herein by reference.
  • the phage infects a pathogenic bacteria (i.e. a bacteria that can cause or be associated with a disease in humans, livestock, crops, or other living organism).
  • a pathogenic bacteria i.e. a bacteria that can cause or be associated with a disease in humans, livestock, crops, or other living organism.
  • the virus e.g. phage
  • the virus is a clinically approved phage.
  • the virus is FDA approved for clinical use.
  • the phage is FDA approved for treatment of an infectious bacterium.
  • the phages of some embodiments of the invention comprise an exogenous or a heterologous polynucleotide encoding an anti-defense system polypeptide.
  • polynucleotide or “nucleic acid sequence”, which are interchangeably used herein, refers to a single or double stranded nucleic acid sequence provided e.g., in the form of an RNA sequence, a DNA and/or a composite polynucleotide sequences (e.g., a combination of the above).
  • a non-limiting example of a polynucleotide encoding Tad3 is provided in SEQ ID NO:
  • a non-limiting example of a polynucleotide encoding Tad4 is provided in SEQ ID NO: 50.
  • a non-limiting example of a polynucleotide encoding Tad5 is provided in SEQ ID NO:
  • a non-limiting example of a polynucleotide encoding Tad6 is provided in SEQ ID NO: 49.
  • a non-limiting example of a polynucleotide encoding Tad7 is provided in SEQ ID NO:
  • a non-limiting example of a polynucleotide encoding Tad8 is provided in SEQ ID NO:
  • a non-limiting example of a polynucleotide encoding Acb3 is provided in SEQ ID NO: 243.
  • the polynucleotide encoding the “Tad3 polypeptide”, “Tad4 polypeptide”, “Tad5 polypeptide”, “Tad6 polypeptide”, “Tad7 polypeptide”, “Tad8 polypeptide” or “Acb3 polypeptide” is at least 70 %, at least 75 %, at least 80 %, at least 81 %, at least 82 %, at least 83 %, at least 84 %, at least 85 %, at least 86 %, at least 87 %, at least 88 %, at least 89 %, at least 90 %, at least 91 %, at least 92 %, at least 93 %, at least 94 %, at least 95 %, at least 96 %, at least 97 %, at least 98 %, at least 99 % or 100 % identical or homologous to the SEQ ID NOs: 42, 50, 43, 49,
  • nucleic acid sequences encoding of the polypeptides of some embodiments of the invention may be optimized for expression in a specific organism.
  • the polynucleotide of the present invention encodes no more than 20, no more than 15, no more than 10 genes expression products.
  • the polynucleotide encodes one of the anti-defense polypeptides (i)-(vii) described herein.
  • the polynucleotide comprises a nucleic acid sequence encoding one of the anti-defense polypeptides (i)-(vii) described herein, whereby a plurality of polynucleotides can be used to assemble several anti-defense polypeptides, as described below.
  • a single polynucleotide encodes at least two, at least three, at least four, at least five or six of the anti-defense polypeptides (i)-(vii) described herein. Further description on expression of multiple polypeptides from a single polynucleotide is provided hereinbelow.
  • heterologous or “exogenous” means the sequence (polynucleotide or polypeptide) is not natively expressed in at least localization (e.g. genomic, cellular compartment and/or cell type) or level/amount or is completely absent from the native counterpart.
  • endogenous means expression of the native sequence (polynucleotide or polypeptide) in its natural location and expression level.
  • the virus is devoid of an endogenous polynucleotide encoding the anti-defense polypeptide.
  • virus of some embodiments of the invention is genetically modified to express the anti-defense system polypeptide.
  • expressing refers to expression at the nucleic acid and/or protein level. Expression can be determined using methods known in the art e.g. but not limited to selectable marker gene, Northern blot analysis, PCR analysis, DNA sequencing, RNA sequencing, Western blot analysis, and Immunohistochemistry.
  • Non-limiting Examples include, homologous recombination, bacteriophage recombineering of electroporated DNA (BRED), CRISPR-Cas-based engineering and rebooting viruses using assembled vims genomic DNA.
  • One Exemplary' method of obtaining the genetically modified virus is by recombination- mediated genetic exchange between at least 2 distinct viruses, as described for example in International Patent Application Publication No. W02023/100189, the contents of which are fully incorporated herein by reference.
  • the vims (e.g. phage) comprises genomic segments of a distinct virus (e.g. phage) integrated in a genome of said vims (e.g. phage).
  • these segments are about 1000 - 60,000, 2000 - 50,000 or 10,000 - 30, 000 long.
  • these segments constitute about 0.5 - 50 % of the viral genome. According to specific embodiments, these segments constitute less than 40 %, less than 30 %, less than 20 %, less than 10 % of the viral genome.
  • these segments constitute more than 1%, more than 5 %, more than 10 %, more than 20 % of the viral genome.
  • these segments constitute less than 1% of the viral genome.
  • the virus may also be genetically engineered to change its host range, convert a temperate phage to a lytic phage, encode an anti-defense system polypeptide and the like.
  • Non-limiting examples of modifications that can be introduced to a virus include genetic engineering of host-range-determining regions (HRDRs) in the tail fiber protein [see e.g. Yehl et al. Cell (2019) 179(2):459-469.e9 doi: 10.1016/j.cell.2019.09.015, the contents of which are fully incorporated herein by reference] or receptor-binding proteins [see e.g. Lenneman et al. Curr Opin Biotechnol (2021) 68: 151-159 doi: 10.1016/j.copbio.2020.11.003, the contents of which are fully incorporated herein by reference].
  • HRDRs host-range-determining regions
  • the vims disclosed herein has an increased infectivity to at least one cell as compared to a virus of the same species not comprising said exogenous polynucleotide.
  • the phage disclosed herein has an increased infectivity to at least one bacteria as compared to a phage of the same species or strain not comprising said exogenous polynucleotide.
  • Various modalities may be used to introduce or express an exogenous or heterologous polynucleotide encoding the anti-defense polypeptide in the virus, as further described hereinabove and below.
  • a method of producing a virus comprising introducing into a virus an exogenous polynucleotide encoding the anti-defense system polypeptide, thereby producing the virus.
  • the method comprises introducing the polynucleotide into the virus, under conditions which allow integration of the polynucleotide in a genome of the virus.
  • Various methods known within the art can be used to introduce the polynucleotides into a virus or a cell, typically involving contacting (e.g. the virus with the polynucleotide, the cell with the polynucleotide, the cell with the virus etc.) such as by way of genetic engineering.
  • contacting e.g. the virus with the polynucleotide, the cell with the polynucleotide, the cell with the virus etc.
  • Such methods are described for example in Gibb et al. (2021) Pharmaceuticals, 14, 634; Chen et al. (2019) Front. Microbiol. 10:954; and Rustad, et al. (2016) Synthetic Biology, 3(1) ysy002, doi(dot)org/10.1093/synbio/ysy002, the contents of which are fully incorporated herein by reference.
  • the polynucleotides described herein are part of a nucleic acid construct (also referred to herein as an "expression vector” or a “vector”) which facilitates expression of the polynucleotide in a cell, integration of the polynucleotide in a genome of a virus or a cell, and/or selection or detection. Additional description on nucleic acid constructs is provided hereinbelow.
  • the present invention also contemplates cells comprising the anti-defense system polypeptide described herein.
  • a cell comprising an exogenous anti-defense system polypeptide or a polynucleotide encoding same, wherein said anti-defense system polypeptide is selected from the group consisting of:
  • cell refers to a prokaryotic or a eukaryotic cell.
  • Non-limiting examples of cells that can be used in some embodiments of the present invention include, but are not limited to, bacteria, yeast, plant cell or an animal cell.
  • the cell is a prokaryotic cell.
  • the cell is a bacteria.
  • the cell is an eukaryotic cell.
  • the cell is a plant cell.
  • the cell is an animal cell.
  • the cell is a mammalian cell.
  • the cell is a human cell.
  • the cell is a differentiated cell.
  • the cell is a stem or progenitor cell.
  • the cell is a primary cell.
  • the cell is a cell line.
  • cell lines include a Chinese Hamster Ovary (CHO), HEK293, PER.C6, HT1080, NSO, Sp2/0, BHK, Namalwa, COS, HeLa and Vero cell.
  • the cell may be derived from a suitable tissue including but not limited to blood, muscle, nerve, brain, heart, lung, liver, pancreas, spleen, thymus, esophagus, stomach, intestine, kidney, testis, ovary, hair, skin, bone, breast, uterus, bladder, spinal cord, or various kinds of body fluids.
  • a suitable tissue including but not limited to blood, muscle, nerve, brain, heart, lung, liver, pancreas, spleen, thymus, esophagus, stomach, intestine, kidney, testis, ovary, hair, skin, bone, breast, uterus, bladder, spinal cord, or various kinds of body fluids.
  • the cell expresses the defense system polypeptide or a homolog thereof.
  • the cell comprises one of the anti-defense polypeptides (i)-(vii) described herein or polynucleotides encoding same.
  • the cell comprises at least two, at least three, at least four, at least five or six of the anti-defense polypeptides (i)-(vii) described herein or polynucleotide encoding same.
  • the cell has increased sensitivity to infection by at least one virus as compared to a cell of the same species not comprising the exogenous antidefense system polypeptide or polynucleotide encoding same.
  • the cell has an impaired ability to respond to stress as compared to a cell of the same species not comprising the exogenous anti-defense system polypeptide or polynucleotide encoding same.
  • a method of impairing ability of a cell to respond to stress comprising introducing into the cell an anti-defense system polypeptide or a polynucleotide encoding same, wherein said anti-defense system polypeptide is selected from the group consisting of:
  • Various modalities may be used to introduce or express an exogenous anti-defense polypeptide or a polynucleotide encoding same in the cell, as further described hereinabove and below. It will be appreciated that the cell may be comprised inside a particular organism, for example inside a mammalian body or inside a plant.
  • the introducing or the contacting is effected in-vivo.
  • the introducing or the contacting is effected in- vitro or ex- vivo.
  • a method of producing a cell comprising introducing into a cell the anti-defense system polypeptide or the polynucleotide encoding same, thereby producing the cell.
  • the method comprises introducing into the cell the anti-defense system polypeptide.
  • peptide and “polypeptide”, which are interchangeably used, encompass native peptides (either degradation products, synthetically synthesized peptides or recombinant peptides) and peptidomimetics (typically, synthetically synthesized peptides), as well as peptoids and semipeptoids which are peptide analogs, which may have, for example, modifications rendering the peptides more stable while in a body or more capable of penetrating into cells. Such modifications include, but are not limited to N terminus modification, C terminus modification, peptide bond modification, backbone modifications, and residue modification.
  • Natural aromatic amino acids, Trp, Tyr and Phe may be substituted by non-natural aromatic amino acids such as l,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (Tic), naphthylalanine, ring-methylated derivatives of Phe, halogenated derivatives of Phe or O- methyl-Tyr.
  • the peptides of some embodiments of the invention may also include one or more modified amino acids or one or more non-amino acid monomers (e.g. fatty acids, complex carbohydrates etc).
  • modified amino acids e.g. fatty acids, complex carbohydrates etc.
  • amino acid or “amino acids” is understood to include the 20 naturally occurring amino acids; those amino acids often modified post-translationally in vivo, including, for example, hydroxyproline, phosphoserine and phospho threonine; and other unusual amino acids including, but not limited to, 2-aminoadipic acid, hydroxylysine, isodesmosine, nor-valine, nor-leucine and ornithine.
  • amino acid includes both D- and L-amino acids.
  • amino acids of the peptides disclosed herein may be substituted either conservatively or non-conservatively.
  • conservative substitution refers to the replacement of an amino acid present in the native sequence in the peptide with a naturally or non-naturally occurring amino or a peptidomimetics having similar steric properties.
  • side-chain of the native amino acid to be replaced is either polar or hydrophobic
  • the conservative substitution should be with a naturally occurring amino acid, a non-naturally occurring amino acid or with a peptidomimetic moiety which is also polar or hydrophobic (in addition to having the same steric properties as the side-chain of the replaced amino acid).
  • amino acid analogs synthetic amino acids
  • a peptidomimetic of the naturally occurring amino acid is well documented in the literature known to the skilled practitioner.
  • the substituting amino acid should have the same or a similar functional group in the side chain as the original amino acid.
  • non-conservative substitutions refers to replacement of the amino acid as present in the parent sequence by another naturally or non-naturally occurring amino acid, having different electrochemical and/or steric properties.
  • the side chain of the substituting amino acid can be significantly larger (or smaller) than the side chain of the native amino acid being substituted and/or can have functional groups with significantly different electronic properties than the amino acid being substituted.
  • non-conservative substitutions of this type include the substitution of phenylalanine or cycohexylmethyl glycine for alanine, isoleucine for glycine, or -NH-CH [(-CH2)5-COOH] -CO- for aspartic acid.
  • Those non-conservative substitutions which fall under the scope of the present invention are those which still constitute a peptide having anti-bacterial properties.
  • N and C termini of the peptides of the present invention may be protected by function groups.
  • Suitable functional groups are described in Green and Wuts, "Protecting Groups in Organic Synthesis", John Wiley and Sons, Chapters 5 and 7, 1991, the teachings of which are incorporated herein by reference.
  • Preferred protecting groups are those that facilitate transport of the compound attached thereto into a cell, for example, by reducing the hydrophilicity and increasing the lipophilicity of the compounds.
  • a polypeptide into a cell typically involving contacting (e.g. the cell with the polypeptide or composition comprising same).
  • the peptides of the present invention may be attached (either covalently or non-covalently) to- or encapsulated by- a cell penetrating moiety.
  • cell penetrating moiety refers to a heterologous moiety which enhances translocation of the polypeptide across a cell membrane.
  • Non-limiting examples of cell penetrating moieties include cell penetrating peptides and lipid particles.
  • the polypeptide may be incorporated into a particulated delivery vehicle, e.g., a liposome, or a nano- or microparticle, by any of the methods known in the art [e.g. Liposome Technology, Vol. II, Incorporation of Drugs, Proteins, and Genetic Material, CRC Press; Monkkonen, J. et al., 1994, J. Drug Target, 2:299-308; Monkkonen, J. et al., 1993, Calcif. Tissue Int., 53: 139-145; Lasic D D., Liposomes Technology Inc., Elsevier, 1993, 63-105.
  • a particulated delivery vehicle e.g., a liposome, or a nano- or microparticle
  • Liposomes include any synthetic (i.e., not naturally occurring) structure composed of lipid bilayers, which enclose a volume. Liposomes include emulsions, foams, micelles, insoluble monolayers, liquid crystals, phospholipid dispersions, lamellar layers and the like. Liposomes can be of different sizes, may contain a low or a high pH and may be of different charge.
  • the cell penetrating moiety is a cell penetrating peptide.
  • peptide penetrating agents have an amino acid composition containing either a high relative abundance of positively charged amino acids such as lysine or arginine, or have sequences that contain an alternating pattern of polar/charged amino acids and non-polar, hydrophobic amino acids.
  • CPPs that can penetrate cells in a non-toxic and efficient manner and may be suitable for use in accordance with some embodiments of the invention include TAT (transcription activator from HIV-1), pAntp (also named penetratin, Drosophila antennapedia homeodomain transcription factor) and VP22 (from Herpes Simplex virus).
  • Protocols for producing CPPs-cargos conjugates and for infecting cells with such conjugates can be found, for example L Theodore et al. [The Journal of Neuroscience, (1995) 15(11): 7158- 7167], Fawell S, et al. [Proc Natl Acad Sci USA, (1994) 91:664-668], and Jing Bian et al. [Circulation Research. (2007) 100: 1626-1633].
  • the peptides of the present invention may also comprise non-amino acid moieties, such as for example, hydrophobic moieties (various linear, branched, cyclic, polycyclic or hetrocyclic hydrocarbons and hydrocarbon derivatives) attached to the peptides; non-peptide penetrating agents; various protecting groups, especially where the compound is linear, which are attached to the compound’s terminals to decrease degradation.
  • non-amino acid moieties such as for example, hydrophobic moieties (various linear, branched, cyclic, polycyclic or hetrocyclic hydrocarbons and hydrocarbon derivatives) attached to the peptides; non-peptide penetrating agents; various protecting groups, especially where the compound is linear, which are attached to the compound’s terminals to decrease degradation.
  • Chemical (non-amino acid) groups present in the compound may be included in order to improve various physiological properties such; decreased degradation or clearance; decreased repulsion by various cellular pumps, improve immunogenic activities, improve various modes of administration (such as attachment of various sequences which allow penetration through various barriers, through the gut, etc.); increased specificity, increased affinity, decreased toxicity and the like.
  • Attaching the amino acid sequence component of the peptides of the invention to other non- amino acid agents may be by covalent linking, by non-covalent complexion, for example, by complexion to a hydrophobic polymer, which can be degraded or cleaved producing a compound capable of sustained release; by entrapping the amino acid part of the peptide in liposomes or micelles to produce the final peptide of the invention.
  • the association may be by the entrapment of the amino acid sequence within the other component (liposome, micelle) or the impregnation of the amino acid sequence within a polymer to produce the final peptide of the invention.
  • polypeptides of some embodiments of the invention may be synthesized and purified by any techniques known to those skilled in the art of peptide synthesis, such as, but not limited to, solid phase techniques and recombinant techniques such as further described herein.
  • the method comprises introducing into the cell a polynucleotide encoding the anti-defense system polypeptide, under conditions which allow expression of the anti-defense polypeptide in the cell.
  • the polynucleotides described herein are part of a nucleic acid construct (also referred to herein as an "expression vector” or a “vector”) which facilitates expression of the polynucleotide in a cell, integration of the polynucleotide in a genome of a virus or a cell, and/or selection or detection.
  • a nucleic acid construct also referred to herein as an "expression vector” or a “vector” which facilitates expression of the polynucleotide in a cell, integration of the polynucleotide in a genome of a virus or a cell, and/or selection or detection.
  • nucleic acid construct and “expression vector” refer to a nucleic acid vector designed to introduce specific expression products of interest (i.e. polynucleotides) in a virus or a cell.
  • the expression can be transient or consistent, episomal or integrated into the chromosome.
  • the expression is on a transmissible genetic element such as a plasmid.
  • the nucleic acid construct of some embodiments of the invention includes additional sequences which render this vector suitable for replication and integration in prokaryotes, eukaryotes, or preferably both (e.g., shuttle vectors).
  • a typical cloning vector may contain regulatory elements e.g. promoters, enhancers, transcription and translation initiation sequence, transcription and translation terminator, polyadenylation signal transcription termination signals, a multiple cloning site (MCS) etc.
  • nucleic acid construct comprising a polynucleotide encoding an anti-defense polypeptide selected from the group consisting of:
  • the nucleic acid sequence heterologous to the polynucleotide encoding the anti-defense polypeptide is a cis-acting regulatory element for directing expression of the polynucleotide in a cell.
  • Cis-acting regulatory sequences include those that direct constitutive expression of a nucleotide sequence as well as those that direct inducible expression of the polynucleotide only under certain conditions.
  • a promoter sequence for directing transcription of the polynucleotide sequence in the cell in a constitutive or inducible manner is included in the nucleic acid construct.
  • the promoter is active in a specific cell population transformed.
  • Eukaryotic promoters typically contain two types of recognition sequences, the TATA box and upstream promoter elements.
  • the TATA box located 25-30 base pairs upstream of the transcription initiation site, is thought to be involved in directing RNA polymerase to begin RNA synthesis.
  • the other upstream promoter elements determine the rate at which transcription is initiated.
  • Enhancer elements can stimulate transcription up to 1,000 fold from linked homologous or heterologous promoters. Enhancers are active when placed downstream or upstream from the transcription initiation site. Many enhancer elements derived from viruses have a broad host range and are active in a variety of tissues. For example, the SV40 early gene enhancer is suitable for many cell types. Other enhancer/promoter combinations that are suitable for some embodiments of the invention include those derived from polyoma virus, human or murine cytomegalovirus (CMV), the long term repeat from various retroviruses such as murine leukemia virus, murine or Rous sarcoma virus and HIV. See, Enhancers and Eukaryotic Expression, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. 1983, which is incorporated herein by reference.
  • CMV cytomegalovirus
  • Constitutive promoters suitable for use with some embodiments of the invention are promoter sequences which are active under most environmental conditions and most types of cells such as the cytomegalovirus (CMV) and Rous sarcoma virus (RSV).
  • CMV cytomegalovirus
  • RSV Rous sarcoma virus
  • constitutive promoters suitable for use with some embodiments of the invention include T7, Sp6 and T3.
  • inducible promoters suitable for use with some embodiments of the invention include the tetracycline-inducible promoter (Zabala M, et al., Cancer Res. 2004, 64(8): 2799-804), the arabinose metabolic operon promoter (araBAD) or pathogen-inducible promoters.
  • Such promoters include those from pathogenesis-related proteins (PR proteins), which are induced following infection by a pathogen.
  • the promoter is a bacterial promoter.
  • a bacterial promoter is any DNA sequence capable of binding bacterial RNA polymerase and initiating the downstream (3') transcription of a coding sequence into mRNA.
  • a promoter can have a transcription initiation region, which is usually placed proximal to the 5' end of the coding sequence. This transcription initiation region typically includes an RNA polymerase binding site and a transcription initiation site.
  • a bacterial promoter can also have a second domain called an operator, which can overlap an adjacent RNA polymerase binding site at which RNA synthesis begins. The operator permits negative regulated (inducible) transcription, as a gene repressor protein can bind the operator and thereby inhibit transcription of a specific gene. Constitutive expression can occur in the absence of negative regulatory elements, such as the operator.
  • positive regulation can be achieved by a gene activator protein binding sequence, which, if present is usually proximal (5') to the RNA polymerase binding sequence.
  • a gene activator protein is the catabolite activator protein (CAP), which helps initiate transcription of the lac operon in Escherichia coli (Raibaud et al. (1984) Annu. Rev. Genet. 18: 173). Regulated expression can therefore be either positive or negative, thereby either enhancing or reducing transcription. Other examples of positive and negative regulatory elements are well known in the art.
  • Various promoters that can be included in the protein expression system include, but are not limited to, a T7/LacO hybrid promoter, a trp promoter, a T7 promoter, a lac promoter, and a bacteriophage lambda promoter.
  • Sequences encoding metabolic pathway enzymes provide particularly useful promoter sequences. Examples include promoter sequences derived from sugar metabolizing enzymes, such as galactose, lactose (lac) (Chang et al. (1987) Nature 198: 1056), and maltose. Additional examples include promoter sequences derived from biosynthetic enzymes such as tryptophan (trp) (Goeddel et al. (1980) Nucleic Acids Res. 8:4057; Yelverton et al. (1981) Nucleic Acids Res. 9:731; U.S. Pat. No. 4,738,921; EPO Publication Nos. 36,776 and 121,775).
  • trp tryptophan
  • the betalactamase (bla) promoter system (Weissmann, (1981) "The Cloning of Interferon and Other Mistakes," in Interferon 3 (ed. I. Gresser); bacteriophage lambda PL (Shimatake et al. (1981) Nature 292: 128); the arabinose-inducible araB promoter (U.S. Pat. No. 5,028,530); and T5 (U.S. Pat. No. 4,689,406) promoter systems also provide useful promoter sequences. See also Baibas (2001) Mol. Biotech. 19:251-267, where E. coli expression systems are discussed. In addition, synthetic promoters that do not occur in nature also function as bacterial promoters.
  • transcription activation sequences of one bacterial or phage promoter can be joined with the operon sequences of another bacterial or phage promoter, creating a synthetic hybrid promoter (U.S. Pat. No. 4,551,433).
  • the tac (Amann et al. (1983) Gene 25: 167; de Boer et al. (1983) Proc. Natl. Acad. Sci. 80:21) and trc (Brosius et al. (1985) J. Biol. Chem. 260:3539-3541) promoters are hybrid trp-lac promoters comprised of both trp promoter and lac operon sequences that are regulated by the lac repressor.
  • the tac promoter has the additional feature of being an inducible regulatory sequence.
  • expression of a coding sequence operably linked to the tac promoter can be induced in a cell culture by adding isopropyl- l-thio-.beta.-D-galactoside (IPTG).
  • IPTG isopropyl- l-thio-.beta.-D-galactoside
  • a bacterial promoter can include naturally occurring promoters of non-bacterial origin that have the ability to bind bacterial RNA polymerase and initiate transcription.
  • a naturally occurring promoter of non- bacterial origin can also be coupled with a compatible RNA polymerase to produce high levels of expression of some genes in prokaryotes.
  • the phage T7 RNA polymerase/promoter system is an example of a coupled promoter system (Studier et al.
  • a hybrid promoter can also be comprised of a phage promoter and an E. coli operator region (EPO Publication No. 267,851).
  • the promoter is preferably positioned approximately the same distance from the heterologous transcription start site as it is from the transcription start site in its natural setting. As is known in the art, however, some variation in this distance can be accommodated without loss of promoter function.
  • the nucleic acid construct is designed to allow integration of the polynucleotide in a location enabling expression of the polynucleotide from a native promoter.
  • the nucleic acid construct is devoid of a promoter.
  • the polynucleotide sequence is typically not inserted inside an existing viral open reading frame.
  • the nucleic acid construct can additionally contain a nucleic acid sequence encoding the repressor (or inducer) for the promoter.
  • a nucleic acid sequence encoding the repressor (or inducer) for the promoter can regulate transcription from the Lac operator (LacO) by expressing the nucleotide sequence encoding the LacI repressor protein.
  • Other examples include the use of the lexA gene to regulate expression of pRecA, and the use of trpO to regulate ptrp.
  • Alleles of such genes that increase the extent of repression (e.g., laclq) or that modify the manner of induction (e.g., lambda CI857, rendering lambda pL thermo-inducible, or lambda CI+, rendering lambda pL chemo-inducible) can be employed.
  • the construct encodes a polycistronic mRNA comprising the polynucleotides of the present invention; that is the polynucleotides can be cotranscribed as a polycistronic message from a single promoter sequence of the nucleic acid construct.
  • the different polynucleotide segments can be transcriptionally fused via a linker sequence including an internal ribosome entry site (IRES) sequence which enables the translation of the polynucleotide segment downstream of the IRES sequence.
  • IRES internal ribosome entry site
  • a transcribed polycistronic RNA molecule including the coding sequences of different combinations of the polynucleotides of the present invention will be translated from both the capped 5' end and the internal IRES sequence of the polycistronic RNA molecule.
  • the construct has an operon structure.
  • each two nucleic acid sequence segments can be translationally fused via a protease recognition site cleavable by a protease expressed by the cell to be transformed with the nucleic acid construct.
  • a chimeric polypeptide translated will be cleaved by the cell expressed protease.
  • the nucleic acid construct of some embodiments of the invention can include at least two Cis acting regulatory elements (e.g. promoter) each being for separately expressing a distinct polynucleotide. These at least two Cis acting regulatory elements can be identical or distinct.
  • the nucleic acid sequence heterologous to the polynucleotide encoding the anti-defense system polypeptide is an element for expression of multiple polynucleotides from a single construct.
  • the nucleic acid sequence heterologous to the polynucleotide encoding the anti-defense system polypeptide is a transmissible genetic element.
  • the polynucleotide is on a transmissible genetic element
  • a transmissible element comprising a polynucleotide encoding the anti-defense system polypeptide.
  • the term “transmissible element” or “transmissible genetic element”, which are interchangeably used, refers to a nucleic acid sequence that allows the transfer of the polynucleotide from one cell to another, e.g. a plasmid.
  • the nucleic acid sequence heterologous to the polynucleotide encoding the anti-defense system polypeptide is a recombination element.
  • the nucleic acid construct comprises a plurality of cloning sites for ligating a nucleic acid sequence of the invention such that it is under transcriptional regulation of the regulatory elements.
  • the expression vector of some embodiments of the invention may typically contain other specialized elements intended to increase the level of expression of cloned nucleic acids or to facilitate the identification of cells that carry the recombinant DNA.
  • a number of animal viruses contain DNA sequences that promote the extra chromosomal replication of the viral genome in permissive cell types. Plasmids bearing these viral replicons are replicated episomally as long as the appropriate factors are provided by genes either carried on the plasmid or with the genome of the host cell.
  • the vector may or may not include a eukaryotic replicon. If a eukaryotic replicon is present, then the vector is amplifiable in eukaryotic cells using the appropriate selectable marker. If the vector does not comprise a eukaryotic replicon, no episomal amplification is possible. Instead, the recombinant DNA integrates into the genome of the engineered cell, where the promoter directs expression of the desired nucleic acid.
  • selectable markers include those which confer resistance to drugs such as ampicillin, chloramphenicol, erythromycin, kanamycin (neomycin), and tetracycline (Davies et al. (1978) Annu. Rev. Microbiol. 32:469). Selectable markers can also allow a cell to grow on minimal medium, or in the presence of toxic metabolite and can include biosynthetic genes, such as those in the histidine, tryptophan, and leucine biosynthetic pathways.
  • the expression construct of some embodiments of the invention can also include sequences engineered to enhance stability, production, purification, yield or toxicity of the expressed polypeptide.
  • nucleic acid sequences may be optimized for increased expression in the transformed organism.
  • the nucleic acid sequences can be synthesized using preferred codons for improved expression.
  • mammalian expression vectors include, but are not limited to, pcDNA3, pcDNA3.1(+/-), pGL3, pZeoSV2(+/-), pSecTag2, pDisplay, pEF/myc/cyto, pCMV/myc/cyto, pCR3.1, pSinRep5, DH26S, DHBB, pNMTl, pNMT41, pNMT81, which are available from Invitrogen, pCI which is available from Promega, pMbac, pPbac, pBK-RSV and pBK-CMV which are available from Strategene, pTRES which is available from Clontech, and their derivatives.
  • bacterial constructs include the pET series of E. coli expression vectors [Studier et al. (1990) Methods in Enzymol. 185:60-89).
  • yeast a number of vectors containing constitutive or inducible promoters can be used, as disclosed in U.S. Pat. Application No: 5,932,447.
  • vectors can be used which promote integration of foreign DNA sequences into the yeast chromosome.
  • the expression of the coding sequence can be driven by a number of promoters.
  • viral promoters such as the 35S RNA and 19S RNA promoters of CaMV [Brisson et al. (1984) Nature 310:511-514], or the coat protein promoter to TMV [Takamatsu et al. (1987) EMBO J. 3:17-311] can be used.
  • plant promoters such as the small subunit of RUBISCO [Coruzzi et al. (1984) EMBO J.
  • Expression vectors containing regulatory elements from eukaryotic viruses such as retroviruses can be also used.
  • SV40 vectors include pSVT7 and pMT2.
  • Vectors derived from bovine papilloma virus include pBV-lMTHA, and vectors derived from Epstein Bar virus include pHEBO, and p2O5.
  • exemplary vectors include pMSG, pAV009/A + , pMTO10/A + , pMAMneo-5, baculovirus pDSVE, and any other vector allowing expression of proteins under the direction of the SV-40 early promoter, SV-40 later promoter, metallo thionein promoter, murine mammary tumor virus promoter, Rous sarcoma virus promoter, polyhedrin promoter, or other promoters shown effective for expression in eukaryotic cells.
  • the type of vector used by some embodiments of the invention will depend on the cell type transformed.
  • the ability to select suitable vectors according to the virus or cell type transformed is well within the capabilities of the ordinary skilled artisan and as such no general description of selection consideration is provided herein.
  • nucleic acids by viral infection offers several advantages over other methods such as lipofection and electroporation, since higher transfection efficiency can be obtained due to the infectious nature of viruses.
  • nucleic acid transfer techniques include transfection with viral or non-viral constructs, such as adenovirus, lentivirus, Herpes simplex I virus, or adeno- associated virus (AAV) and lipid-based systems.
  • viral or non-viral constructs such as adenovirus, lentivirus, Herpes simplex I virus, or adeno- associated virus (AAV) and lipid-based systems.
  • Useful lipids for lipid-mediated transfer of the gene are, for example, DOTMA, DOPE, and DC-Chol [Tonkinson et al., Cancer Investigation, 14(1): 54-65 (1996)].
  • the most preferred constructs for use in gene therapy are viruses, most preferably adenoviruses, AAV, lentiviruses, or retroviruses.
  • a viral construct such as a retroviral construct includes at least one transcriptional promoter/enhancer or locus-defining element(s), or other elements that control gene expression by other means such as alternate splicing, nuclear RNA export, or post-translational modification of messenger.
  • Such vector constructs also include a packaging signal, long terminal repeats (LTRs) or portions thereof, and positive and negative strand primer binding sites appropriate to the virus used, unless it is already present in the viral construct.
  • LTRs long terminal repeats
  • Other vectors can be used that are non-viral, such as cationic lipids, polylysine, and dendrimers.
  • introducing the polynucleotides or constructs into a cell is effected by contacting the cell with the genetically modified virus disclosed herein.
  • a method of infecting a cell comprising contacting the cell with the virus of some embodiments of the invention.
  • the cell further comprises an exogenous polynucleotide of interest.
  • the method disclosed herein further comprises introducing into the cell an exogenous polynucleotide of interest.
  • the present invention also contemplates uses of the viruses and cells disclosed herein.
  • the phages of some embodiments of the invention comprising the polynucleotide encoding an anti-defense system polypeptide can be used for infecting a bacteria and/or treating a bacterial infection.
  • a method of infecting a bacteria comprising contacting the bacteria with the phage comprising the exogenous polynucleotide encoding the anti-defense system polypeptide disclosed herein, thereby infecting the bacteria.
  • a method of treating a bacterial infection in a subject in need thereof comprising administering to the subject a therapeutically effective amount of the phage comprising the exogenous polynucleotide encoding the anti-defense system polypeptide disclosed herein, thereby treating the bacterial infection in the subject.
  • the phage comprising the exogenous polynucleotide encoding the anti-defense system polypeptide disclosed herein, for use in treating a bacterial infection in the subject in need thereof.
  • treating refers to inhibiting or arresting the development of a pathology (disease, disorder or condition) and/or causing the reduction, remission, or regression of a pathology.
  • pathology disease, disorder or condition
  • bacterial infection may be assessed by, but not limited to, clinical evaluation, urine dipstick tests, throat culture, sputum tests, histology, indirect non-culture-based tests, including C-reactive protein and procalcitonin tests, serological tests and/or nucleic acid amplification tests.
  • the phrase “subject in need thereof’ includes mammals, preferably human beings of any gender and at any age which suffer from the pathology.
  • the bacteria is of a Gram-negative bacteria or Negativicutes that stain negative in Gram stain.
  • Non-limiting examples of Gram-negative bacteria include Acinetobacter calcoaceticus, Actinobacillus actinomycetemcomitans, Aeromonas hydrophila, Alcaligenes xylosoxidans, Bacteroides, Bacteroides fragilis, Bartonella bacilliformis, Bordetella spp., Borrelia burgdorferi, Branhamella catarrhalis, Brucella spp., Campylobacter spp., Chalmydia pneumoniae, Chlamydia psittaci, Chlamydia trachomatis, Chromobacterium violaceum, Citrobacter spp., Eikenella corrodens, Enterobacter aerogenes, Escherichia coli, Flavobacterium meningosepticum, Fusobacterium spp., Haemophilus influenzae, Haemophilus spp., Helicobacter pylori, Klebsiella
  • the bacteria is a gammaproteobacteria (e.g. Escherichia coli, pseudomonas, vibrio and klebsiellA' ) or a Firmicutes (belonging to class Negativicutes that stain negative in Gram stain).
  • a gammaproteobacteria e.g. Escherichia coli, pseudomonas, vibrio and klebsiellA'
  • a Firmicutes belonging to class Negativicutes that stain negative in Gram stain.
  • the bacteria is a Gram-positive bacteria.
  • Non-limiting examples of Gram-positive bacteria include, but are not limited to, Actinomyces spp., Bacillus anthracis, Bifidobacterium spp., Clostridium botulinum, Clostridium perfringens, Clostridium spp., Clostridium tetani, Corynebacterium diphtheriae, Corynebacterium jeikeium, Enterococcus faecalis, Enterococcus faecium, Erysipelothrix rhusiopathiae, Eubacterium spp., Gardnerella vaginalis, Gemella morbillorum, Leuconostoc spp., Mycobacterium abcessus, Mycobacterium avium complex, Mycobacterium chelonae, Mycobacterium fortuitum, Mycobacterium haemophilium, Mycobacterium kansasii, Mycobacterium leprae, Mycobacter
  • the bacteria is a species selected from the group consisting of Escherichia, Shigella, Salmonella, Erwinia, Yersinia, Bacillus, Vibrio, Legionella, Pseudomonas, Neisseria, Bordetella, Helicobacter, Listeria, Agrobacterium, Staphylococcus, Streptococcus, Enterococcus, Clostridium, Corynebacterium, Mycobacterium, Treponema, Borrelia, Francisella, Brucella, Campylobacter, Klebsiella, Frankia, Bartonella, Rickettsia, Shewanella, Serratia, Enterobacter, Proteus, Providencia, Brochothrix, and Brevibacterium.
  • the bacteria is selected from the group consisting of Escherichia coli, Klebsiella pneumoniae, Clostridium difficile and Pseudomonas aeruginosa.
  • the bacterium expresses the bacterial defense system the anti-defense system polypeptide is directed against e.g. Thoeris and/or CBASS.
  • the method comprises determining expression of the bacterial defense system in the bacteria prior to the introducing, the contacting or the treating.
  • Methods of determining expression are well known in the art and include e.g. sequencing, PCR, Western blot etc.
  • the phage therapy of some embodiments of the invention may be combined with one or more non-phage therapeutic and/or prophylactic agents, useful for the treatment and/or prevention of bacterial infections, as described herein and/or known in the art (e.g. one or more traditional antibiotic agents).
  • Other therapeutic and/or prophylactic agents that may be used in combination with the phage(s) of some embodiments of the invention include, but are not limited to, antibiotic agents, anti-inflammatory agents, antiviral agents, antifungal agents, or local anesthetic agents.
  • the methods of the present invention further comprise administering to the subject a therapeutically effective amount of an antibiotic or contacting the bacteria with an antibiotic.
  • the uses of the present invention further comprise an antibiotic.
  • antibiotics include, but are not limited to aminoglycoside antibiotics, cephalosporins, quinolone antibiotics, macrolide antibiotics, penicillins, sulfonamides, tetracyclines and carbapenems.
  • Standard or traditional antibiotic agents that can be administered with the phages described herein include, but are not limited to, amikacin, gentamicin, kanamycin, neomycin, netilmicin, paromomycin, rhodostreptomycin, streptomycin, tobramycin, apramycin, rifamycin, naphthomycin, mupirocin, geldanamycin, ansamitocin, carbacephems, imipenem, meropenem, ertapenem, faropenem, doripenem, panipenem/betamipron, biapenem, PZ-601, cephalosporins, cefacetrile, cefadroxil, cefalexin, cefaloglycin, cefalonium, cefaloridine, cefalotin, cefapirin, cefatrizine, cefazaflur, cefazedone, cefazolin, cefradine, ce
  • ceftobiprole azithromycin, clarithromycin, dirithromycin, erythromycin, roxithromycin, aztreonam, pencillin and penicillin derivatives, actinomycin, bacitracin, colistin, polymyxin B, cinoxacin, flumequine, nalidixic acid, oxolinic acid, piromidic acid, pipemidic acid, rosoxacin, ciprofloxacin, enoxacin, fleroxacin, lomefloxacin, nadifloxacin, norfloxacin, ofloxacin, pefloxacin, rufloxacin, balofloxacin, gatifloxacin, grepafloxacin, levofloxacin, moxifloxacin, pazufloxacin, sparfloxacin, temafloxacin, tosufloxacin, clinafloxacin, garenoxacin, gemifloxacin
  • an article of manufacture or a kit comprising as active ingredients the phage comprising the exogenous polynucleotide encoding the anti-defense system polypeptide disclosed herein; and an antibiotic.
  • the article of manufacture is identified for treating a bacterial infection.
  • the phage and the antibiotic are in separate formulations.
  • the phage and the antibiotic are packaged in separate containers.
  • the phage and the antibiotic are in a coformulation.
  • the phage therapy is the only active agent administered to the subject, e.g. in the absence of a standard or traditional effective antibiotic agent.
  • a method of improving resistance of a plant to biotic stress comprising introducing into the plant an anti-defense system polypeptide or a polynucleotide encoding same, wherein said anti-defense system polypeptide is selected from the group consisting of:
  • Tad3 polypeptide (i) a Tad3 polypeptide, wherein said Tad3 polypeptide binds a homolog of the defense system polypeptide ThsB of a Type I Thoeris set forth in SEQ ID NO: 10 endogenously expressed in said plant;
  • Tad4 polypeptide (ii) a Tad4 polypeptide, wherein said Tad4 polypeptide binds a homolog of the defense system polypeptide ThsB of a Type I Thoeris set forth in SEQ ID NO: 10 endogenously expressed in said plant;
  • Tad5 polypeptide (iii) a Tad5 polypeptide, wherein said Tad5 polypeptide binds a homolog of the defense system polypeptide ThsB of a Type I Thoeris set forth in SEQ ID NO: 10 endogenously expressed in said plant;
  • a Tad6 polypeptide wherein said Tad7 polypeptide binds a homolog of the defense system polypeptide ThsB of a Type I Thoeris set forth in SEQ ID NO: 10 endogenously expressed in said plant
  • a Tad7 polypeptide wherein said Tad7 polypeptide binds a homolog of the defense system polypeptide ThsB of a Type I Thoeris set forth in SEQ ID NO: 228 endogenously expressed in said plant
  • Tad8 polypeptide (vi) a Tad8 polypeptide, wherein said Tad8 polypeptide binds a homolog of the defense system polypeptide ThsB of a Type I Thoeris set forth in SEQ ID NO: 227 endogenously expressed in said plant;
  • an Acb3 polypeptide wherein said Acb3 polypeptide binds a homolog of the bacterial defense system polypeptide cGAS of a CBASS set forth in amino acid sequence selected from the group consisting of SEQ ID NOs: 229-231 and 233 endogenously expressed in said plant, thereby improving resistance of the plant to biotic stress.
  • a method of treating a disease that can benefit from inhibiting a defense system polypeptide in a subject in need thereof comprising administering to the subject a therapeutically effective amount of an anti-defense system polypeptide or a polynucleotide encoding same, wherein: when said defense system polypeptide is a homolog of the defense system polypeptide ThsB of a Type I Thoeris set forth in SEQ ID NO: 10 endogenously expressed in said subject, the anti-defense system polypeptide is a Tad3 polypeptide, a Tad4 polypeptide, a Tad5 polypeptide and/or a Tad6 polypeptide which binds said homolog of SEQ ID NO: 10; when said defense system polypeptide is a homolog of the defense system polypeptide ThsB of a Type II Thoeris set forth in SEQ ID NO: 228 endogenously expressed in said subject, the anti-defense system polypeptide is a Tad
  • the anti-defense system polypeptide or a polynucleotide encoding same for use in treating a disease that can benefit from inhibiting a defense system polypeptide in a subject in need thereof, wherein: when said defense system polypeptide is a homolog of the defense system polypeptide ThsB of a Type I Thoeris set forth in SEQ ID NO: 10 endogenously expressed in said subject, the anti-defense system polypeptide is a Tad3 polypeptide, a Tad4 polypeptide, a Tad5 polypeptide and/or a Tad6 polypeptide which binds said homolog of SEQ ID NO: 10; when said defense system polypeptide is a homolog of the defense system polypeptide ThsB of a Type II Thoeris set forth in SEQ ID NO: 228 endogenously expressed in said subject, the anti-defense system polypeptide is a Tad7 polypeptide which binds said homolog of SEQ ID NO: 2
  • the disease is selected from the group consisting of autoimmune disease, interferonopathy and a disease associated with neuronal degeneration.
  • the disease is an autoimmune disease.
  • autoimmune diseases include, but are not limited to, rheumatoid arthritis (RA), lupus (SLE), atherosclerosis, multiple sclerosis (MS), hashimoto disease, type I diabetes, autoimmune pancreatitis, graft-versus-host disease (GVHD), sepsis, Ebola, avian influenza, smallpox, systemic inflammatory response syndrome (SIRS), hemophagocytic lymphohistiocytosis, Crohn’s and ulcerative colitis, familial Mediterranean fever (FMF), TNF receptor-associated periodic syndrome (TRAPS), hyperimmunoglobulinemia D with periodic fever syndrome (HIDS), familial cold autoinflammatory syndrome (FCAS), the Muckle-Wells syndrome (MWS), neonatal-onset multisystem inflammatory disease (NOMID), deficiency of ADA2 (DADA2), NLRC4 inflammasomopathies, X-linked lymphoproliferative type 2 disorder (
  • the disease is associated with neuronal degeneration.
  • the injury may be brought about by a disease e.g. a neurodegenerative disease, stroke or by an injury per se, such as a traumatic brain injury, a spinal cord injury, a peripheral nerve injury or an eye injury.
  • a disease e.g. a neurodegenerative disease, stroke or by an injury per se, such as a traumatic brain injury, a spinal cord injury, a peripheral nerve injury or an eye injury.
  • exemplary neurodegenerative diseases include, but are not limited to Amyotrophic Lateral Sclerosis (ALS), Parkinson's disease, Multiple System Atrophy (MSA), Huntington's disease, Alzheimer's disease, Rett Syndrome and Multiple Sclerosis (MS).
  • the therapeutic agent described herein may be used per se or as part of a pharmaceutical composition, where it is mixed with suitable carriers or excipients.
  • a "pharmaceutical composition” refers to a preparation of one or more of the active ingredients described herein with other chemical components such as physiologically suitable carriers and excipients.
  • the purpose of a pharmaceutical composition is to facilitate administration of a compound to an organism.
  • physiologically acceptable carrier and “pharmaceutically acceptable carrier” which may be interchangeably used refer to a carrier or a diluent that does not cause significant irritation to an organism and does not abrogate the biological activity and properties of the administered compound.
  • An adjuvant is included under these phrases.
  • excipient refers to an inert substance added to a pharmaceutical composition to further facilitate administration of an active ingredient.
  • excipients include calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils and polyethylene glycols.
  • Suitable routes of administration may, for example, include topical, oral, rectal, transmucosal, especially transnasal, intestinal or parenteral delivery, including intramuscular, subcutaneous and intramedullary injections as well as intrathecal, direct intraventricular, intracardiac, e.g., into the right or left ventricular cavity, into the common coronary artery, intravenous, intraperitoneal, intranasal, or intraocular injections.
  • the phage may be administered directly into an infected area or tissue of the subject.
  • compositions of some embodiments of the invention may be manufactured by processes well known in the art, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping, spray drying, coating or lyophilizing processes.
  • compositions for use in accordance with some embodiments of the invention thus may be formulated in conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries, which facilitate processing of the active ingredients into preparations which, can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen.
  • the active ingredients of the pharmaceutical composition may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hank’s solution, Ringer’s solution, or physiological salt buffer.
  • physiologically compatible buffers such as Hank’s solution, Ringer’s solution, or physiological salt buffer.
  • penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.
  • the pharmaceutical composition can be formulated readily by combining the active compounds with pharmaceutically acceptable carriers well known in the art.
  • Such carriers enable the pharmaceutical composition to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and the like, for oral ingestion by a patient.
  • Pharmacological preparations for oral use can be made using a solid excipient, optionally grinding the resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries if desired, to obtain tablets or dragee cores.
  • Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carbomethylcellulose; and/or physiologically acceptable polymers such as polyvinylpyrrolidone (PVP).
  • disintegrating agents may be added, such as cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.
  • Dragee cores are provided with suitable coatings.
  • suitable coatings For this purpose, concentrated sugar solutions may be used which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, titanium dioxide, lacquer solutions and suitable organic solvents or solvent mixtures.
  • Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses.
  • compositions which can be used orally include push-fit capsules made of gelatin as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol.
  • the push-fit capsules may contain the active ingredients in admixture with filler such as lactose, binders such as starches, lubricants such as talc or magnesium stearate and, optionally, stabilizers.
  • the active ingredients may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols.
  • stabilizers may be added. All formulations for oral administration should be in dosages suitable for the chosen route of administration.
  • compositions may take the form of tablets or lozenges formulated in conventional manner.
  • the active ingredients for use according to some embodiments of the invention are conveniently delivered in the form of an aerosol spray presentation from a pressurized pack or a nebulizer with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichloro-tetrafluoroethane or carbon dioxide.
  • a suitable propellant e.g., dichlorodifluoromethane, trichlorofluoromethane, dichloro-tetrafluoroethane or carbon dioxide.
  • the dosage unit may be determined by providing a valve to deliver a metered amount.
  • Capsules and cartridges of, e.g., gelatin for use in a dispenser may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.
  • compositions described herein may be formulated for parenteral administration, e.g., by bolus injection or continues infusion.
  • Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multidose containers with optionally, an added preservative.
  • the compositions may be suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
  • compositions for parenteral administration include aqueous solutions of the active preparation in water-soluble form. Additionally, suspensions of the active ingredients may be prepared as appropriate oily or water based injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acids esters such as ethyl oleate, triglycerides or liposomes. Aqueous injection suspensions may contain substances, which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol or dextran. Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility of the active ingredients to allow for the preparation of highly concentrated solutions.
  • the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile, pyrogen-free water based solution, before use.
  • a suitable vehicle e.g., sterile, pyrogen-free water based solution
  • compositions suitable for use in context of some embodiments of the invention include compositions wherein the active ingredients are contained in an amount effective to achieve the intended purpose. More specifically, a therapeutically effective amount means an amount of active ingredients (e.g. phage, antibiotic) effective to prevent, alleviate or ameliorate symptoms of a disorder (e.g. bacterial infection) or prolong the survival of the subject being treated.
  • active ingredients e.g. phage, antibiotic
  • the therapeutically effective amount or dose can be estimated initially from in vitro and cell culture assays.
  • a dose can be formulated in animal models to achieve a desired concentration or titer. Such information can be used to more accurately determine useful doses in humans.
  • the pharmaceutical composition is delivered to a subject in need thereof so as to provide one or more phages in an amount corresponding to a multiplicity of infection (MOI) of about 0.001 to about 10.
  • MOI is determined by assessing the approximate bacterial load, or using an estimate for a given type of infection; and then providing phage in an amount calculated to give the desired MOI.
  • the composition comprises at least about 10 6 PFU, 10 7 PFU, 10 8 PFU, 10 9 PFU, or even IO 10 PFU or more of the phage disclosed herein.
  • the amount of phage is provided so as to reduce the amount of bacteria by at least 10 %, 20 %, 30 %, 40 %, 50 %, 60 %, 70 %, 80 %, 90 % or even 100 %.
  • Toxicity and therapeutic efficacy of the active ingredients described herein can be determined by standard pharmaceutical procedures in vitro, in cell cultures or experimental animals.
  • the data obtained from these in vitro and cell culture assays and animal studies can be used in formulating a range of dosage for use in human.
  • the dosage may vary depending upon the dosage form employed and the route of administration utilized.
  • the exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition. (See e.g., Fingl, et al., 1975, in "The Pharmacological Basis of Therapeutics", Ch. 1 P-l).
  • Dosage amount and interval may be adjusted individually to provide levels of the active ingredient are sufficient to induce or suppress the biological effect (minimal effective concentration, MEC).
  • MEC minimum effective concentration
  • the MEC will vary for each preparation, but can be estimated from in vitro data. Dosages necessary to achieve the MEC will depend on individual characteristics and route of administration. Detection assays can be used to determine plasma concentrations. Depending on the severity and responsiveness of the condition to be treated, dosing can be of a single or a plurality of administrations, with course of treatment lasting from several days to several weeks or until cure is effected or diminution of the disease state is achieved.
  • compositions to be administered will, of course, be dependent on the subject being treated, the severity of the affliction, the manner of administration, the judgment of the prescribing physician, etc.
  • doses of the antibiotic may be lower (e.g. 20 % lower, 30 % lower, 40 % lower, 50 % lower, 60 % lower, 70 % lower, 80 % lower or even 90 % lower) than their gold standard dose or in a sub-efficacious dose when administered as a single agent.
  • compositions of some embodiments described herein may comprise a single phage strain or a cocktail of multiple distinct phages wherein as least one of the phages is the phage disclosed herein.
  • compositions described herein comprise more than one phage strain.
  • the composition comprises 2 phage strains, 3 phage strains, 4 phage strains, 5 phage strains or more.
  • the phage cocktails of some embodiments comprise phages that target a single bacteria species or subspecies.
  • the phage cocktail comprises phages that target multiple bacteria species or subspecies, each phage with a distinct host range.
  • compositions of some embodiments of the invention may, if desired, be presented in a pack or dispenser device, such as an FDA approved kit, which may contain one or more unit dosage forms containing the active ingredient.
  • the pack may, for example, comprise metal or plastic foil, such as a blister pack.
  • the pack or dispenser device may be accompanied by instructions for administration.
  • the pack or dispenser may also be accommodated by a notice associated with the container in a form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals, which notice is reflective of approval by the agency of the form of the compositions or human or veterinary administration.
  • Such notice for example, may be of labeling approved by the U.S. Food and Drug Administration for prescription drugs or of an approved product insert.
  • compositions comprising a preparation of the invention formulated in a compatible pharmaceutical carrier may also be prepared, placed in an appropriate container, and labeled for treatment of an indicated condition, as is further detailed above.
  • the phages, phage cocktails and articles of manufacture of some embodiments of the invention can be also used in anti-infective compositions for controlling the growth of bacteria on a surface contacted therewith.
  • the phages of some embodiments of the invention may be incorporated into compositions that are formulated for application to biological surfaces, such as the skin and mucus membranes, as well as for application to non-biological surfaces.
  • Anti-infective formulations for use on biological surfaces include, but are not limited to, gels, creams, ointments, sprays, and the like.
  • the anti-infective formulation is used to sterilize a surgical field, or the hands and/or exposed skin of healthcare workers and/or patients.
  • Anti-infective formulations for use on non-biological surfaces include sprays, solutions, suspensions, wipes impregnated with a solution or suspension and the like.
  • the anti-infective formulation is used on solid surfaces in hospitals, nursing homes, ambulances, etc., including, e.g., appliances, countertops, and medical devices, hospital equipment.
  • the non-biological surface is a surface of a hospital apparatus or piece of hospital equipment.
  • the non- biological surface is a surgical apparatus or piece of surgical equipment.
  • compositions, method or structure may include additional ingredients, steps and/or parts, but only if the additional ingredients, steps and/or parts do not materially alter the basic and novel characteristics of the claimed composition, method or structure.
  • a compound or “at least one compound” may include a plurality of compounds, including mixtures thereof.
  • range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.
  • a numerical range is indicated herein, it is meant to include any cited numeral (fractional or integral) within the indicated range.
  • the phrases “ranging/ranges between” a first indicate number and a second indicate number and “ranging/ranges from” a first indicate number “to” a second indicate number are used herein interchangeably and are meant to include the first and second indicated numbers and all the fractional and integral numerals therebetween.
  • method refers to manners, means, techniques and procedures for accomplishing a given task including, but not limited to, those manners, means, techniques and procedures either known to, or readily developed from known manners, means, techniques and procedures by practitioners of the chemical, pharmacological, biological, biochemical and medical arts.
  • sequences that substantially correspond to its complementary sequence as including minor sequence variations, resulting from, e.g., sequencing errors, cloning errors, or other alterations resulting in base substitution, base deletion or base addition, provided that the frequency of such variations is less than 1 in 50 nucleotides, alternatively, less than 1 in 100 nucleotides, alternatively, less than 1 in 200 nucleotides, alternatively, less than 1 in 500 nucleotides, alternatively, less than 1 in 1000 nucleotides, alternatively, less than 1 in 5,000 nucleotides, alternatively, less than 1 in 10,000 nucleotides.
  • a database of short phage proteins of unknown function To construct a database of short phage proteins of unknown function, the IMG/VR v3 database 38 was downloaded. Identical protein sequences were removed, resulting in ⁇ 32 million non-identical protein sequences. Next, the 32 million proteins were clustered into groups of homologs via an iterative clustering process, to thereby reduce computational load. Clustering was based on sequence identity, wherein homologs were eventually defined as sequences having an e-value ⁇ 0.001 when aligned to each other; and the clusters were defined as groups of homologs in which there is a representative sequence (“center” of the cluster) having an e-value ⁇ 0.001 to each member in the cluster.
  • the protein sequences were first clustered using the “cluster” option of MMseqs2 release 12-113e3 54 with default parameters. Then, a representative sequence from each cluster was extracted using the “createsubdb” option of MMseqs2, and the representative sequences were aggregated into groups using the “cluster” option of MMseqs2 with the parameter “-c 0”. Next, the 2 sets of clusters were merged using the “mergeclusters” option of MMseqs2. Merged-clusters that include at least 40 members and at least one group of homologs from the first step of clustering with an MMseqs2-as signed representative sequence shorter than 200 amino acids were retained for downstream analysis.
  • the MMseqs2-assigned sequence representing the largest number of proteins in the first step of clustering that is also shorter than 200 amino acids was selected as the representative sequence of each merged cluster.
  • the structure of the representative sequence was modeled using AlphaFold2 version 2.2 55 with default parameters, with the addition of the IMG/VR v3 database to the default protein databases searched through the AlphaFold2 pipeline to collect homologous sequences. Proteins that were modeled with an average pLDDT score (a a confidence score provided by AlphaFold) lower than 80 were removed from the analysis.
  • proteins with a higher number of homologs detected in the MGnify database (a general metagenomic database) 56 than the IMG/VR v3 database (a database of phage proteins) were removed, as these proteins were considered as not primarily carried on phage genomes.
  • the retaining proteins were searched against an annotated database of proteins constructed previously 4 using the “search” option of MMseqs2 with default parameters, and the top hit of each phage protein was extracted. Phage proteins similar to a protein with a known function were removed, resulting in a final database of -38,700 protein sequences, each representing a family of short phage proteins enriched for proteins with unknown functions.
  • Each one of the -38,700 proteins was modeled as a homodimer using AlphaFold2-Multimer version 2.2 37 , and predictions with an average PAE score lower than 5 were considered as homodimers for downstream analyses. Prediction of phage-encoded proteins that inhibit type I Thoeris - To discover phage proteins that bind the type I Thoeris system, each one of the -38,700 phage protein sequences was predicted as a protein complex with the ThsA and ThsB proteins of type I Thoeris in an iterative process using AlphaFold2-Multimer version 2.3 37 . First, each one of the phage proteins was modeled together with each immune protein generating one predicted complex using the first model of AlphaFold2-Multimer ran with default parameters.
  • Predicted interactions with a co-folding model confidence score above 0.8 in at least 15 of the 25 predicted complexes were defined as final candidate anti-defense proteins, and were taken for experimental verification.
  • phage proteins that were predicted to form a homodimer were presented in two copies when modeled as a protein complex with an immune protein.
  • Multiple sequence alignments presented in Figures 6 and 7A-B were computed with MAFFT version 7.490 57 and visualized using Jalview 58 .
  • Bacterial strains and growth conditions - E. coll and B. subtilis strains were grown in magnesium manganese broth (MMB; LB + O.l mM MnCh + 5 mM MgCh) at 37 °C while shaking at 200 RPM. Whenever applicable, the appropriate antibiotics were added at the following concentrations: For B. subtilis strains spectinomycin (100 pg ml -1 ) and chloramphenicol (5 pg mF 1 ), and for E. coll strains ampicillin (100 pg ml” 1 ) and kanamycin (50 pg ml” 1 ).
  • the type I and type II Thoeris systems, as well as the type I CBASS system, were cloned under their native promoters into the amyE locus of the B. subtilis BEST7003 genome, as described in 2,41 (SEQ ID NOs: 1-3, respectively).
  • the type III CBASS system was synthetized and cloned with its native promoter into plasmid pSGl-CBASS, as described in 11 (SEQ ID NO: 4).
  • Phage strains - The B. subtilis phages SBSphiJ (GenBank: LT960608.1) and SBSphiC (GenBank: LT960610.1) were isolated as described in 2 .
  • the E. coll phage BAS 18 from the BASEL phage collection was described in 59 .
  • Phages were propagated on either E. coll MG1655 or B. subtilis BEST7003 by picking a single phage plaque into a liquid culture grown at 37 °C to an optical density at 600 nm (ODeoonm) of 0.3 in MMB broth until culture collapse (or 3 hours in the case of no lysis). The culture was then centrifuged for 10 minutes at 3,200 g and the supernatant was filtered through a 0.2-pm filter to get rid of remaining bacteria and bacterial debris.
  • Cloning of candidate anti-defense genes - Anti-defense genes were synthesized and cloned by Genscript Corp.
  • the anti-defense candidates for type I and type II Thoeris and type I CBASS were cloned into the pSG-thrC-Phspank vector 22 and transformed into NEB 5-alpha competent cells.
  • the cloned vector was subsequently transformed into B. subtilis BEST7003 cells containing the respective defense system integrated into the amyE locus 2,41 , resulting in cultures expressing both a defense system and the corresponding anti-defense gene candidate, integrated into the amyE and rArC loci, respectively.
  • the anti-defense candidate for type III CBASS was cloned into the pBbS8k vector (Addgene #35276) and subsequently transformed into NEB 5-alpha competent cells.
  • the cloned vector was further transformed into E. coli MG1655 cells containing the pSGl-CBASS system 11 .
  • transformants with an identical plasmid containing RFP instead of the anti-defense gene were used.
  • Plaque assays - Phage titer was determined using the small drop plaque assay method 61 .
  • a 300 pl (E. coli) or 400 ul (B. subtilis) volume of overnight culture of bacteria was mixed with 0.5 % agar and 30 ml MMB and poured into a 10-cm square plate followed by incubation for 1 hour at room temperature.
  • 1 mM IPTG (B. subtilis) or 0.2 % arabinose (E. coli) was added to the 30 ml MMB 0.5 % agar.
  • Tenfold serial dilutions in MMB were carried out for each of the tested phages and 10-pl drops were put on the bacterial layer.
  • Plaque-forming units were determined by counting the derived plaques after overnight incubation and lysate titer was determined by calculating PFUs per milliliter. When no individual plaques could be identified, a faint lysis zone across the drop area was considered to be 10 plaques. The efficiency of plating was measured by comparing plaque assay results for control bacteria and those for bacteria containing the defense system and/or a candidate anti-defense gene. Protein co-expression for biochemical assessment of metabolites - B. subtilis BEST7003 cultures, co-expressing a genomically-integrated B.
  • ThsANii2AB 7 (SEQ ID NO: 12298, as sequence in which the effector of the defense system is mutated, so the Thoeris system produces the immune signal but does not lead to activation of the effector and cell death) under native promoter and either Tad3 (SEQ ID NO: 15), Tad4 (SEQ ID NO: 16), Tad5 (SEQ ID NO: 17, or Tad6 (SEQ ID NO: 18) under the Physpank promoter were grown in 50 ml MMB media supplemented with ImM IPTG. Cultures were grown for ⁇ 2 hours at 37 °C, 200 RPM, until reaching an ODeoo of 0.3.
  • Human SARM1 TIR domain (position 561-724, NCBI Ref seq: NP_055892.2, SEQ ID NOs: 5-6) and BdTIR (Brachypodium distachyon TIR, NCBI ref seq: XP_003560074.3, SEQ ID NOs: 7-8) were co-expressed with Tad4 (SEQ ID NO: 16) in E. coli MG1655.
  • Cultures were grown in 50 ml MMB media with ampicillin (100 pg ml 1 ) and chloramphenicol (30 pg ml’ 1 ). Initially, cultures were grown for ⁇ 2 hours at 37°C, 200 RPM, until reaching an ODeoo of 0.3.
  • Enzymatic assays The ThsA protein (SEQ ID NO: 7) was expressed and purified as described in 12 , and ThsA-based NADase activity assay for the detection gcADPR was carried out as described in 12 .
  • the NAD/NADH-Glo (Promega) kit was used for directly measuring the NAD + levels in filtered cell lysates. The lysates were diluted 1 : 150 in 0.1 M Na-phosphate buffer, pH 8.0. Reactions were performed in a volume of 10 pl (5 pl of sample + 5 pl of reaction mixture) according to ratios recommended in the manufacturer’s instructions. Luciferin signal, proportional to the amount of NAD + , was detected using the kit luciferase enzyme. Tecan Infinite 200 PRO plate reader was used to monitor the developing luminescent signal. NAD + concentrations were calculated from the calibration curve using a set of NAD + standards with known concentrations.
  • Protein expression and purification - ThsB, BdTIR, and SARMITIR sequences were codon optimized for E. Coli (SEQ ID NOs: 11-13), synthesized (Integrated DNA Technologies), and cloned into a custom pET-based expression vector with an N-terminal 6xHis-SUMO2 tag.
  • a spacer (AAAGAGGAGAAATTAACT, SEQ ID NO: 14) containing a second ribosome binding site was inserted directly downstream of ThsB, BdTIR, and SARMITIR, and codon- optimized sequences for Tad3-6 (SEQ ID NOs: 15-18) were cloned into the second open reading frame for co-expression studies.
  • Expression plasmids were transformed into RIL cells (Agilent) and plated on MDG plates (1.5 % Bacto agar, 0.5 % glucose, 25 mM Na 2 HPO4, 25 mM KH2PO4, 50 mM NH4CI, 5 mM Na 2 SO4, 0.25 % aspartic acid, 2-50 pM trace metals, 100 pg ml -1 ampicillin, 34 pg ml -1 chloramphenicol). Colonies were picked into 30 ml MDG liquid media and grown overnight at 37 °C with shaking.
  • Resin was then washed with 20 ml lysis buffer, 50 ml wash buffer (20 mM HEPES-KOH pH 7.5, 1 M NaCl, 10 % glycerol, 30 mM imidazole, 1 mM TCEP). Bound protein was eluted in 20 ml elution buffer (20 mM HEPES-KOH pH 7.5, 400 mM NaCl, 10% glycerol, 300 mM imidazole, 1 mM TCEP).
  • SDS-PAGE analysis -_Protein purity and complex formation were assessed by SDS- PAGE by mixing 10 pl of protein (containing 1-10 pg of protein sample) with 4 pl of loading buffer. The samples were run on a 15 % gel for 45 minutes at 200 V. Proteins were stained using BrilliantBlue Coomassie stain (VWR) and visualized using a ChemiDoc MP Imaging system (BioRad).
  • Cloning was earned out using the NEBuilder HiFi DNA Assembly cloning kit (NEB, number E5520S) and the cloned vector was transformed into NEB 5-aIpha competent cells. The cloned vector was subsequently transformed into the thrC site of B. subtilis BEST7003. The TaJ3-containing B. subtilis BEST7003 strain was then infected with phage SBSphiJ with a multiplicity of infection (MOI) of 0.1 and cell lysate was collected. Tad3 lysate was used to infect a Thoeris-containing B. subtilis culture in two consecutive rounds with an MOI of 2 in each round at 30 °C.
  • MOI multiplicity of infection
  • homologs of anti-defense candidates for binding analysis were identified in the IMG/VR v3 database using the “search” option of MMseqs2 release 12-113e3 with the parameter “-c 0.8”. Then, the homologs of each candidate anti-defense protein were separated into 10 bins based on their sequence identity percentage to the query anti-defense candidate. A random sequence was selected from each bin and predicted as a complex with the relevant immune protein using AlphaFold2-Multimer version 2.3, generating five predictions per each one of the five AlphaFold2-Multimer models.
  • Prediction of phage encoded proteins that inhibit type II Thoeris or CBASS - Inhibitors of type II Thoeris and CBASS were predicted by analyzing the ThsB and ThsA proteins of type II Thoeris and the CD-NTase protein from E. coli KTE188 together with each of the phage proteins as described above, with the exception that two additional filtering steps were applied to the results. First, interactions that were predicted to have less than 25 residues of the immune protein interacting with the candidate inhibitor based on an analysis in the RING version 4 server 66 were removed.
  • homologs of each predicted binder were selected and modeled as a complex with the immune protein using AlphaFold2-Multimer as described in the “Selection of homologs of anti-defense candidates for binding analysis” part hereinabove.
  • Candidate inhibitors that did not have diverse homologs predicted to bind the immune protein with an average co-folding model confidence score higher than 0.75 were removed.
  • the homolog having the highest co-folding model confidence score was selected for experimental verification.
  • the downloaded proteins from IMG/VR v4 were clustered using the “cluster” option of MMseqs2 with default parameters.
  • structure-based homologs were defined as sequences that have a predicted structure that is significantly similar (probability of 1.0 in foldseek, a program that aligns protein structures and provides homology probability scores) to the predicted structure of the anti-defense protein.
  • a representative sequence was extracted from each cluster containing at least 30 non-identical members, and its structure was predicted using AlphaFold2 version 2.3 with default parameters, resulting in 182,179 phage protein structures.
  • LC-MS polar metabolite analysis Samples were centrifuged twice (20,800g) and transferred to HPLC vials. Sample evaluation was conducted according to the method described by Zheng et al. 68 with minor modifications as described below. Briefly, analysis was performed using Acquity I class UPLC System combined with mass spectrometer Q Exactive Plus OrbitrapTM (Thermo Fisher Scientific), operated in a negative ionization mode with a scan range of 70 - 1050 m/z. The LC separation was done using the SeQuant Zic-pHilic (150 mm x 2.1 mm) with the SeQuant guard column (20 mm x 2.1 mm) (Merck). Mobile phase consisted of two different mobile phases: The first phase (A) consisting of acetonitrile, and the second phase
  • AlphaFold2-Multimer 37 could be used to discover phage proteins that inhibit bacterial immunity, the type I Thoeris defense system from Bacillus cereus MSX- D12, a two-gene system that protects against a broad array of phages and whose function is well understood 7,22 , was considered.
  • the Thoeris system encodes ThsB, a TIR-domain protein that generates a signaling molecule once it detects phage infection, and ThsA, an effector NAD + - cleaving protein that is activated by the signaling molecule.
  • phage proteins whose predicted co-folding scores with one of the Thoeris proteins passed the cutoff threshold were detected. Fifteen of these proteins were predicted to bind ThsB, the TIR-do main-containing protein responsible for sensing phage infection 7,22 , and one was predicted to bind the Thoeris immune effector ThsA (Table 2 hereinbelow).
  • the DNA sequence for each of the candidate immune inhibitor proteins was synthesized together with an inducible promoter and integrated into the genome of a Bacillus subtilis strain also carrying the Thoeris system 2 .
  • ThsB senses phage infection and then produces the immune signaling molecule 1"— 3' gcADPR 22 . This molecule binds and activates the NADase activity of ThsA, which then depletes the cell of the essential molecule NAD + 7 ’ 22 ’ 39 ( Figure ID).
  • NAD + 7 ’ 22 ’ 39 Figure ID.
  • the infected cells were lysed, filtered to include only small molecules, and incubated with a purified ThsA protein in vitro.
  • the filtered lysates extracted from infected cells that expressed only ThsB were able to induce the NADase activity of ThsA, but lysates from cells also expressing Tad3, Tad4, Tad5 or Tad6 failed to activate ThsA ( Figure IE).
  • Tad3 was engineered into the genome of SBSphiJ, a phage normally blocked by Thoeris, under the control of the native promoter of Tadl, a previously identified Thoeris inhibitor 22 .
  • SBSphiJ knocked-in for Tad3 became fully resistant to Thoeris, showing that tad3 expression from the phage genome is sufficient for Thoeris inhibition ( Figure 2A).
  • Tad3, Tad4 and Tad6 directly block the active site pocket of ThsB via a loop that interacts with the active site residues (Figure 2B).
  • the amino acid residues that block the ThsB active site pocket are highly conserved among homologs of each of Tad3, Tad4 and Tad6, suggesting that blockage of the active site of ThsB is a conserved function of these anti-Thoeris proteins ( Figures 6 and 7A-B).
  • Tad5 is also predicted to interact with E85 and other active site residues in ThsB but does not completely block the active site pocket (Figure 2B).
  • ThsB TIR-domain protein that generates a histidine conjugated to ADPR (His-ADPR) as an immune signaling molecule 40 .
  • the Ths A effector protein of type II Thoeris encodes a Macro domain capable of binding His-ADPR, and a transmembrane- spanning domain that likely impairs membrane integrity once activated by the signaling molecule 2,40 .
  • the ThsB TIR-domain protein of the type II Thoeris system (SEQ ID NO: 228) is substantially different to that of type I Thoeris (SEQ ID NO: 10), with no detectable sequence similarity between the two and only little structural similarity ( Figure 9).
  • AlphaFold2-Multimer did not predict high scoring interactions between ThsB of type II Thoeris and the anti type I proteins identified in this study, and, consistently, inhibitors of type I Thoeris did not inhibit type II Thoeris defense when tested experimentally ( Figure 10).
  • Tad7 binds the ThsB protein of type II Thoeris and inserts a loop into the active site pocket ( Figures 4C-D).
  • Tad8 is predicted to form a homodimer and insert loops into the Macro domain of ThsA. These loops occupy the same space that would be otherwise occupied by the His-ADPR immune signaling molecule ( Figures 4E-G).
  • the present inventors set out to detect phage proteins that inhibit the bacterial CBASS defense system 41,42 .
  • the pipeline was applied on the cGAS-like protein (CD-NTase) from the type III CBASS of Escherichia coli KTE188 11 (SEQ ID NO: 229).
  • the computational pipeline retrieved only one phage protein predicted to interact with the bacterial CD-NTase (Table 6 hereinbelow).
  • the phage protein inhibited its ability to defend against phages, verifying the pipeline prediction ( Figure 4H).
  • This CBASS inhibitor referred to herein as “Acb3” (anti-CBASS 3).
  • the MGV database 32 an independent database containing -190,000 genome scaffolds representing sequenced and partially sequenced phage genomes, was also examined (Table 7 hereinbelow).
  • the anti-Thoeris proteins detected in this study were found in 4,356 of these genome scaffolds (2.3% of the scaffolds), with Tad3 being the most abundant anti-Thoeris protein also in this set, occurring in 3,627 scaffolds.
  • Acb3 was underrepresented in this set of phage genomes, detected in only 8 scaffolds.
  • the present inventors first focused on the TIR domain protein from the plant Brachypodium distachyon (BdTIR), which is known to produce l''-2' gcADPR and l''-3' gcADPR molecules 46 .
  • BdTIR plant Brachypodium distachyon
  • the interaction between each of the anti-TIR phage proteins discovered (Tad3-Tad7) and the BdTIR protein was modeled using AlphaFold2-Multimer and high- scoring predicted interactions were found between Tad4 and BdTIR, suggesting that Tad4 may bind BdTIR (Figure 13A).
  • Tad4 was co-expressed in cells also expressing a 6xHis-SUMO2-tagged BdTIR.
  • BdTIR is known to constitutively produce l"-2' gcADPR and l"-3' gcADPR molecules when expressed in E. cold' ( ⁇ and indeed, filtered cell lysates derived from cells expressing BdTIR activated Ths A from type I Thoeris, a protein triggered by l"-3' gcADPR ( Figure 5B).
  • SARM1 Sterile alpha and TIR motif-containing 1 protein
  • SARM1 is an essential protein within a pathway that leads to axonal death in response to neuronal injury, and it was shown that the SARM1 TIR domain degrades cellular NAD + when activated following neuron insult 47,48 .
  • Prokaryotic viperins produce diverse antiviral molecules. Nature 589, 120-124. 10.1038/s41586-020-2762-2. Johnson, A.G., Wein, T., Mayer, M.L., Duncan-Lowey, B., Yirmiya, E., Oppenheimer- Shaanan, Y., Amitai, G., Sorek, R., and Kranzusch, P.J. (2022). Bacterial gasdermins reveal an ancient mechanism of cell death. Science 375, 221-225. 10.1126/science.abj8432. Wein, T., and Sorek, R. (2022). Bacterial origins of human cell-autonomous innate immune mechanisms. Nat. Rev.
  • Phage anti-CBASS and anti-Pycsar nucleases subvert bacterial immunity. Nature 605, 522-526. 10.1038/s41586-022-04716-y. Leavitt, A., Yirmiya, E., Amitai, G., Lu, A., Garb, J., Herbst, E., Morehouse, B.R., Hobbs, S.J., Antine, S.P., Sun, Z.-Y.L, et al. (2022). Viruses inhibit TIR gcADPR signalling to overcome bacterial defence. Nature 611, 326-331. 10.1038/s41586-022-05375-9.
  • Single phage proteins sequester TIR- and cGAS -generated signaling molecules. Preprint in bioRxiv. 10.1101/2023.11.15.567273.
  • Phage anti-CBASS protein simultaneously sequesters cyclic trinucleotides and dinucleotides. Mol. Cell 84, 375-385. e7. 10.1016/j.molcel.2023.11.026. Jenson, J.M., Li, T., Du, F., Ea, C.-K., and Chen, Z.J. (2023). Ubiquitin-like conjugation by bacterial cGAS enhances anti-phage defence. Nature 616, 326-331. 10.1038/s41586- 023-05862-7. Alawneh, A.M., Qi, D., Yonesaki, T., and Otsuka, Y. (2016).
  • An anti-CRISPR protein disables type V Casl2a by acetylation. Nat. Struct. Mol. Biol. 26, 308-314.
  • IMG/VR v4 an expanded database of uncultivated virus genomes within a framework of extensive functional, taxonomic, and ecological metadata. Nucleic Acids Res. 57, D733-D743. 10.1093/nar/gkacl037.
  • IMG/VR v3 an integrated ecological and evolutionary framework for interrogating genomes of uncultivated viruses. Nucleic Acids Res. 49, D764-D775. 10.1093/nar/gkaa946. Tamulaitiene, G., Sabonis, D., Sasnauskas, G., Ruksenaite, A., Silanskas, A., Avraham, C., Ofir, G., Sorek, R., Zaremba, M., and Siksnys, V. (2024). Activation of Thoeris antiviral system via SIR2 effector filament assembly.
  • Plant and prokaryotic TIR domains igenerate distinct cyclic ADPR NADase products. Sci. Adv. 9, eade8487. 10.1126/sciadv.ade8487. Essuman, K., Summers, D.W., Sasaki, Y., Mao, X., DiAntonio, A., and Milbrandt, J. (2017).
  • the SARM1 Toll/Interleukin-1 Receptor Domain Possesses Intrinsic NAD+ Cleavage Activity that Promotes Pathological Axonal Degeneration. Neuron 93, 1334- 1343. e5. 10.1016/j.neuron.2017.02.022. Figley, M.D., and DiAntonio, A.
  • the SARM1 axon degeneration pathway control of the NAD+ metabolome regulates axon survival in health and disease.
  • the SARM1 TIR domain produces glycocyclic ADPR molecules as minor products. Preprint at bioRxiv. 10.1101/2023.08.10.552750.
  • MMseqs2 enables sensitive protein sequence searching for the analysis of massive data sets. Nat. Biotechnol. 35, 1026-1028. 10.1038/nbt.3988. Jumper, J., Evans, R., Pritzel, A., Green, T., Figumov, M., Ronneberger, O., Tunyasuvunakool, K., Bates, R., Zfdek, A., Potapenko, A., et al. (2021). Highly accurate protein structure prediction with AlphaFold. Nature 596, 583-589. 10.1038/s41586-021- 03819-2.

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Abstract

L'invention concerne des protéines anti-défense, des virus les comprenant ainsi que des procédés de production et d'utilisation de telles protéines et virus. L'invention concerne également des procédés d'identification de polypeptides de système anti-défense putatifs.
PCT/IL2025/050306 2024-04-08 2025-04-08 Polypeptides de système anti-défense et leurs utilisations Pending WO2025215636A1 (fr)

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