NOVEL PHAGE ENDOLYSIN AND ITS USES

Abstract

The present invention relates to a novel endolysin isolated from a phage and its therapeutic use such as in the treatment of skin pathologies caused by Staphylococcus, in particular pyoderma in dogs or cats. [Fig. **]

Description

Title of the invention: NOVEL PHAGE ENDOLYSIN AND ITS USES Technical field

[0001] The present invention relates to a novel endolysin and its use for its anti-staphylococcal activity. It can thus be used in a disinfectant solution, in food safety or in therapy, for example in the treatment of pathologies caused by Staphylococcus such as skin pathologies like pyoderma in dogs or cats. Prior art

[0002] Phages, also called "bacteriophages", are viruses that only infect bacteria. Considered the most abundant entities in nature, they have a more or less significant specificity, allowing them to infect several bacterial strains within the same species, but they are only rarely capable of infecting several bacterial species. This aspect makes phages particularly interesting for the development of personalized antibacterial therapies.

[0003] Following attachment to the bacterium, phages inject their genomes into the bacterium and hijack the bacterial machinery to produce the viral proteins necessary for their replication and the generation of new phages. In the final stages of the cycle, phages infecting Gram-positive bacteria produce two proteins involved in the lysis of their host: endolysins and holins. Holins form holes in the bacterial membrane to allow endolysins to access and cut the bacterial wall, resulting in lysis and release of the viral progeny.

[0004] Most endolysins contain one or two N-terminal enzyme active domains (EADs) that cleave bonds in bacterial peptidoglycan and a cell wall binding domain (CBD) involved in bacterial recognition. The two EAD and CBD domains are connected by a linker.

[0005] The enzymatic activity of endolysins is influenced by the composition of the cell walls. For Gram-positive bacteria, the cell wall consists of a cytoplasmic cell membrane that is surrounded by a thick layer of peptidoglycan. For exogenous applications, endolysins easily access the peptidoglycan layer and hydrolyze the basic bonds of the peptidoglycan (at the glycan or amide bond), resulting in osmotic lysis and bacterial death.

[0006] The EADs which make up endolysins are thus classified into several groups according to their cleavage sites: - Glycosidases cleaving the glycan part of the peptidoglycan (MurNAc-GlcNAc); - Amidases cleaving the amide bond between the glycan fragment (MurNAc) and the peptide fragment (L-alanine); - Endopeptidase, cleaving the peptide bond between two amino acids; - transglycosylases degrade peptidoglycan by cleaving the glycosidic bond |3-(1—>4) between MurNAc and GlcNAc residues; - CHAP (Cysteine, Histidine-dependent Amidohydrolase / Peptidase): This type of enzyme domain is not classified according to the peptidoglycan binding it cleaves, but by their catalytic mechanism. As its name suggests, it has a cysteine ​​and histidine residue in its active site involved in the cleavage.

[0007] Endolysins can be identified using various methods of phage genome analysis. It is then possible to clone the gene coding for endolysins into an inducible bacterial expression vector and to produce the protein and purify it using affinity tags (polyhistidines, GST, etc.).

[0008] They have shown promising results in the elimination of bacteria in different areas: food safety, surface disinfection, therapeutics, particularly to treat antibiotic-resistant bacteria. It is important to note that some endolysins are also effective in eliminating intracellular bacteria and in destroying bacterial biofilms (1,2).

[0009] Lys38 has been identified for anti-Staphylococcus pseudintermedius activity.

[0010] Canine pyoderma is one of the most frequently consulted skin infections by veterinarians. It results in redness, lesions, pain and inflammation. The bacterium S. pseudintermedius is the most common pathogen associated with skin infections, predominating in up to 92% of cases of canine pyoderma. The rate of methicillin-resistant S. pseudintermedius (MRSP) can be up to 59% of cases of canine pyoderma (3).

[0011] It was therefore necessary to find a new effective treatment for pathologies caused by S. pseudintermedius.

[0012] In the literature, endolysins have often been cited for anti-Slaphylococcus application in general or for targeted application to particular Staphylococcus species such as Staphylococcus aureus ( S. aureus ) in most cases, or other species such as Staphylococcus homilis or Staphylococcus epidermis, but rarely to target S. pseudintermedius.

[0013] So far, the activity of endolysins of phages specific to S. pseudintermedius has not been demonstrated on either S. pseudintermedius or S. aureus.

[0014] The inventors were able to identify from a bacteriophage, a new endolysin of sequence SEQ ID NO: 1, also called hereinafter Lys38, which exhibits remarkable anti-S. pseudintermedius activity. This new endolysin is very promising for the development of a new effective treatment against S. pseudintermedius.

[0015] In particular, the results demonstrated that Lys38 has a specific activity on S. pseudintermedius strains at low concentrations and that at certain concentrations it is also active on S. aureus. Summary

[0016] In a first aspect, the present disclosure relates to an endolysin comprising a sequence having a percentage identity of at least 80% with the sequence SEQ ID NO: 2 and a sequence having a percentage identity of at least 80% with the sequence SEQ ID NO: 3.

[0017] In a second aspect, the present disclosure relates to the use of an endolysin as described above as a medicament.

[0018] In a third aspect, the present disclosure relates to an endolysin as described above for use in the treatment of an infection caused by a staphylococcus, in particular by S. aureus or by S. pseudintermedius.

[0019] In a fourth aspect, the present disclosure relates to the non-therapeutic use of an endolysin as described above as an anti-staphylococcal agent, in particular anti-Staphylococcus aureus, in particular anti-Staphylococcus pseudintermedius.

[0020] In a fifth aspect, the present disclosure relates to the non-therapeutic use of an endolysin as described above as an antibacterial compound.

[0021] In a sixth aspect, the present disclosure relates to the non-therapeutic use of an endolysine as described above in an antibiofilm solution.

[0022] In a seventh aspect, the present disclosure relates to the non-therapeutic use of an endolysin as described above in food safety.

[0023] In an eighth aspect, the present disclosure relates to the non-therapeutic use of an endolysin as described above in a cosmetic product or in a pharmaceutical preparation. Brief description of the drawings Fig.l

[0024] [Fig-1] shows an SDS-Page analysis of a production of the endolysin of sequence SEQ ID NO: 1 (Lys38). Detection of the band at approximately 50 kDa (indicated by the arrow). Fig. 2

[0025] [Fig.2] shows the comparison of the activity of endolysin of sequence SEQ ID NO: 1 (Lys38) with the activity of phage K endolysin on different strains of Staphylococcus aureus and Staphylococcus pseudintermedius by assessment of the optical density (OD) at 600 nm (ordinate) after 30 min of incubation at 37°C. Fig. 3

[0026] [Fig.3] shows the comparison of the average activity of the endolysin of sequence SEQ ID NO: 1 (Lys38) with the average activity of the endolysin of phage K on 23 strains of Staphylococcus aureus and 23 strains of Staphylococcus pseudintermedius by assessment of the optical density (OD) at 600 nm (on the ordinate) after 30 min of incubation at 37°C. Averages of the results of [Fig.2]. Concentration of Lys38 and Lys K: 1 pg / ml. Fig. 4

[0027] [Fig.4] shows the activity of Lys38 activity on different bacterial species (on the abscissa). The optical density (OD) is read at 600 nm (on the ordinate). Lys38 concentration: 1 pg / ml. Fig. 5

[0028] [Fig.5] shows the comparison of the activity of the endolysin of sequence SEQ ID NO: 1 (Lys38) with the activity of the endolysin of phage K on different strains of Staphylococcus aureus, by assessing the decrease in optical density (OD) at 600 nm (on the ordinate), initially at 1, after 30 min of incubation at 37°C. Concentration of Lys38: 10pg / ml. Fig. 6

[0029] [Fig.6] shows the comparison of the activity of the endolysin of sequence SEQ ID NO: 1 (Lys38) with the activity of the endolysin of phage K on different strains of Staphylococcuspseudintermedius by assessing the decrease in optical density (OD) at 600 nm (on the ordinate), initially at 1, after 30 min of incubation at 37°C. Concentration of Lys38: 10pg / ml. Fig. 7

[0030] [Fig.7] shows the reduction of S. pseudintermedius after treatment with endolysin of sequence SEQ ID NO: 1 (Lys38) on mice presenting a lesion induced by an infection with S. pseudintermedius and treated with two doses of 75 pg per day, in topical application. Fig. 8

[0031] [Fig-8] shows the decrease in the size of the lesion induced in mice by a S. pseudintermedius infection during the 9 days of treatment (DI to D9) with the administration of two doses of 75pg per day of endolysin of sequence SEQ ID NO: 1 (Lys38), in topical application. Fig. 9

[0032] [Fig.9] shows the activity results of the different domains of endolysin Lys38 (SEQ ID NO: 1), ie CHAP (SEQ ID NO: 3), CHAP_Am (SEQ ID NO: 2), Am (SEQ ID NO: 4) and Am_SH3 (SEQ ID NO: 5), tested at concentrations of 18nM, 90nM, 180nM, 450nM and 900nM for 1h30 on a culture of the SP1 strain of S. pseudintermedius. Fig. 10

[0033] [Fig. 10] shows the activity results of the different domains of endolysin Lys38 (SEQ ID NO: 1), ie CHAP (SEQ ID NO: 3), CHAP_Am (SEQ ID NO: 2), Am (SEQ ID NO: 4) and Am_SH3 (SEQ ID NO: 5), tested at concentrations of 18nM, 90nM, 180nM, 450nM and 900nM for 1h30 on a culture of the C24 strain of S. aureus. Description of the invention

[0034] According to a first aspect, the present invention relates to an endolysin comprising the sequence SEQ ID NO: 2 and the sequence SEQ ID NO: 3 or its variants comprising a sequence having a percentage identity of at least 80% with the sequence SEQ ID NO: 2 and a sequence having a percentage identity of at least 80% with the sequence SEQ ID NO: 3.

[0035] SEQ ID NO: 2

[0036] MVASLSKKEFISFLKSSEGKQFNEDGWYGYQCFDFANTGWLKLFGHKLMGL GAKDIPFNSINKNYFKTEAKVYSNTPEFLAEAGDMVVFGENYGGGYGHVAW VIEATLDYIVVLEQNWLGGGWTDGIEQPGSGWESVTRRKHAYDFPMWFIRP NFK

[0037] SEQ ID NO: 3

[0038] KLNYIRDEIKGYNMDKRGYKPRGIVLHNDAGSAG ATV

[0039] For the purposes of the present application, a percentage identity of at least 80% may in particular be at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%. This applies in particular to sequences of variants having at least 80% identity with the sequences SEQ NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4.

[0040] The percent identity between two amino acid sequences can be determined using the algorithm of E. Meyers and W. Miller (Comput. Appl. Biosci., 4:11-17, 1988) which has been incorporated into the ALIGN program (version 2.0), using a residue table of weight PAM120, a gap length penalty of 12 and a gap penalty of 4. Alternatively, the percent identity between two amino acid sequences can be determined using published techniques and widely available computer programs, such as BLASTP, FASTA (Atschul et al., J. Molecular Biol. 215:403, 1990) or the algorithm of Needleman and Wunsch (J. Mol, Biol. 48:444-453, 1970) which has been incorporated into the GAP program of the GCG software package (Devereux et al.), Nucleic Acids Res. 12:387, 1984, generally available at http: / / www.gcg.com), using either a Blossom 62 die or a PAM250 die, and a space weight of 16, 14, 12, 10, 8, 6 or 4 and a length weight of 1, 2, 3, 4, 5 or 6. .

[0041] By variant is meant an endolysin comprising the sequence SEQ ID NO:2 and the sequence SEQ ID NO:3 comprising at least one mutation by substitution, deletion or addition of amino acids. In particular, the invention relates to functional variants which retain an activity similar to that of the protein comprising the sequence SEQ ID NO:2 and the sequence SEQ ID NO:3, according to the activity test presented in the last example of the present application

[0042] This activity test includes the following steps: - Culture of a strain of S. pseudintermedius or a strain of S. aureus to obtain an OD = 1; - Collection of 300 pl and addition of the variant at different concentrations: 1, 5, 10, 25, 50 pg / mL; - Incubation of the mixture for 90 minutes at 37°C; - Transfer to ice to stop the reaction; - Measurement of DO; - Comparison with the OD obtained for a protein comprising the sequence SEQ ID NO: 2 and the sequence SEQ ID NO: 3, in particular comparison with the OD of the protein SEQ ID NO: 4 or the OD of the protein SEQ ID NO: 1, measured within the same test.

[0043] Preferably, the functional variants comprise only substitution mutations, preferably conservative substitution.

[0044] In the present application, the expression "conservative substitution of an amino acid" means that a given amino acid can be replaced by a residue having similar physiochemical characteristics, for example by substituting one aliphatic residue for another (such as Ile, Val, Leu or Ala), or by substituting one polar residue for another (such as between Lys and Arg; Glu and Asp; or Gln and Asn). OthersConservative substitutions of this type, e.g., substitutions of entire regions with similar hydrophobicity characteristics, are known. Polypeptides comprising conservative amino acid substitutions may be tested in one of the assays described herein to confirm that a desired activity, e.g., the antigen-binding activity and specificity of a native or reference polypeptide, is retained. Amino acids may be grouped according to similarities in the properties of their side chains (in A.L. Lehninger, in Biochemistry, second edition, pp. 73-75, Worth Publishers, New York (1975)): (1) nonpolar: Gly (G), Ala (A), Val (V), Leu (L), Ile (I), Pro (P), Phe (F), Trp (W), Met (M); (2) uncharged polar: Ser (S), Thr (T), Cys (C), Tyr (Y), Asn (N), Gln (Q); (3) acidic: Asp (D), Glu (E); (4) basic: Lys (K), Arg (R), His (H).Naturally occurring residues can also be divided into groups based on common side-chain properties: (1) hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile; (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues that influence chain orientation: Gly, Pro; (6) aromatic: Trp, Tyr, Phe. Nonconservative substitutions involve the exchange of a member of one of these classes for a member of another class. Particular conservative substitutions include, for example, Ala to Gly or Ser; Arg to Lys; Asn to Gln or His; Asp to Glu; Cys to Ser; Gln to Asn; Glu to Asp; Gly to Ala or Pro; His to Asn or Gln; Ile to Leu or Val; Leu in Island or Valley; Lily in Arg, Gin or Glu; Met in Leu, Tire or Island; Phe in Met, Leu or Tyr; Ser in Thr; Thr to Ser; Trp in Tyre; Tyr to Trp; and / or Phe en Val, Ile or Leu. .

[0045] Preferred variants are variants comprising conservative substitutions on 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids of the sequence.

[0046] Thus, according to a first aspect, the present invention relates to an endolysin comprising a sequence having a percentage identity of at least 80% with the sequence SEQ ID NO: 2 and a sequence having a percentage identity of at least 80% with the sequence SEQ ID NO: 3.

[0047] The present invention also relates to an endolysin comprising a sequence SEQ ID NO: 4 or its variants comprising a sequence having a percentage identity of at least 80% with the sequence SEQ ID NO: 4.

[0048] SEQ ID NO: 4

[0049] MVASLSKKEFISFLKSSEGKQFNEDGWYGYQCFDFANTGWLKLFGHKLMGL GAKDIPFNSINKNYFKTEAKVYSNTPEFLAEAGDMVVFGENYGGGYGHVAW VIEATLDYIVVLEQNWLGGGWTDGIEQPGSGWESVTRRKHAYDFPMWFIRP NFKSETTLLSAQSVTKQKSKQPAKKPKKLNYIRDEIKGYNMDKRGYKPRGI VLHNDAGSAGATAEAYHKGLVNADYNRLEKGVAHSYVSGSTVYQALPEGKI AWHVANRIGNRDYYGIEICQSVGATDKQFLANEQSAFQEAARMLKKWGLPA NRNTVRLHVEFYNTACPHRSMLLHTGFDPTVKGIPPQAMQLKLKDYFIKQI RAYMDGKVPVATV

[0050] By variant is meant an endolysin of sequence SEQ ID NO: 4 comprising at least one mutation by substitution, deletion or addition of amino acids. In particular, the invention relates to functional variants which retain an activity similar to that of the protein of sequence SEQ ID NO: 4 according to the activity test presented in the last example of the present application.

[0051] The present invention also relates to an endolysin of sequence SEQ ID NO: 1 or its variants comprising a sequence having a percentage identity of at least 70% with the sequence SEQ ID NO: 1.

[0052] SEQ ID NO: 1

[0053] MVASLSKKEFISFLKSSEGKQFNEDGWYGYQCFDFANTGWLKLFGHKLMGL GAKDIPFNSINKNYFKTEAKVYSNTPEFLAEAGDMVVFGENYGGGYGHVAW VIEATLDYIVVLEQNWLGGGWTDGIEQPGSGWESVTRRKHAYDFPMWFIRP NFKSETTLLSAQSVTKQKSKQPAKKPKKLNYIRDEIKGYNMDKRGYKPRGI VLHNDAGSAGATAEAYHKGLVNADYNRLEKGVAHSYVSGSTVYQALPEGKI AWHVANRIGNRDYYGIEICQSVGATDKQFLANEQSAFQEAARMLKKWGLPA NRNTVRLHVEFYNTACPHRSMLLHTGFDPTVKGIPPQAMQLKLKDYFIKQI RAYMDGKVPVATVSNKTSASSNTVKPIAGAWQVNSYGTYYMKESATFVCGN KPITTRTVGPFTTCPEGYMFQPGGWCEYDEVMLQDGHVWIGYDWQGQRYYL PIRKWNGVAPPNQGLGDLWGTIK

[0054] For the purposes of the present application, the variants may have a percentage identity with the sequence SEQ ID NO: 1 of at least 70%, in particular at least 75%, at least 80%, at least 85%, more particularly at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.

[0055] By variant is meant an endolysin of sequence SEQ ID NO: 1 comprising at least one mutation by substitution, deletion or addition of amino acids. In particular, the invention relates to functional variants which retain an activity similar to that of the protein of sequence SEQ ID NO: 1 according to the activity test presented in the last example of the present application.

[0056] In particular, the present application relates to an endolysin comprising a sequence having a percentage identity of at least 70% with the sequence SEQ ID NO: 1, and comprising a sequence having at least 80% identity with the sequence SEQ ID NO: 2 and a sequence having at least 80% identity with the sequence SEQ ID NO: 3.

[0057] In a more particular embodiment, the present application relates to an endolysin comprising a sequence having a percentage identity of at least 70% with the sequence SEQ ID NO: 1, and comprising a sequence having at least 80% identity with the sequence SEQ ID NO: 4.

[0058] In a more particular embodiment, the present application relates to an endolysin comprising a sequence having a percentage identity of at least 70% with the sequence SEQ ID NO: 1, and comprising the sequence SEQ ID NO: 2 and the sequence SEQ ID NO: 3.

[0059] In a more particular embodiment, the present application relates to an endolysin comprising a sequence having a percentage identity of at least 70% with the sequence SEQ ID NO: 1, and comprising the sequence SEQ ID NO: 4.

[0060] In a more particular embodiment, the present invention relates to an endolysin of sequence SEQ ID NO: 1.

[0061] The protein of sequence SEQ ID NO: 1 is an endolysin isolated from a bacteriophage, and is also referred to as Lys38 in the present application.

[0062] The present invention also relates to a pharmaceutical composition comprising an endolysin which comprises a sequence having a percentage identity of at least 80% with the sequence SEQ ID NO: 2 and a sequence having a percentage identity of at least 80% with the sequence SEQ ID NO: 3.

[0063] The present invention also relates to a pharmaceutical composition comprising an endolysin which comprises a sequence having a percentage identity of at least 80% with the sequence SEQ ID NO: 4.

[0064] The present invention also relates to a pharmaceutical composition comprising an endolysin which comprises a sequence having a percentage identity of at least 70% with the sequence SEQ ID NO: 1.

[0065] In particular, it relates to a pharmaceutical composition comprising an endolysin which comprises a sequence having a percentage identity of at least 70% with the sequence SEQ ID NO: 1 and which comprises a sequence having at least 80% identity with the sequence SEQ ID NO: 2 and a sequence having at least 80% identity with the sequence SEQ ID NO: 3.

[0066] In particular, the pharmaceutical composition comprises an endolysin which comprises a sequence having a percentage identity of at least 70% with the sequence SEQ ID NO: 1 and which comprises a sequence having at least 80% identity with the sequence SEQ ID NO: 4.

[0067] In particular, the pharmaceutical composition comprises an endolysin which comprises a sequence having a percentage identity of at least 70% with the sequence SEQ ID NO: 1 and which comprises the sequence SEQ ID NO: 2 and the sequence SEQ ID NO: 3.

[0068] In particular, the pharmaceutical composition comprises an endolysin which comprises a sequence having a percentage identity of at least 70% with the sequence SEQ ID NO: 1 and which comprises the sequence SEQ ID NO: 4.

[0069] In a more particular embodiment, said composition comprises the endolysin of sequence SEQ ID NO: 1.

[0070] The present application also relates to the non-therapeutic use of an endolysin comprising a sequence having a percentage identity of at least 80% with the sequence SEQ ID NO: 2 and a sequence having a percentage identity of at least 80% with the sequence SEQ ID NO: 3, as an anti-staphylococcus agent, in particular anti-S. aureus, in particular anti-S. pseudintermedius.

[0071] The present application also relates to the non-therapeutic use of an endolysin comprising a sequence having a percentage identity of at least 80% with the sequence SEQ ID NO: 4, as an anti-staphylococcus agent, in particular anti-S. aureus, in particular anti-S. pseudintermedius.

[0072] The present application also relates to the non-therapeutic use of an endolysin comprising a sequence having a percentage identity of at least 70% with the sequence SEQ ID NO: 1 as an anti-staphylococcus agent, in particular anti-S. aureus, in particular anti-S. pseudintermedius.

[0073] For the purposes of the present application, by "anti-staphylococcal agent" is meant that said endolysin has an anti-staphylococcal activity, that is to say that it makes it possible to inhibit the growth of this bacterial species, or even to destroy these bacteria in a non-therapeutic context.

[0074] In particular, the present application relates to the non-therapeutic use of an endolysin comprising a sequence having a percentage identity of at least 70% with the sequence SEQ ID NO: 1 and which comprises a sequence having at least 80% identity with the sequence SEQ ID NO: 2 and a sequence having at least 80% identity with the sequence SEQ ID NO: 3, as an anti-staphylococcus agent, in particular anti-S. aureus, in particular anti-S. pseudintermedius.

[0075] In particular, the present application relates to the non-therapeutic use of an endolysin comprising a sequence having a percentage identity of at least 70% with the sequence SEQ ID NO: 1 and which comprises a sequence having at least 80% identity with the sequence SEQ ID NO: 4, as an anti-staphylococcus agent, in particular anti-S. aureus, in particular anti-S. pseudintermedius.

[0076] In particular, the present application relates to the non-therapeutic use of an endolysin comprising a sequence having a percentage identity of at least 70% with the sequence SEQ ID NO: 1 and which comprises the sequence SEQ ID NO: 2 and the sequence SEQ ID NO: 3, as an anti-staphylococcus agent, in particular anti-S. aureus, in particular anti-S. pseudintermedius.

[0077] In particular, the present application relates to the non-therapeutic use of an endolysin comprising a sequence having a percentage identity of at least 70% with the sequence SEQ ID NO: 1 and which comprises the sequence SEQ ID NO: 4, as an anti-staphylococcus agent, in particular anti-S. aureus, in particular anti-S. pseudintermedius.

[0078] In particular, the present application relates to the non-therapeutic use of an endolysin of sequence SEQ ID NO: 1 as an anti-staphylococcal agent, in particular anti-S. aureus, in particular anti-S. pseudintermedius.

[0079] The present invention also relates to the non-therapeutic use of a composition comprising an endolysin which comprises a sequence having a percentage identity of at least 80% with the sequence SEQ ID NO: 2 and a sequence having a percentage identity of at least 80% with the sequence SEQ ID NO: 3, as an anti-staphylococcal agent, in particular anti-S. aureus, in particular anti-S. pseudintermedius.

[0080] The present invention also relates to the non-therapeutic use of a composition comprising an endolysin which comprises a sequence having a percentage identity of at least 80% with the sequence SEQ ID NO: 4, as an anti-staphylococcus agent, in particular anti-S. aureus, in particular anti-S. pseudintermedius.

[0081] The present invention also relates to the non-therapeutic use of a composition comprising an endolysin which comprises a sequence having a percentage identity of at least 70% with the sequence SEQ ID NO: 1, as an anti-staphylococcus agent, in particular anti-S. aureus, in particular anti-S. pseudintermedius.

[0082] In particular, the present application relates to the non-therapeutic use of a composition comprising an endolysin which comprises a sequence having a percentage identity of at least 70% with the sequence SEQ ID NO: 1 and which comprises a sequence having at least 80% identity with the sequence SEQ ID NO: 2 and a sequence having at least 80% identity with the sequence SEQ ID NO: 3, as an anti-staphylococcal agent, in particular anti-S. aureus, in particular anti-S. pseudintermedius.

[0083] In particular, the present application relates to the non-therapeutic use of a composition comprising an endolysin which comprises a sequence having a percentage identity of at least 70% with the sequence SEQ ID NO: 1 and which comprises a sequence having at least 80% identity with the sequence SEQ ID NO: 4, as an anti-staphylococcus agent, in particular anti-S. aureus, in particular anti-S. pseudintermedius.

[0084] In particular, the present application relates to the non-therapeutic use of a composition comprising an endolysin which comprises a sequence having a percentage identity of at least 70% with the sequence SEQ ID NO: 1 and which comprises the sequence SEQ ID NO: 2 and the sequence SEQ ID NO: 3, as an anti-staphylococcal agent, in particular anti-S. aureus, in particular anti-S. pseudintermedius.

[0085] In particular, the present application relates to the non-therapeutic use of a composition comprising an endolysin which comprises a sequence having a percentage identity of at least 70% with the sequence SEQ ID NO: 1 and which comprises the sequence SEQ ID NO: 4, as an anti-staphylococcus agent, in particular anti-S. aureus, in particular anti-S. pseudintermedius.

[0086] In a more particular embodiment, the present application relates to the non-therapeutic use of a composition comprising the endolysin of sequence SEQ ID NO: 1, as an anti-staphylococcal agent, in particular anti-S. aureus, in particular anti-S. pseudintermedius.

[0087] According to another aspect, the present application relates to the non-therapeutic use of a phage endolysin in a disinfectant solution.

[0088] By disinfectant solution is meant a solution which destroys microorganisms such as bacteria, in particular S. aureus or S. pseudintermedius, found on inert surfaces such as medical equipment.

[0089] Thus, the present application also relates to the non-therapeutic use of a phage endolysin as an antibacterial compound.

[0090] According to another aspect, the present application relates to the non-therapeutic use of a phage endolysin in an anti-biofilm solution.

[0091] A biofilm is a structured mass of microorganisms adhering to each other and to a surface and marked by the secretion of an adhesive and protective matrix. It generally forms in water or in an aqueous medium. An anti-biofilm solution is a solution which, after its application, destroys or limits the formation of biofilms.

[0092] According to another aspect, the present application relates to the non-therapeutic use of a phage endolysin in food safety, as a food preservative for example, in order to combat the development of staphylococci such as S. aureus, in agri-food products.

[0093] According to another aspect, the present application relates to the non-therapeutic use of a phage endolysin in cosmetics, for example in a cosmetic product as a cosmetic preservative, in order to combat the development of staphylococci in cosmetic products.

[0094] According to another aspect, the present application relates to the non-therapeutic use of a phage endolysin in a pharmaceutical preparation, as a preservative for example, in order to combat the development of staphylococci in pharmaceutical products.

[0095] In one embodiment, said phage endolysin for its non-therapeutic use in a disinfectant solution, as an antibacterial compound, in an anti-biofilm solution, in food safety, in cosmetics, or in a pharmaceutical preparation, is an endolysin comprising a sequence having a percentage identity of at least 80% with the sequence SEQ ID NO: 2 and a sequence having a percentage identity of at least 80% with the sequence SEQ ID NO: 3.

[0096] In one embodiment, said phage endolysin for its non-therapeutic use in a disinfectant solution, as an antibacterial compound, in an anti-biofilm solution, in food safety, in cosmetics, or in a pharmaceutical preparation, is an endolysin comprising a sequence having a percentage identity of at least 80% with the sequence SEQ ID NO: 4.

[0097] In one embodiment, said phage endolysin for its non-therapeutic use in a disinfectant solution, as an antibacterial compound, in an anti-biofilm solution, in food safety, in cosmetics, or in a pharmaceutical preparation, is an endolysin comprising a sequence having a percentage identity of at least 70% with the sequence SEQ ID NO: 1.

[0098] In one embodiment, said phage endolysin for its non-therapeutic use in a disinfectant solution, as an antibacterial compound, in an anti-biofilm solution, in food safety, in cosmetics or in a pharmaceutical preparation, is an endolysin comprising a sequence having a percentage identity of at least 70% with the sequence SEQ ID NO: 1 and which comprises a sequence having at least 80% identity with the sequence SEQ ID NO: 2 and a sequence having at least 80% identity with the sequence SEQ ID NO: 3.

[0099] In one embodiment, said phage endolysin for its non-therapeutic use in a disinfectant solution, as an antibacterial compound, in an anti-biofilm solution, in food safety, in cosmetics or in a pharmaceutical preparation, is an endolysin comprising a sequence having a percentage identity of at least 70% with the sequence SEQ ID NO: 1 and which comprises a sequence having at least 80% identity with the sequence SEQ ID NO: 4.

[0100] In one embodiment, said phage endolysin for its non-therapeutic use in a disinfectant solution, as an antibacterial compound, in an anti-biofilm solution, in food safety, in cosmetics or in a pharmaceutical preparation, is an endolysin comprising a sequence having a percentage at least 70% identity with the sequence SEQ ID NO: 1 and which includes the sequence SEQ ID NO: 2 and the sequence SEQ ID NO: 3.

[0101] In one embodiment, said phage endolysin for its non-therapeutic use in a disinfectant solution, as an antibacterial compound, in an anti-biofilm solution, in food safety, in cosmetics or in a pharmaceutical preparation, is an endolysin comprising a sequence having a percentage identity of at least 70% with the sequence SEQ ID NO: 1 and which comprises the sequence SEQ ID NO: 4.

[0102] In one embodiment, said phage endolysin for its non-therapeutic use in a disinfectant solution, as an antibacterial compound, in an anti-biofilm solution, in food safety in cosmetics or in a pharmaceutical preparation, is an endolysin of sequence SEQ ID NO: 1.

[0103] The present application also relates to the non-therapeutic use of a composition as described above, in a disinfectant solution, as an antibacterial compound, in an anti-biofilm solution, in food safety, in cosmetics or in a pharmaceutical preparation.

[0104] According to another aspect, the present application relates to the therapeutic use of an endolysin comprising a sequence having a percentage identity of at least 80% with the sequence SEQ ID NO: 2 and a sequence having a percentage identity of at least 80% with the sequence SEQ ID NO: 3.

[0105] The present application also relates to the therapeutic use of an endolysin comprising a sequence having a percentage identity of at least 80% with the sequence SEQ ID NO: 4.

[0106] The present application also relates to the therapeutic use of an endolysin comprising a sequence having a percentage identity of at least 70% with the sequence SEQ ID NO: 1.

[0107] In particular, it relates to the therapeutic use of an endolysin comprising a sequence having a percentage identity of at least 70% with the sequence SEQ ID NO: 1 and which comprises a sequence having at least 80% identity with the sequence SEQ ID NO: 2 and a sequence having at least 80% identity with the sequence SEQ ID NO: 3, in particular an endolysin comprising a sequence having a percentage identity of at least 70% with the sequence SEQ ID NO: 1 and which comprises a sequence having at least 80% identity with the sequence SEQ ID NO: 4, more particularly the endolysin of SEQ ID NO: 1.

[0108] The present application thus relates to an endolysin comprising a sequence comprising a sequence having a percentage identity of at least 80% with the sequence SEQ ID NO: 2 and a sequence having a percentage identity of at least 80% with the sequence SEQ ID NO: 3, for its use as an active ingredient in a therapeutic solution.

[0109] The present application therefore also relates to an endolysin comprising a sequence having a percentage identity of at least 80% with the sequence SEQ ID NO: 4 for its use as an active ingredient in a therapeutic solution.

[0110] The present application also relates to an endolysin comprising a sequence having a percentage identity of at least 70% with the sequence SEQ ID NO: 1 for its use as an active ingredient in a therapeutic solution.

[0111] In particular, it relates to an endolysin comprising a sequence having a percentage identity of at least 70% with the sequence SEQ ID NO: 1 and which comprises a sequence having at least 80% identity with the sequence SEQ ID NO: 2 and a sequence having at least 80% identity with the sequence SEQ ID NO: 3, in particular an endolysin comprising a sequence having a percentage identity of at least 70% with the sequence SEQ ID NO: 1 and which comprises a sequence having at least 80% identity with the sequence SEQ ID NO: 4, more particularly the endolysin of SEQ ID NO: 1, for its use as an active ingredient in a therapeutic solution.

[0112] Thus, the present application relates to an endolysin comprising a sequence having a percentage identity of at least 80% with the sequence SEQ ID NO: 2 and a sequence having a percentage identity of at least 80% with the sequence SEQ ID NO: 3, for its use as a medicament.

[0113] The present application therefore also relates to an endolysin comprising a sequence having a percentage identity of at least 80% with the sequence SEQ ID NO: 4, for its use as a medicament.

[0114] The present application therefore also relates to an endolysin comprising a sequence having a percentage identity of at least 70% with the sequence SEQ ID NO: 1, for its use as a medicament.

[0115] In particular, it relates to an endolysin comprising a sequence having a percentage identity of at least 70% with the sequence SEQ ID NO: 1 and which comprises a sequence having at least 80% identity with the sequence SEQ ID NO: 2 and a sequence having at least 80% identity with the sequence SEQ ID NO: 3, in particular an endolysin comprising a sequence having a percentage identity of at least 70% with the sequence SEQ ID NO: 1 and which comprises a sequence having at least 80% identity with the sequence SEQ ID NO: 4, more particularly the endolysin of SEQ ID NO: 1, for its use as a medicament.

[0116] In particular, it is a therapeutic use in the treatment of an infection caused by a staphylococcus.

[0117] By "infection" we mean the penetration and proliferation in the body of a microorganism such as bacteria or virus, capable of causing a health problem. An infection can be local or generalized (septicemia).

[0118] In one embodiment, said infection is caused by S. aureus.

[0119] In one embodiment, said infection is caused by S. pseudintermedius.

[0120] This can be in humans or pets such as dogs or cats.

[0121] In particular, said infection caused by a staphylococcus is a skin infection such as pyoderma.

[0122] In a particular embodiment, it concerns the treatment of pyoderma in dogs or cats, in particular caused by S. pseudintermedius.

[0123] Endolysin can be administered topically, orally, or parenterally.

[0124] In particular, it is administered topically, that is to say it is administered to an external point of the body, on a body surface such as the skin or mucous membranes. This is also called cutaneous application.

[0125] When applied topically, it may be in the form of a lotion, cream, gel, hydrogel, emulsion, balm or spray.

[0126] In one embodiment, it is applied at a concentration between 10 pg / mL and 500 pg / mL, in particular between 50 and 250 pg / mL, more particularly between 100 and 150 pg / mL.

[0127] In one embodiment, the daily dose of endolysin administered is between 10 pg and 300 pg, in particular between 100 and 250 pg, more particularly between 150 and 200 pg. The daily dose can be administered at once or divided into several doses over the day. It can, for example, be divided into 2 doses. The doses are spaced between 6 and 8 hours apart. Typically, the treatment can last between one and two weeks.

[0128] A person skilled in the art will know how to adapt the dose and duration of treatment according to the severity of the symptoms and the effectiveness of the treatment over time.

[0129] The present disclosure also relates to a method of treating an infection caused by a staphylococcus comprising administering a phage endolysin to the subject to be treated.

[0130] The embodiments relating to a phage endolysin for its use in the treatment of an infection caused by staphylococcus described above, all apply without exception to this method of treatment.

[0131] The present application also relates to an endolysin comprising a sequence having a percentage identity of at least 80% with the sequence SEQ ID NO: 2 and a sequence having a percentage identity of at least 80% with the sequence SEQ ID NO: 3, for its therapeutic use in a disinfectant washing solution for cleaning the skin or mucous membranes.

[0132] The present application also relates to an endolysin comprising a sequence having a percentage identity of at least 70% with the sequence SEQ ID NO: 1, for its therapeutic use in a disinfectant washing solution for cleaning the skin or mucous membranes.

[0133] In particular, it relates to an endolysin comprising a sequence having a percentage identity of at least 70% with the sequence SEQ ID NO: 1 and which comprises a sequence having at least 80% identity with the sequence SEQ ID NO: 2 and a sequence having at least 80% identity with the sequence SEQ ID NO: 3, in particular an endolysin comprising a sequence having a percentage identity of at least 70% with the sequence SEQ ID NO: 1 and which comprises a sequence having at least 80% identity with the sequence SEQ ID NO: 4, more particularly the endolysin of SEQ ID NO: 1, for its therapeutic use in a disinfectant washing solution for cleaning the skin or mucous membranes. Examples

[0134] Materials and Methods

[0135] Culture media, culture dishes and antibiotics

[0136] Brain Heart Infusion (BHI; BIOKAR #BK015HA) and Lysogeny Broth (LB; BD Difco #240230) culture media are prepared according to the manufacturers' instructions and autoclaved.

[0137] Cloning and plasmid constructions

[0138] The lysines are cloned by in vitro assembly (Garcia-Nafria et al. 2016) into a pET28a (+) plasmid to generate recombinant proteins tagged with a hexahistidine tag at the N-terminal position and under the activity of the T7 promoter which is controlled by a lac operon.

[0139] Overexpression and purification of proteins

[0140] BL21(DE3)Star bacteria transformed with the plasmid encoding Lys38 were used to produce and purify endolysin. Induction is performed when the bacteria reach an optical density ~ 0.5. After induction with IPTG, the bacteria are incubated at 18°C ​​overnight, with shaking. The cultures are centrifuged, then resuspended and concentrated in a lysis buffer and lysed with a sonicator. Subsequently, soluble proteins are separated from insoluble proteins by centrifugation and then purified using a Ni-NTA resin. Finally, before use, the elution buffer containing imidazole is exchanged for 1X PBS using a PD-10 column. The dialyzed fractions are then centrifuged at 21100g at 4°C, the supernatants are collected and pooled then stored at -80°C. The proteins are quantified by Nanodrop and the degree of purity by SDS-PAGE using Image J software. <h2 style=";text-align:left;direction:ltr">

[0141] Comparison of the specification of Lys 38 and Lys K (reference protein) (Figures 2, 3, 5 and 6)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0142] The protein LysK from the sequence SEQ ID NO: 6, is an endolysine containing 40% of the homolog with Lys38.<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0143] SEQ ID NO:6<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0144] MAKTQAEINKRLDAYAKGTVDSPYRVKKATSYDP<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> SFGVMEAGAIDADGYYHAQCQDLITDYVLWLTDNKVRTWGNAKDQIKQSYG TGFKIHENKPSTVPKKGWIAVFTSGSYEQWGHIGIVYDGGNTSTFTILEQN WNGYANKKPTKRVDNYYGLTHFIEIPVKAGTTVKKETAKSASKTPAPKKK ATLKVSKNHINYTMDKRGKKPEGMVIHNDAGRSSGQQYENSLANAGYARYA NGIAHYYGSEGYVWEAIDAKNQIAWHTGDGTGANSGNFRFAGIEVCQSMSA SDAQFLKNEQAVFQFTAEKFKEWGLTPNRKTVRLHMEFVPTACPHRSMVLH TGFNPVTQGRPSQAIMNKLKDYFIKQIKNYMDKGTSSSTVVKDGKTSSAST PATRPVTGSWKKNQYGTWYKPENATFVNGNQPIVTRIGSPFLNAPVGGNLP AGATIVYDEVCIQAGHIWIGYNAYNGNRVYCPVRTCQGVPPNQIPGVAWGV FK<h2 style=";text-align:left;direction:ltr">

[0145] From a preculture made overnight, the different bacterial strains are cultured for 3 hours at 37°C with shaking, in BHI medium. It is then centrifuged and resuspended to obtain an OD of 1 in buffer (20mM Tris, 2.5mM CaCl2, 100mM NaCl, pH 7.5). In spectrophotometer cuvettes, 1 μL of bacterial suspension is deposited with 1 pg / mL of purified total proteins to obtain Figures 2 and 3, and with 10 pg / mL of purified total proteins to obtain Figures 5 and 6. The bacteria are incubated at 37°C and an OD reading is taken at 30 minutes. The activity of endolysins Lys38 and LysK on each bacterial strain is reflected by the decrease in OD.

[0146] Evaluation of the antimicrobial activity of Lys38 on Staphylococcus aureus and Staphylococcuspseudintermedius at 10 pg / mL ([Fig.4])

[0147] Similarly, the different strains are prepared as described above and diluted to obtain a bacterial OD of 1 in the buffer (20 mM Tris, 2.5 mM CaCl2, 100 mM NaCl, pH 7.5). Endolysin Lys38 is added at 10 g / mL. The bacteria are incubated for 30 minutes at 37 °C and are placed on ice to stop the reaction. The activity of endolysin Lys38 on each bacterial strain is reflected by the decrease in OD.

[0148] Toxicological study of Lys38 on animal model (mouse)

[0149] All animal experiments were carried out by a CRO (contract research company) and in accordance with their protocols developed and approved by an ethics committee.

[0150] Seven-week-old male BALB / c mice were obtained from Janvier Labs (France) and identified by a unique number. They were housed in ventilated and enriched cages with irradiated sawdust as bedding under controlled conditions of light (12 hours day and night), temperature (22 ± 2 °C) and humidity (50 ± 10%). Housing parameters were recorded daily, and mice had free access to food and water. Mice were fed the same diet before and during the experiment. All bedding materials, food and water were checked for microbial contamination. Acclimation of the animals lasted 1 week before dosing.

[0151] For the toxicology study, the mice were subsequently divided into 4 random experimental groups receiving various treatments via subcutaneous injection: - Group 1: Vehicle alone (PBS IX) (n=3) - Group 2: Lys38 protein at 25pg / 200pL, twice daily (total of 50pg / day) (n=3) - Group 3: Lys38 protein at 50pg / 200pL, twice daily (total of 100pg / day) (n=3) - Group 4: Lys38 protein at 100pg / 200pL, twice daily (total of 200pg / day) (n=3)

[0152] The Lys38 molecule was diluted in the vehicle (PBS 1X) to reach a final volume of 200pL for each treatment, with two injections per day at an interval of 8 hours. The duration of the study was 7 days. All animals were maintained under identical conditions in accordance with regulations.

[0153] A visual examination of the injection site to observe any inflammation, as well as a monitoring of weight and mortality over 7 days was finally carried out to observe the absence of toxicity of the molecule in mice.

[0154] Study of the efficacy of Lys38 on animal model (mouse) (Figures 7 and 8)

[0155] To evaluate the efficacy of the Lys38 molecule for cutaneous treatment of Staphylococcus pseudintermedius infection, BALB / c mice were anesthetized with isoflurane and then shaved along the dorsal midline 24 hours before infection and then infected with the SP1 strain of VETOPHAGE. For infection, 5x109 CFU / mL were injected into the dorsal subcutaneous tissue just behind the interscapular region. The injection was made with a 26-gauge needle equipped with a 5 mm needle guide to ensure a standardized injection depth.

[0156] During the study, the evolution of weight and the length of the lesion observed on the longest axis, as well as for any clinical manifestation of disease, were recorded in raw data. Photos were taken every day to monitor the evolution of the lesions. The appearance and behavior of the animals were also evaluated at least once a day from the beginning to the end of the experimental phase.

[0157] For skin treatment, two different applications were carried out the day after infection, for each group, for 7 days: - Group 1: Vehicle alone (PBS IX) (n=5) - Group 2: 75 pg of Lys38 per treatment, for a total of 150 pg per day (n=5). There is an 8-hour interval between the first and last application. Each 50 μl application is divided into three drops applied to the infection-induced lesion.

[0158] After the 7 days of treatment, the animals were kept for 2 additional days of observations. At the end of the experiment, the animals were euthanized and skin lesions excised to determine the bacterial load. These skin samples were suspended in 1 ml of PBS in lysis matrix D tubes containing 1.4 mm ceramic spheres. Homogenization of the skin samples was performed by pulsing at 4.0 m / s twice, for 20 seconds per pulse, with a 1-minute pause between pulses. Serial dilutions were then prepared from the homogenate and inoculated for bacterial load counting.

[0159] Activity test (Figures 9 and 10)

[0160] The Lys38 protein was divided into several domains of activity, which were then compared for their effectiveness.

[0161] The sequence SEQ ID NO:4 contains the sequences SEQ ID NO:2 and SEQ ID NO:3. The sequence SEQ ID NO:1 contains the sequence SEQ ID NO:4, and therefore also contains the sequences SEQ ID NO:2 and SEQ NO:3.

[0162] [Tables 1] <h2 style=";text-align:left;direction:ltr">Lys33 CHAP -- Amidase ....... SH3 SEQ ID NO : 1 WASLSKKEFISFLKSSEGKQFNEDGWYGYQCFC-FANTGWLKLBGHKLMGLGA^ TEAKVYSmPEFLAEAGDMWFGENYGGGYGHVAVWiEATLDYSVVLEQNWLGGGWmGiEQPGSGi WESVTRRKHAYDFPMWRRPNFKSETTLLSAQSVTKQKSKQPAKKPKKLfWRDE^KGYNMDKRGYKP RGiVLHWDAGSAGATAEAYHKGLVNADYNRLEKGVAHSWSGSPA'QALPEGKÎAWHyANRlGNRDY YGîË}CQSVGÂTDKQFLANËQSAFQEAARMLKKVVGLPANR?rrVR^ 2 WASLSKKEFISFLKSSEGKaFNEDGWYGY<æFDFANTGWLKLFGHKLMGLGAKDIPFNSINK^^ TEAKWSNTPEFLAEAGDMWFGENYGGGYGHyAVWÏEATLDYiWLEQm'LGGGVVYDGiEQPGSG,<h2 style=";text-align:left;direction:ltr">WESVtRRkmYDFPkW Am Amidase SEQID NO : 3 KUWRDEfKGY^DkRGYKPRGK^NDAGSAGATAEAYHKGLVNADYNRŒKGVAHSYVSGSIVY QAEPEGfôAWhVAMRiGNRDYYGiEiCQSVGATDKQFLANEQSÀFQEAARMLKKWGLPANRNTVRLH VEFYNTACPHRSMLLHTGFDPTYKGlPPœfcK3ÉkLkD#ÎKQ!^ ÇBAFLAm CHAP Amidase SEQ !D NO: 4 MVASLSKKEFISFLKSSEGKQFN'EDG'AYGYQCFDFANTGWLKLFGHKLMGEGAKDIPFNSI^KNYFK TÊAKVYSNTPE F LAEAG DM WFGENYGGGYGHWW1 EAYLDYiWLEQ NWLGGGWTDGÏEQPGSG WESŸTRRKHAYDFPMWFiRP^FKSETTLESAQSVTKQKSKQPAKKPKKLNYJRDEiKGYNMDKRGYKP RGiVLHNDAGSAGATAEAYHKGLVNADYNRLEKGVAHSWSGSTWQALpEGkfÀWHVANRJGNRDY YGîEiCQSVGAtDKQFUANEQSAFQEAAR^LkkWGLPÀNRNTVRLHVEFYNtAGPHRSMLlHTGFDP TVKGiPPQAMQLKLKDYFiKQiRAYMSGKyPVAW Am_SH3 Amidase .......... SH3 SEQ 1D NO : 5 KLNYSRDEiKGYWDKRGYKPRGfVLHNDAGSAGATAEAYHKGLVfiADYNREEKGVAHSYVSGSTV¥ QALPEGKÎAWHVANR!GNRD¥YG!ElCGSVGATOkQFLANEQSAFQÉAARm <KwâLPAN^ VEFYNTACPHRSMLLHTGFDPTVXGIPPQAMQLKLKDYRKQSRAYMDGKVPVATVSNKTSASSNTVK RAGAVO / NSYGTYYMKESATFVCGNKPITTRTVGPFTTCREGYm2PGGWCEYîÆVMLQDGHVW GYDWQGQRYYLPÎRKWNGVAPPNQGLGDLWGTIKl , <h2 style=";text-align:left;direction:ltr">

[0163] The antimicrobial activity of these different parts of the Lys38 protein was evaluated on a Staphylococcus aureus strain (C24) and on a Staphylococcus pseudintermedius strain (SP1) at different concentrations: 1.5, 10, 25, 50pg / mL which correspond respectively to 18nM, 90nM, 180nM, 450nM, 900nM.

[0164] From a preculture made overnight, the two bacterial strains were cultured for 3 hours at 37°C with shaking, in BHI medium. They were then centrifuged and resuspended to obtain an OD of 1 in buffer (20mM Tris, 2.5mM CaCl2, 100mM NaCl, pH 7.5). 300pL of this bacterial solution was then placed in a 96-well culture plate. Each protein was added at different concentrations: 1, 5, 10, 25, 50pg / mL. The solution is incubated for 90 minutes at 37°C and put on ice to stop the reaction. The lysine activity on each bacterial strain is reflected by the decrease in OD.

[0165] RESULTS

[0166] Overexpression and purification of proteins

[0167] Following induction, an intense band migrating around 50 kDa is visible ( [Fig.l]) and corresponds to Lys38.

[0168] Comparison of the specificity of Lys 38 and Lys K (reference protein)

[0169] In order to determine the specificity of Lys38, several strains of several bacterial species were tested with a Lys38 concentration of 1pg / ML. For Gram-negative bacteria, Escherichia coli (n=5) was used as a model species. Several endolysins have previously shown a broader spectrum of activity than the phages from which they were derived. Therefore, in addition to strains of Staphylococcus xylosus (n=2), Staphylococcus epidermis (n=2), Staphylococcus haemolyticus (n=2), Staphylococcus aureus (n=23), Staphylococcus pseudintermedius (n=23), strains of Streptoccocus uberis were also tested (n=5). Note that for strains from the species Escherichia coli and Streptococcus uberis, 100g / mL of Lys38 were used to ensure that there were no effects on these species. The results are then normalized to the control (initial OD = approximately 1) and presented in the form of a histogram ([Fig.2]).

[0170] The results showed that LysK has similar activity between Staphylococcus aureus (SA) and Staphylococcus pseudintermedius (SP) bacteria while Lys38 is more active on SP strains and less active on SA strains (Figures 2 and 3).

[0171] The results in [Fig.4] confirm that Lys38 has specificity for the species Staphylococcus pseudintermedius at a low concentration, e.g., Ipg / mL.

[0172] The results in [Fig.5] and 6 confirm that Lys 28 at a concentration of 10 pg / mL is active on the bacterial strains Staphylococcus aureus (SA) and Staphylococcus pseudintermedius (SP).

[0173] Toxicological study of Lys38 on animal model (mouse)

[0174] During the experiment, the molecule in the form of subcutaneous injection showed no toxicity at all doses tested. In fact, all the animals survived during the experiment, and all gained weight in a similar way to the control group. Analysis of the weight curves shows that even at the highest dose, the mice continuously gained weight ranging from 1% of the initial weight gained up to 13.4%.

[0175] No redness or inflammation was detected at the injection site (data not shown) during the study time. These results demonstrate that the injection is well tolerated and does not appear to be toxic.

[0176] Study of the efficacy of Lys38 on animal model (mouse)

[0177] The analysis of the evolution of the size of the lesions shows a significant reduction in these, with on average a reduction of 40% in their size after 9 days of treatment compared to the condition treated without endolysin (p-value 0.023; Mann-Whitney test) (Figures 7 and 8). This effect is also observed when quantifying the bacterial load, where an average loss of 93.76% of the bacterial load was observed compared to the control condition (p-value = 0.007, Mann-Whitney test). These results allow us to conclude on a significant efficacy of the Lys38 molecule in the context of cutaneous treatment of infection caused by a strain of Staphylococcus pseudintermedius.

[0178] Identification of the activity site of the Lys38 protein

[0179] [Fig.9] presents the results of the activity of each part of the Lys38 protein on Staphylococcus pseudintermedius (SP1) and shows that: - The best activity is obtained for the entire protein Lys 38 (SEQ ID NO: 1); - The sequence responsible for the lysis of S. pseudintermedius is CHAP-Am (SEQ ID NO: 2); - CHAP retains lytic activity but remains less effective than CHAP-Am (SEQ ID NO: 3); - Am (SEQ ID NO:4) or AM-SH3 (SEQ ID NO:5) demonstrates only slight activity on S. pseudintermedius.

[0180] [Fig. 10] presents the results of the activity of each part of the Lys38 protein on Staphylococcus aureus (C24) and shows that: - The best activity is obtained for the entire protein Lys 38 (SEQ ID NO: 1); - The sequence responsible for the lysis of S. aureus is CHAP-Am (SEQ ID NO: 2); - CHAP retains lytic activity but less than CHAP-Am (SEQ ID NO: 3); - Am (SEQ ID NO:4) or AM-SH3 (SEQ ID NO:5) demonstrates only weak activity on S. aureus. Liste des documents cités

[0181] 1. Yang Shen et al. A bactériophage endolysin that éliminâtes intracellular streptococci. eLife. 2016; 5: el3152 2. Mikhail V. Fursov et al. Antibiofilm Activity of a Broad-Range Recombinant Endolysin LysECD7: In Vitro and In Vivo Study. Viruses. 2020 May; 12(5): 545 3. Stéphanie A Lynch and Karla J Helbig. The Complex Diseases of Staphylococcus pseudintermedius in Canines: Where to Next? Veterinary Sciences. 2021 Jan 18;8(1):11. 4. Garcia-Nafria et al. A single-tube universal cloning System exploiting bacterial In Vivo Assembly. Nature, scientific reports. 2016 June; volume 6, Article number: 27459

Claims

Claims

1. Endolysin comprising a sequence having a percentage identity of at least 80% with the sequence SEQ ID NO: 2 and a sequence having a percentage identity of at least 80% with the sequence SEQ ID NO:

3.

2. Endolysin according to claim 1, comprising a sequence having a percentage identity of at least 70% with the sequence SEQ ID NO: 1 and which comprises a sequence having at least 80% identity with the sequence SEQ ID NO: 2 and a sequence having at least 80% identity with the sequence SEQ ID NO:

3.

3. Endolysin according to claim 1 or claim 2, for use as a medicament.

4. Endolysin according to claim 1 or claim 2, for use in the treatment of an infection caused by a staphylococcus, in particular by S. aureus or by S. pseudintermedius.

5. Endolysin for use according to claim 4, wherein said infection caused by a staphylococcus is a skin infection, such as pyoderma.

6. Endolysin for use according to claim 4 or 5, wherein the infection is caused in a pet animal such as a dog or cat.

7. Endolysin for use according to one of claims 3 to 6, characterized in that said endolysin is in a form suitable for topical administration, for example in the form of lotion, cream, gel, hydrogel, emulsion, balm or spray.

8. Non-therapeutic use of an endolysin according to claim 1 or claim 2, as an anti-staphylococcal agent, in particular anti-Staphylococcus aureus, in particular anti-Staphylococcus pseudintermedius.

9. Non-therapeutic use of an endolysin according to claim 1 or claim 2 as an antibacterial compound.

10. Non-therapeutic use of an endolysin according to claim 1 or claim 2 in an anti-biofilm solution.

11. Non-therapeutic use of an endolysin according to claim 1 or claim 2 in food safety.

12. Non-therapeutic use of an endolysin according to claim 1 or claim 2 in a cosmetic product or in a pharmaceutical preparation.

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