Bacteriophage lytic enzyme or chimera and use thereof

Abstract

Provided are a bacteriophage lytic enzyme or a chimera and a use thereof. The lytic enzyme or chimera thereof comprises: (1) a natural bacteriophage lytic enzyme, the natural bacteriophage lytic enzyme being selected from the amino acid sequences of GNL14, GNL24, GNL25, GNL46, GNL53, GNL66, GNL68, GNL73, GNL80, or GNL82; (2) an antimicrobial peptide, the antimicrobial peptide being selected from the amino acid sequences of GNP6, GNP24, GNP27, GNP30, or GNP45; and (3) a linker, the linker being selected from the amino acid sequences of GNLK1, GNLK2, GNLK4, GNLK5, GNLK6, GNLK7, GNLK8, GNLK9, or GNLK11. The lytic enzyme or chimera thereof can be used for treating or preventing bacterial infections caused by Gram-negative bacteria, particularly infections of Acinetobacter baumannii, Klebsiella, and Pseudomonas.

Description

Phage lytic enzyme or chimera thereof and use thereof Technical Field

[0001] The present invention relates to the field of biomedicine. Specifically, the present invention relates to a phage lytic enzyme or chimera thereof with anti-Gram-negative bacteria and its application, including a lytic enzyme or chimera thereof having antibacterial activity against Acinetobacter baumannii, Pseudomonas aeruginosa and Klebsiella pneumoniae. Background Art

[0002] Bacterial antimicrobial resistance (AMR) has emerged as one of the major public health threats of our generation. In China, Acinetobacter baumannii (AB), Klebsiella pneumoniae (KP), and Pseudomonas aeruginosa (PA) are three of the top five most commonly isolated clinical strains. They cause infections in various sites of the human body, including the respiratory tract, urinary tract, bloodstream, central nervous system, skin, and wounds. Antibiotic resistance in these bacteria poses an urgent public health threat, as multiply, extensively, and pandrug-resistant strains have been isolated in hospital-acquired infections.

[0003] Phage lytic enzymes are a new class of antimicrobial agents targeting drug-resistant bacteria. Lytic enzymes are enzymes that bacteriophages use to destabilize the bacterial cell wall, lysing the host bacterium and releasing phage progeny from within. Phage lytic enzymes are peptidoglycan hydrolases that mediate bacterial lysis by cleaving specific bonds within the peptidoglycan structure. Lytic enzymes are classified based on their specific enzymatic activities: glucosaminidase and muramamidase cleave the peptidoglycan sugar backbone, L-alanine amidase cleaves the amide bond connecting the sugar backbone to the peptide, and endopeptidases cleave the peptide portion of the peptidoglycan (stem peptide and / or cross-bridges). While lytic enzymes function naturally within phage-infected cells, recent work has demonstrated that lytic enzymes can be recombinantly expressed, purified, and used to kill target bacteria when applied externally. Recombinant lytic enzymes against Gram-positive bacteria have been shown to be effective bactericidal agents, causing hypotonic lysis. With few exceptions, each lytic enzyme targets bacteria belonging to a single genus or species. Because lytic enzymes have withstood billions of years of selective evolutionary pressure to maintain their ability to infect bacteria, the target peptidoglycan bonds are highly conserved and unlikely to be easily altered by bacteria. This characteristic minimizes resistance to lytic enzymes in target bacteria, making them suitable for long-term use.

[0004] To date, no bacterial resistance to the corresponding phage lytic enzymes has been identified. While Gram-positive bacteria have an exposed peptidoglycan cell wall, making it accessible to externally applied endolytic lytic enzymes, Gram-negative bacteria have an outer membrane that prevents lytic enzymes from directly accessing the peptidoglycan. Therefore, to be effective as therapeutic agents, Gram-negative lytic enzymes must also be functionally capable of crossing this barrier. Certain lytic enzymes possess intrinsic antimicrobial peptide (AMP)-like domains that facilitate this function.

[0005] Although some progress has been made in the research and development of lytic enzymes, the research progress of lytic enzyme drugs effective against Gram-negative bacteria in clinical settings is still slow, especially the research on phage lytic enzymes with antibacterial activity and low toxicity against Acinetobacter baumannii, Klebsiella pneumoniae and Pseudomonas aeruginosa.

[0006] Bioinformatics tools were used to identify phage lytic enzyme genes from known published genomes. Although genomic data were available, many genes were not initially annotated as phage lytic enzymes and therefore had to be putatively identified using tools such as BLAST. Even when annotated as phage lytic enzymes, the vast majority of naturally occurring phage lytic enzymes we tested lacked intrinsic antimicrobial activity against target bacteria. This lack of antimicrobial activity was attributed to several factors, including incorrect gene annotation, low expression, low activity, and, most importantly, an inability to cross the outer membrane of Gram-negative bacteria. This further demonstrates that identifying clinically applicable phage lytic enzymes is neither easy nor straightforward, as the vast majority of naturally occurring phage lysozymes do not readily achieve the desired antimicrobial activity, stability, host range, and production yield characteristics required of such enzymes.

[0007] As mentioned above, it is more challenging to find lytic enzymes that can attack and kill Gram-negative organisms from the outside because most of these enzymes can only evolve to cut cell wall peptidoglycan and cannot pass through the outer membrane of the cell. In most phages, outer membrane permeability is carried out by a separate mechanism, such as using transmembrane proteins, while passing through the inner membrane requires the assistance of cave proteins. Therefore, natural lytic enzymes are generally unable to pass through any bacterial membrane. In addition, even if natural lytic enzymes can pass through the outer membrane, most lytic enzymes have evolved to function from the inside to the outside, while clinical requirements require that lytic enzymes work from the outside to the inside, which is a more difficult task considering the ubiquitous presence of lipopolysaccharides (LPS) on the outer layer of the bacterial outer membrane. In short, the process of identifying and modifying phage lytic enzymes for clinical use, especially those related to Gram-negative bacteria, is very difficult.

[0008] US20200376096 - Lyticase polypeptides active against Gram-negative bacteria. In this patent, the authors describe five lysase sequences and related lysase-derived amphiphilic peptides. The described lysases are capable of killing Pseudomonas aeruginosa in buffer solution, but have no antibacterial activity in serum alone.

[0009] WO2021180892 - New recombinant lytic enzymes and their use in treating Gram-negative bacterial infections. This patent describes the discovery of a phage lytic enzyme that is effective against Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii, and Pseudomonas aeruginosa at very high concentrations, but its activity in serum or biological matrices is not disclosed. Summary of the Invention

[0010] In view of the above technical status, the present invention provides a novel recombinant phage lytic enzyme or a chimera thereof for resisting Gram-negative bacteria.

[0011] The present invention provides a phage lytic enzyme chimera for resisting Gram-negative bacteria, wherein the phage lytic enzyme chimera is selected from GNL1810 shown in SEQ ID NO:46.

[0012] 1. In one aspect, the present invention provides a phage lytic enzyme or a chimera thereof for use against Gram-negative bacteria, wherein the lytic enzyme comprises:

[0013] (1) a natural phage lytic enzyme, the natural phage lytic enzyme being selected from the amino acid sequence of GNL14 shown in SEQ ID NO: 1, GNL24 shown in SEQ ID NO: 2, GNL25 shown in SEQ ID NO: 3, GNL46 shown in SEQ ID NO: 4, GNL53 shown in SEQ ID NO: 5, GNL66 shown in SEQ ID NO: 6, GNL68 shown in SEQ ID NO: 7, GNL73 shown in SEQ ID NO: 8, GNL80 shown in SEQ ID NO: 9, or GNL82 shown in SEQ ID NO: 10;

[0014] 2. Another aspect of the present invention provides a chimera of a phage lytic enzyme and an antimicrobial peptide for use against Gram-negative bacteria, the chimera comprising:

[0015] (1) a natural phage lytic enzyme selected from the group consisting of GNL14 shown in SEQ ID NO: 1, GNL24 shown in SEQ ID NO: 2, GNL25 shown in SEQ ID NO: 3, GNL46 shown in SEQ ID NO: 4, GNL53 shown in SEQ ID NO: 5, GNL66 shown in SEQ ID NO: 6, GNL68 shown in SEQ ID NO: 7, GNL73 shown in SEQ ID NO: 8, GNL80 shown in SEQ ID NO: 9, or GNL82 shown in SEQ ID NO: 10; and

[0016] (2) an antimicrobial peptide, wherein the antimicrobial peptide is selected from the amino acid sequence of GNP6 shown in SEQ ID NO: 19, GNP24 shown in SEQ ID NO: 20, GNP27 shown in SEQ ID NO: 21, GNP30 shown in SEQ ID NO: 22, or GNP45 shown in SEQ ID NO: 23.

[0017] 3. In another aspect, the present invention provides a chimera of an anti-Gram-negative phage lytic enzyme, an antimicrobial peptide, and a linker, the chimera comprising:

[0018] (1) having an amino acid sequence selected from the group consisting of GNL14 shown in SEQ ID NO: 1, GNL24 shown in SEQ ID NO: 2, GNL25 shown in SEQ ID NO: 3, GNL46 shown in SEQ ID NO: 4, GNL53 shown in SEQ ID NO: 5, GNL66 shown in SEQ ID NO: 6, GNL68 shown in SEQ ID NO: 7, GNL73 shown in SEQ ID NO: 8, GNL80 shown in SEQ ID NO: 9, or GNL82 shown in SEQ ID NO: 10;

[0019] (2) an antimicrobial peptide, the antimicrobial peptide being selected from the amino acid sequence of GNP6 shown in SEQ ID NO: 19, GNP24 shown in SEQ ID NO: 20, GNP27 shown in SEQ ID NO: 21, GNP30 shown in SEQ ID NO: 22, or GNP45 shown in SEQ ID NO: 23; and

[0020] (3) a linker, wherein the linker is selected from the amino acid sequence of GNLK1 shown in SEQ ID NO: 11, GNLK2 shown in SEQ ID NO: 12, GNLK4 shown in SEQ ID NO: 13, GNLK5 shown in SEQ ID NO: 14, GNLK6 shown in SEQ ID NO: 15, GNLK7 shown in SEQ ID NO: 16, GNLK8 shown in SEQ ID NO: 17, GNLK9 shown in SEQ ID NO: 18, or GG.

[0021] The novel recombinant phage lytic enzyme or chimera thereof for resisting Gram-negative bacteria provided by the present invention is applied to, but not limited to, resisting Acinetobacter, Klebsiella or Pseudomonas.

[0022] The phage lysin described in the present invention is also known as an endolysin. Phage lysins are produced by bacteriophages in the late stages of bacterial host infection and lyse the cell wall from the inside out. However, when using lysins for therapeutic purposes, they need to work from the outside in. Therefore, when targeting Gram-negative bacteria, phage lysins need to penetrate the outer membrane to kill the target organism, a function that natural lysins have not yet evolved.

[0023] The novel lytic enzymes, chimeric enzymes, or combinations thereof provided herein are highly effective in killing Gram-negative bacteria. Even more surprisingly, the modified lytic enzymes retain their bactericidal activity in serum, bronchoalveolar lavage fluid, lung extracts, and other biological matrices.

[0024] The phage lytic enzyme or chimera thereof of the present invention, as one embodiment, comprises mutations in one, two or more of the following sites in GNL53:

[0025] R13E, R13Q or R13G;

[0026] R18S;

[0027] K41S or K41P;

[0028] K52M, K52T;

[0029] K116A or

[0030] K141E.

[0031] The phage lytic enzyme or chimera thereof of the present invention, as one embodiment, the GNL25 comprises mutations in one, two or more of the following sites:

[0032] K3Q, R27G, K49D, K56T, R83Q, K106G, R115D, K121D, R124S, R138F, or K155T.

[0033] The phage lytic enzyme or chimera thereof of the present invention, as one embodiment, the GNL80 includes mutations in one, two or more of the following sites: K5A, K22Y, R51Q, K54A, K56D, K77G, R87M, K90A, R93D, K113Q, K129A or K143E.

[0034] The phage lytic enzyme or chimera thereof of the present invention, as one embodiment, the GNL14 includes mutations at one, two or more of the following sites: K27P, H42D, K48T, K67E, K105Q, or E inserted after G156, V159 or K160.

[0035] In the present invention, as one embodiment, the phage lytic enzyme or its chimera includes a natural phage lytic enzyme. The present invention also includes an amino acid sequence having at least 80% identity, preferably 85% identity, even more preferably 90% identity, even more preferably 95% identity, even more preferably 96% identity, even more preferably 97% identity, even more preferably 98% identity, even more preferably 99% identity, even more preferably 99.5% identity, and most preferably 99.7% identity to the phage lytic enzyme or its chimera.

[0036] In the present invention, the antimicrobial peptide (AMP) is also called "membrane permeabilization enhancing peptide"

[0037] Its fusion to a natural lytic enzyme with antibacterial activity can enhance the ability of the natural phage lytic enzyme to penetrate the outer membrane of Gram-negative bacteria, making it more effective in killing target bacteria and able to function effectively in environments containing serum, lung mucosa or other biological matrices.

[0038] In one aspect of the present invention, the antimicrobial peptide of the present invention has one of the following properties: (a) outer membrane permeability, (b) lipopolysaccharide binding protein binding and neutralization, (c) antimicrobial activity in blood and blood components, and (d) anti-biofilm activity.

[0039] As one embodiment, the antimicrobial peptide comprises an amino acid sequence having at least 80% identity, preferably 85% identity, even more preferably 90% identity, even more preferably 95% identity, even more preferably 96% identity, even more preferably 97% identity, even more preferably 98% identity, even more preferably 99% identity, even more preferably 99.5% identity, and most preferably 99.7% identity.

[0040] In the present invention, as one embodiment, the linker connects the phage lytic enzyme and the antimicrobial peptide. The present invention also includes amino acid sequences having at least 80% identity, preferably 85% identity, even more preferably 90% identity, even more preferably 95% identity, even more preferably 96% identity, even more preferably 97% identity, even more preferably 98% identity, even more preferably 99% identity, even more preferably 99.5% identity, and most preferably 99.7% identity.

[0041] In the present invention, as one embodiment, the antimicrobial peptide can be fused to the N-terminus or C-terminus. Preferably, the antimicrobial peptide is fused to the C-terminus of the bacteriophage lytic enzyme. Any linker polypeptide sequence described in the present invention can be inserted between the naturally-occurring bacteriophage lytic enzyme and the membrane permeabilization-enhancing polypeptide for fusion.

[0042] The present invention also includes a fusion protein selected from one of the following combinations: a fusion between natural phage lytic enzymes, a fusion between a natural phage lytic enzyme and an antimicrobial peptide, or a fusion between a natural phage lytic enzyme, a linker sequence and an antimicrobial peptide.

[0043] The fusion protein has killing activity against Acinetobacter and / or Klebsiella and / or Pseudomonas.

[0044] In the present invention, as one embodiment, the phage lytic enzyme or its chimera is selected from the group consisting of GNL993 shown in SEQ ID NO: 24, GNL1142 shown in SEQ ID NO: 25, GNL1146 shown in SEQ ID NO: 26, GNL1159 shown in SEQ ID NO: 27, GNL1166 shown in SEQ ID NO: 28, GNL1249 shown in SEQ ID NO: 29, GNL1250 shown in SEQ ID NO: 30, GNL1252 shown in SEQ ID NO: 31, GNL1255 shown in SEQ ID NO: 32, GNL1261 shown in SEQ ID NO: 33, GNL1263 shown in SEQ ID NO: 34, GNL1292 shown in SEQ ID NO: 35, GNL1300 shown in SEQ ID NO: 36, GNL1301 shown in SEQ ID NO: 37, GNL1302 shown in SEQ ID NO: 38, GNL1342 shown in SEQ ID NO: 39, The amino acid sequence of GNL1347 set forth in SEQ ID NO:40, GNL1350 set forth in SEQ ID NO:41, GNL1366 set forth in SEQ ID NO:42, GNL1771 set forth in SEQ ID NO:43, GNL1780 set forth in SEQ ID NO:44, GNL1799 set forth in SEQ ID NO:45, GNL1810 set forth in SEQ ID NO:46, GNL1815 set forth in SEQ ID NO:47, GNL1847 set forth in SEQ ID NO:48, or GNL1997 set forth in SEQ ID NO:49;

[0045] In the present invention, as one embodiment, the phage lytic enzyme or its chimera is selected from GNL993-v1 shown in SEQ ID NO: 50, GNL993-v2 shown in SEQ ID NO: 51, GNL993-v3 shown in SEQ ID NO: 52, GNL993-v4 shown in SEQ ID NO: 53, GNL993-v5 shown in SEQ ID NO: 54, GNL993-v6 shown in SEQ ID NO: 55, GNL1249-v1 shown in SEQ ID NO: 56, GNL1249-v2 shown in SEQ ID NO: 57, GNL1249-v3 shown in SEQ ID NO: 58, GNL1249-v4 shown in SEQ ID NO: 59, GNL1249-v5 shown in SEQ ID NO: 60, GNL1249-v6 shown in SEQ ID NO: 61, GNL1249-v7 shown in SEQ ID NO: 62, : GNL1250-v5 shown in SEQ ID NO: 71, GNL1250-v6 shown in SEQ ID NO: 72, GNL1250-v7 shown in SEQ ID NO: 73, GNL1250-v8 shown in SEQ ID NO: 74, GNL1250-v9 shown in SEQ ID NO: 75, GNL1250-v10 shown in SEQ ID NO: 76, GNL1250-v11 shown in SEQ ID NO: 77, GNL1250-v1 shown in SEQ ID NO: 78, GNL1250-v2 shown in SEQ ID NO: 79, GNL1250-v3 shown in SEQ ID NO: 80, GNL1250-v4 shown in SEQ ID NO: 81, GNL1250-v5 shown in SEQ ID NO: 82, GNL1250-v7 shown in SEQ ID NO: 83, GNL1250-v8 shown in SEQ ID NO: 84, GNL1250-v9 shown in SEQ ID NO: 85, GNL1250-v10 shown in SEQ ID NO: 86, , GNL1250-v17 shown in SEQ ID NO: 84, GNL1250-v18 shown in SEQ ID NO: 85, GNL1250-v19 shown in SEQ ID NO: 86, GNL1250-v20 shown in SEQ ID NO: 87, GNL1250-v21 shown in SEQ ID NO: 88, GNL1250-v22 shown in SEQ ID NO: 89, GNL1250-v30 shown in SEQ ID NO: 90, GNL1250-v31 shown in SEQ ID NO: 91, GNL1250-v32 shown in SEQ ID NO: 92, GNL1250-v33 shown in SEQ ID NO: 93, GNL1250-v34 shown in SEQ ID NO: 94, GNL1250-v35 shown in SEQ ID NO: 95, GNL1250-v36 shown in SEQ ID NO: 96The amino acid sequence of GNL1799-v2 shown in SEQ ID NO:87, GNL1799-v3 shown in SEQ ID NO:88, GNL1799-v4 shown in SEQ ID NO:89, GNL1799-v5 shown in SEQ ID NO:90, or GNL1799-v6 shown in SEQ ID NO:91.

[0046] The amino acid sequence of the modified phage lytic enzyme against Gram-negative bacteria or its chimera also includes an amino acid sequence with 80%-100% identity.

[0047] Among the modified phage lytic enzymes of the present invention, GNL1249, GNL1250, GNL1255, and GNL1350 exhibit strong bactericidal activity against Acinetobacter baumannii, Pseudomonas aeruginosa, and Klebsiella pneumoniae in a serum environment.

[0048] The present invention also provides a polynucleotide molecule encoding a bacteriophage lytic enzyme or a chimera thereof as described above, wherein the nucleic acid molecule is DNA or RNA. The terms polynucleotide and polynucleotide molecule are used synonymously with the term nucleic acid molecule.

[0049] The present invention also provides a vector comprising the polynucleotide molecule of the present invention. In one embodiment, the vector is an expression vector. Any suitable expression vector known in the art can be used, such as pET and any T7 vector, pBAD and any arabinose inducible vector, and any other suitable vector. For example, the vector can be a plasmid. Therefore, one aspect of the present invention provides a plasmid comprising the polynucleotide of the present invention. It will be appreciated by those skilled in the art that the choice of vector will be determined by the choice of host expression system.

[0050] The host cells provided by the present invention also include those comprising the polynucleotide molecules of the present invention, or the vectors or plasmids according to the present invention. In one embodiment, the host cell is a microorganism, preferably a bacterium. Preferably, the host cell is non-pathogenic. Most preferably, the host cell is Escherichia coli. Therefore, one aspect of the present invention relates to a bacterial host, preferably Escherichia coli, comprising a plasmid of the present invention. The present invention also provides a method for producing a natural phage lytic enzyme of the present invention or a chimera thereof, comprising culturing a host cell population comprising a polynucleotide molecule of the present invention or a plasmid / vector according to the present invention under conditions expressing the natural phage lytic enzyme or a chimera thereof, and isolating therefrom.

[0051] The present invention provides a phage lytic enzyme or a chimera thereof for resisting Gram-negative bacteria, comprising a possible mutation for reducing the surface charge of the lytic enzyme, such as GNL993, GNL1249, GNL1250, GNL1350, GNL53, GNL25, GNL80 or GNL14.

[0052] The present invention provides a composition comprising the anti-Gram-negative bacteria phage lytic enzyme or its chimera, a nucleic acid molecule encoding the anti-Gram-negative bacteria phage lytic enzyme of the present invention, and a host cell containing an expression vector of the nucleic acid sequence of the phage lytic enzyme.

[0053] In the present invention, as one embodiment, the above composition comprises a pharmaceutically acceptable carrier, buffer or excipient.

[0054] As one embodiment, the composition of the present invention can be formulated into a solution, suspension, emulsion, inhalable powder, aerosol, spray, cream, gel, ointment, mouthwash, antibiotic, sustained-release preparation, or a preparation combined with other antibacterial agents, antibiotics or disinfectants.

[0055] As one of the embodiments, the composition of the present invention is for liquid injection, lyophilized injection, inhalation, external use, nasal application or solid form.

[0056] In some embodiments, a polypeptide linker is used to connect the native lytic enzyme and the AMP to preserve the structure, function, and mobility of the lytic enzyme and AMP. Non-limiting examples of linker and antimicrobial peptide sequences are listed in Table 2 (non-limiting examples of linker sequences) and Table 3 (non-limiting examples of peptide sequences), respectively. Modified phage lytic enzymes are created as native phage lytic enzyme-polypeptide linker-antimicrobial peptide constructs.

[0057] The present invention provides a pharmaceutical composition comprising the above-mentioned anti-Gram-negative bacteria or chimeric phage lytic enzyme or chimeric enzyme thereof or a pharmaceutically acceptable carrier.

[0058] The present invention provides a use of any of the above-mentioned anti-Gram-negative phage lytic enzymes or chimeras thereof in the presence of serum, lung extract or other biological matrices, which has bactericidal activity in the presence of serum, lung extract, blood, urinary tract, sputum, mucus or other biological matrices.

[0059] The present invention provides the use of the above composition or pharmaceutical composition in treating or preventing infectious diseases, gastrointestinal infections, bloodstream infections, sexually transmitted infections, multiple microbial infections, infections associated with indwelling devices or diseases caused by bacterial infections.

[0060] The present invention provides the use of any of the above-mentioned anti-Gram-negative phage lytic enzymes or chimeras thereof in the preparation of drugs for treating or preventing infectious diseases, gastrointestinal infections, bloodstream infections, sexually transmitted infections, polymicrobial infections, infections associated with indwelling devices, or diseases caused by bacterial infections.

[0061] The above compositions may be used in combination with other antimicrobial agents, antibiotics, antiseptics or disinfectants.

[0062] The antibiotics may include, but are not limited to, penicillins (e.g., piperacillin-tazobactam), cephalosporins (e.g., ceftriaxone, ceftazidime, cefepime), carbapenems (e.g., meropenem, imipenem, ertapenem), monobactams (e.g., aztreonam), aminoglycosides (e.g., gentamicin, tobramycin, amikacin), fluoroquinolones (e.g., ciprofloxacin, levofloxacin), tetracyclines (e.g., doxycycline, minocycline), sulfonamides (e.g., trimethoprim-sulfamethoxazole, macrolides (e.g., azithromycin, clarithromycin), polymyxins (e.g., colistin, polymyxin B), combinations (e.g., ceftolozane-tazobactam, ceftazidime-avibactam), glycylcyclines (e.g., tigecycline), fosfomycin, or any other antibiotic effective against the target organism, which may be used alone or in combination with the lytic enzymes disclosed in this patent.

[0063] Such preservatives include, but are not limited to, parabens (methylparaben, propylparaben, butylparaben, ethylparaben), benzalkonium chloride, thimerosal, chlorhexidine, benzyl alcohol, phenol, cresol (m-cresol), ethanol, sodium benzoate, sorbic acid and potassium sorbate, phenoxyethanol, formaldehyde and formaldehyde releasers (e.g., quaternium-15, DMDM ​​hydantoin), isothiazolinones (methylisothiazolinone, methylchloroisothiazolinone), triclosan and triclocarban, ethylhexylglycerin, caprylyl glycol, nitrates and nitrites (sodium nitrate and sodium nitrite), sulfites (sodium sulfite, sodium bisulfite, sodium metabisulfite), lactic acid, acetic acid (vinegar), citric acid, diacetyl, lactic acid (sodium lactate, potassium lactate), natamycin, and any other such preservatives commonly used in pharmaceuticals, cosmetics, and foods.

[0064] Compounds considered disinfectants include alcohols (e.g., ethanol, isopropyl alcohol), chlorhexidine, iodophors (e.g., povidone-iodine), quaternary ammonium compounds (e.g., benzalkonium chloride), hydrogen peroxide, chlorine compounds (e.g., sodium hypochlorite, chlorine dioxide), phenolic compounds, hexachlorophene, triclosan, silver sulfadiazine, peracetic acid, glutaraldehyde, formaldehyde, and any other compound commonly used to disinfect skin, inanimate objects in the home, or in a healthcare setting, including medical equipment and surgical tools.

[0065] The present invention provides a method for inhibiting, reducing or killing at least one Gram-negative bacterium, comprising contacting an effective amount of the above-mentioned anti-Gram-negative bacteriolytic enzyme or its chimera with the bacterium.

[0066] In one embodiment disclosed herein, a naturally occurring phage lytic enzyme having at least 80% amino acid identity, or at least 85% amino acid identity, or at least 90% amino acid identity, or at least 95% amino acid identity is administered to a patient suffering from a Gram-negative bacterial infection, such as, but not limited to, Acinetobacter baumannii, Pseudomonas aeruginosa, and Klebsiella pneumoniae.

[0067] The present invention provides the above-mentioned phage lytic enzyme or chimera thereof for resisting Gram-negative bacteria, the above-mentioned nucleic acid molecule, vector or host cell or composition for preparing anti-Acinetobacter baumannii, Aeromonas hydrophila, Bacteroides fragilis, Bartonella henselae, Bordetella pertussis, Borrelia burgdorferi, Brucella abortus, Burkholderia cepacia, Campylobacter fetus, Campylobacter jejuni, Chlamydia pneumoniae, Chlamydia psittaci, Chlamydia trachomatis, Citrobacter freundii, Coxiella burnetii and the like. body, Eikenella corrosa, Enterobacter aerogenes, Enterobacter cloacae, Enterococcus faecalis, Escherichia coli, Francisella tularensis, Fusobacterium nucleatum, Haemophilus influenzae, Helicobacter pylori, Klebsiella (preferably Klebsiella granulosus, Klebsiella oxytoca, Klebsiella pneumoniae), Legionella longbeach, Legionella pneumophila, Leptospira, Moraxella catarrhalis, Neisseria gonorrhoeae, Neisseria meningitidis, Pasteurella multiflora, Pseudomonas aeruginosa, Proteus mirabilis, common Proteus, Rickettsia provazekii, Rickettsia, Salmonella enterica, Serratia marcescens, Shigella dysenteriae, Stenotrophomonas maltophilia, Treponema pallidum, Vibrio cholerae, Yersinia enterocolitica, Yersinia pestis, Aggregatibacter actinomycetemcomitans, Vibrio butzleri, Campylobacter coli, Cardiomycobacterium hominis, Citrobacter tarda, Edwardsiella tarda, Haemophilus ducreyi, Kingella kingii, Leptospira buccae, Mycoplasma genitalium, Prevotella intermedia , Proteus penetrans, Providencia reticularis, Pseudomonas (preferably Pseudomonas aeruginosa, Pseudomonas putida, Pseudomonas stutzeri, Pseudomonas fluorescens, or Pseudomonas menstruata), Serratia liquefaciens, Serratia Fontica, Serratia red, Serratia odorata, Serratia purimica, Sphingomonas, Nitrotrophomonas nitrosotrophomonas, Rhizotrophomonas rhizospermum, Xanthomonas microphylla, Xanthomonas microphylla or Xanthomonas maltifer.

[0068] The present invention can effectively address drug resistance, including multidrug resistance (MDR) or extensive drug resistance (XDR).In one embodiment, the lytic enzyme is used to treat a variety of microbial infections.

[0069] As one of the embodiments, the present invention provides the use of the above-mentioned anti-Gram-negative phage lytic enzyme or its chimera, the above-mentioned nucleic acid molecule, vector or host cell or composition in the preparation of a drug for resisting infections caused by Acinetobacter baumannii, Klebsiella pneumoniae, and Pseudomonas aeruginosa.

[0070] In one embodiment of the present invention, the bacterial infection is a respiratory tract infection such as pneumonia, bronchitis, sinusitis, pharyngitis, tonsillitis, and tuberculosis. Such pulmonary infections include, but are not limited to, patients with lower and / or upper respiratory tract infections.

[0071] The present invention, as one embodiment, provides a lytic enzyme or chimera thereof for use in treating, controlling, or preventing bacterial infection or colonization in patients at increased risk of bacterial infection, or chronic bacterial colonization of the lungs. These patients include, but are not limited to, patients with cystic fibrosis, chronic obstructive pulmonary disease (COPD), patients in intensive care units, patients on ventilators, patients with compromised immune systems, patients taking immunosuppressive medications, and other patients with impaired lung function.

[0072] In one embodiment of the present invention, the infection treated by the lytic enzyme or its chimera is community-acquired pneumonia or hospital-acquired pneumonia.

[0073] In one embodiment of the present invention, the infection treated by the lytic enzyme or its chimera is a complicated or uncomplicated skin and soft tissue infection.

[0074] In one embodiment of the present invention, the infectious diseases treated by the lytic enzyme or its chimera include urinary tract infections, such as cystitis, pyelonephritis, prostatitis and urethritis.

[0075] In one embodiment of the present invention, the disease treated by the lytic enzyme or its chimera is gastrointestinal infection, such as gastroenteritis, enteritis, enterocolitis, food poisoning, typhoid fever, cholera and Helicobacter pylori infection.

[0076] In one embodiment of the present invention, the bacterial infection treated by the lytic enzyme or its chimera is skin and soft tissue infection, such as cellulitis, impetigo, folliculitis, furunculosis, carbuncle, erysipelas, necrotizing fasciitis, myonecrosis and wound infection.

[0077] In one embodiment of the present invention, the infection treated by the lytic enzyme or chimera thereof is a bloodstream infection, such as sepsis, bacteremia, endocarditis and septicemia.

[0078] In one embodiment of the present invention, the infection treated by the lytic enzyme or its chimera is a sexually transmitted infection, such as gonorrhea, chlamydia, syphilis, chancroid and lymphogranuloma venereum.

[0079] The present invention, as one of the embodiments, the lytic enzyme or its chimera for treating or preventing infection refers to meningitis, encephalitis, osteomyelitis, suppurative arthritis, peritonitis, pericarditis, abscess, botulism, tetanus, diphtheria and pertussis infection, or other types of infection with Gram-negative bacteria as the cause.

[0080] The present invention, as one embodiment, the infection prevented or treated by the lytic enzyme or its chimera is an infection associated with an indwelling device. A non-exhaustive list of such devices includes catheters, such as urinary catheters, intravenous catheters, arterial catheters, spinal catheters, peritoneal catheters, dialysis catheters; intravenous (IV) lines, such as peripheral intravenous catheters, central venous catheters, peripherally inserted central catheters (PICCs), midline catheters; ventilators; pacemakers; implantable cardioverter defibrillators (ICDs); central cannulae: tunneled central venous catheters, implanted ports; gastrostomy tubes, such as percutaneous endoscopic gastrostomy (PEG) tubes, radiotherapy tubes. Insertion of a renal inguinal fistula (RIG) tube; tracheostomy tube, e.g., cuffed tracheostomy tube, cuffless tracheostomy tube; neurostimulator, e.g., spinal cord stimulator, deep brain stimulator, vagus nerve stimulator; prosthetic heart valve; vascular access device, e.g., arteriovenous (AV) fistula, AV graft; tympanostomy tube; hemodialysis catheter; feeding tube, e.g., nasogastric tube, nasojejunal tube, jejunostomy tube; drainage tube, e.g., chest tube, abdominal drain tube, biliary drain tube, pancreatic drain tube.

[0081] The present invention also provides the use of any of the above-mentioned anti-Gram-negative bacteria phage lytic enzymes or chimeras thereof as food preservatives and for combating bacterial biofilms. Broadly speaking, the biofilms include but are not limited to bacterial cell outer membranes, cell walls, cell membranes, capsules, etc.

[0082] The present invention, as one embodiment, discloses a lytic enzyme or a chimera thereof for use in treating, controlling or preventing bacterial infection or colonization in patients who are at increased risk of bacterial infection due to a compromised immune system, including but not limited to patients taking immunosuppressive drugs, HIV patients, and patients with genetic abnormalities that lead to a weakened immune system.

[0083] The present invention, as one embodiment, provides a lytic enzyme or chimera thereof for use in preventing infection in individuals at high risk of acquiring Gram-negative bacterial infections, where the risk of these infections is increased. Some non-limiting examples include severe burns, cystic fibrosis, chronic obstructive pulmonary disease (COPD), mechanical ventilation, immunocompromised states, hospitalization, use of a ventilator, indwelling catheters, and surgical procedures.

[0084] In one embodiment of the present invention, the lytic enzyme or chimera thereof is used together with another antimicrobial agent or antiseptic as a combination therapy.

[0085] In one embodiment of the present invention, the lytic enzyme or its chimera is administered together with an agent that has a permeabilizing effect on the bacterial outer membrane.

[0086] In one embodiment of the present invention, the lytic enzyme or its chimera is administered together with an antibacterial agent that has a permeabilizing effect on the bacterial outer membrane (such as colistin or polymyxin B).

[0087] In one embodiment of the present invention, the lytic enzyme or its chimera is used in combination with an antimicrobial disinfectant.

[0088] In one embodiment of the present invention, the lytic enzyme or its chimera is used as an antimicrobial disinfectant.

[0089] In one embodiment of the present invention, the lytic enzyme or its chimera is formulated into a topical formulation for application to the skin or mucosal surface. Such topical formulations include, but are not limited to, gels, creams, ointments, powders, and sprays.

[0090] In one embodiment of the present invention, the lytic enzyme or its chimera is embedded in a wound dressing.

[0091] In one embodiment of the present invention, the lytic enzyme or its chimera is coated onto an implantable device.

[0092] The present invention, as one embodiment, uses one or more of the inventive lyases or chimeras thereof together with an addition lyase from a different source, or any combination thereof in addition to another antimicrobial agent or preservative.

[0093] The present invention, as one embodiment, encapsulates or modifies the lytic enzyme or chimera thereof in a manner that improves its pharmacokinetic properties.

[0094] In one embodiment of the present invention, the lytic enzyme or its chimera is used as a food preservative to prevent or eliminate contamination by target Gram-negative bacteria.

[0095] The present invention, as one embodiment, uses the lytic enzyme of the present invention or its chimera to combat bacterial biofilms, such as biofilms in host tissues, bones, etc., in mucosal tissues, lungs, or associated with indwelling devices or catheters.

[0096] Lyase production

[0097] The present invention includes using various vector expression systems to clone and express the lytic enzyme gene. The example of a suitable vector expression system includes but is not limited to plasmid, phage, virus and artificial chromosome. These vector systems can be used for cloning the lytic enzyme gene into a suitable expression vector, which is then converted into a suitable host cell for expression. The present invention also includes any modification or change to the vector expression system, which is well known to those skilled in the art and which causes the expression, output or purification of the enzyme to be improved. The recombinant cloning of the lytic enzyme gene can be achieved using standard molecular biology techniques (such as PCR, restriction endonuclease digestion and connection). The present invention includes any method for recombinant cloning of the lytic enzyme gene known to those skilled in the art, whether now known or developed in the future. In addition, any modification or change to the lytic enzyme gene sequence known to those skilled in the art, such as site-directed mutagenesis or gene fusion, which causes the expression, output or active improvement of the enzyme is also included within the scope of protection of the present invention.

[0098] The present invention includes various recombinant expression systems for producing enzymes, including but not limited to bacterial systems such as Escherichia coli, Bacillus subtilis and Pseudomonas putida, yeast systems such as Saccharomyces cerevisiae, Pichia pastoris and lactic yeast, plant systems such as Arabidopsis thaliana, tobacco and water chestnut, mustard systems such as Chlamydomonas reinhardtii, Tripterygium wilfordii, Phoebe styraciflua, insect or insect cell culture expression systems, such as baculovirus-based systems, and mammalian systems such as Chinese hamster ovary (CHO) cells, human embryonic kidney (HEK) cells and mouse myeloma cells. These recombinant expression systems can be used to produce enzymes using well-known molecular biology techniques (e.g., DNA cloning, expression vector design and gene transformation).

[0099] The present invention encompasses any method known to those skilled in the art for producing enzymes using a recombinant expression system, whether now known or developed in the future. In addition, any modifications or alterations to a recombinant expression system known to those skilled in the art that result in improved expression, yield, or purification of the enzyme are also encompassed within the scope of the present invention.

[0100] The present invention also includes using a cell-free expression system to produce lyase.The example of a suitable cell-free expression system includes but is not limited to Escherichia coli S30 extract, wheat germ extract and rabbit reticulocyte lysate.These systems can use various modifications and additives (such as chaperones, energy regeneration systems and stabilizers) to optimize lyase expression.The present invention includes any modification or change to the cell-free expression system, and these modifications or changes are well known to those skilled in the art, which cause the expression, output or purification of the enzyme to be improved.

[0101] In addition, the present invention includes any modification or change to the lyase gene sequence known to those skilled in the art, which modification or change causes the expression, output or activity of the enzyme in the cell-free expression system to be improved. These modifications may include, but are not limited to, codon optimization, signal sequence modification and the truncation of non-functional or deleterious regions of the lyase protein. The present invention also includes any method known to those skilled in the art for preparing and optimizing the reaction conditions of the cell-free expression system to improve the expression, yield or purification of the lyase enzyme, whether now known or developed in the future.

[0102] The present invention includes using various vector expression systems to clone and express the lytic enzyme gene. The example of a suitable vector expression system includes but is not limited to plasmid, phage, virus and artificial chromosome. These vector systems can be used for cloning the lytic enzyme gene into a suitable expression vector, which is then converted into a suitable host cell for expression. The present invention also includes any modification or change to the vector expression system, which is well known to those skilled in the art and which causes the expression, output or purification of the enzyme to be improved. The recombinant cloning of the lytic enzyme gene can be achieved using standard molecular biology techniques (such as PCR, restriction endonuclease digestion and connection). The present invention includes any method for recombinant cloning of the lytic enzyme gene known to those skilled in the art, whether now known or developed in the future. In addition, any modification or change to the lytic enzyme gene sequence known to those skilled in the art, such as site-directed mutagenesis or gene fusion, which causes the expression, output or active improvement of the enzyme is also included within the scope of protection of the present invention.

[0103] As will be appreciated by those skilled in the art, several methods are available for purifying enzymes such as lyase, including but not limited to ultrafiltration, dialysis, precipitation, chromatography and centrifugation. Ultrafiltration can be used for removing low molecular weight impurities, and dialysis can be used for buffer exchange and desalination. Precipitation techniques such as salting out or solvent precipitation can be used for concentrating and purifying enzymes. Chromatography, including affinity, ion exchange, size exclusion and hydrophobic interaction chromatography, can be used for separating and purifying enzymes from other proteins, pollutants and impurities. In addition, centrifugation can be used for separating enzymes from cell debris and insoluble matter. Therefore, the present invention encompasses any method for purifying enzymes known to those skilled in the art, whether now known or developed in the future. BRIEF DESCRIPTION OF THE DRAWINGS

[0104] Figure 1: Structure prediction of native and chimeric lyases using AlphaFold2;

[0105] Figure 2: CFU reduction experimental results of partially purified modified lytic enzyme;

[0106] Figure 3: Activities of modified lytic enzymes and their mutants;

[0107] Figure 4: SDS-PAGE analysis of purified modified lysate;

[0108] Figure 5: Experiment on reduction of CFU of Pseudomonas aeruginosa by modified phage lytic enzyme in phosphate buffer and normal saline;

[0109] Figure 6: Experimental study on the reduction of Klebsiella pneumoniae CFU by modified phage lytic enzyme using EDTA, phosphate buffer, and saline in phosphate buffer;

[0110] Figure 7: Experiment on reduction of CFU of Acinetobacter baumannii by modified phage lytic enzyme in normal saline, 50% serum and 90% serum;

[0111] Figure 8: Experimental study on the reduction of Acinetobacter baumannii CFU in bronchoalveolar lavage fluid of bovine and porcine lungs by modified phage lytic enzymes;

[0112] Figure 9: Cytotoxicity assay of multiple lytic enzymes using rabbit erythrocytes;

[0113] Figure 10: In vivo toxicity assay of GNL1250 injected intravenously through the tail vein;

[0114] Figure 11: Treatment of Acinetobacter baumannii infection with GNL1350 in a mouse neutropenic lung infection model;

[0115] Figure 12: SDS-PAGE analysis of GNL1810 purified with the H7MBP tag using a NiNTA column;

[0116] Figure 13: Antibacterial activity of GNL1810 detected by CFU assay;

[0117] Figure 14: Cytotoxicity assay of GNL1810 using rabbit erythrocytes;

[0118] FIG15 : Treatment of Pseudomonas aeruginosa infection with GNL1810 in a mouse neutropenic lung infection model. DETAILED DESCRIPTION

[0119] The present invention is further described in detail by the following examples. These examples are for illustrative purposes only and are not intended to limit the scope of the present invention.

[0120] Example 1. Bacterial strains and growth conditions

[0121] The Pseudomonas aeruginosa strains tested were PAO1, CMCC10211 (GNPW97), and Klebsiella pneumoniae strain CICC21519 (GNPW101). Acinetobacter baumannii strains were clinical isolates from the Air Force Military Medical University (GNPW5), CMCC25001 (GNPW78), and CICC22933 (GNPW102). Strains were cultured in tryptic soy broth (TSB), tryptic soy agar (TSA), or MacConkey agar (Mac). Clinical isolates were confirmed as A. baumannii by morphological examination and 16S rRNA sequencing.

[0122] Example 2. Computer prediction of naturally obtained phage lytic enzymes

[0123] The natural phage lytic enzymes in the present disclosure are identified by the following bioinformatics methods. First, phage prediction algorithms are used to extract phage sequences from the NCBI genome database, such as Moraxellaceae (AB family), Enterobacteriaceae (E. coli family and KP) and Pseudomonadaceae (PA family). Phage lytic enzyme sequences are predicted by performing a BLAST search of the open reading frames (ORFs) in the phage sequences into a local database of known phage lytic enzyme sequences. The predicted sequences are further clustered based on sequence similarity. One to five sequences are selected from each cluster and screened for antimicrobial assays using phage lytic enzymes in clarified lysates, as described below. These active sequences are incorporated into a BLAST search of the NCBI protein database to expand the number of active lytic enzymes. Approximately 170 genes were predicted and synthesized.

[0124] Example 3. Gene synthesis and cloning of naturally derived lytic enzymes

[0125] The lytic enzyme gene was synthesized and cloned into the pET28 plasmid at Sangon Biotech. The plasmid was transformed into the BL21(DE3) E. coli expression strain. Alternatively, the plasmid was first transformed into a temporary host for amplification (DH5α) and then transformed into E. coli BL21(DE3).

[0126] Example 4. Antimicrobial assay using naturally derived phage lytic enzymes in clarified lysates

[0127] The synthesized genes were overexpressed in E. coli and crude lysates were prepared by mechanical and / or chemical lysis of the E. coli. The crude lysates were centrifuged to remove the insoluble fraction. The clarified lysates were used to determine the antimicrobial activity of the lysates against AB, KP, and PA in different matrices, such as saline, saline containing 0.1-0.5 mM EDTA. Of the 170 synthesized, more than 100 naturally derived lysates showed antimicrobial activity against at least one AB, KP, and PA strain in the presence of EDTA, a known outer membrane permeabilizing agent. Table 1 (Sequences of naturally derived lysates with antimicrobial activity) lists nine active lysate sequences. These were further modified to allow for activity in the absence of outer membrane permeabilizing agents in the presence of blood components or bronchoalveolar lavage.

[0128] Table 1: Sequences of naturally occurring lytic enzymes with antimicrobial activity

[0129] Example 5. Modification of naturally occurring phage lytic enzymes with antimicrobial peptides

[0130] The naturally derived lytic enzyme is modified by an N-terminal or C-terminal extension of an antimicrobial peptide (AMP). A linker is used to connect the naturally derived lytic enzyme and the AMP, thereby preserving the structure, function, and mobility of the lytic enzyme and the AMP. The AMPs are selected from previously characterized sequences and have one of the following properties: (a) outer membrane permeabilization; (b) LPS binding and neutralization; (c) antimicrobial activity in blood and blood components; and (d) antibiofilm activity. Non-limiting examples of linker and peptide sequences are listed in Table 2 (non-limiting examples of linker sequences) and Table 3 (non-limiting examples of peptide sequences), respectively. The modified lytic enzyme is created as a naturally derived lytic enzyme-polypeptide linker-antimicrobial peptide construct.

[0131] Table 2: Non-limiting examples of linker sequences

[0132] Table 3: Non-limiting examples of peptide sequences (AMP)

[0133] Example 6. Antibacterial assay using modified lytic enzymes in clarified lysates

[0134] The modified lytic enzymes were cloned into a modified pET28 vector using basic molecular cloning techniques. The plasmids were transformed into an intermediate DH5α E. coli host for amplification and then transformed into BL21 (DE3) for expression. The lytic enzymes were overexpressed in E. coli. Clarified lysates were prepared by mechanical or chemical lysis of E. coli in 20 mM sodium phosphate (pH 7.4) and centrifugation to remove the insoluble fraction. CFU reduction of several AB, KP and PA strains was assessed by clarifying the lysates in the presence of EDTA, saline, 1% human or sheep serum. Table 4 lists non-limiting examples of modified lytic enzymes that showed activity in the presence of serum (non-limiting examples of modified lytic enzymes).

[0135] Table 4: Non-limiting examples of modified lytic enzymes

[0136] Example 7. Structural analysis of naturally derived and modified lytic enzymes

[0137] Peptide sequences were analyzed using Alphafold2 to assess the effects of fusion on the structure of the cleavage enzyme, linker, and peptide. Interestingly, significant differences were observed in the predicted structures of the AMP portion of GNL1249 (pexiganan AMP is unstructured) and GNL993 (pexiganan AMP is in an α-helical conformation). It is well known that pexiganan is unstructured in aqueous solution and adopts an α-helical structure when interacting with membrane lipids. Intramolecular interactions in GNL993, particularly interactions with the α-helical linker found in this molecule, may help stabilize the α-helical conformation, which may promote the increased cleavage enzyme activity after modification. In contrast, GNP27 and GNP30 fold into an α-helical structure regardless of the N-terminal cleavage enzyme or linker. The results of structure prediction of naturally derived and modified cleavage enzymes by AlphaFold2 are shown in Figure 1.

[0138] Example 8. Antibacterial test using partially purified modified lytic enzyme

[0139] Using basic molecular cloning techniques, constructs showing activity in clarified lysates were cloned into the pET28 vector with an N-terminal hepta-histidine and maltose binding protein (H7MBP) tag, and the plasmids were transformed into BL21(DE3) cells for expression. The lyases were overexpressed in E. coli, purified by NiNTA affinity chromatography, and desalted using standard gel filtration methods using Sephadex G-25 media. The tag was cleaved by HRV3C protease. These partially purified lyases were evaluated for the reduction of CFU in the presence of 150 mM NaCl and 0.1 mM EDTA. The results are summarized in Figure 2. Table 4 lists non-limiting examples of modified lyases that showed activity in these assays.

[0140] Partially purified lytic enzymes reduce CFU. Modified lytic enzyme proteins (GNL1771, GNL1780, GNL1799, GNL1815, GNL1847, and GNL1997) were purified using NiNTA and desalted to 20 mM sodium phosphate, pH 7.4, 150 mM NaCl. The affinity tag was cleaved by HRV3C protease. Lytic enzyme activity was evaluated by CFU reduction in 150 mM NaCl and 0.1 mM EDTA. Lytic enzymes that fully or partially reduced the CFU count of Acinetobacter baumannii GNPW78 compared to the control (C) are shown in Figure 2.

[0141] Example 9. Additional mutations to generate lytic enzyme variants

[0142] Using the predicted structures, GNL993, GNL1249, GNL1250, and GNL1350 were further modified to create variants with improved activity in biologically relevant matrices. Previous studies have shown that serum albumin and divalent cations (such as calcium and magnesium) are among the complex components of serum that hinder lytic enzyme activity. It is plausible that reducing the surface charge of serum-active lytic enzymes could achieve two goals: 1) reduce interference with adsorption to negatively charged serum albumin, and 2) reduce electrostatic repulsion of membrane-stabilizing cations on the outer membrane of Gram-negative bacteria. Table 5 lists some possible mutations (possible mutations to reduce surface charge in lytic enzymes). One, two, or any number of mutations can be introduced into the sequence to obtain similar or improved activity.

[0143] Table 6 lists non-limiting examples of modified lytic enzyme sequences, GNL 993, 1249, 1250, and 1799, with some of the mutations listed in Table 5. The sequences were cloned into the pET28 vector with an N-terminal hepta-histidine and maltose binding protein (H7MBP) tag using basic molecular cloning techniques. Partially purified lytic enzymes were prepared as described in Example 10. Concentrations were calculated using the Bradford method. CFU reduction was performed in the presence of 0.1 mM EDTA, saline, and 10% bovine lung extract. The activity of the parent lytic enzyme was not negatively affected by the point mutations, indicating that the surface charge at these positions can be safely modified. Therefore, combining these benign mutations can result in significant changes in surface charge, resulting in molecules with favorable properties (Figure 3). The mutations identified did not impair activity and, when combined, further strongly improved activity.

[0144] Figure 3 shows the activity of modified lyases and their mutations. The activity of modified lyases with surface charge-reducing mutations was compared with that of the parental lyase in the presence of 0.1 mM EDTA (left panel), saline (center panel), and 10% bovine lung extract (right panel). (A) GNL993 and mutations v1 to v6, (B) GNL1249 and mutations v1 to v11, (C) GNL1250 and mutations v1 to v19, and (D) GNL1799 and mutations v1 to v6. The reduction in CFU compared to control C was similar to that observed with the surface charge mutations.

[0145] Table 5. Possible mutations to reduce the surface charge of lyases

[0146] Table 6 Non-limiting examples of improved lyase sequences with mutations

[0147] Example 10. Purification of modified lytic enzyme

[0148] The modified lyase was cloned into a plasmid with an N-terminal seven histidines and a maltose binding protein (H7MBP) tag and expressed in Escherichia coli. The affinity-tagged lyase was purified using a NiNTA column. The tag was cleaved by the HRV3C protease and removed by a second NiNTA column. The homogeneity of the target protein was 50-90%. The production of the modified lyase is not limited to the described method, and any other purification method can be applied to obtain 50-99% pure modified lyase samples. These samples were used for in vitro and in vivo assays.

[0149] Figure 4 shows an SDS-PAGE analysis of the purified modified lyase. The modified lyase was purified by a NiNTA column using an N-terminal hepta-amino acid and maltose binding protein (H7MBP) tag. The tag was cleaved by HRV 3C protease and removed by a second NiNTA column. The target protein purity was 50-90%. The purified lyase sample was loaded into a 4-12% SDS gel and run at 150V for 1 hour. The gel was stained with Coomassie brilliant blue and destained for easy observation.

[0150] Example 11. CFU reduction assay using modified lytic enzymes

[0151] The assay was performed using logarithmically growing bacteria and purified lytic enzymes. Logarithmically growing bacteria were prepared by diluting an overnight culture of GNPW at 1:100 in TSB at 37°C and 200 rpm until the OD 600 0.3-0.8. Centrifuge the culture to collect the cell pellet. Wash the cells twice with physiological saline and adjust the OD 600 Adjust to 0.1-0.5 (~10 7 CFU / mL). OD 0.1-0.5 was diluted 1:100 in the respective matrix and incubated with various concentrations of modified lysate at 37°C, 300 RPM for 1 hour. The assay was then serially diluted 10-fold, 100-fold, and 1000-fold and plated on TSA. The plates were incubated overnight at 37°C, and CFU were counted the next day to compare CFU / mL. The matrices evaluated were buffer (20 mM sodium phosphate, pH 7.4) and saline (0.9% w / v NaCl) for P. aeruginosa GNPW97 (Figure 5); buffer containing 0.5 mM EDTA, buffer, and saline for K. pneumoniae GNPW101 (Figure 6); and saline, sheep serum, human serum, mouse serum, rat serum, horse serum, rabbit serum (Figure 7), bovine lung bronchoalveolar lavage, or porcine lung bronchoalveolar lavage fluid (Figure 8) for Acinetobacter baumannii GNPW 5, 78, and 102. The final concentrations of the purified lysozymes are shown in the figure legends. Predictably, the conditions most favorable to least favorable for lysozyme activity were: the presence of EDTA, the presence of sodium chloride, the presence of lung mucosa, and the presence of serum. All 19 lysozymes were active against A. baumannii in saline, but only five showed a reduction in CFU in sheep serum, equivalent to a complete serum environment (Figure 7).

[0152] Figure 5 shows the reduction of Pseudomonas aeruginosa CFU by modified phage lytic enzymes in phosphate buffer and saline. Assay conditions are as shown. The limit of detection was 100 CFU / mL. Several modified lytic enzymes, particularly GNL1249, demonstrated a 3-log reduction of Pseudomonas aeruginosa at physiological salinity.

[0153] Figure 6 shows the reduction of K. pneumoniae CFU by modified phage lytic enzymes in EDTA, phosphate buffer, and saline in phosphate buffer. Assay conditions are as shown, with a limit of detection of 100 CFU / mL. Several modified lytic enzymes, particularly GNL1249, demonstrated a 3-log reduction of K. pneumoniae at physiological salinity.

[0154] Figure 7 shows the reduction of Acinetobacter baumannii CFU by modified phage lytic enzymes in saline, 50% serum, and 90% serum. Assay conditions are as shown, with a limit of detection of 100 CFU / mL. Several modified lytic enzymes, particularly GNL1250 and GNL1350, demonstrated a 3-log reduction of Acinetobacter baumannii in 90% serum.

[0155] Figure 8 shows the reduction of Acinetobacter baumannii CFU in bronchoalveolar lavage fluid from bovine and porcine lungs by modified phage lytic enzymes. The assay conditions are as shown, with a limit of detection of 100 CFU / mL. Several modified lytic enzymes, particularly GNL1249, GNL1250, and GNL1350, demonstrated a 3-log reduction of Acinetobacter baumannii in 90% of bronchoalveolar lavage fluids.

[0156] Example 12: Minimum inhibitory concentration experiment of modified lytic enzyme

[0157] The activity of the modified lytic enzyme was evaluated using the minimum inhibitory concentration (MIC) method. A single colony of Acinetobacter baumannii GNPW78 cultured overnight on a TSA plate was resuspended in saline to an OD value of 0.1. The bacteria were diluted 1:100 in Mueller-Hinton broth (MHB), and two-fold serial dilutions of the lytic enzyme were added to the MHB. The inoculum size was 5-8 × 10 5 CFU / mL. Cultures were incubated at 35 ± 2°C for 16-20 h. The assay was repeated and the values ​​are shown in Table 7. Since the purity of the lytic enzyme used in the assay is estimated to be 50-90%, the MIC values ​​may vary.

[0158] Table 7. MIC values ​​of lytic enzymes against GNPW78

[0159] Example 13. Hemolysis assay

[0160] One of the risks associated with membrane active peptides and such peptide fusion proteins is that they exhibit cytotoxicity and cause mammalian cell lysis. A hemolytic assay was performed to measure the effect of one of the modified lytic enzymes, GNL1159, GNL1249, GNL 1250, GNL1350, GNL1799, on red blood cells. Red blood cells (RBCs) were collected from fresh blood of rabbits in heparin tubes and suspended in saline to 25%. GNL1159, GNL1249, GNL1250, GNL1350, GNL1799 were serially diluted and 80 μL was added to 20 μL of 25% red blood cells (final concentration of lytic enzyme was 256, 128, 64 μg / mL, red blood cells 5%). Triton X-100 (1%) was used as a positive control. After incubation at 37°C for 1 hour, the intact cells were centrifuged to the bottom and the supernatant was collected to read the absorbance at 405 nm. No hemolysis was observed at 256 μg / mL (the highest concentration in the assay) for GNL1159, GNL1249, GNL1250, GNL1350, and GNL1799.

[0161] Figure 9 shows a cytotoxicity assay for several lytic enzymes using rabbit erythrocytes. Hemolysis assays show that GNL1159, GNL1249, GNL1250, GNL1350, and GNL1799 do not affect erythrocytes. Similar absorbance at 405 nm was detected for all tested concentrations and the 0 μg / mL buffer control. The absorbance was significantly lower than that of the positive control (1% Triton X-100), in which all erythrocytes were lysed.

[0162] Example 14. In vivo toxicity determination of GNL1250 by tail vein injection

[0163] GNL 1250 was administered once intravenously via the tail vein to mice. Survival was followed for 14 days. No adverse effects were observed in mice at doses of 10 or 40 mg / kg.

[0164] Figure 10 shows the in vivo toxicity of GNL 1250. Mice were intravenously injected with GNL 1250 via the tail vein, and their survival was tracked for 14 days, with no adverse effects observed.

[0165] Example 15. Treatment of Acinetobacter baumannii pulmonary infection with GNL1350 in a mouse neutropenic lung infection model

[0166] The potential of GNL1350 to treat Acinetobacter baumannii lung infection was evaluated using a mouse neutropenic lung infection model. Female BALB / c mice aged 6-8 weeks were neutropenic by injection with 150 mg / kg cyclophosphamide on day -4 before infection and 100 mg / kg cyclophosphamide on day -1. Mice were intranasally infected with 1.5 x 10 6 CFU / mouse. Mice were treated intranasally at three time points: 0.5 hours, 3 hours, and 19 hours post-infection with 20 μL of GNL1350 or buffer (200 mM Tris-HCl, pH 7.4). The total dose of GNL1350 was 520 μg. Then, 24 hours post-infection, mice were sacrificed and lungs were removed and placed in 2.5 mL of 2% activated charcoal-containing saline. The bacterial count in the lungs was measured after grinding using a tissue grinder.

[0167] Figure 11 shows the treatment of Acinetobacter baumannii infection in a mouse neutropenic lung infection model using GNL1350. A significant reduction in lung CFU was observed in mice treated with GNL1350 (p < 0.05).

[0168] Example 16: Purification of GNL1810

[0169] GNL1810 (SEQ ID NO: 46) was cloned into a pET28 plasmid containing an N-terminal hepta-histidine and maltose binding protein (H7MBP) tag. The plasmid was transformed into Escherichia coli BL21 (DE3) strain for protein expression. The resulting strain was grown in autoinduction medium at 37°C, 300 RPM for 3.5 hours and then grown overnight at 20°C, 300 RPM. The cells were collected and homogenized in lysis buffer (20 mM sodium phosphate, pH 7.4, 0.5 M NaCl), and the resulting lysate was centrifuged to separate cell debris and soluble components. The soluble fraction was loaded onto an IMAC column and washed with lysis buffer containing 5 mM and 40 mM imidazole. The tagged protein was eluted with lysis buffer containing 350 mM imidazole. To cleave the H7MBP tag, HRV 3C protease was added to the eluted fraction and supplemented with 1 mM DTT. Simultaneously, the solution was dialyzed overnight in lysis buffer. The dialyzed sample was loaded onto the IMAC column again, and the flow-through containing purified GNL1810 was collected ( FIG. 12 ).

[0170] Figure 12 shows SDS-PAGE analysis of GNL1810 purified with an H7MBP tag using a NiNTA column. The modified lysate was purified using a NiNTA column with seven N-terminal histidines and a maltose binding protein (H7MBP) tag. The tag was cleaved by HRV 3C protease and removed by a second NiNTA column. The target protein was ~90% pure. The purified lysate sample was loaded onto a 4-12% SDS gel and run at 150V for 1 hour. The gel was Coomassie stained and destained for visualization.

[0171] Example 17: Minimum inhibitory concentration (MIC) of GNL1810 against Acinetobacter baumannii, Pseudomonas aeruginosa and Klebsiella pneumoniae

[0172] MICs were determined by broth microdilution using Acinetobacter baumannii GNPW78, GNPW102, Pseudomonas aeruginosa GNPW97, and Klebsiella pneumoniae GNPW101. Serial dilutions of purified GNL1810 (Figure 12) were mixed with 1-5 × 10 5 CFU / mL of GNPW78, GNPW102, GNPW97, and GNPW101 were mixed. Each experiment included a sterility control (no lytic enzyme, no bacteria) and a growth control (no lytic enzyme). MIC values ​​are shown in Table 8.

[0173] Table 8: MIC values ​​of GNL1810 against Acinetobacter baumannii, Pseudomonas aeruginosa, and Klebsiella pneumoniae

[0174] Example 18: CFU assay to determine the antibacterial activity of GNL1810

[0175] As described in Example 11, CFU assays were performed using Acinetobacter baumannii GNPW78, Pseudomonas aeruginosa GNPW97, and Klebsiella pneumoniae GNPW101.

[0176] Figure 13 shows the antibacterial activity of GNL1810 as determined by the CFU assay. The assay was performed in 50 mM HEPES, pH 7.0, at 37°C for 1 hour in the presence of various reagents. The dotted line indicates the limit of detection.

[0177] Example 19: In vitro toxicity of GNL1810 - rabbit erythrocyte hemolysis test

[0178] Hemolysis assay was performed as described in Example 13.

[0179] Figure 14 shows a cytotoxicity assay of GNL1810 using rabbit erythrocytes. Hemolysis assays show that GNL1810 does not affect erythrocytes. Similar absorbance at 405 nm was detected for all tested concentrations and the buffer (0 μg / mL) control. The absorbance was significantly lower than that of the positive control (1% Triton X-100), in which all erythrocytes were lysed.

[0180] Example 20: Treatment of Pseudomonas aeruginosa infection with GNL1810 in a mouse neutropenic lung infection model

[0181] The potential of GNL1810 to treat Pseudomonas aeruginosa infection was evaluated using a mouse neutropenic lung infection model. Female C57BL / 6 mice (n=23) aged 6-8 weeks were neutropenic by injection with 100 mg / kg cyclophosphamide 4 days before infection. Each mouse was infected with 4.70 x 10 5 CFU of Pseudomonas aeruginosa strain PAO1. 2 hours after infection, 5 mice were sacrificed, and their lungs were removed and placed in 2.5 mL of 2% activated carbon saline. The lung bacteria count was measured after being ground using a tissue grinder to assess the success of the mouse lung infection model and to determine the true level of lung infection at that time point. 2 hours after infection, CFU / lung reached ~10 5 (Figure 15) Mice were treated intranasally at two time points: 2 and 4 hours post-infection with either 30 μL of 4 mg / mL GNL1810 (n=9) or 300 mM xylitol, 10 mM sodium phosphate buffer, pH 7.4 (n=9). The total dose of GNL1810 was 240 μg. 26 hours post-infection, mice were sacrificed and the bacterial CFU in the lungs was measured using the above method. One mouse in the buffer group succumbed to infection (not counted in the CFU / lung count), while all mice in the GNL1810 group survived.

[0182] Figure 15 shows that treatment of mice with GNL1810 reduced neutrophil counts in P. aeruginosa infection. A significant reduction in lung CFU was observed in GNL1810-treated mice (p value = 0.0056).

Claims

1. A phage lytic enzyme chimera against Gram-negative bacteria, characterized in that: The bacteriophage lytic enzyme chimera is selected from GNL1810 shown in SEQ ID NO:

46.

2. A phage lytic enzyme or a chimera thereof for use against Gram-negative bacteria, characterized in that: The lytic enzymes include: (1) A natural phage lytic enzyme, wherein the natural phage lytic enzyme is selected from the amino acid sequence of GNL14 shown in SEQ ID NO: 1, GNL24 shown in SEQ ID NO: 2, GNL25 shown in SEQ ID NO: 3, GNL46 shown in SEQ ID NO: 4, GNL53 shown in SEQ ID NO: 5, GNL66 shown in SEQ ID NO: 6, GNL68 shown in SEQ ID NO: 7, GNL73 shown in SEQ ID NO: 8, GNL80 shown in SEQ ID NO: 9, or GNL82 shown in SEQ ID NO:

10.

3. A phage lytic enzyme or a chimera thereof for use against Gram-negative bacteria, characterized in that: The lytic enzyme or chimera thereof comprises: (1) a natural phage lytic enzyme selected from the group consisting of the amino acid sequence of GNL14 shown in SEQ ID NO: 1, GNL24 shown in SEQ ID NO: 2, GNL25 shown in SEQ ID NO: 3, GNL46 shown in SEQ ID NO: 4, GNL53 shown in SEQ ID NO: 5, GNL66 shown in SEQ ID NO: 6, GNL68 shown in SEQ ID NO: 7, GNL73 shown in SEQ ID NO: 8, GNL80 shown in SEQ ID NO: 9, or GNL82 shown in SEQ ID NO: 10; more preferably, the amino acid sequence of GNL14 shown in SEQ ID NO: 1, GNL24 shown in SEQ ID NO: 2, GNL25 shown in SEQ ID NO: 3, GNL46 shown in SEQ ID NO: 4, GNL66 shown in SEQ ID NO: 6, GNL68 shown in SEQ ID NO: 7, GNL73 shown in SEQ ID NO: 8, or GNL82 shown in SEQ ID NO: 10; and (2) an antimicrobial peptide, wherein the antimicrobial peptide is selected from the amino acid sequence of GNP6 shown in SEQ ID NO: 19, GNP24 shown in SEQ ID NO: 20, GNP27 shown in SEQ ID NO: 21, GNP30 shown in SEQ ID NO: 22, or GNP45 shown in SEQ ID NO:

23.

4. A phage lytic enzyme or a chimera thereof for use against Gram-negative bacteria, characterized in that: The lytic enzyme or chimera thereof comprises: (1) A natural phage lytic enzyme, wherein the natural phage lytic enzyme is selected from the amino acid sequence of GNL14 shown in SEQ ID NO: 1, GNL24 shown in SEQ ID NO: 2, GNL25 shown in SEQ ID NO: 3, GNL46 shown in SEQ ID NO: 4, GNL53 shown in SEQ ID NO: 5, GNL66 shown in SEQ ID NO: 6, GNL68 shown in SEQ ID NO: 7, GNL73 shown in SEQ ID NO: 8, GNL80 shown in SEQ ID NO: 9, or GNL82 shown in SEQ ID NO: 10; further preferably, the amino acid sequence of GNL14 shown in SEQ ID NO: 1, GNL24 shown in SEQ ID NO: 2, GNL25 shown in SEQ ID NO: 3, GNL46 shown in SEQ ID NO: 4, GNL66 shown in SEQ ID NO: 6, GNL68 shown in SEQ ID NO: 7, GNL73 shown in SEQ ID NO: 8, or GNL82 shown in SEQ ID NO: 10; (2) an antimicrobial peptide, the antimicrobial peptide being selected from the amino acid sequence of GNP6 shown in SEQ ID NO: 19, GNP24 shown in SEQ ID NO: 20, GNP27 shown in SEQ ID NO: 21, GNP30 shown in SEQ ID NO: 22, or GNP45 shown in SEQ ID NO: 23; and (3) a linker, wherein the linker is selected from the amino acid sequence of GNLK1 shown in SEQ ID NO: 11, GNLK2 shown in SEQ ID NO: 12, GNLK4 shown in SEQ ID NO: 13, GNLK5 shown in SEQ ID NO: 14, GNLK6 shown in SEQ ID NO: 15, GNLK7 shown in SEQ ID NO: 16, GNLK8 shown in SEQ ID NO: 17, GNLK9 shown in SEQ ID NO: 18, or GG.

5. The bacteriophage lytic enzyme or chimera thereof according to any one of claims 2 to 4, characterized in that The GNL53 comprises mutations in one, two or more of the following sites: R13E, R13Q or R13G; R18S; K41S or K41P; K52M, K52T; K116A or K141E.

6. The bacteriophage lytic enzyme or chimera thereof according to any one of claims 2 to 4, characterized in that The GNL25 comprises mutations in one, two or more of the following sites: K3Q, R27G, K49D, K56T, R83Q, K106G, R115D, K121D, R124S, R138F, or K155T.

7. The bacteriophage lytic enzyme or chimera thereof according to any one of claims 2 to 4, characterized in that The GNL80 comprises mutations in one, two or more of the following sites: K5A, K22Y, R51Q, K54A, K56D, K77G, R87M, K90A, R93D, K113Q, K129A, or K143E.

8. The bacteriophage lytic enzyme or chimera thereof according to any one of claims 2 to 4, characterized in that The GNL14 comprises mutations in one, two or more of the following sites: K27P, H42D, K48T, K67E, K105Q, or E is inserted after G156, V159, or K160.

9. The bacteriophage lytic enzyme or chimera thereof according to any one of claims 2 to 4, characterized in that The native phage lytic enzyme also includes amino acid sequences or active fragments thereof having at least 80% identity, preferably 85% identity, even more preferably 90% identity, even more preferably 95% identity, even more preferably 96% identity, even more preferably 97% identity, even more preferably 98% identity, even more preferably 99% identity, even more preferably 99.5% identity, and most preferably 99.7% identity.

10. The bacteriophage lytic enzyme or chimera thereof according to claim 3 or 4, characterized in that The antimicrobial peptides also include amino acid sequences or active fragments thereof having at least 80% identity, preferably 85% identity, even more preferably 90% identity, even more preferably 95% identity, even more preferably 96% identity, even more preferably 97% identity, even more preferably 98% identity, even more preferably 99% identity, even more preferably 99.5% identity, and most preferably 99.7% identity.

11. The bacteriophage lytic enzyme or chimera thereof according to claim 4, characterized in that The linkers also include amino acid sequences or active fragments thereof having at least 80% identity, preferably 85% identity, even more preferably 90% identity, even more preferably 95% identity, even more preferably 96% identity, even more preferably 97% identity, even more preferably 98% identity, even more preferably 99% identity, even more preferably 99.5% identity, and most preferably 99.7% identity.

12. The bacteriophage lytic enzyme or chimera thereof according to claim 3 or 4, characterized in that The antimicrobial peptide is fused to the N-terminus or C-terminus of the active peptide. Preferably, the antimicrobial peptide is fused to the C-terminus of the active peptide.

13. The bacteriophage lytic enzyme or chimera thereof according to any one of claims 1 to 12, characterized in that The phage lytic enzyme or chimera thereof is selected from the group consisting of GNL993 shown in SEQ ID NO: 24, GNL1142 shown in SEQ ID NO: 25, GNL1146 shown in SEQ ID NO: 26, GNL1159 shown in SEQ ID NO: 27, GNL1166 shown in SEQ ID NO: 28, GNL1249 shown in SEQ ID NO: 29, GNL1250 shown in SEQ ID NO: 30, GNL1252 shown in SEQ ID NO: 31, GNL1255 shown in SEQ ID NO: 32, GNL1261 shown in SEQ ID NO: 33, GNL1263 shown in SEQ ID NO: 34, GNL1292 shown in SEQ ID NO: 35, GNL1300 shown in SEQ ID NO: 36, GNL1301 shown in SEQ ID NO: 37, GNL1302 shown in SEQ ID NO: 38, GNL1342 shown in SEQ ID NO: 39, 40, GNL1347 shown in SEQ ID NO:40, GNL1350 shown in SEQ ID NO:41, GNL1366 shown in SEQ ID NO:42, GNL1771 shown in SEQ ID NO:43, GNL1780 shown in SEQ ID NO:44, GNL1799 shown in SEQ ID NO:45, GNL1810 shown in SEQ ID NO:46, GNL1815 shown in SEQ ID NO:47, GNL1847 shown in SEQ ID NO:48, or GNL1997 shown in SEQ ID NO:49; or GNL993-v1 shown in SEQ ID NO: 50, GNL993-v2 shown in SEQ ID NO: 51, GNL993-v3 shown in SEQ ID NO: 52, GNL993-v4 shown in SEQ ID NO: 53, GNL993-v5 shown in SEQ ID NO: 54, GNL993-v6 shown in SEQ ID NO: 55, GNL1249-v1 shown in SEQ ID NO: 56, GNL1249-v2 shown in SEQ ID NO: 57, GNL1249-v3 shown in SEQ ID NO: 58, GNL1249-v4 shown in SEQ ID NO: 59, GNL1249-v5 shown in SEQ ID NO: 60, GNL1249-v6 shown in SEQ ID NO: 61, GNL1249-v7 shown in SEQ ID NO: 62, GNL1249-v8 shown in SEQ ID NO: 63, : GNL1250-v5 shown in SEQ ID NO: 71, GNL1250-v6 shown in SEQ ID NO: 72, GNL1250-v7 shown in SEQ ID NO: 73, GNL1250-v8 shown in SEQ ID NO: 74, GNL1250-v9 shown in SEQ ID NO: 75, GNL1250-v10 shown in SEQ ID NO: 76, GNL1250-v11 shown in SEQ ID NO: 77, GNL1250-v2 shown in SEQ ID NO: 78, GNL1250-v3 shown in SEQ ID NO: 79, GNL1250-v4 shown in SEQ ID NO: 80, GNL1250-v5 shown in SEQ ID NO: 81, GNL1250-v6 shown in SEQ ID NO: 82, GNL1250-v7 shown in SEQ ID NO: 83, GNL1250-v8 shown in SEQ ID NO: 84, GNL1250-v9 shown in SEQ ID NO: 85, GNL1250-v10 shown in SEQ ID NO: 86, GNL1250-v11 shown in SEQ ID NO: 87 , GNL1250-v18 shown in SEQ ID NO: 84, GNL1250-v19 shown in SEQ ID NO: 85, GNL1799-v1 shown in SEQ ID NO: 86, GNL1799-v2 shown in SEQ ID NO: 87, GNL1250-v3 shown in SEQ ID NO: 88, GNL1250-v4 shown in SEQ ID NO: 89, GNL1250-v5 shown in SEQ ID NO: 90, GNL1250-v6 shown in SEQ ID NO: 91, GNL1250-v7 shown in SEQ ID NO: 92, GNL1250-v8 shown in SEQ ID NO: 93, GNL1250-v9 shown in SEQ ID NO: 94, GNL1250-v10 shown in SEQ ID NO: 95, GNL1799-v11 shown in SEQ ID NO: 96, GNL1799-v2 shown in SEQ ID NO: 97, GNL1799-v3 shown in SEQ ID NO:The amino acid sequence of GNL1799-v3 shown in SEQ ID NO:88, GNL1799-v4 shown in SEQ ID NO:89, GNL1799-v5 shown in SEQ ID NO:90, or GNL1799-v6 shown in SEQ ID NO:

91.

14. The bacteriophage lytic enzyme or chimera thereof according to claim 13, characterized in that The bacteriophage lytic enzyme or its chimera also includes an amino acid sequence or an active fragment thereof having 80%-100% identity.

15. A nucleic acid molecule encoding the anti-Gram-negative bacteria phage lytic enzyme according to any one of claims 1 to 14, wherein the nucleic acid molecule is DNA or RNA.

16. An expression vector comprising the nucleic acid molecule of the bacteriophage lytic enzyme according to claim 15; preferably, the expression vector is a pET or T7 vector, pBAD or any arabinose-inducible vector, plasmid, phage, virus or artificial chromosome.

17. A host cell comprising the expression vector according to claim 16, characterized in that: The host cell is a microbial cell, preferably a bacterial cell, most preferably an E. coli cell.

18. A composition comprising the anti-Gram-negative bacteria phage lytic enzyme or a chimera thereof according to any one of claims 1 to 14, the nucleic acid according to claim 15, the vector according to claim 16 or the host cell according to claim 17.

19. The composition according to claim 18, characterized in that The composition comprises a pharmaceutically acceptable carrier, buffer or excipient.

20. The composition according to claim 18, characterized in that The composition is formulated as a solution, suspension, emulsion, inhalable powder, aerosol, spray, cream, gel, ointment, mouthwash, antibiotic, sustained release formulation, or a formulation in combination with other antimicrobial agents, antibiotics or disinfectants.

21. The composition according to claim 18, characterized in that The composition is for liquid injection, lyophilized injection, inhalation, external use, nasal application or solid form.

22. The phage lytic enzyme or chimera thereof against Gram-negative bacteria according to any one of claims 1 to 14, the nucleic acid according to claim 15, the vector according to claim 16, the host cell according to claim 17, or the composition according to claims 18 to 21 in the preparation of a phage lytic enzyme against Acinetobacter baumannii, Aeromonas hydrophila, Bacteroides fragilis, Bartonella henselae, Bordetella pertussis, Borrelia burgdorferi, Brucella abortus, Burkholderia cepacia, Campylobacter fetus, Campylobacter jejuni, and Campylobacter jejuni. bacteria, Chlamydia pneumoniae, Chlamydia psittaci, Chlamydia trachomatis, Citrobacter freundii, Coxiella burnetii, Eikenella corrosive, Enterobacter aerogenes, Enterobacter cloacae, Enterococcus faecalis, Escherichia coli, Francisella tularensis, Fusobacterium nucleatum, Haemophilus influenzae, Helicobacter pylori, Klebsiella (preferably Klebsiella granulosus, Klebsiella oxytoca, Klebsiella pneumoniae), Legionella longbeach, Legionella pneumophila, Leptospira, Moraxella catarrhalis, Neisseria gonorrhoeae, Neisseria meningitidis, Pasteurella multiforme, Pseudomonas aeruginosa, Proteus mirabilis, Proteus vulgaris, Rickettsia provazekii, Rickettsia, Salmonella enterica, Serratia marcescens, Shigella dysenteriae, Stenotrophomonas maltophilia, Treponema pallidum, Vibrio cholerae, Yersinia enterocolitica, Yersinia pestis, Aggregatibacter actinomycetemcomitans, Vibrio butzleri, Campylobacter coli, Cardiomycobacterium hominis, Citrobacter, Edwardsiella tarda, Haemophilus ducreyi, Kingella kingii, Leptospira buccal, Use of drugs for infections caused by Mycoplasma colonization, Prevotella intermedia, Proteus pennyroyd, Providencia reticularis, Pseudomonas (Pseudomonas aeruginosa, preferably Pseudomonas putida, Pseudomonas stutzeri, Pseudomonas fluorescens, or Pseudomonas menstruata), Serratia liquefaciens, Serratia Fontica, Serratia red, Serratia odorata, Serratia purimica, Sphingomonas, Nitrotrophomonas nitrosotrophomonas, Rhizotrophomonas rhizosporus, Xanthomonas microphylla, Xanthomonas microphylla or Xanthomonas maltifer.

23. Use of the anti-Gram-negative phage lytic enzyme or chimera thereof according to any one of claims 1 to 14, the nucleic acid according to claim 15, the vector according to claim 16, the host cell according to claim 17, or the composition according to claims 18 to 21 in the preparation of a medicament for treating or preventing infectious diseases, gastrointestinal infections, bloodstream infections, sexually transmitted infections, polymicrobial infections, infections associated with indwelling devices, or diseases caused by bacterial infections; wherein, The infectious disease includes urinary tract infection, further including cystitis, pyelonephritis, prostatitis or urethritis; Diseases caused by gastrointestinal infections include gastroenteritis, enteritis, enterocolitis, food poisoning, typhoid fever, cholera or Helicobacter pylori infection; Diseases caused by the bacterial infection include pneumonia, bronchitis, sinusitis, pharyngitis, tonsillitis and tuberculosis, and also include skin and soft tissue infections, further including cellulitis, impetigo, folliculitis, furunculosis, carbuncle, erysipelas, necrotizing fasciitis, myonecrosis or wound infection; Diseases caused by the bloodstream infection include sepsis, bacteremia, endocarditis and septicemia; Diseases caused by the sexually transmitted infections mentioned include gonorrhea, chlamydia, syphilis, chancroid, and lymphogranuloma venereum; The indwelling device includes a catheter, intravenous line, ventilator, pacemaker, implantable cardioverter-defibrillator, central line, gastrostomy tube, tracheostomy tube, neurostimulator, artificial heart valve, vascular access device, tympanostomy tube, hemodialysis catheter, feeding tube or drainage tube.

24. Use of the anti-Gram-negative phage lytic enzyme or chimera thereof according to any one of claims 1 to 14, the nucleic acid according to claim 15, the vector according to claim 16, the host cell according to claim 17, or the composition according to claims 18 to 21 in the preparation of a medicament for treating or preventing infection caused by meningitis, encephalitis, osteomyelitis, suppurative arthritis, peritonitis, pericarditis, abscess, botulism, tetanus, diphtheria or pertussis.

25. Use of the anti-Gram-negative phage lytic enzyme or chimera thereof according to any one of claims 1 to 14, the nucleic acid according to claim 15, the vector according to claim 16, the host cell according to claim 17, or the composition according to claims 18 to 21 as an antiseptic, disinfectant, bactericide or for combating bacterial biofilms.

26. Use according to claims 22 to 25, characterized in that The anti-Gram-negative phage lytic enzyme or chimera thereof according to any one of claims 1 to 14, the nucleic acid according to claim 15, the vector according to claim 16, the host cell according to claim 17, or the composition according to claims 18 to 21 is used in combination with other antibacterial agents, antibiotics, preservatives, disinfectants, or agents that increase the permeability of the bacterial outer membrane.

27. Use according to claims 22 to 25, characterized in that The anti-Gram-negative phage lytic enzyme or chimera thereof according to any one of claims 1 to 14, the nucleic acid according to claim 15, the vector according to claim 16, or the host cell according to claim 17, or the composition according to claims 18 to 21 has bactericidal activity in serum, lung extract, urinary tract, sputum, or mucus.

28. Use according to claims 22 to 25, characterized in that The anti-Gram-negative phage lytic enzyme or chimera thereof according to any one of claims 1 to 14, the nucleic acid according to claim 15, the vector according to claim 16, the host cell according to claim 17, or the composition according to claims 18 to 21 is contacted with bacteria.

Images