Chimeric endolysin polypeptides

Abstract

The present invention relates to the field of medicine, in particular to the field of treatment of pathologies associated with Staphylococcus infections. The present invention relates to novel endolysin polypeptides that specifically target bacterial Staphylococcus cells. The present invention further relates to said endolysin polypeptides for medical use, preferably for treating individuals suffering from pathologies associated with Staphylococcus infections.

Description

Detailed Description of the Invention

[0001] [Field of the Invention] The present invention relates to the field of medicine, in particular to the field of treatment of pathologies associated with Staphylococcus infections. The present invention relates to novel endolysin polypeptides that specifically target bacterial Staphylococcus cells. The present invention further relates to said endolysin polypeptides for medical use, preferably for treating individuals suffering from pathologies associated with Staphylococcus infections.

[0002] [Background of the invention] The widespread spread of antimicrobial resistance (AMR) genes among pathogenic bacteria has resulted in a health crisis of staggering proportions, leaving affected individuals with few or no treatment options, a problem that is expected to become more severe over time. A recent study analyzing the global burden of AMR in 2019 concluded that it was the leading cause of death, with 1.27 million deaths directly attributable to resistance, and that AMR-related deaths are likely to be more prevalent (ARC., 2022). This is in line with a UK government-funded review of antimicrobial resistance, which estimated that global deaths due to AMR could reach 10 million deaths per year by 2050.

[0003] The WHO has identified six pathogens responsible for the majority of resistance-related deaths: Escherichia coli, followed by Staphylococcus aureus, Klebsiella pneumoniae, Streptococcus pneumoniae, Acinetobacter baumannii, and Pseudomonas aeruginosa. Methicillin-resistant S. aureus alone was the direct cause of 100,000 deaths in 2019 (ARC., 2022).

[0004] Staphylococcal bloodstream infections (SBIs) present significant challenges, particularly with regard to appropriate treatment. Persistent S. aureus colonization of the anterior nares in a substantial portion of the population (approximately 30%) provides opportunities for bacteria to enter the bloodstream, potentially leading to devastating consequences. Sepsis and septic shock occur in a significant number of S. aureus bloodstream infection cases, while endocarditis and other deep tissue infections can occur (Song et al., 2020). Coagulase-negative staphylococci, most notably S. epidermidis, are also frequent causes of SBIs. Possessing fewer virulence genes than S. aureus, SBIs caused by S. epidermidis generally manifest as subacute or chronic but can spread to many parts of the body. Biofilm production is a common feature of many S. epidermidis strains, frequently resulting in the colonization of implanted devices, such as intravascular devices, cerebrospinal fluid shunts, intraocular lenses, artificial joints, and heart valve replacements. Treatment of established SBI requires antimicrobial agents, but removal of affected medical devices is recommended. Methicillin resistance, observed among strains of both bacteria, is becoming more prevalent, reducing the number of available antibiotic treatments. Vancomycin and linezolid treatments can have serious side effects, and cases of increased minimal inhibitory concentrations and even complete resistance to these compounds are becoming more frequent. In fact, multidrug resistance has been observed in 70–85% of nosocomial strains of S. epidermidis (Kleinschmidt et al., 2015).

[0005] Therefore, novel antimicrobial compounds to combat these infections are urgently needed. Peptidoglycan hydrolases (PGHs) can cleave specific bonds within the bacterial peptidoglycan (PG) network and have been shown to be active against biofilms. Such high lytic activity makes PGHs potent antistaphylococcal agents. Endolysins are highly specific phage-derived PGHs that are active against both drug-susceptible and drug-resistant bacteria (Schmelcher et al. 2012). As a potential alternative to antibiotics, they have been studied in vitro and in vivo and are currently being tested in several clinical trials (Kashani et al. 2017). Staphylococcal PGHs exhibit a regular domain-like architecture consisting of an enzymatically active domain (EAD) and a cell wall-binding domain (CBD). The high specificity of staphylococcal PGHs can be attributed to their CBDs, which regularly characterize the SH3b fold. The structure of the staphylococcal endolysin SH3b domain has been elucidated and displays high homology to the bacteriocins lysostaphin (LST) and ALE1, suggesting a consensus recognition site in the PG.

[0006] EADs are more diverse and can be grouped according to their structure and cleavage site within the PG. Cysteine, histidine-dependent amidohydrolase / peptidase (CHAP) domains are frequently found in staphylococcal lytic endolysins, such as phage Twort or phage K (Korndorfer et al. 2006). Depending on the CHAP domain present, cleavage within the PG can occur at various positions, including the amide bond of the sugar backbone to the stem peptide and the linkage of the stem peptide to the peptide crossbridge. Herein, the amidohydrolase / peptidase activity of the CHAP domain is referred to as CHAP activity. The M23 domain has only been found in one endolysin (phage 2638), but is also present in the staphylococcal bacteriocin LST and its homolog ALE1. The M23 domains of LST and ALE1 cleave the pentaglycine cross-bridge connecting adjacent stem peptides in the PG of S. aureus, while the M23 domain of phage 2638 cleaves between the peptide bridge and the stem peptide (Grundling et al. 2006; Schmelcher et al. 2015 JAC). Herein, the peptidase activity of the M23 domain is referred to as M23 peptidase activity or M23 activity. Endolysins from phages that infect Staphylococcus have been shown to potentially control these pathogens. In most cases, the main obstacles to the application of endolysins targeting Staphylococcus species are low enzyme activity, difficult mass production, and / or protein stability. Therefore, there is a need for endolysin polypeptides with improved properties, for example, with respect to antimicrobial activity. Several endolysins are presented in WO 2021 / 213898. In summary, there is a need for single endolysin polypeptides with improved properties in terms of antimicrobial activity and stability, especially against systemic infections and sepsis, as well as against prosthetic devices and catheters.

[0007] [Description of the Invention] The present inventors have demonstrated that the combination of M23 endopeptidase and CHAP domains on a single chimeric endolysin polypeptide provides the desired improved activity.

[0008] Thus, in a first aspect, the present invention relates to an endolysin polypeptide having lytic activity against Staphylococcus, said endolysin polypeptide comprising a polypeptide, the amino acid sequence of which has at least 93% sequence identity to SEQ ID NO: 1, - an endolysin having the amino acid sequence shown in SEQ ID NO: 2, and / or - an endolysin having the amino acid sequence shown in SEQ ID NO: 3 has enhanced lytic activity against Staphylococcus in human serum compared to

[0009] Endolysin polypeptides are referred to herein interchangeably as endolysin polypeptides disclosed herein, endolysins disclosed herein, endolysin polypeptides, and endolysins. Lytic activity can be determined using any method known to those skilled in the art. In embodiments herein, the methods described in the Examples herein are preferably used to determine the (enhanced) lytic activity of an endolysin. In embodiments herein, lytic activity is preferably determined at 37°C. Lytic activity is enhanced when it is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, or at least 100% higher compared to an endolysin having the amino acid sequence set forth in SEQ ID NO: 2 and / or SEQ ID NO: 3.

[0010] In embodiments herein, the endolysin polypeptide preferably has enhanced lytic activity against both coagulase-positive and coagulase-negative species of Staphylococcus, particularly against either or both of Staphylococcus aureus and Staphylococcus epidermidis.

[0011] In embodiments herein, an endolysin may comprise a polypeptide, the amino acid sequence of which has at least 93%, more preferably 94%, 95%, 96%, 97%, 98%, 99% or most preferably 100% sequence identity to SEQ ID NO: 1. In embodiments herein, the amino acid sequence of an endolysin polypeptide may have at least 93%, more preferably at least 94%, 95%, 96%, 97%, 98%, 99% or most preferably 100% sequence identity to SEQ ID NO: 1.

[0012] In embodiments herein, in the endolysin polypeptide, the M23 endopeptidase domain and the CHAP domain are preferably separated by a linker, which may be a peptide of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or most preferably at least 23 amino acids. In embodiments herein, the linker may be a peptide consisting of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or most preferably at least 23 amino acids, wherein the peptide has an amino acid sequence having at least 80%, more preferably 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or most preferably 100% sequence similarity and / or identity to SEQ ID NO:8. In embodiments herein, the linker may be a peptide consisting of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or most preferably at least 23 amino acids, wherein the peptide has an amino acid sequence having at least 80%, more preferably 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or most preferably 100% sequence similarity to SEQ ID NO:8. In embodiments herein, the linker may be a peptide consisting of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or most preferably at least 23 amino acids, wherein the peptide has an amino acid sequence that has at least 80%, more preferably 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or most preferably 100% sequence identity to SEQ ID NO: 8. Preferably, the linker comprises at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or most preferably at least 23 amino acids of SEQ ID NO: 8.Preferably, the linker comprises or consists of one of the peptides shown in Table A.

[0013] [Table 1]

[0014] [Table 2]

[0015] [Table 3]

[0016] In embodiments herein, in the endolysin polypeptide, the M23 domain and the SH3b domain are preferably separated by a linker. In embodiments herein, the linker may be a peptide of 5, 6, 7, 8, 9, 10, 11, 12, or most preferably at least 13 amino acids. In embodiments herein, the linker may be a peptide of 5, 6, 7, 8, 9, 10, 11, 12, or most preferably at least 13 amino acids, wherein the peptide has an amino acid sequence having at least 80%, more preferably 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or most preferably 100% sequence similarity and / or identity to SEQ ID NO: 9. In embodiments herein, the linker may be a peptide of 5, 6, 7, 8, 9, 10, 11, 12, or most preferably at least 13 amino acids, wherein the peptide has an amino acid sequence having at least 80%, more preferably 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or most preferably 100% sequence similarity to SEQ ID NO: 9. In embodiments herein, the linker can be a peptide consisting of 5, 6, 7, 8, 9, 10, 11, 12, or most preferably at least 13 amino acids, wherein the peptide has an amino acid sequence having at least 80%, more preferably 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or most preferably 100% sequence identity to SEQ ID NO: 9. Preferably, the linker comprises at least 5, 6, 7, 8, 9, 10, 11, 12, or most preferably at least 13 amino acids of SEQ ID NO: 9. Preferably, the linker comprises or consists of one of the peptides shown in Table B.

[0017] [Table 4]

[0018] Table 5

[0019] Further provided are polynucleotides encoding the endolysin polypeptides disclosed herein. Such polynucleotides are also referred to herein as the polynucleotides disclosed herein. The polynucleotides can be any type of polynucleotide known to those skilled in the art, such as DNA, RNA, or mRNA. The polynucleotide can be, for example, DNA encoding the endolysin polypeptide, where the DNA is expressed in a host cell or an in vitro transcription / translation system. The polynucleotide can also be mRNA delivered to a host for in vivo transcription / translation, where the host can be a cell or a multicellular organism, such as a mammal. Nucleic acid constructs comprising the polynucleotides disclosed herein are also provided. Such nucleic acid constructs are referred to herein as the nucleic acid constructs disclosed herein. Expression vectors comprising the nucleic acid constructs disclosed herein are also provided. Such expression vectors are referred to herein as the expression vectors disclosed herein. The expression vectors disclosed herein can be recombinant expression vectors. Such vectors can comprise, or be part of, a plasmid, cosmid, bacteriophage, or virus, which can be transformed by introducing a nucleic acid construct or polynucleotide disclosed herein. Such transformation vectors specific to the host organism to be transformed are well known to those skilled in the art and widely described in the literature. To produce the polynucleotides or endolysin polypeptides disclosed herein in a host, the transformation of the host organism and the process of integrating the polynucleotides, nucleic acid constructs, or expression vectors disclosed herein can be suitable. Such transformation can be carried out by any suitable known means widely described in the specialist literature and well known to those skilled in the art. Also provided are host cells comprising the polynucleotides disclosed herein, the nucleic acid constructs disclosed herein, or the expression constructs disclosed herein. Such host cells are referred to herein as the host cells disclosed herein. The host cells disclosed herein can be any microbial, prokaryotic, or eukaryotic cell suitable for expressing the endolysin polypeptides disclosed herein. Preferably, the cell is Escherichia coli (E. coli), such as E. coli XL1 blue MRF, E. coli BL21(DE3).

[0020] Further provided herein is a method for producing an endolysin polypeptide, comprising: - culturing a host cell as disclosed herein under conditions conducive to the production of an endolysin polypeptide; - optionally isolating and purifying the endolysin polypeptide from the culture broth; - optionally freeze-drying or spray-drying the endolysin polypeptide; A method is provided which includes:

[0021] Preferably, Escherichia coli (E. coli) is used in the method for producing an endolysin polypeptide disclosed herein. Preferably, E. coli XL1 blue MRF or E. coli BL21-Gold (DE3) is used in step i). Preferably, when a His tag is used, the isolation and purification step may involve IMAC and an Econo-Pac chromatography column (Biorad) packed with 5 mL low-density nickel-chelating agarose beads (ABT beads) in combination with gravity flow for the purification of the endolysin polypeptide disclosed herein. The eluted polypeptide may be dialyzed against 3 x 1 L lyophilization buffer for 2, 4, and 12 hours, preferably comprising 50 mM phosphate, 500 mM sucrose, 200 mM mannitol, 0.005% polysorbate 20, pH 7.4.

[0022] In one embodiment, no His tag is used, and preferably no tag is used at all, so in one embodiment the endolysin polypeptide does not comprise a His tag or does not comprise a tag.

[0023] Lyophilization and reconstitution preferably refers to dehydration by freeze-drying or spray-drying, followed by sample reconstitution by the addition of water. Lyophilization and reconstitution are preferably carried out by dialysis against three changes of 300 ml aliquots of lyophilization buffer (50 mM phosphate or Tris, 500 mM sucrose, 200 mM mannitol, pH 7.4) and freezing in the gaseous phase of liquid nitrogen. Lyophilization is preferably carried out under standard conditions, preferably at -40°C and a vacuum of 75 mTorr for 60 minutes, followed by a temperature increase to -10°C for 5 hours and an additional 60 minutes at the same vacuum level at -10°C. As a final step, the temperature is preferably increased to 25°C for 10 hours. Spray-drying can be carried out using any method known to those skilled in the art. The endolysin polypeptide is preferably reconstituted by the addition of water.

[0024] Further provided is a method for purifying an endolysin polypeptide disclosed herein having enhanced activity, comprising dialysis of the endolysin polypeptide disclosed herein, said dialysis comprising: i) dialysis against a buffer containing a chelating compound; ii) a step of dialysis against a buffer containing a divalent metal ion, preferably the divalent metal ion being Co 2+ , Cu 2+ , Mg 2+ , Ca 2+ , Mn 2+ and Zn 2+ and Includes.

[0025] A "chelating compound" is defined herein as a compound that binds to metal ions. Well-known chelating compounds are ethylenediaminetetraacetic acid (EDTA) and ethylene glycol tetraacetic acid (EGTA). Preferably, EDTA is used in step i) of the purification method.

[0026] Preferably, the divalent metal ion in step ii) is Mn 2+ , Co 2+ , Cu 2+and more preferably, the divalent metal ion is selected from the group consisting of Mn 2+ and Co 2+ and even more preferably, the divalent metal ion is selected from the group consisting of Mn 2+ is.

[0027] It has previously been demonstrated that substitution of divalent metal ions with any of those defined above increases the lytic activity of Ply2638 by 2-2.5-fold. Lytic activity was assessed as described in the Examples herein. Preferably, the method increases lytic activity by at least 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2-fold compared to the untreated polypeptide. Even more preferably, the method increases lytic activity by at least 2.5-fold.

[0028] Further provided are compositions comprising an endolysin polypeptide as disclosed herein, or a polynucleotide as disclosed herein, or a nucleic acid construct as disclosed herein, or an expression construct as disclosed herein, or a host cell as disclosed herein. Such compositions disclosed herein may comprise a mixture of different polynucleotides, and / or nucleic acid constructs, and / or endolysin polypeptides, and / or vectors, and / or cells disclosed herein or obtainable by the methods disclosed herein. Said compositions are also referred to herein as compositions disclosed herein.

[0029] In embodiments herein, the compositions may further comprise a cosmetically acceptable excipient, i.e., an excipient acceptable for cosmetic use. Such a cosmetically acceptable excipient may be any cosmetically acceptable excipient known to those skilled in the art, such as, but not limited to, emulsifiers, emollients, pigments, colorants, binders, antifoaming agents, surfactants, preservatives, and film formers.

[0030] The compositions disclosed herein may further comprise a pharmaceutically acceptable excipient. Such compositions are referred to herein as pharmaceutical compositions and are preferably used as medicines or medicaments. Preferably, the medicaments are used in the treatment of infectious diseases, preferably infections caused by Staphylococcus, such as Staphylococcus aureus and Staphylococcus epidermidis.

[0031] Therefore, further provided is a pharmaceutical composition comprising an endolysin polypeptide disclosed herein, a polynucleotide disclosed herein, a nucleic acid construct disclosed herein, an expression construct disclosed herein and / or a host cell disclosed herein, said pharmaceutical composition further comprising a pharmaceutically acceptable excipient.

[0032] The compositions or pharmaceutical compositions disclosed herein may further comprise one or more additional active ingredients. Activity is preferably defined as exhibiting lytic activity as defined elsewhere herein. Preferably, the one or more additional active ingredients are selected from the group consisting of bacteriophages or phages, phage endolysins derived from such phages, and antibiotics. Phages encompassed herein can be any phage known in the literature. Preferably, such phages are from the list consisting of, but not limited to, Myoviridae, Siphoviridae, and Podoviridae. Such phages may also be from the list consisting of Tectiviridae, Corticoviridae, Lipothrixviridae, Plasmaviridae, Rudiviridae, Fuselloviridae, Inoviridae, Microviridae, Leviviridae and Cystoviridae. In the context of the present invention, the combination of active ingredients defined herein may be administered sequentially or simultaneously. The compositions defined herein may be liquid, solid, or semi-liquid or semi-solid.

[0033] The compositions or pharmaceutical compositions disclosed herein can be used to treat animals, including humans, infected with Staphylococcus species as defined herein. Any suitable route of administration can be used to administer the compositions, including but not limited to oral, aerosol or other devices for pulmonary delivery, nasal spray, intravenous, intramuscular, intraperitoneal, intrathecal, vaginal, rectal, topical, lumbar puncture, intrathecal, and direct application to the brain and / or meninges.

[0034] Preferably, a disclosed composition or pharmaceutical composition is said to be active, functional, or therapeutically active, or capable of treating, preventing, and / or delaying infectious disease, when it reduces the amount of Staphylococcus spp. present in a patient or in said patient's cells or cell lines or cell-free in vitro systems, preferably meaning that 99%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5% or less of the initial amount of Staphylococcus spp. is still detectable after treatment. Preferably, Staphylococcus spp. is not detectable after treatment. As used herein, the expression "amount of Staphylococcus spp." preferably refers to viable Staphylococcus spp. Staphylococcus species can be detected using standard techniques known to those skilled in the art, such as immunohistochemical techniques using Staphylococcus-specific antibodies, tube coagulase tests to detect staphylococcus or "free coagulase," and detection of surface proteins such as clumping factor (slide coagulase test) and / or protein A (commercial latex test). Viable Staphylococcus species can be detected using standard techniques known to those skilled in the art, such as microbiological bacterial culture techniques and / or real-time quantitative reverse transcription polymerase chain reaction to assay bacterial mRNA. The reduction is preferably assessed in tissues or cells of an individual or patient by comparison with the amount present in the individual or patient prior to treatment with a composition or pharmaceutical composition disclosed herein. Alternatively, the comparison can be made with tissues or cells of the individual or patient that have not yet been treated with a composition or pharmaceutical composition disclosed herein, in cases where the treatment is topical.

[0035] The compositions or pharmaceutical compositions disclosed herein may be administered to a subject or cell, tissue or organ in need thereof or to said patient for at least one day, one week, one month, six months, one year or more.

[0036] Thus, provided herein are compositions or pharmaceutical compositions disclosed herein for use as a medicament for treating a subject in need thereof. Preferably, the composition or pharmaceutical composition is used as a medicament in preventing, delaying, or treating a condition in a subject, the condition being associated with infection with Staphylococcus, e.g., coagulase-positive or coagulase-negative Staphylococcus, preferably Staphylococcus aureus and / or Staphylococcus epidermidis. Such a condition may be a skin infection, a soft tissue infection, e.g., an infected diabetic foot ulcer, mastitis, pneumonia, meningitis, endocarditis, toxic shock syndrome (TSS), sepsis, septicaemia, bacteremia, or osteomyelitis. The skin infection may be selected from the group consisting of pimples, impetigo, boils, furuncles, cellulitis, folliculitis, carbuncles, scalded skin syndrome, atopic dermatitis, and abscesses.

[0037] Further provided is a composition or pharmaceutical composition disclosed herein for use as a medicament, wherein the composition or pharmaceutical composition is for systemic or local administration to a subject.

[0038] Further provided is a composition or pharmaceutical composition disclosed herein for use as a medicament, wherein the condition is selected from the group consisting of bacteremia, infective endocarditis, prosthetic joint infection, osteomyelitis, indwelling medical device infection, and implanted medical device infection.

[0039] Further provided is a composition or pharmaceutical composition disclosed herein for use as a medicament, wherein the composition or pharmaceutical composition is for systemic or local administration to a subject, and the condition is selected from the group consisting of bacteremia, infective endocarditis, prosthetic joint infection, osteomyelitis, indwelling medical device infection, and implanted medical device infection.

[0040] Local administration can be used locally at the site of infection or implantation during surgery, for example. The medical uses disclosed herein can be formulated as the products disclosed herein for use as a medicament for the treatment of the specified condition, but can equally be formulated as a method of treating the specified condition using the products disclosed herein, the products disclosed herein for use in the preparation of a medicament for treating the specified condition, and the use of the products disclosed herein for the treatment of the specified condition. All such medical uses are contemplated by the present invention. The subject in need of treatment, delay, and / or prevention of the listed conditions can be any animal subject, preferably a mammal, more preferably a domestic animal such as a bovine, canine, or feline, or a human subject.

[0041] Further provided is a method for cosmetic treatment of the skin of a subject, comprising administering to the skin of a subject an endolysin polypeptide as disclosed herein, a polynucleotide encoding such an endolysin polypeptide, or a composition as disclosed herein. The subject may be any animal subject, preferably a mammal, more preferably a domestic animal such as a bovine, canine or feline, or a human subject.

[0042] Further provided is the in vitro use of an endolysin polypeptide disclosed herein, or a nucleic acid construct disclosed herein, or an expression construct disclosed herein, or a host cell disclosed herein, or a composition or pharmaceutical composition disclosed herein as an antimicrobial agent, preferably a food additive or disinfectant, preferably for coating or impregnating a medical device. Examples of such uses include, but are not limited to, rinsing the cups of milking equipment with a composition according to the invention before milking to prevent the transmission of staphylococci from cow to cow, cleaning and sanitizing surfaces in the food industry, and cleaning medical articles such as surgical tools such as gastroscopes, laparoscopes, thoracoscopes, arthroscopes, and catheters and tubes that have long or hollow sections and are prone to repeated use by being introduced into the human or animal body. Such use may be combined with the use of any sterilization method or disinfectant known in the art, such as ultrasonic cleaning, irradiation or thermal sterilization, disinfecting solutions such as ethanol, immersion of the device in ammonium, iodine and / or aldehyde disinfectants, or gas sterilization by holding the equipment in a closed atmosphere such as formalin gas or ethylene oxide gas.

[0043] Further provided is an in vitro method for coating or impregnating a medical device with an endolysin polypeptide disclosed herein or a composition or pharmaceutical composition disclosed herein, comprising contacting the medical device with an endolysin polypeptide disclosed herein or a composition or pharmaceutical composition disclosed herein.

[0044] Further provided is the use of an endolysin polypeptide disclosed herein, or a polynucleotide disclosed herein, or a nucleic acid construct disclosed herein, or an expression construct disclosed herein, or a host cell disclosed herein, or a composition or pharmaceutical composition disclosed herein for the detection of Staphylococcus, such as Staphylococcus aureus, in an ex vivo diagnostic application.

[0045] [Definition] "Sequence identity" is defined herein as the relationship between two or more amino acid (peptide, polypeptide, or protein) sequences or two or more nucleic acid (nucleotide, polynucleotide) sequences, as determined by comparing the sequences. In the art, "identity" also means the degree of sequence relatedness between amino acid sequences or nucleotide sequences, as the case may be, as determined by the match between strings of such sequences. "Similarity" between two amino acid sequences is determined by comparing the amino acid sequence and its conserved amino acid substitutes of one peptide or polypeptide with the sequence of a second peptide or polypeptide. In a preferred embodiment, identity or similarity is calculated across all SEQ ID NOs identified herein. "Identity" and "similarity" can be readily calculated by known methods, including, but not limited to, those described in Computational Molecular Biology, Lesk, AM, ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, DW, ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, AM, and Griffin, HG, eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heine, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991; and Carillo, H., and Lipman, D., SIAM J. Applied Math., 48:1073 (1988).

[0046] Preferred methods to determine identity are designed to give the largest match between the sequences tested. Methods to determine identity and similarity are codified in publicly available computer programs. Preferred computer program methods for determining identity and similarity between two sequences include, for example, the GCG program package (Devereux, J., et al., Nucleic Acids Research 12(1):387(1984)), BestFit, BLASTP, BLASTN, and FASTA (Altschul, S., et al., J. Mol. Biol. 215:403-410(1990)). The BLAST X program is publicly available from NCBI and other sources (BLAST Manual, Altschul, S., et al., NCBI NLM NIH Bethesda, MD 20894; Altschul, S., et al., J. Mol. Biol. 215:403-410(1990)). The well-known Smith-Waterman algorithm may also be used to determine identity.

[0047] Preferred parameters for polypeptide sequence comparison include the algorithm: Needleman and Wunsch, J. Mol. Biol. 48:443-453 (1970), comparison matrix: BLOSUM62 from Hentikoff and Hentikoff, Proc. Natl. Acad. Sci. USA. 89:10915-10919 (1992), gap penalty: 12, and gap length penalty: 4. A program useful with these parameters is publicly available as the "Ogap" program from the Genetics Computer Group, Madison, WI. The aforementioned parameters are the default parameters for amino acid comparisons (along with no penalty for end gaps).

[0048] Preferred parameters for nucleic acid comparison include the algorithm: Needleman and Wunsch, J. Mol. Biol. 48:443-453 (1970), comparison matrix: match = +10, mismatch = 0, gap penalty: 50, gap length penalty: 3. Available as the Gap program from the Genetics Computer Group, Madison, Wis. Given above are the default parameters for nucleic acid comparison.

[0049] Optionally, when determining the degree of amino acid similarity, those skilled in the art may also consider so-called "conservative" amino acid substitutions, as would be apparent to those skilled in the art. Conservative amino acid substitutions refer to the interchangeability of residues with similar side chains. For example, amino acids with aliphatic side chains include glycine, alanine, valine, leucine, and isoleucine; amino acids with aliphatic hydroxyl side chains include serine and threonine; amino acids with amide-containing side chains include asparagine and glutamine; amino acids with aromatic side chains include phenylalanine, tyrosine, and tryptophan; amino acids with basic side chains include lysine, arginine, and histidine; and amino acids with sulfur-containing side chains include cysteine ​​and methionine. Preferred conservative amino acid substitution groups are valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, and asparagine-glutamine. Substitutional variants of the amino acid sequences disclosed herein are those in which at least one residue in the disclosed sequences has been removed and a different residue inserted in its place. Preferably, the amino acid changes are conservative. Preferred conservative substitutions for each of the naturally occurring amino acids are as follows: ala to ser, arg to lys, asn to gln or his, asp to glu, cys to ser or ala, gln to asn, glu to asp, gly to pro, his to asn or gln, ile to leu or val, leu to ile or val, lys to arg, gln or glu, met to leu or ile, phe to met, leu or tyr, ser to thr, thr to ser, trp to tyr, tyr to trp or phe and val to ile or leu.

[0050] A "nucleic acid molecule" or "polynucleotide" (these terms are used interchangeably herein) is represented by a nucleotide sequence. A "polypeptide" is represented by an amino acid sequence. A "nucleic acid construct" is defined as a nucleic acid molecule isolated from a naturally occurring gene or modified to contain segments of nucleic acids combined or juxtaposed in a non-naturally occurring manner. A nucleic acid molecule is represented by a nucleotide sequence. Optionally, the nucleotide sequence present in the nucleic acid construct is operably linked to one or more control sequences that direct the production or expression of said peptide or polypeptide in a cell or subject. "Operably linked" is defined herein as a configuration in which a control sequence is suitably positioned relative to a nucleotide sequence encoding a polypeptide of the invention such that the control sequence directs the production / expression of a peptide or polypeptide of the invention in a cell and / or subject. "Operably linked" may also be used to define a configuration in which a sequence is suitably positioned relative to another sequence encoding a functional domain such that a chimeric polypeptide is encoded in a cell and / or subject.

[0051] "Expression" is taken to include any step involved in the production of a peptide or polypeptide, including but not limited to transcription, post-transcriptional modification, translation, post-translational modification and secretion.

[0052] "Control sequences" is defined herein to include all components necessary or advantageous for expression of a polypeptide. At a minimum, control sequences include a promoter and transcriptional and translational stop signals. Optionally, a promoter represented by a nucleotide sequence present in the nucleic acid construct is operably linked to another nucleotide sequence encoding a peptide or polypeptide identified herein.

[0053] The term "transformation" refers to a permanent or transient genetic change induced in a cell following the integration of new DNA (i.e., DNA exogenous to the cell). When the cell is a bacterial cell, as intended in this invention, the term refers to an extrachromosomal, self-replicating vector, usually carrying a selectable antibiotic resistance.

[0054] An "expression vector" can be any vector that can be conveniently subjected to recombinant DNA procedures and can result in the expression of a nucleotide sequence encoding a polypeptide of the present invention in a cell and / or subject. As used herein, the term "promoter" refers to a nucleic acid fragment that functions to control the transcription of one or more genes or nucleic acids, located upstream in the transcriptional direction of the gene's transcription start site. It is related to a binding site for DNA-dependent RNA polymerase, a transcription start site, and any other DNA sequence, including, but not limited to, transcription factor binding sites, repressor and activator protein binding sites, and any other nucleotide sequence known to those skilled in the art that acts to directly or indirectly regulate the amount of transcription from the promoter. In the context of the present invention, a promoter preferably ends at nucleotide -1 of the transcription start site (TSS).

[0055] As used herein, "polypeptide" means any peptide, oligopeptide, polypeptide, gene product, expression product, or protein. A polypeptide is made up of consecutive amino acids. The term "polypeptide" encompasses naturally occurring or synthetic molecules.

[0056] The sequence information provided herein should not be so strictly construed as to require the inclusion of incorrectly identified bases, as one of skill in the art is capable of identifying such incorrectly identified bases and knows how to correct such errors.

[0057] In addition, those skilled in the art know that when expressing proteins, terminal amino acids such as the N-terminal methionine are sometimes cleaved.

[0058] In this document and its claims, the verb "comprise" and its conjugations are used in their open-ended sense, meaning to include items that follow the word, but not to exclude items not specifically listed. Additionally, the verb "consisting of" may be replaced by "consisting essentially of," which means that a nucleic acid construct, vector, or cellular product, or composition, or nucleic acid molecule, or peptide, or polypeptide defined herein may contain additional components other than those specifically identified, without altering the specific properties of the invention. Additionally, reference to an "element" by the indefinite article "a" or "an" does not exclude the possibility that more than one element is present, unless the context clearly requires that only one element be present. Thus, the indefinite article "a" or "an" typically means "at least one." The words "about" or "approximately" when used in connection with a numerical value (e.g., about 10) preferably mean that the value may be 10% greater or less than the given value (10).

[0059] All patents and literature references cited herein are incorporated by reference in their entirety.

[0060] The examples herein are provided for illustrative purposes only and are not intended to limit the scope of the present invention in any way. [Brief explanation of the drawings]

[0061] [Figure 1]Killing of S. aureus NR-46543 / USA300JE2 (MRSA) in human serum by peptidoglycan hydrolase. A quantitative killing assay (QKA) was performed with peptidoglycan hydrolase (PGH) constructs ID453, ID557, and ID558 against methicillin-resistant Staphylococcus aureus (MRSA) NR46543 / USA300JE2 in human serum at 37°C and 180 rpm (25 mm orbit) for 30 minutes. Viable cell counts (CFU / ml) of S. aureus NR46543 / USA300JE2 (y-axis) are shown for the three PGH constructs across a two-fold concentration range (320–0.625 nM) (x-axis) in nM. The depicted negative control (no PGH addition) corresponds to the average individual control of the three test constructs. The limit of detection (LoD) is 200 CFU / ml (gray dashed line). The y-axis is cut at 160 CFU / ml. Three biological replicates (n=3) were made. Error bars for the average control and construct represent the standard error of the mean (SEM). [Figure 2]Killing of S. aureus ATCC 12600 (MSSA) in human serum by peptidoglycan hydrolase. A quantitative killing assay (QKA) was performed with peptidoglycan hydrolase (PGH) constructs ID453, ID557, and ID558 against methicillin-susceptible Staphylococcus aureus (MSSA) ATCC 12600 in human serum at 37°C, 180 rpm (25 mm orbit) for 30 minutes. Viable cell counts (CFU / ml) of S. aureus ATCC 12600 (y-axis) are shown for three PGH constructs over a two-fold concentration range (320 nM to 0.625 nM) (x-axis) in nM. The depicted negative control (no PGH added) corresponds to the average individual control of the three test constructs. The limit of detection (LoD) is 200 CFU / ml (gray dashed line). The y-axis is cut at 160 CFU / ml. Three biological replicates (n=3) were made. Error bars for the mean control and construct represent the standard error of the mean (SEM). [Figure 3] Killing of S. aureus ATCC 12598 (MSSA) in human serum by peptidoglycan hydrolase. A quantitative killing assay (QKA) was performed with peptidoglycan hydrolase (PGH) constructs ID453, ID557, and ID558 against methicillin-susceptible Staphylococcus aureus (MSSA) ATCC 12598 (Cowan I) in human serum at 37°C, 180 rpm (25 mm orbit), for 30 minutes. Viable cell counts (CFU / ml) of S. aureus ATCC 12598 (y-axis) are shown for three PGH constructs over a two-fold concentration range (320 nM to 0.625 nM) (x-axis) in nM. The depicted negative control (no PGH addition) corresponds to the average individual control of the three test constructs. The limit of detection (LoD) is 200 CFU / ml (gray dashed line). The y-axis is cut at 160 CFU / ml. Three biological replicates (n=3) were made. Error bars for the mean control and construct represent the standard error of the mean (SEM). [Figure 4] Killing of S. epidermidis ATCC12228 / DSM1798 (MSSE) in human serum by peptidoglycan hydrolase. A quantitative killing assay (QKA) was performed with peptidoglycan hydrolase (PGH) constructs ID453, ID557, and ID558 against methicillin-susceptible Staphylococcus epidermidis (MSSE) ATCC12228 / DSM1798 in human serum at 37°C, 180 rpm (25 mm orbit) for 30 minutes. Viable cell counts (CFU / ml) of S. epidermidis ATCC12228 / DSM1798 (y-axis) are shown for the three PGH constructs over a two-fold concentration range in nM (320 nM to 0.625 nM) (x-axis). The depicted negative control (no PGH addition) corresponds to the average individual control of the three test constructs. The limit of detection (LoD) is 200 CFU / ml (gray dashed line). The y-axis is cut at 160 CFU / ml. Three biological replicates (n=3) were made, except for concentrations 0.625 and 1.25 nM (n=2). Error bars for the average control and construct represent the standard error of the mean (SEM). [Figure 5]Killing of S. epidermidis ATCC35984 / BB1336 (MRSE) in human serum by peptidoglycan hydrolase. Quantitative killing assays (QKAs) were performed with peptidoglycan hydrolase (PGH) constructs ID453, ID557, and ID558 against methicillin-resistant (MRSE) ATCC35984 / BB1336 in human serum at 37°C, 180 rpm (25 mm orbit) for 30 min. Viable cell counts (CFU / ml) of S. epidermidis ATCC35984 / BB1336 (y-axis) are shown for three PGH constructs over a two-fold concentration range (320 nM to 0.625 nM) (x-axis) in nM. The depicted negative control (no PGH added) corresponds to the average individual control of the three test constructs. The limit of detection (LoD) is 200 CFU / ml (gray dashed line). The y-axis is cut at 160 CFU / ml. Three biological replicates (n=3) were made, except for concentrations of 0.625 and 1.25 nM (n=2). Error bars for the mean control and construct represent the standard error of the mean (SEM).

[0062] [Example] 1. Introduction Phage lysins are phage-encoded cell wall hydrolases that cleave bonds within the bacterial peptidoglycan, leading to localized lysis and ultimately cell death.

[0063] Briefly, these peptidoglycan hydrolases (PGHs) are modular, with one domain responsible for specific binding to the target cell wall and another responsible for cleaving a very specific bond within the peptidoglycan. The modules may be connected by polypeptide linkers. These enzymes exhibit varying degrees of specificity with respect to binding to the target and cleaving certain bonds that may be absent in other bacterial genera or species. By combining and rearranging the different parts that make up the various naturally occurring enzymes, novel molecules with very specific recognition and cleavage patterns can be created, allowing for the creation of highly targeted antimicrobial preparations.

[0064] Here, we present the efficacy profiles in human serum of three such molecules (Table 1). Two of these molecules have already been published in WO2021213898 (ID557 [SEQ ID NO: 41] and ID558 [SEQ ID NO: 5]), while ID453 is a novel molecule presented herein. All three molecules contain the CHAP domain of phage Twort followed by the M23LST domain of the lysostaphin gene of Staphylococcus simulans. Both ID453 and ID557 contain the SH3b domain of the lysostaphin gene, while ID558 contains the SH3b domain from phage 2638A. The origin references of the domains and linkers are shown in Table 2.

[0065] [Table 6]

[0066] [Table 7]

[0067] When targeting SBIs, sufficient activity of the candidate molecule in human serum is a prerequisite. In this setting, we investigated its efficacy against three S. aureus strains, including one methicillin-resistant strain, as well as one methicillin-resistant and one methicillin-susceptible S. epidermidis strain. Interestingly, despite significant similarity between the three molecules, one of the candidate compounds showed a clear advantage over the other two. ID453 performed better than the others, requiring lower doses to achieve complete target cell killing in human serum against two of the three S. aureus strains tested and one of the two S. epidermidis strains examined. It was shown to be at least as effective against the remaining strains as any of the other compounds, with higher killing efficacy at lower concentrations compared to the other constructs. ID453 is an excellent choice for targeting SBIs.

[0068] [2. Results] 2.1 Quantitative Killing Assay S. aureus NR-46543 / USA300JE2, S. aureus ATCC12600, S. aureus ATCC12598 (Cowan Quantitative killing assays (QKAs) were performed in human serum for 30 min at 37 °C and 180 rpm (25 mm orbit) using the peptidoglycan hydrolase (PGH) constructs CHAPTw(L)_M23LST_SH3bLST (ID453), CHAPTw_M23LST_SH3bLST (ID557), and CHAPTw(L)_M23LST_(L)SH3b2638 (ID558) against S. epidermidis ATCC12228 / DSM1798 and S. epidermidis ATCC35984 / BB1336. Constructs were tested over a two-fold concentration range (320 nM to 0.625 nM). The QKA evaluation results (viable cell counts) of the three constructs in human serum are shown in Figures 1-5. Three biological replicates of each construct were utilized for each strain. As shown in Figures 1-5, all three constructs possess staphylococcal lytic activity in each strain within the concentration range tested. Furthermore, a dose response is evident. The further to the left the curve is, the more active the construct is.

[0069] ID453 was the construct with the highest killing activity against the methicillin-resistant S. aureus strain NR-46543 / USA300JE2 (Figure 1) and the methicillin-susceptible S. aureus strain ATCC12598 (Cowan I) (Figure 3), followed by ID557. The difference in activity between these two constructs was less pronounced against the methicillin-susceptible S. aureus strain ATCC12600 (Figure 2), but ID453 still showed higher activity than the other constructs at most of the concentrations tested. Among all three strains, ID558 showed the lowest activity.

[0070] The superiority of ID453 is most pronounced against the methicillin-susceptible S. epidermidis ATCC12228 / DSM1798 strain (Figure 4). In contrast to the results obtained with S. aureus strains, ID557 is the construct with the lowest activity when tested against S. epidermidis strains. ID453 exhibits activity similar to the ID558 construct against the methicillin-resistant S. epidermidis ATCC35984 / BB1336 strain at higher concentrations (>20 nM), but ID453 is clearly superior at lower concentrations (<20 nM) (Figure 5). Overall, our data demonstrated the superiority of ID453 compared to the other tested constructs.

[0071] 3. Materials and Methods 3.1 Materials: Bacteria, media, buffers, equipment, and consumables

[0072] [Table 8]

[0073] [Table 9]

[0074] [Table 10]

[0075] [Table 11]

[0076] [Table 12]

[0077] [Table 13]

[0078] 3.2 Methods: Protocols and Procedures 3.2.1 Quantitative Killing Assay (QKA) A total of three PGH constructs were tested in human serum against three S. aureus strains and two S. epidermidis strains. Aliquots of the constructs were stored in PCR tubes at -80°C (in CIEX PO4 elution buffer). Unless otherwise stated, all steps were performed under sterile conditions. A range of final PGH concentrations were tested for all five strains: 320 nM, 160 nM, 80 nM, 40 nM, 20 nM, 10 nM, 5 nM, 2.5 nM, 1.25 nM, and 0.625 nM. The desired final bacterial control was 1 x 10 6 ~1×10 7 CFU / ml. At least two biological replicates were utilized. One 96-well F-bottom plate was used for each PGH construct tested.

[0079] Precultures were prepared by inoculating 5 ml of tryptic soy broth (TSB) medium with either S. aureus NR-46543 / USA300JE2, S. aureus ATCC 12600, S. aureus ATCC 12598 (Cowan I), S. epidermidis ATCC 12228 / DSM 1798, or S. epidermidis ATCC 35984 / BB1336. Cultures were incubated overnight (O / N) at 37°C and 180 revolutions per minute (rpm) (25 mm orbit).

[0080] Aliquots of human serum (stored at -20°C) were thawed in a 30°C water bath, filtered through a 0.45 μm filter, and stored on ice. A 1:25 dilution of the O / N culture was made by mixing 400 μl of the O / N culture with 10 ml TSB medium. The culture was incubated at 37°C and 180 rpm until an optical density (OD600nm) of 0.5-0.6 was reached. The culture was then placed on ice for approximately 5 minutes to stop growth. One ml of the culture was adjusted to an OD of 0.51 with TSB medium, transferred to a 1.5 ml reaction tube, and placed on ice. During culture growth, 160 μl 1x stop buffer was provided to rows B-H of each 96-well F-bottom plate, and the plates were stored at 4°C until use. Once the bacterial culture was adjusted to the desired OD, an appropriate aliquot (10-20 μl aliquot) of the PGH construct to be tested was thawed on ice and briefly spun down. Human serum was allowed to acclimate to room temperature. 100 μl of human serum was dispensed via reverse pipetting into wells A1 and A3–A12 of a prepared 96-well F-bottom plate (one plate per enzyme tested). In well A2 of each plate, enzyme predilutions of each construct to be tested in human serum were prepared (final volume 200 μl) to reach a concentration twice the highest final concentration tested (320 nM) (640 nM). The enzyme predilutions were mixed well and serial two-fold dilutions were made by transferring 100 μl from well A2 to well A3, mixing six times, then changing pipette tips and continuing the same procedure from wells A3 to A4 and on to well A11. The final 100 μl was discarded from well A11, leaving wells A1 and A12 as negative controls (no enzyme added, only bacterial suspension in serum). Each enzyme predilution and serial dilution in the plate was prepared within 10 min before inoculation to minimize interaction of the enzyme with the walls of the plate wells. The bacterial suspension for inoculation was prepared by mixing 500 μl of vortexed, OD-adjusted culture with 4.5 ml of human serum in a 25 ml reservoir (1:10 dilution).Using a manual multichannel pipette, 100 μl of bacterial suspension from the reservoir was added to wells A1 to A12 of the plate, mixed once by pipetting up and down, and the plate was immediately transferred to an incubator shaker at 37°C and 180 rpm for exactly 30 min.

[0081] After exactly 30 minutes of plate incubation, the plate was removed from the incubator shaker, and 20 μl of 10× stop buffer was immediately added to row A using a multichannel pipette. The suspension in the well was homogenized by pipetting up and down eight times to stop further enzyme activity. A 5-fold serial dilution was then performed with the provided 1× stop buffer by transferring 40 μl from row A to row B, mixing six times, then changing pipette tips, and then transferring 40 μl from rows B to C and then to row H. The final 40 μl from row H was discarded. For each well of the 96-well plate, 5.5 μl was spot-plated onto a pre-dried LB agar rectangular plate. After the spots were dried, the agar plates were placed upside down in a 37°C incubator for O / N incubation (approximately 16 hours for S. aureus strains and approximately 20 hours for S. epidermidis strains).

[0082] The next day, colony-forming units (CFU) / spot were counted. CFU / ml was calculated, and the viable cell count and CFU / ml log reduction across the final concentration range tested (320 nM to 0.625 nM) were visualized graphically (GraphPad Prism 9.2.0). Results from biological replicates were averaged, and the standard error of the mean (SEM) was calculated and displayed.

[0083] [References] Antimicrobial Resistance Collaborators.Global burden of bacterial antimicrobial resistance in 2019:a systematic analysis.Lancet.2022 Feb 12;399(10325):629-655.doi:10.1016 / S0140-6736(21)02724-0.Epub 2022 Jan 19.PMID:35065702;PMCID:PMC8841637. Kleinschmidt S,Huygens F,Faoagali J,Rathnayake IU,Hafner LM.Staphylococcus epidermidis as a cause of bacteremia.Future Microbiol.2015;10(11):1859-79.doi:10.2217 / fmb.15.98.Epub 2015 Oct 30.PMID:26517189. Guo Y,Song G,Sun M,Wang J,Wang Y.Prevalence and Therapies of Antibiotic-Resistance in Staphylococcus aureus.Front Cell Infect Microbiol.2020 Mar 17;10:107.doi:10.3389 / fcimb.2020.00107.PMID:32257966;PMCID:PMC7089872. Grundling,A.,Missiakas,D.M.&Schneewind,O.,2006.Staphylococcus aureus Mutants with Increased Lysostaphin Resistance.Journal of Bacteriology,188(17),pp.6286-6297. Kashani,H.et al.,2017.Recombinant Endolysins as Potential Therapeutics against Antibiotic-Resistant Staphylococcus aureus:Current Status of Research and Novel Delivery Strategies.Clinical Microbiology Reviews,31(1). Korndorfer,I.P.et al.,2006.The Crystal Structure of the Bacteriophage PSA Endolysin Reveals a Unique Fold Responsible for Specific Recognition of Listeria Cell Walls.Journal of Molecular Biology,364(4),pp.678-689. Schmelcher,M.et al.,2015.Evolutionarily distinct bacteriophage endolysins featuring conserved peptidoglycan cleavage sites protect mice from MRSA infection.Journal of Antimicrobial Chemotherapy,70(5),pp.1453-1465. Schmelcher,M.,Donovan,D.M.&Loessner,M.J.,2012.Bacteriophage endolysins as novel antimicrobials.Future Microbiology,7(10),pp.1147-1171.

Claims

1. 1. An endolysin polypeptide having lytic activity against Staphylococcus, comprising a polypeptide, the amino acid sequence of which has at least 93% sequence identity to SEQ ID NO: 1, the endolysin polypeptide comprising: an endolysin having the amino acid sequence shown in SEQ ID NO: 2, and / or an endolysin having the amino acid sequence shown in SEQ ID NO: 3 has enhanced lytic activity against Staphylococcus in human serum compared to An endolysin polypeptide, wherein the M23 endopeptidase domain and the CHAP domain in said endolysin polypeptide are separated by a linker comprising at least 13 amino acids.

2. The endolysin polypeptide of claim 1, wherein the amino acid sequence of the endolysin polypeptide has at least 93% sequence identity with SEQ ID NO:

1.

3. A polynucleotide encoding the endolysin polypeptide of claim 1 or 2.

4. A nucleic acid construct comprising the polynucleotide of claim 3.

5. An expression vector comprising the nucleic acid construct of claim 4.

6. A host cell comprising the polynucleotide of claim 3, the nucleic acid construct of claim 4 or the expression construct of claim 5.

7. 3. A method for producing an endolysin polypeptide according to claim 1 or 2, comprising: - culturing the host cell of claim 6 under conditions conducive to said production of said endolysin polypeptide; - optionally isolating and purifying said endolysin polypeptide from the culture broth; optionally freeze-drying or spray-drying the endolysin polypeptide; A method comprising:

8. 10. A method for producing an endolysin polypeptide according to claim 1 or 2 with enhanced activity, comprising dialysis of an endolysin according to claim 1 or 2, said dialysis comprising: i) dialysis against a buffer containing a chelating compound; ii) a step of dialysis against a buffer containing a divalent metal ion, preferably the divalent metal ion being Co ２＋ , Cu ２＋ , Mg ２＋ , Ca ２＋ , Mn ２＋ and Zn ２＋ and A method comprising:

9. A composition comprising an endolysin polypeptide according to claim 1 or 2, or a polynucleotide according to claim 3, or a nucleic acid construct according to claim 4, or an expression construct according to claim 5, or a host cell according to claim 6.

10. A composition comprising an endolysin polypeptide described in claim 1 or 2, or a polynucleotide described in claim 3, or a nucleic acid construct described in claim 4, or an expression construct described in claim 5, or a host cell described in claim 6, and further comprising a cosmetically acceptable excipient.

11. A pharmaceutical composition comprising an endolysin polypeptide described in claim 1 or 2, or a polynucleotide described in claim 3, or a nucleic acid construct described in claim 4, or an expression construct described in claim 5, or a host cell described in claim 6, further comprising a pharmaceutically acceptable excipient.

12. The composition of any one of claims 9 to 11, further comprising an additional active ingredient.

13. 13. A composition according to any one of claims 9, 11 or 12 for use as a medicament, preferably for use as a medicament in the treatment of pathologies associated with Staphylococcus infections.

14. A method for treating a pathology associated with Staphylococcus infection, comprising administering an endolysin polypeptide described in claim 1 or 2, or a polynucleotide described in claim 3, or a nucleic acid construct described in claim 4, or an expression construct described in claim 5, or a host cell described in claim 6, or a composition described in any one of claims 9, 11 or 12.

15. A method for cosmetic treatment of skin, comprising administering an endolysin polypeptide described in claim 1 or 2, or a polynucleotide described in claim 3, or a nucleic acid construct described in claim 4, or an expression construct described in claim 5, or a host cell described in claim 6, or a composition described in any one of claims 9, 10 or 12.

16. 15. The method of treatment according to claim 14, wherein the condition to be treated is selected from a skin infection, a soft tissue infection, such as an infected diabetic foot ulcer, mastitis, pneumonia, meningitis, endocarditis, toxic shock syndrome (TSS), sepsis, septicemia, bacteremia or osteomyelitis. The skin infection may be selected from the group consisting of pimples, impetigo, boils, furuncles, cellulitis, folliculitis, carbuncles, scalded skin syndrome, atopic dermatitis, and abscesses.

17. 15. The method of claim 14, wherein the condition being treated is selected from the group consisting of bacteremia, infective endocarditis, prosthetic joint infection, osteomyelitis, indwelling medical device infection, and implanted medical device infection.

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