Bacteriophage lysins, chimeras thereof and uses thereof

Abstract

Use of a bacteriophage lysin or a bacteriophage lysin chimera in the manufacture of a medicament, cosmetic, or therapeutic device for preventing, treating, or ameliorating acne or an infection caused by, or a disease associated with, Cutibacterium acnes is provided. Also provided is a bacteriophage lysin chimera.

Description

[Technical field]

[0001] This application claims priority to: Chinese patent application CN202110187030.8, filed February 8, 2021.

[0002] The present invention relates to the field of medical biology, and in particular to bacteriophage lysins having anti-Cutibacterium acnes activity, chimeras thereof and uses thereof. [Background technology]

[0003] Acne is an inflammatory disease of the skin, especially the face, neck, shoulders, chest, back, and upper arms. The pilosebaceous unit becomes inflamed due to blockage from excess oil secretion and accumulation of dead skin cells caused by the proliferation of the pathogenic Cutibacterium acnes (C. acnes). According to the National Institutes of Health, 80% of people between the ages of 11 and 30 have some degree of acne. Acne usually begins during adolescence and can continue into the 40s and 50s. A variety of factors can trigger the outbreak of acne, including stress, changes in hormone levels, and allergens. The 2010 Global Burden of Disease Study estimated that acne affects 9.4% of the world's population, ranking it as the eighth most prevalent disease in the world. The American Academy of Dermatology estimated that in 2013, the costs and lost productivity of treating acne patients exceeded $1.2 billion. While rarely life threatening in itself, it can cause permanent scarring on the skin and considerable emotional distress for the patient.

[0004] Current options for treating mild acne include over-the-counter topical medications containing chemicals such as benzoyl peroxide, resorcinol, salicylic acid, and sulfur, as well as prescription topical medications such as antibiotics, benzoyl peroxide, nonyl diacid, dapsone, corticosteroids, and vitamin A derivatives called retinoids. Moderate to severe disease can be treated with prescription topical or oral medications. The goal of intervention is to unclog the pilosebaceous unit by removing dead skin cells and excess oil, lowering the bacterial load. Acne is a chronic disease, requiring long-term treatment and occasional prevention of recurrence. However, none of the existing treatment options are suitable for long-term treatment. Long-term use of these medications, even the mildest chemicals, can cause allergic reactions and skin irritation, leading to dryness, peeling, and cracking. Antibiotic treatment options are increasingly limited as the resistant bacteria, Cutibacterium acnes, becomes more prevalent worldwide. Moreover, chronic antibiotic use has been shown to disrupt the homeostasis of the microbiota by exerting selective pressure on resistant bacterial species. This leads to a myriad of complications, such as dysbiosis and immune abnormalities. It has been shown in the literature that the staphylococcal flora of the skin of acne patients changes from mostly antibiotic sensitive to mostly resistant during antibiotic treatment. Not only the patients themselves, but also their close contacts are affected by multidrug-resistant bacteria. Notably, acne patients who received long-term antibiotic treatment were two times more likely to develop respiratory infections. Thus, safer, more effective and more suitable therapeutic options for long-term use are urgently needed. One such approach would be pathogen-specific antibacterial agents that specifically kill Cutibacterium acnes, regardless of antibiotic resistance, without affecting other commensal bacteria.

[0005] Additionally, Cutibacterium acnes has been associated with a wide range of invasive infections, including postoperative shoulder infections and intervertebral disc infections. Among such infections, Cutibacterium acnes exhibits a slowly progressive biofilm-like infection. Bacteria found in biofilms are inherently resistant to antibiotics. Cutibacterium acnes antimicrobials with strong anti-biofilm properties are ideal for the treatment of such infections.

[0006] An alternative for pathogen-specific antibacterial agents are bacteriophage endolysins. Bacteriophages use these enzymes to destabilize and lyse the cell wall, releasing the bacteriophage progeny from inside the cell. They are classified as peptidoglycan hydrolases that cleave various bonds in the bacterial peptidoglycan. With very few exceptions, each lysin targets bacteria belonging to a single genus or species. Recombinant lysins against Gram-positive bacteria have been shown to be effective bactericidal agents capable of causing hypotonic lysis (e.g. PlyC against some streptococci, PlyG against Bacillus anthracis, etc.). Given that lysins have been under selective evolutionary pressure for billions of years to continue to successfully infect cells, the peptidoglycan bonds of interest are highly conserved and unlikely to be easily altered by bacteria. This property makes it suitable for long-term therapeutic applications, as there is little chance that the target bacteria will develop resistance to lysin. Bacterial resistance to the respective bacteriophage lysins has not been detected so far. In general, ricins against Gram-positive bacteria consist of one or two N-terminal catalytic domains (CD) and a C-terminal cell wall binding domain (BD), with linkers of different lengths between the domains. Ricins are modular proteins, and chimeric ricins can be made by combining catalytic and binding domains from different ricins. The linkers are also variable in length and sequence. However, the main bottleneck in the development of ricins as antibacterial agents is the inability to soluble express highly active ricins. Indeed, so far, no ricin has been reported that has high levels of soluble expression and effectively kills Cutibacterium acnes.

[0007] Chinese Patent Publication CN102482655A discloses an antibacterial agent composed of an endolysin having activity of degrading the cell wall of Gram-positive bacteria and an amphipathic peptide segment fused to the endolysin at the N-terminus or C-terminus or both ends, which can be used for the treatment or prevention of Gram-positive bacterial infections, as a diagnostic tool or as a cosmetic substance. However, the patent describes that PA6 has anti-P.acnes DSMZ1897 and DSMZ16379 strain activity, but does not provide initial cfu / mL; moreover, PA6 cannot solve the problem of increasing soluble expression level.

[0008] In summary, it is clear that new treatments against Cutibacterium acnes are highly desirable, but to date, no bacteriophage lysin with high Cutibacterium acnes-killing activity suitable for industrial-scale production has been reported. The ability of bacteriophage lysin to specifically and efficiently kill Cutibacterium acnes while leaving other commensal bacteria intact may provide a safe and effective acne solution for long-term use without the high risks of dysbiosis and resistance development. Summary of the Invention

[0009] Contents of the invention In order to address the above technical situation, the present invention provides a bacteriophage lysin having anti-Cutibacterium acnes activity, its chimera and use thereof. The details of the present invention are as follows: DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0010] The present invention provides the use of a bacteriophage lysin or chimera thereof in the manufacture of a medicament, cosmetic, or medical device for preventing, treating, or ameliorating acne or an infection caused by, or a disease associated with, Cutibacterium acnes, wherein the bacteriophage lysin comprises a bacteriophage lysin derived from a bacterium of the family Nocardioidaceae or Propionibacteriaceae.

[0011] In one embodiment of the use of the present invention, the Nocardioidaceae bacteria include bacteria of the genera Micropruina, Propionicimonas, Propionicicella, and Friedmanniella; the Propionibacteriaceae bacteria include bacteria of the genera Propioniferax, Mariniluteicoccus, Granulicoccus, Naumannella, Propioniciclava, Auraticoccus, Microlunatus, Aestuariimicrobium, and the like. m), Luteococcus, Tessaracoccus, Brooklawnia, Propionimicrobium, Propionibacterium, Cutibacterium, Acidipropionibacterium, or Pseudopropionibacterium bacteria, preferably Propionibacterium, Cutibacterium, Acidipropionibacterium, or Pseudopropionibacterium bacteria.

[0012] In one embodiment of the use of the present invention, the bacteriophage lysin is selected from the group consisting of Cutibacterium acnes, Propionibacterium humerusii, Cutibacterium avidum, Cutibacterium granulosum, Acidipropionibacterium thoenii, Acidipropionibacterium jensenii, Acidipropionibacterium acidipropionici, Aestuariimicrobium kwangyangse, and the like. kwangyangense, Granulicoccus phenolivorans, Microlunatus phosphovorus, Pseudopropionibacterium propionicum, Tessaracoccus sp., Propionicicella superfundia, Propionibacterium freudenreichii, Propionibacterium freudenreichii subsp. freudenreichii, Propionibacterium freudenreichii subsp. shamanii, Propionibacterium freudenreichii subsp.shermanii, Propionibacterium acidifaciens, Propionibacterium lymphophilum, Propionibacteriaceae bacterium, Propionibacterium sp. oral taxon 192, Propioniferax innocua, Naumannella halotolerans, Propioniciclava tarda, Micropruina glycogenica, Propionicimonas paldicola These include bacteriophage lysins from Auraticoccus paludicola, Auraticoccus monumenti, Luteococcus japonicus, Tessaracoccus oleiagri, Tessaracoccus bendigoensis, Tessaracoccus lapidicaptus, Acidipropionibacterium microaerophilum, Acidipropionibacterium olivae, and Acidipropionibacterium damnosum.

[0013] In one embodiment of the use of the present invention, the bacteriophage lysin is selected from the group consisting of Acidipropionibacterium jensenii, Acidipropionibacterium thoenii, Acidipropionibacterium acidipropionici, Acidipropionibacterium microaerophilum, Acidipropionibacterium olivae, Acidipropionibacterium damnosum, Cutibacterium acnes, and the like. These include the bacteriophage lysins from Cutibacterium acnes, Cutibacterium avidum, Cutibacterium granulosum, and Pseudopropionibacterium propionicum.

[0014] In one embodiment of the use of the present invention, the bacteriophage lysin has an amino acid sequence as set forth in any one of SEQ ID NO: 1 to SEQ ID NO: 28;

[0015] In one embodiment of the use of the present invention, the bacteriophage lysin has a nucleotide sequence as set forth in any one of SEQ ID NO: 29 to SEQ ID NO: 56;

[0016] In one embodiment of the present invention, the bacteriophage lysin preferably has the amino acid sequence PACL10 shown in SEQ ID NO: 10;

[0017] In one embodiment for use according to the invention, the bacteriophage lysin is preferably PACL10 having the nucleotide sequence shown in SEQ ID NO:38.

[0018] In one embodiment of the use of the present invention, the chimera comprises a catalytic domain derived from a bacteriophage lysin, or a combination of a catalytic domain and a binding domain derived from a bacteriophage lysin.

[0019] In one embodiment of the use of the present invention, the catalytic domain has a complete C-terminal linker, a half C-terminal linker, no C-terminal linker, or any part of said linker; the binding domain has a complete N-terminal linker, a half N-terminal linker, no N-terminal linker, or any part of said linker.

[0020] In the use of the present invention, as one embodiment, a synthetic linker may be used, which also includes synthetic linkers. Non-limiting examples of possible linkers derived from bacteriophage lysin are shown in Table 3 below. For non-limiting examples of synthetic linkers, see Table 4 below.

[0021] In one embodiment of the use of the present invention, chimeras are formed by pairing catalytic and binding domains from different bacteriophage lysins.

[0022] In one embodiment of the use of the present invention, the chimera comprises one or two catalytic domains without a binding domain;

[0023] In one embodiment, the chimera comprises one or more catalytic domains and one or more binding domains;

[0024] In one embodiment, the chimera may be one or more catalytic domains connected to a single binding domain;

[0025] In one embodiment, the chimera may be a single catalytic domain connected to a single binding domain;

[0026] In one embodiment, the chimera can be a single catalytic domain at the N-terminus of ricin linked to a single binding domain at the C-terminus of ricin.

[0027] Moreover, one or more catalytic domains can be used without the presence of binding domain.Furthermore, for special use such as detection, one or more binding domains can be used without the presence of catalytic domain and fused to different molecules to facilitate detection.Such molecules include but are not limited to biotin, flag tag, myc tag, avidin, streptavidin, ovalbumin, firefly luciferase, biotin, fluorescent molecules such as FITC, TRITC, Alexafluor, etc.

[0028] In one embodiment of the use of the invention, the chimera further comprises a linker between the catalytic domain and the binding domain, said linker comprising: 1) a linker region, or any portion thereof, derived from the parent ricin molecule from which the catalytic domain is derived; In one embodiment of the use of the present invention, the linker region derived from the parent ricin molecule has the amino acid sequence shown in any one of SEQ ID NO:343 to SEQ ID NO:367.

[0029] 2) a linker region, or any portion thereof, derived from the parent ricin molecule from which the binding domain is derived; In one embodiment of the use of the present invention, the linker region derived from the parent ricin molecule has any of the nucleotide sequences set forth in SEQ ID NO: 368 to SEQ ID NO: 392; or

[0030] 3) A synthetic linker domain having an amino acid sequence as set forth in any one of SEQ ID NOs: 393 to 406, or an amino acid sequence of (GGGS)n, (GGGGS)n, (GGGGGS)n, (Gly)3-8, (EAAAK)n, (Ala-Pro)n, A(EAAAK)nALEA(EAAAK)nA (wherein 1≦n≦15, and n is an integer), where n can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15. Includes.

[0031] In one embodiment, the chimera for use according to the invention comprises a catalytic domain having the amino acid sequence as set forth in SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:77, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:91, SEQ ID NO:93, SEQ ID NO:95, SEQ ID NO:97, SEQ ID NO:98, SEQ ID NO:99, SEQ ID NO:100, SEQ ID NO:102, SEQ ID NO:104, SEQ ID NO:105, SEQ ID NO:106, SEQ ID NO:107, SEQ ID NO:108, SEQ ID NO:110, SEQ ID NO:111, SEQ ID NO:113, SEQ ID NO:114, SEQ ID NO:117, SEQ ID NO:118, SEQ ID NO:121, SEQ ID NO:122, SEQ ID NO:125, SEQ ID NO:127, SEQ ID NO:128, SEQ ID NO:131, SEQ ID NO:132, SEQ ID NO:133, SEQ ID NO:135 or SEQ ID NO:136; The binding domain has the amino acid sequence as set forth in SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:68, SEQ ID NO:72, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:92, SEQ ID NO:94, SEQ ID NO:96, SEQ ID NO:101, SEQ ID NO:103, SEQ ID NO:109, SEQ ID NO:112, SEQ ID NO:115, SEQ ID NO:116, SEQ ID NO:119, SEQ ID NO:120, SEQ ID NO:123, SEQ ID NO:124, SEQ ID NO:126, SEQ ID NO:129, SEQ ID NO:130, SEQ ID NO:134, SEQ ID NO:137, SEQ ID NO:138 or SEQ ID NO:139.

[0032] In one embodiment of the use of the present invention, the catalytic domain is selected from the group consisting of SEQ ID NO:140, SEQ ID NO:141, SEQ ID NO:142, SEQ ID NO:144, SEQ ID NO:146, SEQ ID NO:147, SEQ ID NO:148, SEQ ID NO:149, SEQ ID NO:150, SEQ ID NO:152, SEQ ID NO:153, SEQ ID NO:154, SEQ ID NO:156, SEQ ID NO:157, SEQ ID NO:160, SEQ ID NO:163, SEQ ID NO:164, SEQ ID NO:168, SEQ ID NO:169, SEQ ID NO:170, SEQ ID NO:174, SEQ ID NO:176, SEQ ID NO:178, SEQ ID NO: 180, SEQ ID NO:181, SEQ ID NO:182, SEQ ID NO:183, SEQ ID NO:185, SEQ ID NO:187, SEQ ID NO:188, SEQ ID NO:189, SEQ ID NO:190, SEQ ID NO:191, SEQ ID NO:193, SEQ ID NO:194, SEQ ID NO:196, SEQ ID NO:197, SEQ ID NO:200, SEQ ID NO:201, SEQ ID NO:204, SEQ ID NO:205, SEQ ID NO:208, SEQ ID NO:210, SEQ ID NO:211, SEQ ID NO:214, SEQ ID NO:215, SEQ ID NO:216, SEQ ID NO:218, SEQ ID NO:219; The binding domain has the nucleotide sequence as set forth in SEQ ID NO:143, SEQ ID NO:145, SEQ ID NO:151, SEQ ID NO:155, SEQ ID NO:158, SEQ ID NO:159, SEQ ID NO:161, SEQ ID NO:162, SEQ ID NO:165, SEQ ID NO:166, SEQ ID NO:167, SEQ ID NO:171, SEQ ID NO:172, SEQ ID NO:173, SEQ ID NO:175, SEQ ID NO:177, SEQ ID NO:179, SEQ ID NO:184, SEQ ID NO:186, SEQ ID NO:192, SEQ ID NO:195, SEQ ID NO:198, SEQ ID NO:199, SEQ ID NO:202, SEQ ID NO:203, SEQ ID NO:206, SEQ ID NO:207, SEQ ID NO:209, SEQ ID NO:212, SEQ ID NO:213, SEQ ID NO:217, SEQ ID NO:220, SEQ ID NO:221, or SEQ ID NO:222.

[0033] In one embodiment of the use of the present invention, the chimera has an amino acid sequence as set forth in any one of SEQ ID NO: 223 to SEQ ID NO: 282; preferably, an amino acid sequence as set forth in SEQ ID NO: 258, SEQ ID NO: 259, SEQ ID NO: 260, SEQ ID NO: 271 or SEQ ID NO: 272; more preferably, an amino acid sequence as set forth in SEQ ID NO: 260 or SEQ ID NO: 271;

[0034] In one embodiment of the use of the present invention, the chimera has a nucleotide sequence as set forth in any one of SEQ ID NO:283 to SEQ ID NO:342; preferably, the nucleotide sequence as set forth in SEQ ID NO:316, SEQ ID NO:317, SEQ ID NO:318, SEQ ID NO:329 or SEQ ID NO:330; more preferably, the nucleotide sequence as set forth in SEQ ID NO:318 or SEQ ID NO:329.

[0035] In one embodiment of the use of the present invention, the infection caused by Cutibacterium acnes includes an invasive infection, a post-operative infection and / or a device-associated infection.

[0036] In one embodiment of the use of the present invention, device-associated infections include artificial joints, shunt tubing and artificial heart valve-associated infections.

[0037] In the use of the present invention, in one embodiment, the infections include bone and / or joint infections, particularly post-operative shoulder infections, as well as oral, eye, disc and brain infections.

[0038] In one embodiment of the use of the present invention, the disease associated with Cutibacterium acnes includes cancerous prostatitis, SAPHO (synovitis, acne, impetigo, hyperplasia, osteitis) syndrome, sarcoidosis, or sciatica.

[0039] In one embodiment of the use of the present invention, the medical device includes any device for releasing ricin or its chimeras to the affected area, preferably a clamp or patch or spray applied to the skin surface, a device using microneedles to enhance skin penetration of ricin or its chimeras, fine needles used by beauty professionals to apply ricin or its chimeras to hair follicles, particularly those affected by acne, or other similar devices.

[0040] In one embodiment of the use of the present invention, the medical device comprises a prosthetic device having ricin or a chimera thereof immobilized in a location susceptible to Cutibacterium acnes infection, preferably a prosthetic implant for use in shoulder surgery, which is particularly susceptible to Cutibacterium acnes infection.

[0041] The present invention also provides a bacteriophage lysin chimera as described in the above-mentioned use. In one embodiment, the chimera has an amino acid sequence as set forth in any one of SEQ ID NOs: 223 to 282; preferably, an amino acid sequence as set forth in SEQ ID NO: 258, SEQ ID NO: 259, SEQ ID NO: 260, SEQ ID NO: 271, or SEQ ID NO: 272; more preferably, an amino acid sequence as set forth in SEQ ID NO: 260 or SEQ ID NO: 271.

[0042] In one embodiment of the use of the present invention, the chimera has a nucleotide sequence as set forth in any one of SEQ ID NO:283 to SEQ ID NO:342; preferably, the nucleotide sequence as set forth in SEQ ID NO:316, SEQ ID NO:317, SEQ ID NO:318, SEQ ID NO:329 or SEQ ID NO:330; more preferably, the nucleotide sequence as set forth in SEQ ID NO:318 or SEQ ID NO:329.

[0043] The present invention also provides (1) synthesizing a domain sequence and a primer for amplifying the domain sequence; (2) using Taq DNA polymerase PCR to amplify domain sequences; (3) gel purifying the PCR product and ligating it into the expression plasmid pET28; (4) introducing the recombinant plasmid into E. coli BL21(DE3); (5) culturing E. coli BL21(DE3) containing the recombinant plasmid, inducing expression, harvesting the cells by centrifugation, lysing and purifying to obtain the chimera; The present invention provides a method for producing the aforementioned chimera, comprising:

[0044] In one embodiment of the method of the present invention, the method further comprises: E. coli BL21(DE3) containing the recombinant chimeric lysine expression plasmid was cultured in autoinduction medium at 37 °C and 300 rpm for OD 600 Incubate until the pH reaches 0.6-0.8, then continue incubation at 18°C ​​and 300 rpm for 16-18 hours. Harvest the cells by centrifugation, resuspend in 50 mM sodium phosphate, pH 7.4, and lyse to homogeneity under high pressure. Centrifuge the lysate again to collect the soluble crude lysate. Mix the soluble fraction with an equal volume of 5 M NaCl, and load the mixture onto the hydrophobic column. After sample loading, wash the column with 5 column volumes of 20 mM sodium phosphate, pH 7.4, 2.5 M NaCl. Then elute the recombinant chimeric lysin with 10 mM sodium phosphate, pH 7.4. Alternatively, expression and purification can be optimized by one skilled in the art, and ricin can be expressed in LB using different concentrations of IPTG at different temperatures, aeration and induction times. Cells can be resuspended in various buffers to enhance solubility and lysed by mechanical or chemical means. Various protein chromatography methods can be used to purify ricin. These variables can be optimized for better ricin protein yield.

[0045] The invention also provides formulations containing the chimeras which further comprise antibiotics, other lysines, or inert excipients.

[0046] The present invention also provides amino acid sequences encoding the aforementioned chimeras, or amino acid sequences having 80% or more, 85% or more, 90% or more, 95% or more, or 99% or more similarity thereto, or alternative amino acid sequences having the same functional groups. As illustrative examples, the present invention also provides amino acid sequences having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% similarity thereto.

[0047] In one embodiment, the alternative amino acid sequence is a conservative substitution using amino acids in the same amino acid group, the amino acid group being: Aliphatic: glycine, alanine, valine, leucine, isoleucine; Hydroxyl or sulfur / selenium containing: serine, cysteine, threonine, methionine; Cyclic: Proline; Aromatic: phenylalanine, tyrosine, tryptophan; Basic: histidine, lysine, arginine; Acids and their amides: Aspartic acid, glutamic acid, asparagine, glutamine Includes.

[0048] The present invention also provides nucleotide sequences encoding the above-mentioned chimeras, or synonymous codon sequences thereof.

[0049] The present invention also provides the use of the aforementioned chimeras as reagents for bacterial lysis for DNA extraction and typing using PCR-based kits.

[0050] The present invention also provides the use of the aforementioned chimeras as disinfectants or sterilants on non-biological surfaces to eliminate Cutibacterium acnes in planktonic form or in biofilm, preferably to prevent infections by disinfecting surgical or prosthetic implant devices during surgical procedures.

[0051] The present invention also provides the use of the above-described chimera, a single binding domain therein, or a series of combinations of similar or different binding domains in the chimera, in the manufacture of a diagnostic tool for Cutibacterium acnes, wherein the lysine, chimera or binding domain is used in combination with a detection marker.

[0052] In one embodiment, the lysine, chimera or binding domain and the signal molecule form a fusion through genetic fusion or chemical coupling.

[0053] In one embodiment, the fusion is used for direct detection of Cutibacterium acnes on a microscope slide by fluorescence or other means, for labeling Cutibacterium acnes by immunohistochemistry, as a detection reagent in an ELISA assay, as a detection reagent in a Western blot, for attachment to magnetic beads in a MACS or other pull-down assay, or as a detection reagent in an assay in which an antibody is used as a detection reagent. In one embodiment, the signal molecule comprises a protein or chemical fluorescent dye, protein tag, enzyme, avidin, streptavidin, ovalbumin, biotin, click chemistry labeling sensitive tag, intein, or other molecule that can trigger recruitment of a secondary protein or molecule to generate a signal, wherein The fluorescent dye comprises GFP, RFP, mCherry, FITC, TRITC, Alexafluor 488, Cy3 or Cy5; The protein tag includes a Flag tag, a myc tag, a halo tag, a his tag, or any other tag that can bind to an antibody or other high affinity molecule to generate a signal; Such enzymes include firefly luciferase, β-lactamase, alkaline phosphatase, horseradish peroxidase, or any other enzyme that produces light, a color change, deposition of a substrate, or other reaction that can be detected in the test.

[0054] The present invention also provides the use of a catalytic domain in the above-described chimera in the manufacture of a medicament for the treatment of Cutibacterium acnes infections, wherein the catalytic domain is combined with a targeting module.

[0055] The present invention provides compositions and methods for preventing and treating Cutibacterium acnes infections and skin colonization, and acne lesions associated with such infections or colonization. In a broad aspect, the present invention provides the application and use of lysine, which has broad killing activity against skin-associated microorganisms (including, but not limited to, bacteria of the genera Propionibacterium, Cutibacterium, Acidipropionibacterium, and Pseudopropionibacterium) that are involved in the development or exacerbation of acne lesions, and secondary infections of acne lesions. In particular, the present invention describes methods for decolonization, dispersion, and elimination of established bacterial flora in the skin, with a particular focus on Cutibacterium acnes (formerly known as Propionibacterium acnes). Additionally, the present invention provides methods for producing chimeric lysines that contain catalytic and binding domains from different lysines that provide a variety of advantageous properties, including, but not limited to, improved activity, host range, expression, solubility, stability or better suitability for commercialization.

[0056] According to the present invention, the bacteriophage lysins used in the methods and uses of the present invention are derived from the bacteria Propionibacterium, Cutibacterium, Acidipropionibacterium, Pseudopropionibacterium. Table 2 provides the lysins and polypeptides used in the present invention.

[0057] In one embodiment of the present invention, the lysines shown in SEQ ID NO:1 to SEQ ID NO:28 are encoded by the nucleotide sequences shown in SEQ ID NO:29 to SEQ ID NO:56.

[0058] The present invention also provides methods for producing chimeras between binding and catalytic domains of different bacteriophage lysins. SEQ ID NO:57 to SEQ ID NO:139 show a non-limiting sequence listing providing examples of polypeptide sequences encoding catalytic and binding domains from bacteriophage lysins of the present invention.

[0059] In one embodiment of the present invention, the catalytic domains and binding domains shown in SEQ ID NO:57 to SEQ ID NO:139 are encoded by the nucleotide sequences shown in SEQ ID NO:140 to SEQ ID NO:222.

[0060] The present invention also provides non-limiting examples of chimeric lysins produced by pairing catalytic and binding domains of different lysins as described herein. SEQ ID NOs:223-282 provide a non-limiting sequence listing of chimeric lysins active against Cutibacterium acnes produced by the methods of the present invention.

[0061] In one aspect of the present invention, the nucleotide sequences set forth in SEQ ID NO: 283 to SEQ ID NO: 342 are used to produce the chimeric lysines described in the present invention.

[0062] In one embodiment of the invention, the polypeptide sequence is encoded by a nucleotide sequence inserted into the expression vector pET28.

[0063] In one embodiment of the invention, an expression vector encoding a polypeptide of the invention is expressed in E. coli strain BL21(DE3).

[0064] Additionally, the present invention provides methods for screening and evaluating the activity of bacteriophage lysins against Cutibacterium acnes.

[0065] Furthermore, the present invention demonstrates the activity of a defined bacteriophage lysin against Cutibacterium acnes.

[0066] In one embodiment of the present invention, the lysine for treating Propionibacterium is PACL10. The present invention provides a method for expressing and purifying PACL10. Furthermore, the present invention demonstrates that PACL10 has higher antibacterial activity against Cutibacterium acnes.

[0067] The activity of PACL10 against a series of Cutibacterium acnes strains from different clades was evaluated by minimum inhibitory concentration (MIC) and time-kill tests, and the results consistently showed that PACL10 had high efficacy.

[0068] The bacteriophage lysin and its derivatives produced in the present invention are suitable for industrial-scale production, can specifically and effectively kill Cutibacterium acnes while leaving other commensal bacteria intact, and can provide a safe and effective acne solution suitable for long-term use without high risks of dysbiosis and resistance acquisition. The bacteriophage lysin and its derivatives produced in the present invention have the following advantages: (1) It can kill Cutibacterium acnes (C. acnes) without damaging the natural bacterial flora. (2) Rapid activity (within minutes) compared with antibiotics, which take a long time to become effective. (3) No drug resistance. Bacteria quickly acquire resistance to antibiotics, so lysine is the solution. C. acnes is a microorganism that strongly secretes biofilms. Even microorganisms that are sensitive to antibiotics can become resistant to antibiotics if they are found in the form of biofilms. Lysine is highly effective against biofilms. (4) The present invention is more efficient than other ricins, with lower MICs and higher activity in other assays. (5) Soluble expression is possible, the yield is high, and it is suitable for mass production and industrial production. (6) It is effective against all clades of P. acnes.

[0069] definition A non-limiting example of a comparison of the old and new names following the reclassification of the genus Propionibacterium by Scholz et al. is shown in Table 1 below:

[0070] [Table 1]

[0071] Those skilled in the art should understand that the reclassification, old and new names, and Chinese names of bacteria should not affect the identification of bacteria. Whether the new name or the old name is adopted in the present invention, it represents the same bacterial species.

[0072] Non-limiting examples of sequences relevant to the present invention are set forth in Table 2 below.

[0073] [Table 2-1]

[0074] [Table 2-2]

[0075] [Table 2-3]

[0076] [Table 2-4]

[0077] [Table 2-5]

[0078] [Table 2-6]

[0079] Non-limiting examples of natural linkers as described in the present invention are shown in Table 3 below:

[0080] [Table 3]

[0081] Non-limiting examples of synthetic linkers described in this invention are shown in Table 4 below:

[0082] [Table 4] [Brief description of the drawings]

[0083] [Figure 1] Example 3 shows the effective killing effect of selected ricins against Cutibacterium acnes. Ricin was expressed in E. coli BL21(DE3) on LB agar containing IPTG and released by osmotic lysis of the E. coli. A clear zone or halo around the E. coli indicates that the expressed ricin inhibits the growth or kills Cutibacterium acnes embedded in the agar overlay. [Diagram 2] Example 3 shows clearing zones formed on Cutibacterium acnes overlays by crude E. coli lysates containing induced selected lysines. [Diagram 3] Example 4 demonstrates that PACL10 can be soluble expressed and purified. All samples were run in a 5-12% Tris-glycine gel at 150 V for 50 min and stained with Coomassie Brilliant Blue. PACL10 was expressed in E. coli BL21(DE3) using the pET expression system and purified by hydrophobic interactions. The eluted fraction shows purified PACL10 at 29.4 kDa. [Figure 4]Example 7 shows the kinetics of PACL10 in reducing cfu / mL of Cutibacterium acnes strain 34A. With a MIC of 6.4 μg / mL and 2×MIC of 12.8 μg / mL, the cfu / mL could be below the detection limit within 3 hours and 2 hours, respectively. [Diagram 5] Single-step purified ricin for MIC determination. Ricin concentrations loaded onto a 5-12% Tris-HCl SDS gel were: 0.62 mg / mL PACL10, 1.41 mg / mL PACL390, 2.12 mg / mL PACL391, 2.10 mg / mL PACL392, 1.49 mg / mL PACL403, and 1.60 mg / mL PACL404. [Figure 6] 1 shows PACL392 purified by mixed mode and ion exchange chromatography. [Figure 7] 1 shows the bactericidal activity of PACL392 compared to strain 10. [Figure 8] 1 shows PACL403 purified by mixed-mode chromatography. [Figure 9] Bactericidal activity of PACL403 against strain 10 - Reduction in OD is shown. [Figure 10] Bactericidal activity of PACL403 against strain 10 - CFU reduction. [Figure 11] 1 shows the bactericidal activity of PACL403 compared to biofilm-associated strain 10. EXAMPLES

[0084] The following examples and / or experimental examples are only used to further illustrate the present invention and are not intended to limit the scope of the present invention in any way. The experiments were carried out using standard laboratory methods and gave the results shown in the examples.

[0085] Example 1 By analyzing the sequences of prophages in the genomes of Propionibacteria, we identified a Cutibacterium acnes bacteriophage lysin that was capable of soluble expression and purification. The gene sequence expressing the bacteriophage lysin was synthesized by Sangon and ligated into the expression plasmid pET28. Each recombinant plasmid was then transformed into the E. coli BL21(DE3) strain.

[0086] Example 2 Chimeras were constructed as an N-terminal catalytic domain (CD) and a C-terminal binding domain (BD) with a linker of variable length between them. The domains were identified by sequence analysis using the NCBI constant region database and the RaptorX web server for in silico protein structure prediction. The catalytic domains had a full C-terminal linker, half a C-terminal linker, or no C-terminal linker. Similarly, the binding domains had a full N-terminal linkage, half an N-terminal linkage, or no N-terminal linkage. Primers to amplify the domain sequences were synthesized by GeneCreate. The domain sequences were amplified using Taq DNA polymerase PCR with an annealing temperature of 55°C. The PCR products were gel purified and ligated with the expression plasmid pET28. The recombinant plasmids were then introduced into E. coli BL21(DE3).

[0087] Example 3 The antibacterial activity of ricin was confirmed by Cutibacterium acnes coverage test. Briefly, E. coli BL21(DE3) clones containing the recombinant ricin plasmid were plated on LB agar with IPTG for induction. Upon overexpression of ricin, the E. coli clones osmotically release ricin. The soft agar embedded with Cutibacterium acnes was overlaid and incubated for the growth of Cutibacterium acnes. In the presence of active ricin, a clear zone or halo is formed around the E. coli. The experimental results are shown in Figure 1.

[0088] In a similar experiment, E. coli BL21(DE3) clones containing the recombinant ricin plasmid were induced in liquid medium. Cells were harvested by centrifugation and sonicated in 50 mM sodium phosphate, pH 7.4 buffer. The lysate was centrifuged to separate soluble and insoluble fractions. The soluble crude lysate was spotted onto soft agar embedded with Cutibacterium acnes. The active ricin in the soluble crude lysate formed a clearing zone after incubation for the growth of Cutibacterium acnes. The experimental results are shown in Figure 2.

[0089] Example 4 Expression and purification of PACL10 Expression and purification of PACL10 was as follows. Briefly, E. coli BL21(DE3) containing the recombinant PACL10 expression plasmid was cultured in autoinduction medium at 37°C and 300 rpm until OD 600 The culture was continued until the pH reached 0.6-0.8, and then culture was continued at 18°C ​​and 300 rpm for 16-18 h. The cells were collected by centrifugation, resuspended in 50 mM sodium phosphate, pH 7.4, and homogenized under high pressure. The lysate was centrifuged again to collect the soluble crude lysate. The soluble fraction was mixed with an equal volume of 5 M NaCl, and the mixture was loaded onto the hydrophobic column. After sample loading, the column was washed with 5 column volumes of 20 mM sodium phosphate, pH 7.4, 2.5 M NaCl. PACL10 was then eluted with 10 mM sodium phosphate, pH 7.4. The experimental results are shown in Figure 3.

[0090] Example 5 Chimeric lysin was expressed and purified in a similar manner to PACL10. E. coli BL21(DE3) containing the recombinant chimeric lysin expression plasmid was cultured at OD at 37 °C and 300 rpm in autoinduction medium. 600The cells were cultured until the NA reached 0.6-0.8, and then culture was continued at 18 °C and 300 rpm for 16-18 h. The cells were collected by centrifugation, resuspended in 50 mM sodium phosphate, pH 7.4, and homogenized under high pressure. The lysate was centrifuged again to collect the soluble crude lysate. The soluble fraction was mixed with an equal volume of 5 M NaCl, and the mixture was loaded onto a hydrophobic column. After sample loading, the column was washed with 5 column volumes of 20 mM sodium phosphate, pH 7.4, 2.5 M NaCl. The recombinant chimeric lysin was then eluted with 10 mM sodium phosphate, pH 7.4.

[0091] Example 6 Determination of minimum inhibitory concentration The method for measuring the minimum inhibitory concentration (MIC) is as follows. First, the colony suspension is diluted with OD 600 Prepare in 0.9% saline to a concentration of 0.05 and 10 per mL 7 Prepare an inoculum equivalent to 5 × 10 colony forming units (cfu / mL). The suspension is diluted in Reinforced Clostridial Medium (RCM) without agar at 10 °C for 1 h. 5 The inoculum was diluted 20-fold to 100 cfu / mL. Two-fold serial dilutions of PACL10 or vancomycin were prepared in 0.9% saline and added to no more than one-tenth of the total culture. Cultures were grown for 3 days at 37°C under anaerobic conditions. The MIC is the lowest concentration of PACL10 or vancomycin at which no growth of Cutibacterium acnes was observed with the naked eye. The minimum bactericidal concentration (MBC) was determined by subculturing the above cultures on Reinforced Clostridium Agar (RCA) plates containing 0.1% Tween-80. The MBC is the lowest concentration of PACL10 or vancomycin at which no Cutibacterium acnes colonies were observed on the Reinforced Clostridium Agar (RCA) plates.

[0092] Experimental results: Table 6-1 shows the results of IA compared with vancomycin. 1 , I.A. 2The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of PACL10 to 12 strains of Cutibacterium acnes, which belong to clade II, are shown.

[0093] [Table 6-1]

[0094] Example 7 Time Killing Test The time-kill test method is as follows: Cutibacterium acnes 34A colonies were cultured at OD 600 is 0.05(10 7 The suspension is resuspended in 0.9% saline until it is approximately 10 cfu / mL. 6 cfu / mL. Two-fold serial dilutions of PACL10 were spiked into strain 34A at 0, 0.5x, 1x, and 2x the MIC, respectively. Ten-fold serial dilutions (0, 1, 2, and 3 log) from the study were plated on Reinforced Clostridium Agar (RCA) at each time point and incubated at 37°C for 3 days under anaerobic conditions. The experimental results are shown in Figure 4.

[0095] Example 8 Minimum inhibitory concentrations of selected ricins after one-step purification. PACL10, 390, 391, 392, 403 and 404 are expressed according to the methods of Examples 1-4, and the following one-step purification is performed: E. coli BL21(DE3) containing each recombinant expression plasmid was cultured in autoinduction medium at 37°C and 300 rpm for 3 hours, then at 18°C ​​and 300 rpm for 16-18 hours. The cells were collected by centrifugation and homogenously lysed under high pressure. The cleared lysate from 1 L of E. coli culture medium expressing the protein of interest was loaded onto a 26 mm / 200 mm column containing 70 mL of purification resin. The lysines were purified according to the conditions in Table 8-1, and the purity measurement of the lysines after one-step purification is shown in Figure 5. These lysines were used to determine the minimum inhibitory concentration (MIC).

[0096] The method for measuring the minimum inhibitory concentration (MIC) is as follows. Cutibacterium acnes was streaked on enriched clostridial agar (enriched clostridial medium RCM with 1.5% agar; RCM was prepared according to a revised recipe; per liter - 10 g acid hydrolyzed casein, 10 g beef extract, 3 g yeast extract, 5 g D-glucose, 5 g sodium chloride, 3 g sodium acetate, 0.5 g L-cysteine ​​hydrochloride). The plates were incubated anaerobically at 37°C for 72 h. First, the inoculum for MIC measurement was prepared by resuspending a single clone from RCA in 0.9% saline at 10 per mL. 7 OD, which corresponds to colony forming units (cfu / mL) 600 The suspension was diluted 20-fold in RCM to a concentration of 5 × 10 5 The inoculum consisted of 1000 cfu / mL of ricin. Two-fold serial dilutions of each ricin were prepared in 0.9% saline and added to no more than one-tenth of the total culture volume. Cultures were grown at 37°C under anaerobic conditions for 48-72 hours. The MIC was the lowest concentration of ricin at which no growth of Cutibacterium acnes was observed with the naked eye. The MIC results are shown in Table 8-2.

[0097] [Table 8-1]

[0098] [Table 8-2]

[0099] Example 9 Purification and bactericidal activity of PACL392 PACL392 was expressed by placing E. coli BL21(DE3) containing the recombinant expression plasmid in autoinduction medium and culturing at 37°C and 300 rpm for 3 h, then at 18°C ​​and 300 rpm for 16-18 h. Cells were harvested by centrifugation and homogenously lysed in 20 mM sodium phosphate, pH 7.4 under high pressure. The lysate was centrifuged at 12,000 rpm and 4°C for 1 h. The supernatant was mixed with an equal volume of 20 mM sodium phosphate, pH 7.4, 1 M ammonium sulfate, and centrifuged again at 12,000 rpm and 4°C for 1 h. The supernatant was filtered through a 0.22 μm filter membrane and loaded onto a mixed-mode chromatography column (26 mm / 200 mm column of 70 mL purification resin) equilibrated with 20 mM sodium phosphate, pH 7.4, and 0.5 M ammonium sulfate. After sample loading, the column was washed with a series of buffers: 20 mM sodium phosphate, pH 7.4, 0.5 M ammonium sulfate; 20 mM sodium phosphate, pH 7.4, 2.5 M NaCl; 20 mM sodium phosphate, pH 7.4, and 50 mM piperazine, pH 10.02, 50 mM NaCl. PACL392 was eluted with 50 mM piperazine, pH 10.02, and 750 mM NaCl. The eluate was dialyzed against 50 mM sodium phosphate, pH 7.4 to neutralize the pH and remove cations. The dialyzed sample was filtered and loaded onto an ion exchange column (26 mm / 200 mm column of 70 mL purification resin). The final result of PACL392 purification is shown in Figure 6.

[0100] The bactericidal activity of PACL392 was evaluated in the presence of NaCl, CaCl 2 , MgCl 2 The activity was measured by the reduction of CFU in 50 mM HEPES, pH 7.0 in the presence of a range of additives (concentrations shown in Figure 7), including EDTA, DTT, Tween-20, Tween-80 and hyaluronic acid.

[0101] Example 10 Purification and bactericidal activity of PACL403 PACL403 was expressed by placing E. coli BL21(DE3) containing the recombinant expression plasmid in autoinduction medium and culturing at 37°C and 300 rpm for 3 h, then at 18°C ​​and 300 rpm for 16-18 h. Cells were harvested by centrifugation and homogenously lysed in 20 mM sodium phosphate, pH 7.4 under high pressure. The lysate solution was adjusted to 20 mM sodium phosphate, pH 7.4, 1 M NaCl, and centrifuged at 12,000 rpm, 4°C for 1 h. The supernatant was filtered through a 0.22 μm filter membrane and loaded onto a mixed-mode chromatography column (26 mm / 200 mm column of 70 mL purification resin) equilibrated with 20 mM sodium phosphate, pH 7.4, and 1 M NaCl. After sample loading, the column was washed with a series of buffers: 20 mM sodium phosphate, pH 7.4, 1 M NaCl; 20 mM sodium phosphate, pH 7.4, 2.5 M NaCl; 20 mM sodium phosphate, pH 7.4; 20 mM sodium phosphate, pH 7.4, 0.1% Triton X-114, and again with 20 mM sodium phosphate, pH 7.4. PACL403 was eluted with 50 mM piperazine, pH 9.5, 1 M NaCl. The high pH was neutralized by adding 5 volumes of 500 mM sodium phosphate, pH 7.4. The eluate was then dialyzed against 50 mM sodium phosphate, pH 7.4 to remove cations. The dialyzed sample was filtered. The final result of PACL403 purification is shown in FIG. 8.

[0102] The bactericidal activity of PACL403 was measured by the reduction in OD in 50 mM sodium phosphate, pH 7.0 in the presence of Tween-20 and Tween-80 at the concentrations shown in FIG.

[0103] The bactericidal activity of PACL403 was measured by CFU reduction in 50 mM HEPES, pH 7.0 in the presence of Tween-20 and Tween-80 at the concentrations shown in FIG.

[0104] PACL403 also killed biofilm-associated Cutibacterium acnes (Figure 11). OD 600Strain 10 with an A.D. of 1.0 was added to a 96-well polystyrene plate to inoculate the biofilms overnight in anaerobic gas mixture. The supernatant was removed and the wells were gently rinsed with 0.9% NaCl. Biofilm formation medium (RCM + 5% glucose) was added and the biofilms were grown overnight in anaerobic gas mixture. The medium was removed and the wells were gently rinsed with 0.9% NaCl. The biofilms were then treated with buffer or PACL403 overnight at 25°C under anaerobic conditions. The buffer or PACL403 was removed and the wells were rinsed with 0.9% NaCl. Biofilm-associated CFUs were counted by resuspending in 0.9% NaCl and plating on RCM agar.

Claims

1. A bacteriophage lysin chimera having the amino acid sequence shown in SEQ ID NO:

260.

2. A bacteriophage lysin chimera having the amino acid sequence shown in SEQ ID NO:223, SEQ ID NO:253, SEQ ID NO:255, SEQ ID NO:259, SEQ ID NO:260, SEQ ID NO:263, or SEQ ID NO:

265.

3. A bacteriophage lysin chimera for use in preventing, treating, or ameliorating acne or an infection caused by, or a disease associated with, Cutibacterium acnes, wherein the bacteriophage lysin chimera can be used in the manufacture of a pharmaceutical, cosmetic, medical device, or disinfectant; the bacteriophage lysin chimera comprises a catalytic domain and a binding domain, optionally wherein the catalytic domain and the binding domain are connected by a linker region; the catalytic domain has the amino acid sequence set forth in SEQ ID NO:86, optionally followed by a short linker derived from SEQ ID NO:353 to give the combined sequence set forth in SEQ ID NO:87, or optionally followed by a long linker which is SEQ ID NO:353 to give the combined sequence set forth in SEQ ID NO:85; the binding domain of the bacteriophage lysin chimera has the amino acid sequence set forth in SEQ ID NO: 138, optionally preceded by a short linker derived from SEQ ID NO: 367 resulting in the combined sequence set forth in SEQ ID NO: 139, optionally preceded by a long linker that is SEQ ID NO: 367 resulting in the combined sequence set forth in SEQ ID NO: 137; Preferably, the bacteriophage lysin chimera comprises a catalytic domain, a binding domain, and a linker; The catalytic domain with a short linker has the amino acid sequence shown in SEQ ID NO:87, A bacteriophage lysin chimera, wherein the binding domain has the amino acid sequence set forth in SEQ ID NO:

138. Claim 4: The catalytic domain has a nucleotide sequence as set forth in SEQ ID NO:169, optionally including a short linker region resulting in the combined sequence as set forth in SEQ ID NO:170, or optionally including a long linker region resulting in the combined sequence as set forth in SEQ ID NO:168; the binding domain has a nucleotide sequence as set forth in SEQ ID NO: 221, optionally including a short linker region resulting in the combined sequence as set forth in SEQ ID NO: 222, or a long linker region resulting in the combined sequence as set forth in SEQ ID NO: 220; Preferably, the catalytic domain with a short linker has the nucleotide sequence shown in SEQ ID NO: 170; The bacteriophage lysin chimera of claim 3 , wherein the binding domain has the nucleotide sequence set forth in SEQ ID NO:

221.

5. the catalytic domain has a complete C-terminal linker, a half C-terminal linker, no C-terminal linker, or any part of said linker; the binding domain has a complete N-terminal linker, a half N-terminal linker, no N-terminal linker, or any part of said linker; Preferably, the linker connected to the C-terminus of the catalytic domain has the amino acid sequence set forth in SEQ ID NO: 353 or a portion thereof, and the linker connected to the N-terminus of the binding domain has the amino acid sequence set forth in SEQ ID NO: 367 or a portion thereof. A bacteriophage lysin chimera as described in claim 3.

6. The catalytic domain has any or all of the following mutations with respect to the amino acid sequence set forth in SEQ ID NO:86: L29M, R33K, I51S, V62I, A70G, D72N, S82A, R83H, K100S, N103K, S107T, Q129K, Q132S, T139V, and W163F; Preferably, the mutated sequence comprises all of these mutations as shown in SEQ ID NO: 133, and optionally, the mutated catalytic domain is followed by a linker sequence resulting in the combined sequence shown in SEQ ID NO: 131; The mutated catalytic domain is combined with a binding domain according to claim 3, wherein the binding domain of the bacteriophage lysin chimera has the amino acid sequence shown in SEQ ID NO: 138, optionally preceded by a short linker derived from SEQ ID NO: 367, resulting in the combined sequence shown in SEQ ID NO: 139, or optionally preceded by a long linker which is SEQ ID NO: 367, resulting in the combined sequence shown in SEQ ID NO: 137, Preferably, the mutated catalytic domain with a linker has the amino acid sequence set forth in SEQ ID NO: 133; The bacteriophage lysin chimera of claim 3 , wherein the binding domain has the amino acid sequence set forth in SEQ ID NO:

138.

7. The mutated catalytic domain having a linker has a nucleotide sequence as set forth in SEQ ID NO:216, or has a nucleotide sequence as set forth in SEQ ID NO:214, the binding domain has a nucleotide sequence as set forth in SEQ ID NO: 221, optionally including a short linker region resulting in the combined sequence as set forth in SEQ ID NO: 222, or optionally including a long linker region resulting in the combined sequence as set forth in SEQ ID NO: 220; Preferably, the catalytic domain with a linker has the nucleotide sequence set forth in SEQ ID NO:216; The bacteriophage lysin chimera of claim 6 , wherein the binding domain has the nucleotide sequence set forth in SEQ ID NO:

221.

8. The resulting mutant lysine chimera can have the amino acid sequence set forth in SEQ ID NO:225, SEQ ID NO:257, SEQ ID NO:258, SEQ ID NO:262, SEQ ID NO:266, or SEQ ID NO:272; 7. The bacteriophage lysin chimera of claim 6, wherein the mutated lysin chimera may have the amino acid sequence shown in SEQ ID NO:

258.

9. 7. The bacteriophage lysin chimera of claim 3 or 6, wherein the catalytic domain comprises one, two or more than two catalytic domains.

10. 7. The bacteriophage lysin chimera of claim 3 or 6, wherein the binding domain comprises one, two or more than two binding domains.

11. The chimera further comprises a linker between the catalytic domain and the binding domain, the linker comprising, in whole or in part: 1) an amino acid sequence as set forth in any one of SEQ ID NOs: 343 to 367, preferably SEQ ID NO: 353, SEQ ID NO: 367, or SEQ ID NO: 366; 2) a nucleotide sequence as set forth in any one of SEQ ID NOs: 368 to 392, preferably SEQ ID NO: 378, SEQ ID NO: 392 or SEQ ID NO: 391; 3) an amino acid sequence set forth in any one of SEQ ID NOs: 393 to 406; or 4) (GGGS)n, (GGGGS)n, (GGGGGS)n, (Gly)3-8, (EAAAK)n, (Ala-Pro)n, or A(EAAAK)nALEA(EAAAK)nA, where 1≦n≦15 and n is an integer.

7. The bacteriophage lysin chimera of claim 3 or 6, comprising:

12. The bacteriophage lysin chimera of claim 3 or 6, wherein the infection caused by Cutibacterium acnes comprises an invasive infection, a post-operative infection and / or a device-associated infection.

13. 13. The bacteriophage lysin chimera of claim 12, wherein the device-associated infections include artificial joints, shunt tubing and artificial heart valve-associated infections.

14. 13. The bacteriophage lysin chimera of claim 12, wherein the infections include bone and / or joint infections, in particular post-operative shoulder infections, as well as oral cavity, eye, disc and brain infections.

15. 7. The bacteriophage lysin chimera of claim 3 or 6, wherein the Cutibacterium acnes associated disease comprises cancerous prostatitis, SAPHO (synovitis, acne, impetigo, hyperplasia, osteitis) syndrome, sarcoidosis, or sciatica.

16. The bacteriophage lysin chimera of claim 3 or 6, wherein the medical device comprises any device for releasing the chimera to the affected area, preferably a clamp or patch or a spray applied to the skin surface, a device using microneedles to enhance skin penetration of the chimera, fine needles used by beauty professionals to apply the chimera especially to hair follicles affected by acne, or other similar devices.

17. 7. The bacteriophage lysin chimera of claim 3 or 6, wherein the medical device comprises a prosthetic device for immobilizing the chimera in a location susceptible to Cutibacterium acnes infection, preferably a prosthetic implant for use in shoulder surgery, particularly in shoulder surgery, which is susceptible to Cutibacterium acnes infection.

18. A method for producing a bacteriophage lysin chimera according to any one of claims 1 to 16, said method comprising: (1) synthesizing a domain sequence and a primer for amplifying the domain sequence; (2) using Taq DNA polymerase PCR to amplify the domain sequences; (3) gel purifying the PCR product and ligating it into the expression plasmid pET28; (4) introducing the recombinant plasmid into E. coli BL21(DE3); (5) culturing E. coli BL21(DE3) containing the recombinant plasmid, inducing expression, harvesting the cells by centrifugation, lysing and purifying to obtain the chimera; A method comprising:

19. E. coli BL21(DE3) containing the recombinant chimeric lysine expression plasmid was grown in autoinduction medium at 37°C and 300 rpm to an OD ６００ 19. The method of claim 18, further comprising culturing the cells until a chromatin density of the cells reaches 0.6-0.8, and then continuing the culture at 18° C. and 300 rpm for 16-18 hours; harvesting the cells by centrifugation, resuspending them in 50 mM sodium phosphate, pH 7.4, and lysing them to homogeneity under high pressure; centrifuging the lysate again to collect a soluble crude lysate; mixing the soluble fraction with an equal volume of 5 M NaCl and loading the mixture onto a hydrophobic column; washing the column with 5 column volumes of 20 mM sodium phosphate, pH 7.4, 2.5 M NaCl after sample loading; and then eluting the recombinant chimeric lysine with 10 mM sodium phosphate, pH 7.

4.

20. 17. A formulation containing the bacteriophage lysin chimera of any one of claims 1 to 16, said formulation further comprising an antibiotic, another lysin or lysin chimera, or an inert excipient.

21. 20. An amino acid sequence encoding a bacteriophage lysin chimera according to any one of claims 1 to 18, or an amino acid sequence having 80% or more, 85% or more, 90% or more, 95% or more, or 99% or more similarity, or an alternative amino acid sequence having the same functional group.

22. The alternative amino acid sequence is a conservative substitution using amino acids in the same amino acid group, the amino acid group being: Aliphatic: glycine, alanine, valine, leucine or isoleucine; Hydroxyl or sulfur / selenium containing: serine, cysteine, threonine or methionine; Cyclic: proline; Aromatic: phenylalanine, tyrosine or tryptophan; Basic: histidine, lysine, or arginine; or Acids and their amides: Aspartic acid, glutamic acid, asparagine, glutamine 22. The amino acid sequence of claim 21 .

23. 17. A bacteriophage lysin chimera according to any one of claims 1 to 16, used as a reagent for bacterial lysis, said bacterial lysis being used for DNA extraction and typing using a PCR-based kit.

24. 19. A bacteriophage lysin chimera according to any one of claims 1 to 18, for use as a disinfectant or sterilant on non-biological surfaces, said disinfectant or sterilant preventing infection by eliminating Cutibacterium acnes in planktonic form or in a biofilm, preferably in surgical devices or prosthetic implant devices during surgery.

25. A bacteriophage lysin chimera as described in any one of claims 1 to 18, or a single binding domain therein, or a series of combinations of similar or different binding domains therein, for use as a diagnostic tool for Cutibacterium acnes, wherein the lysin chimera or binding domain is used in combination with a signal molecule.

26. 26. The bacteriophage lysin chimera of claim 25, wherein the fusion of the lysin chimera or binding domain and signal molecule is formed through genetic fusion or chemical coupling.

27. 27. The bacteriophage lysin chimera of claim 26, wherein the fusion is used for direct detection of Cutibacterium acnes on microscope slides by fluorescence or other means, for labeling Cutibacterium acnes by immunohistochemistry, as a detection reagent in ELISA assays and / or Western blots, for attachment to magnetic beads in MACS or other pull-down assays, or as a detection reagent in assays in which an antibody is used as the detection reagent.

28. The signal molecule comprises a protein or chemical fluorescent dye, a protein tag, an enzyme, avidin, streptavidin, ovalbumin, biotin, a click chemistry labeling sensitive tag, an intein, or other molecule that can trigger the recruitment of a secondary protein or molecule that generates a signal, wherein: The fluorescent dye comprises GFP, RFP, mCherry, FITC, TRITC, Alexafluor 488, Cy3 or Cy5; The protein tag includes a Flag tag, a myc tag, a halo tag, a his tag, or any other tag that can bind to an antibody or other high affinity molecule to generate a signal; The enzymes include firefly luciferase, β-lactamase, alkaline phosphatase, horseradish peroxidase, or any enzyme that produces a reaction, e.g., light, a color change, deposition of a substrate, or other enzyme that can be detected during the test.

28. The bacteriophage lysin chimera of claim 27.

29. 20. A bacteriophage lysin chimera according to any one of claims 1 to 18, for use in the manufacture of a medicament for the treatment of Cutibacterium acnes infections, wherein the catalytic domain is combined with a targeting module.

30. The bacteriophage lysin chimera of claim 1, wherein the bacteriophage lysin chimera has a nucleotide sequence as set forth in SEQ ID NO:

318.

31. The bacteriophage lysin chimera of claim 2, wherein the bacteriophage lysin chimera has a nucleotide sequence as shown in SEQ ID NO:283, SEQ ID NO:311, SEQ ID NO:313, SEQ ID NO:317, SEQ ID NO:318, SEQ ID NO:321 or SEQ ID NO:

323.

32. The bacteriophage lysin chimera having a nucleotide sequence as set forth in SEQ ID NO:285, SEQ ID NO:315, SEQ ID NO:316, SEQ ID NO:320, SEQ ID NO:324, or SEQ ID NO:330; Preferably, the bacteriophage lysin chimera has the nucleotide sequence shown in SEQ ID NO:

316. The bacteriophage lysin chimera of claim 8.

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