Chimeric endolysin polypeptides

Abstract

The present invention relates to the field of medicine, in particular to the treatment of conditions associated with Staphylococcus infection. The invention relates to a novel endolysin polypeptide specifically targeting bacterial staphylococcus cells. The invention further relates to said endolysin polypeptide for medical use, preferably for treating an individual suffering from a condition associated with a Staphylococcus infection.

Description

Technical Field

[0001] The present invention relates to the field of medicine, in particular to the field of treatment of conditions associated with staphylococcal infections. The present invention relates to a novel endolysin polypeptide that specifically targets cells of the bacterium Staphylococcus. The present invention further relates to said endolysin polypeptide for medical use, preferably for treating a subject suffering from a condition associated with a staphylococcal infection. Background Art

[0002] The widespread spread of antimicrobial resistance (AMR) genes in pathogenic bacteria has led to a health crisis of staggering proportions, leaving affected patients with few treatment options, and the problem is expected to worsen over time. A recent study analyzing the global burden of AMR in 2019 concluded that AMR had become the leading cause of death, with 1.27 million deaths directly attributable to resistance and many more AMR-related deaths (ARC, 2022). This is consistent with the UK government-funded Review on Antimicrobial Resistance, which estimated that by 2050, the global death toll from AMR could reach 10 million per year.

[0003] The World Health Organization has identified six pathogens that cause the most deaths due to drug resistance: Escherichia coli, followed by Staphylococcus aureus, Klebsiella pneumoniae, Streptococcus pneumoniae, Acinetobacter baumannii, and Pseudomonas aeruginosa. In 2019, methicillin-resistant Staphylococcus aureus alone was directly responsible for 100,000 deaths (ARC, 2022).

[0004] Staphylococcal bloodstream infections (SBIs), in particular, present significant challenges for adequate treatment. A significant proportion of the population (approximately 30%) harbors permanent S. aureus colonization of the anterior nares, allowing the bacteria to enter the bloodstream with potentially devastating consequences. A significant number of S. aureus BSIs develop sepsis and septic shock, but they can also lead to endocarditis and other deep tissue infections (Song et al., 2020). Coagulase-negative staphylococci, most notably Staphylococcus epidermidis (S. epidermidis), are also common causes of SBIs. S. epidermidis, which has fewer virulence genes than S. aureus, typically causes subacute or chronic SBIs but can spread to many sites in the body. Biofilm formation is a common characteristic of many S. epidermidis strains, and they frequently colonize implanted devices such as intravascular devices, cerebrospinal fluid shunts, intraocular lenses, prosthetic joints, and heart valve replacements. Although removal of the affected medical device is recommended, treatment of confirmed SBIs requires antimicrobial agents. Resistance to methicillin among both bacterial strains is becoming increasingly common, reducing the number of available antibiotic treatments. Treatment with vancomycin and linezolid can have serious side effects, and cases showing increased minimum inhibitory concentrations or even complete resistance to these compounds are becoming increasingly common. In fact, multidrug resistance has been observed in 70%-85% of nosocomial Staphylococcus epidermidis strains (Kleinschmidt et al., 2015).

[0005] Therefore, new antimicrobial compounds are urgently needed to combat these infections.

[0006] Peptidoglycan hydrolases (PGHs) cleave specific bonds within the bacterial peptidoglycan (PG) network and have been shown to be active against biofilms. Their high lytic activity makes PGHs potent antistaphylococcal agents. Endolysins are highly specific phage-derived PGHs that are effective against both drug-sensitive and drug-resistant bacteria (Schmelcher et al. 2012). As potential alternatives to antibiotics, they have been studied in vitro and in vivo and are being tested in several clinical studies (Kashani et al. 2017). Staphylococcal PGHs typically exhibit a domain-like structure consisting of an enzymatically active domain (EAD) and a cell wall-binding domain (CBD). The high specificity of staphylococcal PGHs may be attributed to their CBDs, which are often characterized by an SH3b fold. The structure of the SH3b domain of staphylococcal endolysins has been resolved and shows high homology with the bacteriocins lysostaphin (LST) and ALE1, suggesting a shared recognition site within PGs.

[0007] EADs are more diverse and can be grouped based on their structure and cleavage site within the PG. Cysteine, histidine-dependent amidohydrolase / peptidase (CHAP) domains are frequently found in staphylococcal lytic endolysins, such as in bacteriophage Twort or phage K ( et al. 2006). Depending on the presence of the CHAP domain, cleavage may occur at different locations in the PG, including the amide bond between the sugar backbone and the stem peptide and the connection between the stem peptide and the peptide cross bridge. Herein, the amidohydrolase / peptidase activity of the CHAP domain is referred to as CHAP activity. The M23 domain is found only 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 S. aureus PG, while the M23 domain of phage 2638 cleaves between the peptide bridge and the stem peptide (Gründling 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.

[0008] 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 enzymatic activity, difficulty in mass production and / or protein stability. Therefore, there is a need for endolysin polypeptides with improved characteristics (such as antimicrobial activity). Several endolysins have been presented in WO 2021 / 213898. In summary, especially for systemic infections and sepsis and for prosthetic devices and catheters, there is a need for a single endolysin polypeptide with improved antimicrobial activity and stability characteristics. DETAILED DESCRIPTION

[0009] The inventors have determined that the combination of the M23 endopeptidase and CHAP domains on a single chimeric endolysin polypeptide provides the desired improved activity.

[0010] Thus, in a first aspect, the present invention relates to an endolysin polypeptide having lytic activity against Staphylococci, the endolysin polypeptide comprising a polypeptide, wherein the amino acid sequence of the polypeptide has at least 93% sequence identity to SEQ ID NO: 1, and wherein the endolysin polypeptide has enhanced lytic activity against Staphylococci in human serum compared to:

[0011] - an endolysin having the amino acid sequence shown in SEQ ID NO: 2, and / or

[0012] - an endolysin having the amino acid sequence shown in SEQ ID NO: 3.

[0013] Endolysin polypeptides are referred to herein interchangeably as endolysin polypeptides as disclosed herein, endolysins as disclosed herein, endolysin polypeptides, and endolysins.

[0014] Any method known to those skilled in the art can be used to determine lytic activity.

[0015] In the embodiments herein, the methods as described in the Examples herein are preferably used to determine the (enhanced) lytic activity of an endolysin. In the embodiments herein, the lytic activity is preferably determined at 37°. The 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 than an endolysin having an amino acid sequence as shown in SEQ ID NO: 2 and / or SEQ ID NO: 3.

[0016] In the examples herein, the endolysin polypeptide preferably has enhanced lytic activity against both coagulase-positive and coagulase-negative Staphylococcus species, in particular, has enhanced lytic activity against one or both of Staphylococcus aureus and Staphylococcus epidermidis.

[0017] In the embodiments herein, the endolysin may comprise a polypeptide, wherein the amino acid sequence of the polypeptide has at least 93%, more preferably 94%, 95%, 96%, 97%, 98%, 99% or most preferably 100% sequence identity to SEQ ID NO: 1. In the embodiments herein, the amino acid sequence of the 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.

[0018] In the embodiments herein, in the endolysin polypeptide, the M23 endopeptidase domain and the CHAP domain are preferably separated by a linker. In the embodiments herein, the linker can 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. In the embodiments herein, the linker can 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 the embodiments herein, the linker can 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 the embodiments herein, the linker can 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 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.

[0019] Table A: Preferred linkers between CHAP domain and M23 domain

[0020]

[0021]

[0022]

[0023]

[0024] In the embodiments herein, in the endolysin polypeptide, the M23 domain and the SH3b domain are preferably separated by a linker. In the 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. In the 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 similarity and / or identity to SEQ ID NO: 9. In the 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 similarity to SEQ ID NO: 9. In the 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 that is 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.

[0025] Table B: Preferred linkers between the M23 domain and the SH3b domain

[0026]

[0027]

[0028] Further provided are polynucleotides encoding endolysin polypeptides as disclosed herein. The polynucleotides are also referred to herein as polynucleotides as disclosed herein. The polynucleotides can be any type of polynucleotide known to those skilled in the art, such as DNA, RNA, or mRNA. The polynucleotides can be, for example, DNA encoding an endolysin polypeptide, which will be expressed in a host cell or in an in vitro transcription / translation system. The polynucleotides can also be mRNA that is delivered to a host for in vivo transcription / translation, wherein the host can be a cell or a multicellular organism (e.g., a mammal). Also provided are nucleic acid constructs comprising polynucleotides as disclosed herein. The nucleic acid constructs are referred to herein as nucleic acid constructs as disclosed herein. Also provided are expression vectors comprising nucleic acid constructs as disclosed herein. The expression vectors are referred to herein as expression vectors as disclosed herein. The expression vectors as disclosed herein can be recombinant expression vectors. Such vectors can constitute plasmids, cosmids, phages, or viruses, or parts thereof, which are transformed by introducing nucleic acid constructs or polynucleotides as disclosed herein. Such transformation vectors that are specific for the host organism to be transformed are well known to those skilled in the art and are widely described in the literature. In order to produce the polynucleotides or endolysin polypeptides as disclosed herein in a host, it may be appropriate to transform the host organism and integrate the polynucleotides, nucleic acid constructs or expression vectors as disclosed herein. Such transformation can be carried out by any suitable known means, which are widely described in the professional literature and are well known to those skilled in the art. Also provided are host cells comprising the polynucleotides as disclosed herein, nucleic acid constructs as disclosed herein or expression constructs as disclosed herein. The host cell is referred to as a host cell as disclosed herein in this article. The host cell as disclosed herein can be any microbial cell, prokaryotic cell or eukaryotic cell suitable for expressing the endolysin polypeptides as disclosed herein. Preferably, the cell is Escherichia coli, such as Escherichia coli XL1blue MRF, Escherichia coli BL21 (DE3).

[0029] Further provided is a method for producing an endolysin polypeptide as disclosed herein, the method comprising:

[0030] - cultivating a host cell as disclosed herein under conditions favorable for the production of the endolysin polypeptide,

[0031] - optionally isolating and purifying the endolysin polypeptide from the culture broth, and

[0032] - Optionally freeze-drying or spray-drying the endolysin polypeptide.

[0033] Preferably, E. coli is used in the method for producing an endolysin polypeptide as disclosed herein. Preferably, E. coli XL1blueMRF or E. coli BL21-Gold (DE3) is used in step i). Preferably, when a His tag is used, in the separation and purification steps, an IMAC and an Econo-Pac column (Biorad) filled with 5 mL of low-density nickel-chelated agarose beads (ABT beads) can be used in combination with gravity flow to purify an endolysin polypeptide as disclosed herein. The eluted polypeptide can be dialyzed for 2, 4, and 12 hours with 3 x 1 l of lyophilization buffer, preferably comprising 50 mM phosphate, 500 mM sucrose, 200 mM mannitol, 0.005% polysorbate 20, pH 7.4.

[0034] In embodiments, no His tag is used; preferably no tag is used at all; thus, in embodiments, the endolysin polypeptide does not comprise a His tag or comprises no tag.

[0035] Lyophilization and reconstruction are preferably interpreted as dehydration by freeze drying or spray drying and subsequently by adding water to reconstruct the sample. Preferably, freeze drying and reconstruction are carried out by the following: 300ml lyophilization buffer (50mM phosphate or Tris, 500mM sucrose, 200mM mannitol, pH 7.4) aliquots replaced 3 times are dialyzed and frozen in the gas phase of liquid nitrogen. Lyophilization is preferably carried out under standard conditions, preferably at -40 ℃ and 75mTorr vacuum for 60 minutes, then the temperature is increased to -10 ℃ in 5 hours, and at -10 ℃ under the same vacuum for another 60 minutes. As the last step, preferably within 10 hours, the temperature is increased to 25 ℃. Spray drying can be carried out using any method known to those skilled in the art. Preferably, the endolysin polypeptide is reconstructed by adding water.

[0036] Further provided is a method for purifying an endolysin polypeptide as disclosed herein having enhanced activity, the method comprising dialyzing the endolysin polypeptide as disclosed herein, the dialysis comprising the steps of:

[0037] i) dialysis against a buffer containing a chelating compound, and

[0038] ii) dialysis with a buffer containing a divalent metal ion, preferably a divalent metal ion selected from the group consisting of: Co 2+ 、Cu 2+ Mg 2+ , Ca 2+ 、Mn 2+ and Zn 2+ .

[0039] A "chelating compound" is defined herein as a compound that binds 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 method for purification.

[0040] Preferably, the divalent metal ion in step ii) is selected from the group consisting of Mn 2+ 、Co 2+ 、Cu 2+ More preferably, the divalent metal ion is selected from the group consisting of Mn 2+ and Co 2+ Even more preferably, the divalent metal ion is Mn 2+ .

[0041] It has been previously demonstrated that substitution of a divalent metal ion by any of the above-defined substitutions results in a 2-2.5-fold increase in the lytic activity of Ply2638. The lytic activity is assessed as described in the Examples herein. Preferably, the method results in an increase in lytic activity of 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 results in an increase in lytic activity of at least 2.5-fold.

[0042] Further provided is a composition 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 a composition as disclosed herein may comprise a mixture of different polynucleotides and / or nucleic acid constructs and / or endolysin polypeptides and / or vectors and / or cells as disclosed herein or obtainable by a method as disclosed herein. The composition is also referred to herein as a composition as disclosed herein.

[0043] In the embodiments herein, the composition may further comprise a cosmetically acceptable excipient, i.e., an acceptable excipient for cosmetic use. Such cosmetically acceptable excipients may be any cosmetically acceptable excipient known to those skilled in the art, such as, but not limited to, emulsifiers, emollients, dyes, colorants, binders, defoaming agents, surfactants, preservatives, and film formers.

[0044] 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 medicines are used to treat infectious diseases, preferably staphylococcal infections such as Staphylococcus aureus and Staphylococcus epidermidis.

[0045] Therefore, further provided is a pharmaceutical composition comprising an endolysin polypeptide as disclosed herein, a polynucleotide as disclosed herein, a nucleic acid construct as disclosed herein, an expression construct as disclosed herein and / or a host cell as disclosed herein; the pharmaceutical composition further comprises a pharmaceutically acceptable excipient.

[0046] Compositions or pharmaceutical compositions as 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: bacteriophage (bacteriophage or phage), endolysins derived from such phage, and antibiotics. The phage encompassed herein may be any phage known in the literature. Preferably, such phage is, but is not limited to, a family from the following list consisting of: Myoviridae, Siphoviridae, and Podoviridae. Such phage can also come from the section of 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 as defined herein can be used sequentially or simultaneously. Compositions as defined herein can be in liquid, solid or semi-liquid or semi-solid form.

[0047] The compositions of pharmaceutical compositions as 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 delivery to the lungs, nasal spray, intravenous, intramuscular, intraperitoneal, intrathecal, vaginal, rectal, topical, lumbar puncture, intrathecal, and direct application to the brain and / or meninges.

[0048] When the disclosed composition or pharmaceutical composition reduces the amount of Staphylococcus species present in a patient or in the patient's cells or in a cell line or in a cell-free in vitro system, the composition or pharmaceutical composition is preferably considered to be active, functional or therapeutically active or capable of treating, preventing and / or delaying an infectious disease, and preferably means that 99%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5% or less of the initial amount of Staphylococcus species can still be detected after treatment. Preferably, there are no detectable Staphylococcus species after treatment. Herein, the expression "amount of Staphylococcus species" preferably means viable Staphylococcus species. Staphylococcus species can be detected using standard techniques known to those skilled in the art (e.g., immunohistochemistry using Staphylococcus-specific antibodies, tube coagulase tests to detect Staphylococcus coagulase or "free coagulase", detection of surface proteins such as clumping factors (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 (e.g., microbial bacterial culture techniques and / or real-time quantitative reverse transcription polymerase chain reaction for measuring bacterial mRNA). Preferably, the reduction is assessed in tissues or cells of the individual or patient by comparison with the amount present in the individual or patient before treatment with a composition or pharmaceutical composition as disclosed herein. Alternatively, if the treatment is local, the comparison can be made with tissues or cells of the individual or patient that have not been treated with a composition or pharmaceutical composition as disclosed herein.

[0049] A composition or pharmaceutical composition as disclosed herein can be administered to a subject in need thereof or to cells, tissues or organs of said patient for at least one day, one week, one month, six months, one year or longer.

[0050] Therefore, provide composition or pharmaceutical composition as disclosed herein, for use as the medicine of experimenter in need for the treatment.Preferably, composition or pharmaceutical composition are used as the medicine for preventing, delaying or treating the disease of experimenter, wherein this disease is relevant to staphylococcus (for example coagulase positive or coagulase negative staphylococcus, preferably staphylococcus aureus and / or staphylococcus epidermidis) infection.Such disease can be skin infection, soft tissue infection such as infected diabetic foot ulcer, mastitis, pneumonia, meningitis, endocarditis, toxic shock syndrome (TSS), sepsis, septicemia, bacteremia or osteomyelitis.Skin infection can be the one selected from the group consisting of: acne, impetigo, furuncle, furuncle, cellulitis, folliculitis, carbuncle, scalded skin syndrome, atopic dermatitis and abscess.

[0051] Further provided is a composition or pharmaceutical composition as disclosed herein for use as a medicament, wherein the composition or pharmaceutical composition is for systemic or local administration to a subject.

[0052] Further provided is a composition or pharmaceutical composition as 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.

[0053] Further provided is a composition or pharmaceutical composition as disclosed herein for use as a medicament, wherein the composition or pharmaceutical composition is for systemic or local administration to a subject, and 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.

[0054] Topical administration can be used locally at the site of infection or at the site of implantation, for example, during surgery.

[0055] The medical uses disclosed herein can be formulated as products disclosed herein for use as medicaments for treating the conditions, as well as methods of using the products disclosed herein for treating the conditions, as well as products disclosed herein for use in the preparation of medicaments for treating the conditions, and uses of the products disclosed herein for treating the conditions. These medical uses are all 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 cattle, a domestic animal such as a dog or cat, or a human subject.

[0056] Further provided is a method of cosmetically treating the skin of a subject, the method comprising applying to the skin of the subject an endolysin polypeptide as disclosed herein, a polynucleotide encoding such an endolysin polypeptide, or a composition as disclosed herein. The subject can be any animal subject, preferably a mammal, more preferably a cattle, livestock such as a dog or cat, or a human subject.

[0057] Further provided is the in vitro use of an endolysin polypeptide 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, or a composition or pharmaceutical composition as disclosed herein as an antimicrobial agent, preferably as a food additive, or as a disinfectant, preferably for coating or impregnating a medical device. Examples of such uses are, but are not limited to, rinsing the cups of milking apparatus with a composition according to the invention before milking to prevent the spread of staphylococci between cows, cleaning surfaces in the food industry, and cleaning surgical tools (such as gastrointestinal cameras, peritonoscopes, thoracoscopes and arthroscopes) and medical supplies (such as catheters and tubes, which have long tubes or hollow parts and are intended to be repeatedly used by being introduced into the human or animal body). Such uses can be combined with any sterilization method or disinfectant known in the art, such as ultrasonic cleaning, irradiation or heat sterilization, by immersing the device in a disinfectant solution such as ethanol, ammonium, iodine and / or aldehyde disinfectants, or by using gas sterilization, by keeping the device in a closed atmosphere such as formaldehyde gas or ethylene oxide gas.

[0058] Further provided is an in vitro method of coating or impregnating a medical device with an endolysin polypeptide as disclosed herein or a composition or pharmaceutical composition as disclosed herein, the method comprising contacting the medical device with an endolysin polypeptide as disclosed herein or a composition or pharmaceutical composition as disclosed herein.

[0059] Further provided is the use of 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, or a composition or pharmaceutical composition as disclosed herein for detecting Staphylococci (e.g., Staphylococcus aureus) in an ex vivo diagnostic application.

[0060] definition

[0061] "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 refers to the degree of sequence relatedness between amino acid 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 of one peptide or polypeptide and its conservative amino acid substitutes to the sequence of a second peptide or polypeptide. In preferred embodiments, identity or similarity is calculated over the entire SEQ ID NO as 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, NJ, 1993. Press, Stockton, New York, 1991; and Carillo, H. and Lipman, D., SIAM J. Applied Math. 48:1073 (1988).

[0062] Preferred methods for determining identity are designed to obtain the maximum match between the test sequences. Methods for determining identity and similarity are incorporated into 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, SF 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 can also be used to determine identity.

[0063] Preferred parameters for polypeptide sequence comparison include the following: 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 that can be used with these parameters is publicly available as the "Ogap" program from the Genetics Computer Group in Madison, Wisconsin. The foregoing parameters are the default parameters for amino acid comparisons (no penalty for terminal gaps).

[0064] Preferred parameters for nucleic acid comparisons include the following: 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 in Madison, Wisconsin. Default parameters for nucleic acid comparisons are given above.

[0065] Optionally, when determining the degree of amino acid similarity, one may also consider so-called "conservative" amino acid substitutions, as will be clear to one skilled in the art. Conservative amino acid substitutions refer to the interchangeability of residues with similar side chains. For example, a group of amino acids with aliphatic side chains is: glycine, alanine, valine, leucine, and isoleucine; a group of amino acids with aliphatic-hydroxy side chains is serine and threonine; a group of amino acids with amide-containing side chains is asparagine and glutamine; a group of amino acids with aromatic side chains is phenylalanine, tyrosine, and tryptophan; a group of amino acids with basic side chains is lysine, arginine, and histidine; and a group of amino acids with sulfur-containing side chains is cysteine ​​and methionine. Preferred conservative amino acid substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, and asparagine-glutamine. Substitution variants of the amino acid sequences disclosed herein are those in which at least one residue in the disclosed sequence has been removed and a different residue inserted in its place. Preferably, the amino acid changes are conservative. Preferred conservative substitutions for each naturally occurring amino acid 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.

[0066] 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 a nucleic acid molecule that has been modified to contain nucleic acid segments combined or juxtaposed in a manner not found in nature. 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 the peptide or polypeptide in a cell or subject.

[0067] "Operably linked" is defined herein as a configuration in which a control sequence is appropriately positioned relative to a nucleotide sequence encoding a polypeptide of the invention such that the control sequence directs the production / expression of the 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 appropriately positioned relative to another sequence encoding a functional domain such that a chimeric polypeptide is encoded in a cell and / or subject.

[0068] "Expression" is interpreted to include any step involved in the production of the peptide or polypeptide including, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

[0069] "Control sequences" are defined herein to include all components necessary or advantageous for the expression of a polypeptide. At a minimum, control sequences include a promoter and transcriptional and translational stop signals. Optionally, the promoter represented by the nucleotide sequence present in the nucleic acid construct is operably linked to another nucleotide sequence encoding a peptide or polypeptide as identified herein.

[0070] The term "transformation" refers to a permanent or transient genetic change induced in a cell following the incorporation of new DNA (i.e., DNA exogenous to the cell). When the cell is a bacterial cell, as contemplated in the present invention, the term typically refers to an extrachromosomal self-replicating vector that confers selectable antibiotic resistance.

[0071] "Expression vector" can be any vector that can be easily subjected to recombinant DNA procedures and can cause the nucleotide sequence encoding the polypeptide of the present invention to be expressed in cells and / or subjects. As used herein, the term "promoter" refers to a nucleic acid fragment for controlling the transcription of one or more genes or nucleic acids, which is located upstream of the transcription start site of the gene in the direction of transcription. It involves binding sites identified by the presence of binding sites for DNA-dependent RNA polymerase, transcription start sites, and any other DNA sequences, including but not limited to transcription factor binding sites, repressor and activator protein binding sites, and any other nucleotide sequences known to those skilled in the art that act directly or indirectly to regulate the amount of transcription of the promoter. In the context of the present invention, the promoter preferably terminates at nucleotide -1 of the transcription start site (TSS).

[0072] As used herein, "polypeptide" refers to any peptide, oligopeptide, polypeptide, gene product, expression product or protein. A polypeptide is composed of consecutive amino acids. The term "polypeptide" encompasses naturally occurring or synthetic molecules.

[0073] The sequence information as provided herein should not be narrowly interpreted as necessarily including misidentified bases. Those skilled in the art are able to identify such misidentified bases and know how to correct such errors.

[0074] Additionally, it is known to those skilled in the art that when proteins are expressed, terminal amino acids (such as the N-terminal methionine) are sometimes cleaved off.

[0075] In this document and its claims, the verb "to comprise" and its conjugations are used in its non-limiting sense, meaning that the items following the word are included, but items not specifically mentioned are not excluded. In addition, the verb "consisting of" can be replaced by "consisting essentially of", meaning that the product or composition or nucleic acid molecule or peptide or polypeptide or nucleic acid construct or vector or cell as defined herein may contain one or more additional components in addition to the components specifically identified; said additional component or components do not alter the unique characteristics of the invention. In addition, reference to an element by the indefinite article "a" or "an" does not exclude the possibility that more than one of the element is present, unless the context clearly requires the presence of one and only one element. Thus, the indefinite article "a" or "an" generally means "at least one". The word "about" or "approximately" when used in conjunction with a numerical value (e.g., about 10) preferably means that the value may be the given value (10) plus or minus 10% of that value.

[0076] All patents and literature references cited in this specification are hereby incorporated by reference in their entirety.

[0077] The examples herein are for illustrative purposes only and are not intended to limit the scope of the invention in any way. BRIEF DESCRIPTION OF THE DRAWINGS

[0078] Figure 1 . Effect of peptidoglycan hydrolase on Staphylococcus aureus NR-46543 / USA300 JE2 (MRSA) in human serum Killing.

[0079] A quantitative killing assay (QKA) was performed in human serum against methicillin-resistant Staphylococcus aureus (MRSA) NR-46543 / USA300 JE2 using peptidoglycan hydrolase (PGH) constructs ID453, ID557, and ID558 at 37°C, 180 rpm (25 mm orbital) for 30 min. Viable cell counts (CFU / ml) (y-axis) of S. aureus NR-46543 / USA300 JE2 are shown for the three PGH constructs over a two-fold concentration range (in nM) (x-axis) (320–0.625 nM). The depicted negative control (no PGH added) corresponds to the average individual control for the three tested constructs. The limit of detection (LoD) was 200 CFU / ml (dashed gray line). The y-axis cutoff was 160 CFU / ml. Three biological replicates (n=3) were performed. Error bars for the average control and constructs represent the standard error of the mean (SEM).

[0080] Figure 2 . Killing of Staphylococcus aureus ATCC 12600 (MSSA) in human serum by peptidoglycan hydrolases.

[0081] Quantitative killing assay (QKA) of peptidoglycan hydrolase (PGH) constructs ID453, ID557, and ID558 against methicillin-sensitive Staphylococcus aureus (MSSA) ATCC 12600 was performed in human serum at 37°C and 180 rpm (25 mm orbital) for 30 min. Viable cell counts (CFU / ml) (y-axis) of S. aureus ATCC 12600 are shown for the three PGH constructs at a 2-fold concentration (in nM) range (x-axis) (320 nM–0.625 nM). The depicted negative control (no PGH added) corresponds to the average individual control of the three test constructs. The limit of detection (LoD) was 200 CFU / ml (grey dashed line). The y-axis cutoff value was 160 CFU / ml. Three biological replicates (n=3) were performed. The error bars for the average control and constructs represent the standard error of the mean (SEM).

[0082] Figure 3 . Killing of Staphylococcus aureus ATCC 12598 (MSSA) in human serum by peptidoglycan hydrolases.

[0083] Quantitative killing assay (QKA) of peptidoglycan hydrolase (PGH) constructs ID453, ID557, and ID558 was performed in human serum against methicillin-sensitive Staphylococcus aureus (MSSA) ATCC 12598 (Cowan I) at 37°C and 180 rpm (25 mm orbital) for 30 min. Viable cell counts (CFU / ml) (y-axis) of Staphylococcus aureus ATCC 12598 are shown for the three PGH constructs at a 2-fold concentration (in nM) range (x-axis) (320 nM–0.625 nM). The depicted negative control (no PGH added) corresponds to the average individual control of the three test constructs. The limit of detection (LoD) was 200 CFU / ml (grey dashed line). The y-axis cutoff value was 160 CFU / ml. Three biological replicates (n=3) were performed. The error bars for the average control and constructs represent the standard error of the mean (SEM).

[0084] Figure 4 . Killing effect of peptidoglycan hydrolase on Staphylococcus epidermidis ATCC 12228 / DSM1798 (MSSE) in human serum hurt.

[0085] Peptidoglycan hydrolase (PGH) constructs ID453, ID557, and ID558 were subjected to a quantitative killing assay (QKA) in human serum against methicillin-sensitive Staphylococcus epidermidis (MSSE) ATCC 12228 / DSM1798 at 37°C and 180 rpm (25 mm orbital) for 30 min. Viable cell counts (CFU / ml) (y-axis) of S. epidermidis ATCC 12228 / DSM1798 are shown for the three PGH constructs over a 2-fold concentration range (in nM) (x-axis) (320 nM–0.625 nM). The depicted negative control (no PGH added) corresponds to the average individual control of the three tested constructs. The limit of detection (LoD) was 200 CFU / ml (grey dashed line). The y-axis cutoff value was 160 CFU / ml. Three biological replicates (n=3) were performed, except for concentrations of 0.625 and 1.25 nM (n=2). Error bars for the mean of controls and constructs represent the standard error of the mean (SEM).

[0086] Figure 5 . Killing effect of peptidoglycan hydrolase on Staphylococcus epidermidis ATCC 35984 / BB1336 (MRSE) in human serum hurt.

[0087] Peptidoglycan hydrolase (PGH) constructs ID453, ID557, and ID558 were subjected to a quantitative killing assay (QKA) in human serum against methicillin-resistant Staphylococcus epidermidis (MRSE) ATCC 35984 / BB1336 at 37°C and 180 rpm (25 mm orbital) for 30 min. Viable cell counts (CFU / ml) (y-axis) of Staphylococcus epidermidis ATCC 35984 / BB1336 are shown for the three PGH constructs over a 2-fold concentration (in nM) range (x-axis) (320 nM–0.625 nM). The depicted negative control (no PGH added) corresponds to the average individual control of the three test constructs. The limit of detection (LoD) was 200 CFU / ml (grey dashed line). The y-axis cutoff value was 160 CFU / ml. Three biological replicates (n=3) were performed, except for concentrations of 0.625 and 1.25 nM (n=2). Error bars for the mean of controls and constructs represent the standard error of the mean (SEM).

[0088] Examples

[0089] 1. Introduction

[0090] Bacteriophage lysins are phage-encoded cell wall hydrolases that cleave bonds within bacterial peptidoglycan, leading to localized lysis and ultimately cell death.

[0091] In short, these peptidoglycan hydrolases (PGHs) are modular, with one domain responsible for specific binding to the target cell wall and the other parts responsible for cutting highly specific bonds within the peptidoglycan. The modules can be connected by polypeptide linkers. These enzymes exhibit varying degrees of specificity in binding to their targets and cutting certain bonds that may not be present in other bacterial genera or species. By combining and rearranging different parts of various naturally occurring enzymes, novel molecules with highly specific recognition and cleavage patterns can be generated, allowing the manufacture of highly targeted antimicrobial agents.

[0092] Here we show the efficacy profiles of three such molecules in human serum (Table 1). Two of those molecules have been disclosed in WO 2021213898 (ID557 [SEQ ID NO: 41] and ID558 [SEQ ID NO: 5]), and ID453 is a novel molecule presented herein. All three molecules include the CHAP domain of bacteriophage Twort, followed by the M23LST domain that mimics the lysostaphin gene of Staphylococcus simulans. ID453 and ID557 both contain the SH3b domain of the lysostaphin gene, while ID558 contains the SH3b domain from phage 2638A. The domain and linker source references are shown in Table 2.

[0093] Table 1. Overview of the constructs compared in the experiments.

[0094] ID Construct SEQ ID ID453 CHAPTw(L)_M23LST_SH3bLST SEQ ID NO: 1 ID557 CHAPTw_M23LST_SH3bLST SEQ ID NO:2 ID558 CHAPTw(L)_M23LST_(L)SH3b2638 SEQ ID NO:3

[0095] The IDs are listed together with the corresponding construct names and domain configurations. ID557 and ID558 are disclosed in WO2021213898 and are listed together with the corresponding sequence IDs herein.

[0096] Table 2 Domains used to engineer constructs and their sources.

[0097]

[0098] The different domains and their organismal origin are listed.

[0099] When targeting SBI, sufficient activity of the candidate molecule in human serum is a prerequisite. In this case, efficacy was investigated against three strains of Staphylococcus aureus (including one methicillin-resistant strain) and one methicillin-resistant and one methicillin-sensitive Staphylococcus epidermidis strain.

[0100] Interestingly, despite the remarkable similarities among the three molecules, one candidate compound demonstrated clear superiority over the other two. ID453 outperformed the other constructs, requiring a lower dose to achieve complete killing of target cells in human serum against two of the three S. aureus strains tested and one of the two S. epidermidis strains studied. This suggests that it was at least as effective as the other compounds against the remaining strains, with higher killing potency at lower concentrations compared to the other constructs. For targeting SBI, ID453 is a superior choice.

[0101] 2. Results

[0102] 2.1 Quantitative killing assay

[0103] Quantitative killing assays (QKA) were performed in human serum using the peptidoglycan hydrolase (PGH) constructs CHAPTw(L)_M23LST_SH3bLST (ID453), CHAPTw_M23LST_SH3bLST (ID557), and CHAPTw(L)_M23LST_(L)SH3b2638 (ID558) at 37°C and 180 rpm (25 mm orbital) for 30 min against S. aureus NR-46543 / USA300JE2, S. aureus ATCC 12600, S. aureus ATCC 12598 (Cowan I), S. epidermidis ATCC 12228 / DSM1798, and S. epidermidis ATCC 35984 / BB1336. The constructs were tested at a 2-fold concentration range (320 nM–0.625 nM). The results of QKA (viable cell count) evaluation of the three constructs in human serum were shown in Figures 1 to 5 Three biological replicates were performed for each construct in each strain. Figure 1-Figure 5 As shown in Figure 3, all three constructs exhibited staphylococcal lytic activity in each strain across the concentration range tested. Furthermore, a dose response was evident. The further to the left the curve was positioned, the more active the construct.

[0104] ID453 is a strain of methicillin-resistant Staphylococcus aureus NR-46543 / USA300 JE2 ( Figure 1 ) and methicillin-sensitive Staphylococcus aureus ATCC 12598 (Cowan I) ( Figure 3 ) strain, followed by ID557. Although these two constructs were effective against methicillin-sensitive Staphylococcus aureus ATCC 12600 ( Figure 2) strains were not significantly different in activity, but at most concentrations tested, ID453 still showed higher activity than the other constructs. Among all three strains, ID558 showed the lowest activity.

[0105] The superiority of ID453 against methicillin-sensitive Staphylococcus epidermidis ATCC 12228 / DSM1798 ( Figure 4 ) strain. In contrast to the results obtained against S. aureus strains, ID557 was the least active construct when tested against S. epidermidis strains. Although ID453 showed similar activity to construct ID558 against methicillin-resistant S. epidermidis ATCC 35984 / BB1336 strain at higher concentrations (>20 nM), ID453 was significantly superior at lower concentrations (<20 nM). Figure 5 Overall, our data demonstrate the superiority of ID453 compared to the other constructs tested.

[0106] 3. Materials and Methods

[0107] 3.1 Materials: Bacteria, Culture Medium, Buffer, Devices, and Consumables

[0108] Table 3. Bacteria used in experiments.

[0109]

[0110]

[0111] Bacterial strains are listed with the corresponding product number and supplier.

[0112] Table 4. List of culture media and buffers used in the experiments.

[0113]

[0114]

[0115] For each medium and buffer, the components, corresponding amounts per liter, pH, and protocol are listed.

[0116] Table 5. Chemicals used in experiments.

[0117]

[0118] Chemicals are listed with the corresponding product number, supplier, and batch number.

[0119] Table 6. List of equipment and consumables used in the experiment.

[0120]

[0121]

[0122]

[0123] Each device / consumable is listed with its corresponding model, brand, and serial / article number.

[0124] 3.2 Methods: Protocol and Procedure

[0125] 3.2.1 Quantitative Killing Assay (QKA)

[0126] In total, three PGH constructs were tested against three S. aureus and two S. epidermidis strains in human serum. Aliquots of the constructs were stored in PCR tubes at -80°C (in CIEX PO4 elution buffer). All steps were performed under sterile conditions unless otherwise mentioned. A final PGH concentration range of 320 nM, 160 nM, 80 nM, 40 nM, 20 nM, 10 nM, 5 nM, 2.5 nM, 1.25 nM and 0.625 nM was tested against all five strains. The desired final bacterial control was 1 x 10 6 –1x 10 7 CFU / ml. At least two biological replicates were performed. One 96-well F-bottom plate was used for each PGH construct to be tested.

[0127] Precultures were prepared by inoculating 5 ml of tryptone soy broth (TSB) medium with Staphylococcus aureus NR-46543 / USA300 JE2, Staphylococcus aureus ATCC 12600, Staphylococcus aureus ATCC 12598 (Cowan I), Staphylococcus epidermidis ATCC 12228 / DSM1798, or Staphylococcus epidermidis ATCC 35984 / BB1336. The cultures were incubated overnight (O / N) at 37° C. and 180 revolutions per minute (rpm) (25 mm orbit).

[0128] Aliquots of human serum (stored at -20°C) were thawed in 30°C water baths, filtered using a 0.45 μm filter and stored on ice. 1:25 diluted overnight cultures were prepared by mixing 400 μl overnight cultures with 10ml TSB culture medium, and the cultures were incubated at 37°C and 180rpm until an optical density (OD600nm) of 0.5-0.6 was reached. The cultures were then placed on ice for approximately 5 minutes to stop growth. 1 ml of the culture was adjusted to OD of 0.51 with TSB culture medium, transferred to a 1.5 ml reaction tube and placed on ice. When the cultures grew, 160 μl 1x stop buffer was provided in the BH rows 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, the appropriate aliquots (10-20 μl aliquots) of the PGH construct to be tested were thawed on ice and centrifuged briefly. Human serum was adapted to RT. 100 μl of human serum was provided by reverse pipetting into wells A1 and A3-A12 of a prepared 96-well F-bottom plate (one plate for each enzyme to be tested). In well A2 of each plate, an enzyme pre-dilution of the corresponding construct to be tested in human serum (final volume 200 μl) was prepared to achieve a concentration (640 nM) twice as high as the highest final concentration to be tested (320 nM). The enzyme pre-dilution was thoroughly mixed, and a 2-fold dilution series was prepared by transferring 100 μl from well A2 to well A3, mixing six times and changing the pipette tip, continuing the same procedure from well A3 to well A4, and so on to well A11. The last 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 pre-dilution and dilution series in the plate was prepared no more than 10 minutes before inoculation to minimize interaction of the enzyme with the plate well walls. The bacterial suspension for inoculation was prepared by mixing 500 μl of vortexed, OD-adjusted culture with 4.5 ml of human serum (1:10 dilution) in a 25 ml reservoir. Using a manual multichannel pipette, 100 μl of bacterial suspension from the reservoir was added to wells A1-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 a full 30 min.

[0129] After incubating the plate for exactly 30 minutes, remove the plate from the incubator shaker and immediately add 20 μl of 10x stop buffer to row A using a multichannel pipette. Homogenize the suspension in the wells by pipetting up and down eight times to stop further enzyme activity. Then, prepare a 5-fold dilution series in the provided 1x stop buffer by transferring 40 μl from row A to row B, mixing six times, changing the pipette tip, and then transferring 40 μl from row B to row C, and so on until row H. Discard the last 40 μl from row H. Spot 5.5 μl from each well of the 96-well plate onto a pre-dried LB agar square plate. After the spot has dried, incubate the agar plate upside down in a 37°C incubator overnight (approximately 16 hours for S. aureus strains and approximately 20 hours for S. epidermidis strains).

[0130] The next day, colony-forming units (CFU) were counted for each spot. CFU / ml was calculated, and the viable cell count and log reduction in CFU / ml were visualized in a graph (GraphPad Prism 9.2.0) over the final concentration range tested (320 nM–0.625 nM). Results from biological replicates were averaged, and the standard error of the mean (SEM) was calculated and displayed.

[0131] References

[0132] Antimicrobial Resistance Collaborators.Global burden of bacterialantimicrobial resistance in 2019:a systematic analysis.Lancet.2022Feb 12;399(10325):629-655.doi:10.1016 / S0140-6736(21)02724-0.Epub 2022Jan 19.PMID:35065702; PMCID:PMC8841637.

[0133] Kleinschmidt S, Huygens F, Faoagali J, Rathnayake IU, HafnerLM. Staphylococcus epidermidis as a cause of bacteremia. Future Microbiol. 2015;10(11):1859-79.doi:10.2217 / fmb.15.98.Epub 2015Oct 30.PMID:26517189.

[0134] Guo Y,Song G,SunM,Wang J,Wang Y.Prevalence and Therapies ofAntibiotic-Resistance in Staphylococcus aureus.Front CellInfectMicrobiol.2020Mar 17;10:107.doi:10.3389 / fcimb.2020.00107.PMID:32257966;PMCID:PMC7089872.

[0135] Grundling,A.,Missiakas,D.M.&Schneewind,O.,2006.Staphylococcus aureusMutants withIncreasedLysostaphinResistance.Journal of Bacteriology,188(17),pp.6286–6297.

[0136] Kashani,H.etal.,2017.RecombinantEndolysins as Potential TherapeuticsagainstAntibiotic-ResistantStaphylococcus aureus:Current Status of Researchand Novel Delivery Strategies.Clinical Microbiology Reviews,31(1).

[0137] I.P.et al.,2006.The Crystal Structure of the BacteriophagePSA Endolysin Reveals a Unique Fold Responsible for Specific Recognition ofListeria Cell Walls.Journal of Molecular Biology,364(4),pp.678–689.

[0138] Schmelcher,M.et al.,2015.Evolutionarily distinct bacteriophageendolysins featuring conserved peptidoglycan cleavage sites protect mice fromMRSA infection.Journal of Antimicrobial Chemotherapy,70(5),pp.1453–1465.

[0139] Schmelcher,M.,Donovan,D.M.&Loessner,M.J.,2012.Bacteriophageendolysins as novel antimicrobials.Future Microbiology,7(10),pp.1147–1171.

Claims

1. An endolysin polypeptide having lytic activity against Staphylococci, the endolysin polypeptide comprising a polypeptide, wherein the amino acid sequence of the polypeptide has at least 93% sequence identity to SEQ ID NO: 1; wherein the endolysin polypeptide has enhanced lytic activity against Staphylococci in human serum compared to: - 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; and The M23 endopeptidase domain and the CHAP domain in the endolysin polypeptide are separated by a linker comprising at least 13 amino acids. 2 . The endolysin polypeptide according to claim 1 , wherein the amino acid sequence of the endolysin polypeptide has at least 93% sequence identity with SEQ ID NO:

1. A polynucleotide encoding the endolysin polypeptide according to claim 1 . A nucleic acid construct comprising the polynucleotide according to claim 3 . An expression vector comprising the nucleic acid construct according to claim 4 .

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

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

8. A method for producing an endolysin polypeptide according to claim 1 or 2 having enhanced activity, the method comprising dialyzing the endolysin according to claim 1 or 2, the dialysis comprising the following steps: i) dialysis against a buffer containing a chelating compound, and ii) dialysis with a buffer containing a divalent metal ion, preferably a divalent metal ion selected from the group consisting of: Co 2+ 、Cu 2+ Mg 2+ , Ca 2+ 、Mn 2+ and Zn 2+ .

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

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

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

12. A composition according to claim 9, 10 or 11 further comprising an additional active ingredient.

13. A composition according to claim 9, 11 or 12 for use as a medicament, preferably for use as a medicament for the treatment of conditions associated with Staphylococcal infection.

14. A method of treating a condition associated with a Staphylococcal infection, the method comprising administering 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, or a composition according to claim 9, 11 or 12.

15. A method of cosmetic skin treatment comprising administering 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, or a composition according to claim 9, 10 or 12.

16. The method of claim 14, wherein the condition to be treated is selected from the group consisting of skin infection, soft tissue infection such as infected diabetic foot ulcer, mastitis, pneumonia, meningitis, endocarditis, toxic shock syndrome (TSS), sepsis, septicemia, bacteremia, or osteomyelitis. The skin infection may be one selected from the group consisting of acne, impetigo, furuncle, furuncle, cellulitis, folliculitis, carbuncle, scalded skin syndrome, atopic dermatitis, and abscess.

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

Images