WO2023282776A1

WO2023282776A1 - Peptidoglycan hydrolase, compositions comprising it, uses thereof, and a method of hydrolysis utilizing it

Info

Publication number: WO2023282776A1
Application number: PCT/PL2022/050043
Authority: WO
Inventors: Izabela SABAŁA; Piotr Henryk MAŁECKI; Karolina TROCHIMIAK; Elżbieta JAGIELSKA; Weronika AUGUSTYNIAK
Original assignee: Międzynarodowy Instytut Biologii Molekularnej i Komórkowej w Warszawie
Priority date: 2021-07-09
Filing date: 2022-07-06
Publication date: 2023-01-12
Also published as: PL438431A1; EP4367230A1; PL243304B1

Abstract

The object of the invention is the active form of AurR - a peptidoglycan hydrolase capable of digesting the walls of Gram(+) bacteria having interpeptide bridges within peptidoglycan composed of glycine and/or glycine and serine, compositions comprising it, uses thereof, and a method of peptidoglycan hydrolysis. The active form of the AurR protein overcomes the resistance of Gram(+) bacteria to known enzymes capable of only hydrolyzing cell walls with glycine interpeptide bridges in peptidoglycan and digests cell walls with glycine-serine interpeptide bridges.

Description

Peptidoglycan hydrolase, compositions comprising it, uses thereof, and a method of hydrolysis utilizing it

TECHNICAL FIELD

The object of the invention is the active form of the AurR protein, which is a peptidoglycan hydrolase capable of digesting the walls of Gram(+) bacteria having interpeptide bridges within peptidoglycan composed of glycine and/or glycine and serine, compositions comprising it, uses thereof, and a method of peptidoglycan hydrolysis utilizing it. The active form of the AurR protein overcomes the resistance of Gram(+) bacteria to known enzymes capable of only hydrolyzing cell walls with glycine interpeptide bridges within peptidoglycan.

STATE OF THE ART

Infections caused by bacteria of the genus Staphylococcus, in particular Staphylococcus aureus, are becoming more and more difficult to treat due to the widespread drug resistance. For these reasons, it is increasingly important to develop new therapies to combat staphylococcal infections and eliminate harboring (by carriers), especially among medical personnel, but also to develop more effective methods of eliminating this bacterium from the environment, including hospitals. Bacteria of the genus Staphylococcus (staphylococci) are responsible for a wide variety of infections in humans and animals, and many Staphylococcus species are resistant to antibiotics; it is estimated that 30-50% of people are carriers of these bacteria.

Dermatological and inflammatory diseases can be caused by various species of bacteria, including staphylococci. They can appear spontaneously or, for example, in a wound, ulcer or bum. Infected wounds can be life-threatening. Additionally, biofilm-forming bacteria may be present in infected wounds or wounds may become secondarily infected with them. Infections with antibiotic- resistant Staphylococcus aureus, such as MRSA (methicillin-resistant Staphylococcus aureus ) strains, are particularly dangerous. Staphylococcal infections are especially common in the case of diabetic foot, pressure ulcers and bums.

It is known that staphylococci, in particular S. aureus, are often the cause of food poisoning, because they produce peptidic, thermostable enterotoxins causing intoxication. Due to the large- scale nature of the food industry production, it is possible to use enzymes that destroy staphylococci to improve the microbiological quality of food only when such enzymes are inexpensive and are of high availability.

It is also known that bacteria of the genus Staphylococcus, including S. simulans, are pathogens of animals such as cows, sheep, goats and horses. Like S. aureus, S. simulans is commonly associated with bovine mastitis. Although human infections with S. simulans are rare, they are found in people handling livestock. S. simulans is also associated with osteoarticular infections, endocarditis or diabetic osteitis [8],

One of the novel approaches to eliminate pathogenic bacteria, especially those resistant to antibiotics, is the lysis of bacterial cells using lytic enzymes. There are known methods of lysis of bacterial cells or damaging the walls of bacterial cells, requiring the impairment of the wall structure with the use of specific bacteriolytic enzymes - peptidoglycan hydrolases.

This is especially true for Gram(+) bacteria, due to the specific structure of their cell walls. It is known that the cell walls of Gram(+) bacteria differ in the type and amount of amino acids found in the bridges linking the peptidoglycan chains. For their activity, peptidoglycan hydrolases may require the presence of a specific amino acid composition and a minimum number of amino acids in the interpeptide bridges of peptidoglycans. For most staphylococci, the peptidoglycan interpeptide bridge consists of five glycines, and it is a substrate for lytic enzymes. There are known peptidoglycan hydrolases such as lysostaphin and LytM, which cut the pentoglycine bridges characteristic of Staphylococcus peptidoglycans, e.g. 5. aureus , and due to this property they are interesting as potential anti-staphylococcal agents.

The biological role of lysostaphin is well known. Lysostaphin is a bacteriocin secreted by Staphylococcus simulans biovar staphylolyticus . The mature protein is inactive to the organism that produces it, but is very effective at cutting the walls of Staphylococcus aureus. Mature lysostaphin is a monomeric protein, for which the optimum temperature for enzyme activity is around 37-40 °C, pH 7.5, and its isoelectric point pi is 9.5 [1, 2], Lysostaphin has been used to destroy S. aureus and S. epidermidis biofilms on artificial surfaces and has been tested for coating of catheters [3]. In a mouse model, lysostaphin was used to remove biofilms from catheterized jugular veins and to treat systemic infections [4], In in a rat model (cotton rat), a cream with lysostaphin effectively eliminated S. aureus from the noses of infected rats. [5], In humans, lysostaphin has only been used at an experimental level to treat aortic heart valve inflammation caused by methicillin-resistant Staphylococcus aureus [6].

Another, commercially available enzyme, Auresine, which is the catalytic domain of LytM autolysin from Staphylococcus aureus, has a specificity and mechanism of action similar to that of lysostaphin [7], Auresine is an endopeptidase capable of digesting the bonds in the pentaglycine bridges of staphylococcal cell walls. It has unique properties - it is active in conditions of low ionic strength (even in water) and at low temperatures. It is successfully used to lyse staphylococcal cells during the isolation of DNA, RNA and proteins. Due to the capability of quick lysis, both Auresine and other enzymes may be used in diagnostic tests for the identification and determination of staphylococci.

Some bacteria, however, developed a resistance mechanism to these enzymes, e.g. Staphylococcus simulans biovar staphylolyticus to the lysostaphin they produce. [9,10]. This is a very serious restriction in the commercialization of lysostaphin and ways to overcome this problem are still being sought [11]. The lysostaphin gene is located on the pACKl plasmid. The resistance gene is encoded on the same plasmid (endopeptidase resistance gene, epr or lysostaphin immunity factor, lif). The presence of the lif gene, also in other species of staphylococci, increases the serine content and reduces the glycine content in the bacterial cell wall. The substitution of one or more glycines with serine residues in the pentaglycine bridge is believed to determine resistance to the activity of lysostaphin [12, 13]. As with antibiotics, genes for lysostaphin resistance are encoded on mobile genetic elements. Therefore, there is a possibility of spread of the resistance. Studies have shown that staphylococcal species sensitive to the activity of lysostaphin, after receiving the construct containing the resistance gene, became less sensitive or completely resistant to the activity of this enzyme [16, 17]. The emergence of potential resistance to lysostaphin and other lytic enzymes with the same specificity is one of the main factors limiting the use of these enzymes in the control of staphylococci. For this reason, it is extremely important to search for new bacteriolytic enzymes that would have an extended specificity of activity and would be equally effective in lysing staphylococci, also with modified interpeptide bridges. Bacteria, which instead of typical pentaglycine bridges have formed bridges with substituted serine, are resistant to the commonly used lysostaphin or various forms of M23 hydrolases, including LytMCD described in WO2012/144912 or various forms of LytMCD_LssCWT described as Chimeras A and Chimeras B in WO2021/0759888. Lysostaphin is not active against strains with serine in interpeptide bridges, such as: S. simulans, S. epidermidis, S. intermedius, S. haemolyticus or S. warneri or S. aureus with serine introduced into the interpeptide bridges.

An enzyme used to combat staphylococcal infections in humans and animals should have a wide spectrum of activity against bacteria having peptidoglycan interpeptide bridges and exhibit peptidoglycan hydrolase activity against glycine interpeptide bridges, as well as against interpeptide bridges in which one or two glycines have been substituted with serine, i.e. also glycine-serine bridges - such as those found in S. simulans. However, neither lysostaphin nor Auresine are active against strains of staphylococci having serine in their interpeptide bridges. Also the chimeras described in WO2021/0759888, which consist of the LytM catalytic domain and the lysostaphin cell wall binding domain, although active under physiological conditions, are not capable of lysing staphylococci with serines in interpeptide bridges. Therefore, enzymes that would be capable of cutting not only glycine bridges, but also glycine bridges with substituted serine/serines (glycine-serine bridges) are sought and extremely needed.

DISCLOSURE OF THE INVENTION

In view of the described state of the art, the object of the present invention is to overcome the indicated disadvantages and to provide a lytic enzyme with endopeptidase activity, acting against the walls of Gram(+) bacteria containing both pentaglycine bridges, as well as the substitution of glycine residues by serine residues in the interpeptide bridges linking the peptidoglycan chains, in particular against S. aureus resistant to the activity of the available glycyl- glycine endopeptidases; such enzyme will be produced efficiently, will be stable, will provide high substrate specificity and activity, as well as will work effectively in a wide pH range.

The KXA44996 gene, encoding the 410 amino acid AurR protein from Staphylococcus simulans DSM20322, has been identified in the genome of Staphylococcus simulans DSM20322 [18], isolated, cloned and sequenced by the inventors of the present invention. The full-length AurR protein consists of three functional domains (Fig. 1.): (1) an N-terminal regulatory domain, (2) a catalytic domain AurR i.e. CD^AurR with a sequence characteristic for proteins of the M23 family, different from lysostaphin, and (3) CWT^AurR domain -a C-terminal, bacterial cell wall binding domain (CTW, from Cell Wall Targeting). SP- is a 36 amino acid long signal peptide, L - a linker located between CD^AurR and CWT^AurR.

It was unexpectedly found that the active form of AurR is a peptidoglycan hydrolase with extended substrate activity, which, not only like LytM or lysostaphin, cleaves glycine interpeptide bridges in the staphylococcal cell wall, leading to the lysis of bacterial cells, but unlike the enzymes mentioned above, the active form of AurR also cleaves bridges in which one or two glycines have been substituted with serine/s (glycine-serine bridges).

Therefore, peptidoglycan hydrolase in the active form of AurR overcomes the mechanism of bacterial resistance to glycyl-glycine hydrolases, when the interpeptide bridges in peptidoglycan are made of glycine and serine.

The inventors have surprisingly found that the stable and active form of the Staphylococcus simulans AurR protein, containing the CD^AurR catalytic domain, overcomes the indicated limitations, as it lyses both pentaglycine-containing bacterial walls, as well as those containing a serine or two serines in the interpeptide bridge. The active form of AurR is capable of effective degradation of the walls of Gram(+) bacteria, especially S. aureus and S. simulans , under a wide range of conditions, including a physiological-like environment with both low and high conductivity, in a wide range of salt concentrations, including high salt concentration, as well as being effective over a wide pH range.

The inventors have surprisingly found that the active form of the Staphylococcus simulans AurR protein, containing the CD^AurR catalytic domain, lyses both pentaglycine-containing bacteria and those with serine/serines in the interpeptide bridge, and is capable of effective degradation of the walls of Gram(+) bacteria, especially of S. aureus and S. simulans. The active forms of the AurR protein, according to the invention, also show high stability.

The essence of the present invention is thus based on finding the active form of the AurR protein encoded by KXA44996 gene from Staphylococcus simulans showing new, so far unknown enzymatic properties, and demonstrating that it is possible to use the active form of AurR for the proteolysis of a peptide substrate, for example the walls of Gram(+) bacteria containing a serine residue, in addition to glycine residues.

The invention relates to a peptidoglycan hydrolase capable of digesting the walls of Gram(+) bacteria having both glycine and glycine-serine peptidoglycan interpeptide bridges containing within peptidoglycan, comprising the active form of the AurR protein, which comprises CD^AurR as the catalytic domain, wherein the amino acid sequence of the CD^AurR catalytic domain is in at least 80% identical with the amino acid sequence shown in SEQ ID NO: 1, more preferably the CD^AurR sequence is in at least 85%, 90%, more preferably 95%, most preferably at the level of 98% or more identical to the amino acid sequence shown in SEQ ID NO: 1; wherein the interpeptide bridges consist of at least three glycines or glycines and serine/serines.

In a preferred peptidoglycan hydrolase, the active form of the AurR protein is in the form of a single domain active form of AurR, comprising the CD^AurR catalytic domain, but not comprising the CWT^AurR bacterial cell wall binding domain.

In a preferred peptidoglycan hydrolase, the active form of the AurR protein in the form of a single domain is the catalytic domain AurR^158'295 having the SEQ ID NO: 1.

In a preferred peptidoglycan hydrolase, the active form of the AurR protein in the form of a single domain is AurR- having the SEQ ID NO: 4.

In a preferred peptidoglycan hydrolase, the active form of the AurR protein is in the form of a two- domain active form of AurR, comprising the CD^AurR catalytic domain and the CWT^AurR bacterial cell wall binding domain, wherein the amino acid sequence of the CWT^AurR catalytic domain is in at least 80% identical with the amino acid sequence shown in SEQ ID NO: 2, more preferably the CWT^AurR sequence is in at least 85%, 90%, more preferably 95%, most preferably at the level of 98% or more identical with the amino acid sequence shown in SEQ ID NO: 2. In a preferred peptidoglycan hydrolase, the amino acid sequence of the CD^AurR catalytic domain is linked to the sequence of the CWT^AurR bacterial cell wall binding domain by the amino acid sequence of a linker, wherein the preferred sequence of the linker are the amino acids 139 -159 of the SEQ ID NO: 3.

In a preferred peptidoglycan hydrolase, the active form of AurR in the two-domain form is AurR^158'410 having the SEQ ID NO: 3.

In a preferred peptidoglycan hydrolase the active form of AurR in the two-domain form is AurR+ having the SEQ ID NO: 5.

The invention also relates to a genetic construct encoding a peptidoglycan hydrolase, according to the invention.

The invention also relates to a composition for the proteolysis of Gram(+) bacterial cell walls, which comprises a peptidoglycan hydrolase and a carrier, wherein the composition is intended for the proteolysis of cell walls of Gram(+) bacteria having glycine and/or glycine-serine interpeptide bridges within the peptidoglycan.

The invention also relates to a pharmaceutical composition comprising a peptidoglycan hydrolase of the invention and a pharmaceutically acceptable excipient for use as a medicament in the treatment of diseases and/or inflammations caused by Gram(+) bacteria having glycine and/or glycine-serine interpeptide bridges within the peptidoglycan, wherein, preferably, the Gram(+) bacteria are selected from the genus Staphylococcus, and preferably are S. aureus, S. simulans, S. epidermidis, S. intermedius, S. haemolyticus , S. warneri. A preferred pharmaceutical composition is in the form of a liquid, emulsion, gel, spraying liquid, lotion, wet wipe or dressing, preferably the dressing is in the form of a plaster with a dressing, a plaster with a hydrogel, gauze with the composition applied, hydrocolloid dressing, hydrofibre dressing, an alginate dressing, a bandage with the composition applied.

The invention also relates to a cosmetic or care composition for cosmetic, care and hygiene uses in humans and/or animals, which comprises the peptidoglycan hydrolase of the invention, wherein the composition additionally comprises at least one carrier which is approved for cosmetic, care, and hygiene uses in humans and/or animals, and is intended for external use.

The invention also relates to a composition comprising a peptidoglycan hydrolase of the invention and a carrier for use as an antiseptic agent, antibacterial agent, an agent for disinfection of surfaces used against Gram(+) bacteria having glycine and/or glycine-serine interpeptide bridges within the peptidoglycan, wherein, preferably, the composition is used against Gram(+) bacteria of the genus Staphylococcus, preferably selected from S. aureus, S. simulans, S. epidermidis, S, intermedius, S. haemolyticus, S. warneri. Preferably, the composition for use as an antiseptic, antibacterial agent, surface disinfectant, is in the form of a liquid, emulsion, gel, spray, lotion, wet wipe or dressing, preferably the dressing is in the form of a patch with a dressing, a plaster, with a hydrogel, gauze with the composition applied, a hydrocolloid dressing, a hydrofiber dressing, an alginate dressing, a bandage supplemented with the composition.

The invention also relates to the use of a peptidoglycan hydrolase of the invention and/or compositions for the proteolysis of the cell walls of Gram(+) bacteria of the invention for the proteolysis of cell walls of Gram(+) bacteria having glycine and/or glycine-serine interpeptide bridges within the peptidoglycan, wherein, preferably, the Gram(+) bacteria are bacteria of the genus Staphylococcus, preferably S. aureus, S. simulans, S. epidermidis, S. intermedins, S. haemolyticus, S. warneri.

The invention also relates to the use of a peptidoglycan hydrolase of the invention and/or a composition for the proteolysis of the cell walls of Gram(+) bacteria of the invention as a bacteriostatic or bactericidal agent against Gram(+), particularly of the genus Staphylococcus, preferably S. aureus, S. simulans, S. epidermidis, S. intermedius, S. haemolyticus, S. warneri. having glycine and/or glycine-serine interpeptide bridges within the peptidoglycan, preferably the agent is used in the food industry particularly as a food additive for humans and/or animals or for disinfecting surfaces and rooms that come into contact with food or food intermediates, more preferably the agent is used in the dairy industry and milk products.

In a preferred use of the peptidoglycan hydrolase according to the invention and/or the composition for the proteolysis of the cell walls of Gram(+) bacteria of the invention as a bacteriostatic or bactericidal agent against Gram(+) bacteria, the agent is used in the cosmetics industry, preferably as an additive to cosmetics improving their microbiological quality, as an additive to liquids, creams, milks, lotions.

In a preferred use of the peptidoglycan hydrolase of the invention and/or the composition for the proteolysis of the cell walls of Gram(+) bacteria of the invention as a bacteriostatic or bactericidal agent against Gram(+) bacteria, the agent is used in health care for decontamination of the surfaces of tools and instruments used in medicine and diagnostics, as well as other surfaces, in particular hospital and laboratory surfaces.

In a preferred use of the peptidoglycan hydrolase of the invention and/or the composition for cell wall proteolysis of Gram(+) bacteria of the invention as a bacteriostatic or bactericidal agent against Gram(+) bacteria, the agent is used as a bacteriostatic or bactericidal agent in the form of a liquid, emulsion, gel, spraying liquid, lotion, wet wipe or a dressing, preferably used as a bacteriostatic or bactericidal agent, the dressing is in the form of a patch with a dressing, a patch with a hydrogel, a gauze with an applied composition, a hydrocolloid dressing, a hydrofiber dressing, an alginate dressing, a bandage with applied composition.

The invention also relates to the method of hydrolyzing peptidoglycans having glycine and/or glycine-serine interpeptide bridges, wherein it includes the following steps, in which a) the active form of AurR, of the invention, is contacted in an aqueous medium with a peptide substrate having glycine and/or glycine-serine interpeptide bridges within the peptidoglycan; b) the peptide substrate having glycine and/or glycine-serine interpeptide bridges within the peptidoglycan is being hydrolyzed.

In a preferred method of hydrolyzing peptidoglycans, the peptidoglycans of the cell walls of Gram(+) bacteria, preferably of the genus Staphylococcus, preferably S. aureus, S. simulans, S. epidermidis, S. intermedins, S. haemolyticus , S. warneri, are hydrolyzed.

In a preferred method of peptidoglycan hydrolysis, step b) is carried out at a temperature of 4 °C to 45 °C.

DETAILED DESCRIPTION OF THE INVENTION

The term "active form of AurR” as used herein means proteins, polypeptides, peptides or recombinant proteins, polypeptides and peptides having peptidoglycan hydrolase activity capable of digesting the walls of Gram(+) bacteria having both glycine and glycine-serine peptidoglycan interpeptide bridges, which include a CD^AurR catalytic domain with an amino acid sequence identical or highly identical to the amino acid sequence containing amino acids from positions 158 to 295 in the starting AurR protein encoded by KXA44996 (which corresponds to SEQ ID NO: 1, and aa 1-138 of SEQ ID NO: 2), preferably at a level of at least 80%, 85%, 90%, more preferably 95%, most preferably at a level of 98% or more identity.

The exemplary, as described herein in the embodiments, active forms AurR relating to AurR^158'295 ⁽SEQ ID NO: 1), AurR^{158'410 (}SEQ ID NOG), AurR- (SEQ ID NO: 4), AurR+ (SEQ ID NO: 5), exhibit endopeptidase proteolytic activity against glycine interpeptide bridges, also with serine/serine substitutions. It is obvious that certain changes in the amino acid sequence of the active form of AurR polypeptide or in the nucleotide sequence encoding such a polypeptide resulting in a change in the amino acid sequence will not affect the activity of the polypeptide, especially if these changes do not affect the active site of the protein.

The active form of AurR may be (i) a single domain active form of AurR, when it only has the catalytic domain of CD^AurR - preferably at a level of at least 80%, 85%, 90%, more preferably 95%, most preferably 98% or more identity with SEQ ID. NO: 1, for example the single domain active form of AurR is AurR^158-295 with SEQ ID NO: 1, AurR- with SEQ ID NO: 4, or (ii) the two-domain active form of AurR, when it has both the CD^AurR catalytic domain, showing at the amino acid level at least 80%, 85%, 90%, more preferably 95%, most preferably 98% or more identity with SEQ ID NO: 1, and a bacterial cell wall binding domain CWT^AurR having a sequence showing, at the amino acid level, at least 80%, 85%, 90%, more preferably 95%, most preferably

98% or more identity to SEQ ID NO: 2, or being AurR^317'410 SEQ ID NO: 2, an exemplary two- domain active form of AurR is AurR^158-410 having SEQ ID NO: 3, AurR+ having SEQ ID NO: 5.

The two-domain active form of AurR thanks to the CWT^AurR domain, retains its activity in a wider range of pH or ionic strength compared to lysostaphin or the active form of AurR without the CWT^AurR domain. On the other hand, the single-domain active form of AurR is more stable when stored at 37 °C.

Conditions with low conductivity should be understood as conductivity below 10 mS/cm, conditions with high conductivity should be understood as conductivity above 10 mS/cm.

As "peptide substrate" or "protein substrate" for the active form of AurR as used herein is to be understood a peptide, polypeptide or protein composed of at least three glycines or glycines and serine/serines, which are recognized and cleaved by the active form of AurR. The peptide substrate for the active form of AurR will therefore be, in particular, glycine or glycine-serine bridges in the peptidoglycans of Gram(+) - S. simulans , S. epidermidis, S. aureus as well as those containing serine in interpeptide bridges, Staphylococcus hominis, Staphylococcus intermedius, Staphylococcus haemolyticus and others.

Gram(+) bacteria that contain glycines and possibly serines in interpeptide bridges include some bacteria of the genus Staphylococcus , which include the species S. aureus, S. epidermidis, S. roseus, S. carnosus, S. lactis, S. saprophyticus and of the genus Micrococcus such as M caseolyticus, M. candidans, M. naucinus, M. vernae. Biochemical analysis showed that the full-length AurR protein is inactive. Only the active form of the AurR protein is active, i.e. the form of the enzyme (i) containing a CD^AurR catalytic domain with an attached CW^TAurR domain binding bacterial cell walls, for example AurR¹⁵⁸⁴¹⁰, AurR+, which is active under reaction conditions in a high conductivity environment; and in conditions of low conductivity, (ii) active forms of the AurR protein containing only the CD^AurR catalytic domain, for example AurR^158"295, Aur-, which are the most active under conditions of low conductivity. A preferred active form of the AurR protein is the polypeptide AurR^158'295, AurR¹⁵⁸⁴¹⁰, AurR^", AurR+.

In a method of proteolysis according to the invention, in particular of Gram(+) bacterial cell walls, an active form of the AurR protein is contacted with a peptide substrate, preferably with the walls of Gram(+) bacteria, in an aqueous medium of varying conductivity. In a preferred method of proteolysis, contacting is carried out at a temperature ranging from about 0 °C, more preferably from about 4 °C to about 45 °C, more preferably from 21 °C to 37 °C. Further, the pFI of the reaction medium is preferably in the range from about 6 to about 9, more preferably in the range from about 7 to about 9. Preferably the Gram(+) bacteria are bacteria belonging to the genus Staphylococcus, preferably species selected from the group consisting of: S. aureus, S. simulans, S. epidermidis, S. intermedins, S. roseus, S. haemolyticus, S. warneri.

A composition of the invention with an active form of the AurR protein is used as a bacteriostatic or bactericidal agent, especially against Gram(+) bacteria, in particular of the genus Staphylococcus, containing pentaglycine bridges aa well as those with serine substitution. A preferred composition is to be used for surface disinfection, preferably in the form of a liquid, emulsion, gel, spray, lotion or wet wipe. The composition can be supplemented with a suitable carrier, preservative, perfume, buffer, as well as other ingredients useful in the elimination of bacteria, in particular detergents, solvents, antibiotics, bacteriocins.

According to the invention, the composition with the active form of the AurR protein is used as a bacteriostatic or bactericidal agent in the food industry. The agent is used especially as an additive to food for humans and animals or to disinfect surfaces.

According to the invention, the composition with the active form of the AurR protein is used as a bacteriostatic or bactericidal agent in health care. The agent is used to disinfect surfaces instruments and appliances used in medicine and diagnostics, and other surfaces, in particular hospital and laboratory surfaces, preferably, it is used against Gram(+) bacteria, especially belonging to the genus Staphylococcus, containing both pentaglycine bridges, as well as the substitution of glycine residues by serine residues in the interpeptide bridges linking the peptidoglycan chains.

According to the invention, the active form of the AurR protein is used to isolate cellular structures from Gram(+) bacteria. Such isolation of cellular structures is possible, inter alia, for bacteria of the genus Staphylococcus, containing both pentaglycine bridges as well as the substitution of glycine residues by serine residues in the bridges binding the peptidoglycan chains, especially from species selected from the group consisting of: S. aureus, S. simulans, S. epidermidis, S. intermedins, S. roseus, S. haemolyticus, S. warneri.

According to the invention, the active form of the AurR protein is used in the diagnosis of Gram(+) bacteria, in particular bacteria of the genus Staphylococcus.

According to the invention, the active form of the AurR protein is used to impregnate or cover surfaces exposed to contact with Gram(+) bacteria. The active form of the AurR protein of the invention is used to impregnate or cover surfaces exposed to contact with Gram(+) bacteria. Lysis of the bacteria is carried out to isolate the cellular structures of Gram(+) bacteria, in particular DNA, RNA, proteins, peptides, glycopeptides, lipids or useful metabolites.

According to the invention, the active form of AurR can be used as a bacteriostatic or bactericidal agent in medicine, veterinary medicine and diagnostics. A bacteriostatic or bactericidal agent comprising an active form of AurR will be used against Gram(+) bacteria, preferably belonging to the genus Staphylococcus, containing both pentaglycine bridges as well as the substitution of glycine residues by serine residues in the bridges linking peptidoglycan chains. Such a bacteriostatic or bactericidal agent will be used to disinfect the surfaces of tools and instruments used in medicine, veterinary and diagnostic, hospital and laboratory surfaces, and as a surfactant on surfaces that may be contaminated with bacteria. Such an agent may be used alone or in combination with other ingredients useful in tbe elimination of bacteria, in particular detergents, solvents, antibiotics, bacteriocins or other enzymes. The agent used may include a suitable carrier, stabilizer, buffer, or other additives. The active form can be used as a bacteriostatic or bactericidal agent in the form of a liquid, emulsion, gel, spray, lotion, wet wipe, and the like.

The active form of AurR can be used as a diagnostic agent for certain species of Gram(+) bacteria, preferably belonging to the genus Staphylococcus, especially species such as, for example,: S. aureus, S. simulans, S. epidermidis, S. intermedius, S. roseus, S. haemolyticus, S. warneri. The active form of AurR can be used as a tool for specific protolysis of bacteria for direct diagnostics of bacterial species or strains as well as in the stage of initial cell lysis for further diagnostics, for example by methods such as PCR, nucleic acid hybridization, immunological methods, and immunofluorescence, ELISA and methods based on the content of bacterial cells such as enzymatic assessments and others.

The active form of AurR can be used as a tool to disrupt the cell walls of Gram(+) bacteria, for example to isolate cell structures from Gram(+) bacteria. Impairment of the cell walls of bacteria for the purpose of their lysis can be aided by the addition of detergents or other factors weakening the structure of the cell wall, such as other enzymes. Impairment of cell walls can also be earned out in order to obtain protoplasts, allow the transformation of a bacterial cell, isolate genetic material, proteins as well as useful metabolites, for example long-chain carbohydrates.

The active form of AurR will therefore be included in and used in kits intended to disrupt the cell walls of Gram(+) bacteria, for example to isolate cell structures from Gram(+) bacteria. Such kits are also within the scope of the invention.

The active form of AurR can be used as a bacteriostatic or bactericidal agent in the food industry. Such a bacteriostatic or bactericidal agent will be used as an additive to food for humans and animals, for disinfecting surfaces that come into contact with food, in particular equipment, tools and equipment used in the food industry as well as rooms that come into contact with food or intermediates. The active form of AurR will be especially used in the dairy industry and dairy products.

The active form of AurR will also be used to impregnate or cover surfaces exposed to contact with Gram(+) bacteria. The active form of AurR can be used combined with or added to polymers, copolymers or nanocarriers such as nanospheres or nanotubes, for example carbon nanotubes. The surfaces to be coated or impregnated may concern various surfaces, for example rooms, tools, machines, devices, medical, diagnostic or laboratory equipment. Such surfaces covered or impregnated with a layer comprising an active form of AurR will have a long-term bactericidal and/or bacteriostatic effect on Gram(+) bacteria.

The active form of AurR can be used as a bacteriostatic or bactericidal agent in the cosmetics industry. A bacteriostatic or bactericidal agent will be used as an additive to cosmetics improving their microbiological quality, as an addition to liquids, creams, lotions, lotions, or as a surface disinfectant, or to disinfect various surfaces, equipment, tools and equipment used in the cosmetics industry.

Mature form of the AurR protein (KXA44996)

Both lysostaphin and the native protein AurR are produced in a three-domain form, and show, at most, very low activity. AurR is active in its mature form, consisting of two domains - the catalytic domain (CD^AurR) and a cell wall binding domain (CWT^AurR). AurR + was produced and used in the examples in two-domain form, while Aur- is a single-domain protein with a catalytic domain.

Extended activity of the active form of AurR

Both lysostaphin and the active form of AurR are active against bacteria that contain peptidoglycan pentaglycines, such as those in S. aureus. What was not obvious and surprising was the additional activity of the active form of the AurR protein against staphylococci with cell walls, in which the interpeptide bridges are made of both glycines and serines, e.g. in the strain S. simulans CCM3583.

Lack of cell wall binding domain CWT^AurR in the active form of AurR

Both lysostaphin and the active form of AurR are active against bacteria that contain pentaglycine bridges in peptidoglycans, such as those in S. aureus. These proteins recognize the bridges as such, possibly at least in part through interaction with a groove in the active site. Lysostaphin has a CWT domain as does the two-domain active form of AurR +, which also has its own cell wall binding domain CWT^AurR, thanks to which it maintains activity in a wider range of pH or ionic strength compared to lysostaphin or the active form of AurR - without the domain CWT^AurR. In turn, a single-domain protein, an active form of AurR- with a domain CD^AurR, but without a domain CWT^AurR, is more stable when stored at 37°C and works faster under optimal conditions of pH and ionic strength.

The active form of AurR effectively lyses viable S. aureus cells

The inventors showed that active forms of AurR administered externally effectively lysed live Gram(+) bacterial cells, both S. aureus and S. simulans, binding and cleaving peptide substrates of their cell walls. Externally administered, active form of the AurR proteins, one-domain or two- domain, inhibits the further growth of staphylococci and acts as a bacteriostatic and bactericidal agent leading to the lysis of S. aureus and S. simulans cells. This was confirmed in the conducted cell wall lysis tests by measuring the change in the optical density of the cell suspension and monitoring the reduction in the number of colonies formed. Previous experiments showed the ability of lysostaphin to degrade only pentaglycine-containing cell walls. Tests have shown that single and double domain active forms of AurR are active against S. simulans CCM3583 strain, which has interpeptide bridges comprising glycine residues and serine residues and lyse bacterial cells with much greater efficiency than lysostaphin.

The activity of the active form of AurR and lysostaphin depends differently on the pH

The activity of a specific peptidoglycan hydrolase was determined in the S. aureus and S. simulans cell wall lysis test by measuring changes in the optical density of the cell suspension.

Probably due to the remaining enzymatic activity of other enzymes in the cell walls, a slight reduction in turbidity was also observed in the control, in the absence of exogenously added enzyme. Therefore, all OD values measured at 595nm were expressed as percent of the control. A value close to 100% indicates very little activity, while a low OD indicates high enzyme activity. Lysostaphin was very effective at about pH 7-9 and slightly less at pH 10 against S. aureus cells. The range of activity of the two-domain active form of the AurR protein on S. aureus cells is much wider and covers the entire tested range from pH 5.4 to pH 10.9, being most active at pH 7-10. The single-domain active form of the AurR protein is active in the range of pH 6 to 8, retaining its greatest activity at pH 6 to7. A greater restriction is observed in the lysis of S. simulans cells, where lysostaphin has little activity in the pH range of 5.4-8, while the two-domain active form of the AurR protein lyses S. simulans cells significantly effective at pH 5.4-8, with an optimum at pH 7 to 8. The single-domain active form of the AurR protein shows high activity in the pH range of 5.4 to 8, the highest activity at around pH 7.

Dependence of the activity of the active form of the AurR protein and lysostaphin on the conductivity ( conductivity) of the reaction environment It was unexpectedly found that the efficiency of bacterial lysis by the active form of the AurR protein obviously depends on the conductivity (specific conductivity) of the reaction environment (Fig. 4), and this dependence varies depending on the type of lysed cells, S. aureus or S. simulans. In low conductivity buffers, degradation of S. aureus cell walls by the single-domain active form of AurR is more effective, in contrast to the two-domain active form of AurR which works best in buffers with higher conductivity and is less effective in buffers with low conductivity.

The conductivity reflects both parameters together, i.e. the concentration of ions and their mobility. The effect of conductivity on the activity of single-domain and double-domain active AurR in solutions with varying ionic strength was tested by changing the concentration of NaCl in the range from 0 to 250 mM. The lysis of S. simulans cells by the two-domain active form of AurR is most effective at moderate and low ionic strength, from 0.1 mS/cm to 10 mS/cm, while the efficiency of lysis of S. aureus cells differs only slightly from 0.1 mS/cm up to 20 mS/cm. Temperature dependence of the active form of AurR activity

Both single and double domain active forms of AurR have been shown to be stable proteins, acting effectively over a wide temperature range from about 4 °C to about 45 °C. These enzymes work even at 4 °C.

Publications cited in the description, and the references given therein, are also incorporated herein as references.

BRIEF DESCRIPTION OF FIGURES

For a better understanding of the invention, it has been illustrated in the embodiments and in the accompanying figures, in which:

Fig. 1 shows (A) schematically the organization of the domains in the full-length AurR protein, encoded by the gene from the KXA44996 locus: SP - signal peptide, ND - N-terminal domain, CD - catalytic domain, CWT - cell wall recognition domain, L- linker connecting CD and CWT. The bracketed ranges represent the domain extent of the proteins shown: AurR - single-domain active form of AurR consisting of the catalytic domain CD^AurR and AurR+ two-domain active form of AurR consisting of catalytic domain CD^AwR, linked by a linker (L) to the cell wall recognition domain CWT^AurR; (B) detailed domain range and AurR+ sequence. (C) shows the alignment of the amino acid sequences of Chimera A (described in WO2021/0759888) with AurR+ and (D) Lss with AurR+ giving the statistics of the sequence alignment results obtained.

Fig. 2 shows the dependence of the decrease in the number of colonies and the decrease in OD₅₉₅ due to the activity of the enzyme AurR- at three different concentrations compared to lysostaphin (Lss), C - control - no enzyme added. (A) Decrease in CFU of S. simulans shown on a logarithmic scale at 5, 15, 30, 70 and 125 min. (B) Lysis of S. simulam cells monitored at OD₅₉₅. The results are expressed as % of the initial OD (%OD₅₉₅) of the S. simulam cell suspension. The graph shows the results obtained for the reactions run for 125 min., measured every 2.5 minutes in optimal enzyme buffers. (C) Decrease in CFU of S. aureus shown on a logarithmic scale at 5, 15, 30, 70 and 125 min. (D) Lysis of S. aureus cells monitored at OD₅₉₅. The results are expressed as % of the initial OD (%OD₅₉₅) of the S. aureus cell suspension. The graph shows the results obtained for the reactions carried out for 125 min, measured every 2.5 minutes in optimal enzyme buffers.

Fig. 3 shows the dependence of the decrease in the number of colony forming units (CFU) and the decrease in OD₅₉₅ due to the activity of the enzyme AurR- at three different concentrations compared to lysostaphin (Lss). C - control (no enzyme). (A) Decrease in CFU of S. simulans is shown on a logarithmic scale at 5, 15, 30, 70 and 125 min. (B) Lysis of S. simulans cells monitored at OD₅₉₅. The results are expressed as % of the initial OD (%OD₅₉₅) of the S. simulans cell suspension. The graph shows the results obtained for the reactions caried out for 125 min, measured every 2.5 minutes in optimal enzyme buffers. (C) Decrease in CFU of S. aureus shown in logarithmic scale at 5, 15, 30, 70 and 125 min. (D) Lysis of S. aureus cell monitored at OD₅₉₅. The results are expressed as % of the initial OD (%OD₅₉₅) of the £. aureus cell suspension. The graph shows the results obtained for the reactions carried out for 125 min, measured every 2.5 minutes in optimal enzyme buffers.

Fig. 4 shows the effect of the ionic strength of the reaction buffer on the lytic activity of AurR- (solid line) and AurR+ (dashed line) on S. aureus. Lysis was performed in 50 mM glycine buffer, pH 8.0, supplemented with NaCl at concentrations ranging from 0 to 250 mM. Due to the steady trend, results to up to 150 mM NaCl are shown. The conductivity of the reaction solution was measured at room temperature. The results are expressed as % of the initial OD (%OD₅₉₅) of the cell suspension - 100%. The presented results were collected after 45 min of carrying out the lysis at room temperature.

Fig. 5 shows a comparison of the activity of Lss (A), AurR- (B) and AurR+ (C) against S. aureus under different pH conditions. The results are expressed as % of the initial OD (%ODs9s) of the suspension of S. aureus cells. The graph shows the results obtained after 60 min. of the reaction . Fig. 6 shows a comparison of the activities of Lss (A), AurR- (B) and AurR+ (C) against S. simulans under different pH conditions. The results are expressed as % of the initial OD (%OD₅₉₅) of the suspension of S. simulans cells. The graph shows the results obtained after 100 min of the reaction.

Fig. 7shows a comparison of the activity of AurR- and AurR+ against S. aureus (A) and (B), and AurR- and AurR+ against S. simulans (C) and (D) in the presence of a metal chelating agent - phenanatroline. Various concentrations of phenanthroline (0.12 mM - 2 mM) were used, and the enzyme activity without phenanthroline - 0 was also shown for comparison. The results are expressed as % of the initial OD (%OD₅₉₅) of the cell suspension tested. The graph shows the results obtained after 60 min of the reaction.

Fig. 8 shows a comparison of the activity of AurR- and AurR+ against S. aureus (A) and (B) and AurR- and AurR+ against S. simulans (C) and (D) in the presence of various concentrations of a metal chelating agent - EDTA. Various concentrations of EDTA (0.6 raM - 250 mM) were used, and the enzyme activity without EDTA - 0 was also shown for comparison. The figure shows the concentration range at which activity changes were observed. The results are expressed as % of the initial OD (%OD₅₉₅) of the cell suspension tested. The graph shows the results obtained after 60 min. of the reaction.

Fig. 9 shows a comparison of the effect of enzyme storage temperature on the lysis of bacterial cells: AurR+ on S. aureus and S. simulans (A) and (B), and AurR- on S. aureus and S. simulans (C) and (D). The enzymes were stored at -80 °C, 4 °C, 21 °C and 37 °C, respectively, for 2, 6, 16, 44 days. The results are expressed as % value of the fresh enzyme activity, expressed as 100%. The graph shows the results obtained after 60 min of the reaction.

Fig. 10. Shows a comparison of the effect of temperature for the enzymes AurR- and AurR+ on the lysis of bacterial cells of S. aureus (A) and S. simulans (B). Reactions were carried out at the temperature of 4 °C, 21 °C, 37 °C and 45 °C for 60 min. The results are expressed as % of the initial OD (%ODs95) of the cell suspension tested.

METHODS OF CARRYING OUT THE INVENTION

The following examples were presented only to illustrate the invention and to clarify its various aspects, but are not intended to be limitative, and should not be equated with all its scope, which is defined in the appended claims.

EXAMPLES

Example 1

Production of various forms of AurR, the product of the KXA44996 gene.

Based on the analysis of the predicted sequence of the AurR protein and its comparison to other known domains, the presence of at least two domains was defined: catalytic (CD) and binding (CWT) linked by a short peptide (Fig. 1A, B). The amino acid sequence of AurR was compared to sequences of mature lysostaphin and Chimera A consisting of catalytic domains and the CWT binding domain (Fig. 1 C, D). In both cases, the level of identity was approximately 64% and the level of similarity was approximately 72%. Genomic DNA of Staphylococcus simulans DSM 20322 has been isolated. DNA fragments corresponding to the catalytic domain 158-295 of the native AurR protein and the catalytic domain with the binding domain 158-410 of the native protein AurR were amplified by PCR using genomic DNA of Staphylococcus simulans DSM 20322 as a template, and inserted into the pET30 vector. The coding sequence was preceded by a His-tag with the MHHHHHH amino acid sequence combined with a sequence recognized by the TEV protease. Both the single domain and the two domain active form of AurR were expressed in the form of inclusion bodies, so an MBP (Maltose Binding Domain) fusion domain was introduced to increase protein solubility. The soluble form of the AurR- and AurR+ proteins was obtained by expression of the constructs in E.coli strain BL21(DE3). The expression was induced during the logarithmic phase of bacterial growth in LB medium at an OD₅₉₅ of about 1 using 1 mM IPTG and it was continued for 16 h at 18 °C. Recombinant proteins were purified by affinity chromatography on a Ni-NTA agarose column (Qiagen), the His-tag in the case of the two-domain active form of AurR, named AurR +, and His- tagged MBP in the case of the single-domain active form of AurR, called AurR- was cut off using TEV protease, and the proteins were then purified by gel filtration on a Superdex™ 75 pg column (GE Healthcare) according to manufacturers' recommendations. Following the cutting off of the His-tag and His-tagged MBP sequences, respectively, the obtained purified preparations of AurR+ with SEQ ID NO: 5 and AurR- with SEQ ID NO: 4 were used in further experiments as stable active forms of the AurR protein.

The mature form of lysostaphin (Lss) used in the comparative examples, was also produced in the same way[19].

Example 2

The effect of AurR + and AurR- enzymes on bacterial cells of S. aureus and S. simulans.

The cell wall lysis test was carried out by measuring changes in the optical density of the cell suspension (i.e. turbidity reduction assay) with simultaneous determination of the number of bacteria by measuring colony forming units (CFU).

Bacterial cells of S. aureus and S. simulans grown in TSB medium at 37 °C with shaking at 80 rpm) were harvested in the logarithmic growth phase, centrifuged, washed and suspended in assay buffer (50 mM glycine, pH 8) to obtain an OD₅₉₅ of approximately 1. The enzyme AurR and AurR+ obtained in Example 1 was added to the final concentrations of 200 nM, 100 nM and 50 nM and lysostaphin - to 200 nM. 200 mΐ of the reaction mixture was transferred to a microtitration plate. The plates were incubated at room temperature with 10 seconds of shaking every 2.5 min. The OD of the suspension was measured at 595 nm for 125 minutes from the beginning of the reaction. Bacteria treated with lysostaphin and suspended in the buffer without the enzyme were used as controls. Each experiment was performed two times in triple replications. The results of the measurements are shown in Fig. 2 B, D and Fig. 3B, D.

Parallelly, 1 ml of mixtures with the same composition were incubated at room temperature. 100 mΐ of the solution were collected at the time of 5, 15, 30, 70 and 125 min. Serial dilutions from 10 to 10⁷ were performed by taking 20 mΐ of the sample and mixing it with 180 mΐ of buffer supplemented with 2 mM of a metallopeptidase inhibitor - phenanthroline. 5 mΐ of each dilution were collected and applied to Petri dishes with TSB medium with agar, which were then incubated at 37 °C for 24 h. Colonies were counted and CFU was determined. The results of the measurements are shown in Fig. 2 A, C and Fig. 3 A, C. The summarized results for S. simulans CFU reduction tests are shown in Table 1, and S. aureus CFU reduction - in Table 2. The orders of magnitude of decrease in the number of cells and the percent of reduction of the initial number of bacterial cells over time are shown.

The dependence of changes in the density of the cell suspension with a decrease in the number of bacterial cells in the presence of enzymes was demonstrated by measuring the OD. Thus, the effectiveness of the enzymes AurR- and AurR+ in the lysis of S. aureus cells having pentaglycine bridges, as well as S. simulans cells comprising serine/s in interpeptide bridges was demonstrated. Much higher activity of the enzymes AurR- and AurR+ against S. simulans , compared to lysostaphin, was demonstrated.

Table 1. Mean of the order of magnitude of reduction in the number of cells and mean of % reduction of the initial number 5. simulans cells by the enzymes AurR- and AurR+ over time ± standard deviation

Table 2. Mean of the order of magnitude of reduction in the number of cells and mean of % reduction of the initial number S. aureus cells by the enzymes AurR- and AurR+ over time and standard deviation

Example 3

The effect of the conductivity of the reaction environment on the lytic activity of the active forms of AurR- and AurR+ based on the cell wall lysis test, by measuring the changes in the optical density of the cell suspension

Bacterial cells of S. aureus and S. simulans grown in TSB medium at 37 °C with shaking were harvested in the logarithmic growth phase, washed and suspended in double-distilled water to an OD₅₉₅ of approximately 2, and 50 mΐ were applied to a microtitration plate. 50 mΐ of AurR+, AurR- and lysostaphin obtained in Example 1, all dissolved in double-distilled water, were applied to the microtitration plate with the bacteria samples. The final concentration of the enzymes was 100 nM. 100 mΐ of 50 mM glycine buffer with pH 8.0, supplemented with various concentrations of NaCl, from 0 - 500 mM were added to each well. The conductivity of the buffers was measured using the Mettler-Toledo Seven Compact Conductivity S230 conductometer. Conductivity measurements were carried out at room temperature. The OD of the suspension was measured at 595 nm at the beginning of the reaction and every 2.5 min, for 45 min. Lytic activity was calculated as the percent of OD₅₉₅ of the controls (same samples as for the reaction, but without the enzyme). Each experiment was performed two times in triple replications. The results for the time of 45 min for the enzymes AurR- and AurR+ against bacterial cells of S. aureus are shown in Fig. 4. It was found that the active forms of AurR in the form of the enzymes AurR- and AurR+ are active in buffers with different conductivity. The activity of AurR- against S. aureus was particularly high in the reaction environment with the conductivity between 0.5 and 2 mS/cm, reducing the OD of the suspension by 80% in 15 min. In the case of AurR+, high activity against S. aureus was observed for all the measured conditions above 0.08 mS/cm.

Example 4

The effect of buffer conditions on the lytic efficacy of the active form of AurR-, AurR+ and lysostaphin vs. S. aureus.

The bacterial cells and enzymes were prepared as in Example 3. The enzyme activity was tested in buffers with different pH and similar initial conductivity of approximately 2 mS/cm, with the addition of an optimal salt concentration - 3 mM for AurR- and 100 mM for AurR+ and Lss. The cell wall lysis was assayed by measuring the changes in optical density of the cell suspension performed as in Examples 2 and 3. AurR+ was found to work over the widest pH spectrum, from 7 to 10.9. The obtained results are shown in Fig. 5: (A) for Lss, (B) for AurR + and (C) AurR-. It was shown that the activity of Lss against S. aureus is high in buffers with pH from 7 to 9. The single-domain AurR shows high performance in buffers of pH 8 to 9.

Example 5

The effect of buffer conditions on the lytic efficacy of the active form of AurR-, AurR+ and Iysostaphin vs. S. simulans.

The cell wall lysis assay by measuring the changes in optical density of the cell suspension was performed as in the above examples. The bacterial cells and enzymes were prepared as in Example 3. Buffers with different pH and similar initial conductivity of approximately 2 mS/cm, with the addition of an optimal salt concentration - 3 mM for AurR- and 100 mM for AurR+ and Lss were tested. The obtained results are shown in Fig. 6: (A) for Lss, (B) for AurR + and (C) AurR-. It was shown that the activity of Lss against S. simulans is very low in buffers with pH 5.4 to 8. The two- domain AurR+ proved to work best at pH 7 to 8, but activity was also observed in a buffer at pH 5.4 and pH 6. The single-domain AurR- works in the range of pH 5.4 to 8.0, and shows the highest activity in a buffer with pH 7 .

Example 6

The effect of metal ion complexing agents, EDTA and phenanthroline, on the effectiveness of the active form of AurR-, AurR+ vs. S. simulans and S. aureus.

The cell wall lysis assay by measuring the changes in optical density of the cell suspension at different concentrations of complexing agents was performed as in the above examples. The bacterial cells and enzymes were prepared as in Example 3. 100 mΐ of solutions - 50 mM glycine buffer with pH 8.0 supplemented with optimal salt concentrations for enzymes and additionally various concentrations of metal ion complexing agents were added to a microtitration plate. For phenanthroline, the concentrations were 0 to 2 mM for both AurR- and AurR+. For EDTA, the concentrations were from 0 to 250 mM for the AurR+ enzyme and from 0 to 31 mM for AurR-. Bacteria were added to the plate, and the enzymes were added to a final concentration of 100 nM just before measurement. Lytic activity was calculated as the percent of OD₅₉₅ of the controls (same samples as for the reaction, but without the enzyme). Each experiment was performed in triple replications. Phenanthroline inhibited the lysis of S. aureus and S. simulans cells by AurR- even at very low concentrations, while AurR+ showed slightly better tolerance to this inhibitor (Fig. 7 A and B). In the case of the reaction with EDTA, both enzymes showed similar trends, but at the same time much higher tolerance than for phenanthroline (Fig. 8). Example 7

Effect of enzyme storage temperature on their bacterial cell wall lysis properties.

The bacterial cells and enzymes were prepared as in Example 3. The enzymes were stored at various temperature conditions: -80 °C, 4 °C, 21 °C and 37 °C for 2, 6, 16 and 44 days. Lytic activity was calculated as the percent of the density of the controls (same samples as for the reaction, but without the enzyme), measured as OD₅₉₅. Each experiment was performed two times in triple replications. The results were normalized to the activity of the freshly prepared enzymes AurR- and AurR+, Fig 9A and B. The lytic reaction was performed on S. aureus cells for 60 minutes at room temperature. Both enzymes showed high stability during storage in various temperature conditions, even for up to 44 days.

Example 8

The effect of the temperature of reaction with the active forms of AurR: AurR- and AurR+ on the lysis of bacterial cells of S. aureus and S. simulans.

The enzymes AurR- and AurR+ lyse the bacterial cells of A aureus and S. simulans very efficiently at room temperature and higher, remaining active also at 45 °C. The enzymes also showed activity at low temperatures (+4 °C), in particular against S. aureus.

Example 9

The activity of the enzyme AurR+ in the serum of various organisms.

The activity of the enzyme AurR+ in serum was tested in comparison with the enzymes Lss and LytMCD_LssCWT (Chimera A, shown as the sequence SEQ ID NO: 5 in the publication WO2021/075988). 100 nM of the enzymes were incubated in 50 mM glycine buffer pH 8.0, 100 mM NaCl or fetal bovine serum, human serum, horse serum or rabbit serum in the presence of an initial number of cells of lxlO⁶ CFU/ml. Strains of S. aureus with interpeptide bridges comprising GGGGG or GGSGG and S. simulans CCM 3583, which comprises serine in the interpeptide bridges (GGSGG) were used in the experiment. Incubations were carried out at 37 °C for 4 hours. The number of bacterial cells was determined by serial dilutions on TSB agar plates (spot test). The results are shown in Table 3 as a decrease of log 10 (mean ± SD). The experiment was repeated two or three times. The enzyme AurR+ shows a much higher effectiveness in eliminating staphylococci with both glycine interpeptide bridges and bridges comprising serine, compared to lysostaphin and LytMCD_LssCWT.

Table 3

Order of magnitude of reduction in the number of bacterial cells (± standard deviation)

Example 10

Pharmaceutical composition

Bacterial cells of S. aureus and S. simulans, as well as the enzymes were prepared as in Example 3. AurR+ was added to 10 ml of a pharmaceutically acceptable milk-saccharose carrier and to 10 ml of a liposomal earner, to a final concentration of 1 mM. Staphylococcal cells (10⁶ CFU/ml) were added to the so-prepared formulations. The effectiveness of the enzyme activity in the compositions was monitored by checking the number of bacterial cells (CFU) after 4 hours. Serial dilutions of the collected samples were plated on TSB agar plates and the number of bacteria was counted after 24 hours of incubation at 37 °C (spot test) as in Example 9. The composition showed antibacterial activity in both forms of carriers.

Example 11

Cosmetic composition Bacterial cells of S. aureus and S. simulans, as well as the enzymes were prepared as in Example 3. AurR+ was added to a 1 % solution of hyaluronic acid buffer with pH 7, to the final concentration of 1 mM. The cell wall lysis assay by measuring the changes in optical density of the cell suspension was performed as in Examples 2 and 3. The composition showed antibacterial activity as in Example 5.

Example 12 Antiseptic composition

The bacterial cells were prepared as in Example 3. The S. aureus and S. simulans cultures were plated as a lawn on composite agar plates with Baird Parker medium. AurR+ solution was added to 10 ml of sterile saline to the final concentration of 1 mM and sprayed onto freshly inoculated media. The control plates were not sprayed with the composition. Bacterial cultures were incubated for 24 h. The composition showed antibacterial activity against S. aureus and S. simulans, inhibiting their growth on the test plates.

Example 13

In vitro antibacterial activity

The bacterial cells and enzymes were prepared as in Example 3.

The AurR+ enzyme was added to 10 ml of PBS buffer to the concentration of 1 mM. The antimicrobial activity assay was performed on Baird Parker and Baird Parker-RPR media. Bacteria: S. aureus, S. simulans, S. epidermidis, S. intermedins, S. haemolyticus, S. warneri were plated on solidified media as a lawn. 100 mΐ of the composition was applied to a disc placed in the center of a Petri dish and antimicrobial activity was observed. The composition showed antibacterial activity against the tested bacteria, indicated by a large growth inhibition zone.

Example 14 Dressing

Bacterial cells of S. aureus and S. simulans, as well as the enzymes were prepared as in Example 3. AurR+ was added to the PBS buffer to the concentration of 1 mM and 0.2 ml was applied to a dressing with a hydrogel layer with an area of 1 cm² and thickness of 0.25 mm, until absorbed. The dressing was stored in sterile conditions for 30 days and after this time the activity against S. aureus and S. simulans was verified by applying the hydrogel-excised disc to a medium with bacteria plated on it, as in Example 13. A bacteria growth inhibition zone was observed around the AurR+ discs. In this way, a dressing with an antibacterial effect was produced. Literature:

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14. J Bacteriol. 1997 Jul; 179(13):4311-8. doi: 10.1128/jb.179.13.4311 -4318.1997. epr, which encodes glycylglycine endopeptidase resistance, is homologous to femAB and affects serine content of peptidoglycan cross bridges in Staphylococcus capitis and Staphylococcus aureus, M Sugai , T Fujiwara,

K Ohta, H Komatsuzawa, M Ohara, H Suginaka, PMID: 9209049 PMCID: PMC 179255 DOI: 10.1128/jb,179.13.4311-4318.1997 15. J Bacteriol. 2006 Sep; 188(17):6286-97. doi: 10.1128/JB.00457-06. Staphylococcus aureus mutants with increased lysostaphin resistance, Angelika Grundling , Dominique M Missiakas, Olaf Schneewind, PMID: 16923896, PMCID: PMC1595375 DOI: 10.1128/JB.00457-06

16. Antimicrob Agents Chemother. 2007 Feb;51(2):475-82. doi: 10.1128/ AAC.00786-06. Epub 2006 Nov 13. Lysostaphin-resistant variants of Staphylococcus aureus demonstrate reduced fitness in vitro and in vivo, Caroline Kusuma , Anna Jadanova, Tanya Chanturiya, John F Kokai-Kun, PMID: 17101683 PMCID: PMC 1797764 DOI: 10.1128/AAC.00786-06

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19. FEBS J. 2014 Sep;281(18):4112-22. doi: 10.1111/febs.12929. Crystal structure of the antimicrobial peptidase lysostaphin from Staphylococcus simulans

I Sabala, E Jagielska, P T Bardelang, H Czapinska, S O Dahms, J A Sharpe, R James, M E Than, N R Thomas, M Bochtler. PMID: 25039253; PMCID: PMC4286107

Explanation and listing of the sequences;

SEQ ID NO: 1 AurR^138-293-- the amino acid sequence of the single-domain active form of AurR protein, which corresponds to 138 amino acids from 158 to 295 of the AurR protein encoded by the KXA44996 gene; CD domain from AurR:

MEPYASAQWLTKYQLTAGYGHYNLNINNGMHYGADFAMPIGTPVRAITGGKIIEAGWSPYGGGNQIGVKEPDGSHYQ WYMHLSQLNVRVGDYISTGQ11GKSGSTGFSTGPHLHFQRMVGGLGNNYAQNPIPFLKQYG

SEQ ID NO: 2 AurR^317-410- - amino acid sequence of the CWT domain - the cell wall binding domain of AurR, which corresponds to 94 amino acids 317-410 of the sequence of the initial protein AurR:

STYKVDGKGTYYKAESASFTANYDIKTRLNGPFRSNPQSGVLHPGQTIKYDTVMKQDGHVWVVYTGYSGKRIYLPVR TWDKNSNTLGPLWGIIN

SEQ ID NO: 3 AurR^138-410 - the amino acid sequence of the two-domain active form of AurR, which corresponds to amino acids 158-410 of the AurR protein encoded by the KXA44996 gene: amino acids 1-138 correspond to the CD domain of AurR (SEQ ID NO: 1); the L-linker is made of 21 amino acids 139-159 of SEQ P) NO: 3, what corresponds to amino acids 196-316 of the sequence of the initial AurR protein - underlined and in italics, CWT - the cell wall binding domain of AurR is made of 94 amino acids 160-253 of SEQ ID NO: 3, what corresponds to amino acids 317-410 of the sequence of the initial AurR protein:

MEPYASAQWLTKYQLTAGYGHYNLNINNGMHYGADFAMPIGTPVRAITGGKIIEAGWSPYGGGNQIGVKEPDGSHYQ

WYMHLSQLNVRVGDYISTGQIIGKSGSTGFSTGPHLHFQRMVGGLGNNYAQNPIPFLKQYGYGSNTSGYTPPWWVg

APSTNSTYKVDGKGTYYKAESASFTANYDIKTRLNGPFRSNPQSGVLHPGQTIKYDTVMKQDGHVWVVYTGYSGKRI

YLPVRTWDKNSNTLGPLWGIIN SEQ ID NO: 4 AurR- - the amino acid sequence of the single-domain active form of AurR, which includes SEQ ID NO: 1 - here as AA from 4 to 141 with three amino acids remaining at the N- terminus after MBP is cut off from the vector - highlighted in grey, below:

SEQ ID NO: 5 AurR+ - the amino acid sequence of the two-domain active form of AurR from S. simulans. It comprises the AurR^158-410 sequence along with a fragment of the vector, which is made of amino acids 1 -3 at the N terminus - highlighted in grey, below:

Claims

1. Peptidoglycan hydrolase capable of digesting the walls of Gram(+) bacteria having both glycine and glycine-serine interpeptide bridges within peptidoglycan, comprising the active form of the AurR protein, which comprises CD^AurR as the catalytic domain, wherein the amino acid sequence of the CD^AurR catalytic domain is in at least 80% identical with the amino acid sequence shown in SEQ ID NO: 1, more preferably the CD^AurR sequence is in at least 85%, 90%, more preferably 95%, most preferably at the level of 98% or more identical with the amino acid sequence shown in SEQ ID NO: 1; wherein the interpeptide bridges consist of at least three glycines or glycines and serine/serines.

2. Peptidoglycan hydrolase according to claim 1, characterized in that the active form of the AurR protein is in the form of a single domain active form of AurR, comprising the CD^AurR catalytic domain, but not comprising the CWT^AurR bacterial cell wall binding domain.

3. Peptidoglycan hydrolase according to claims 1-2, characterized in that the active form of the AurR protein in the form of a single domain is the catalytic domain AurR^158'295 having the SEQ ID NO: 1.

4. Peptidoglycan hydrolase according to claims 1-2, characterized in that the active form of the AurR protein in the form of a single domain is AurR^' having the SEQ ID NO: 4.

5. Peptidoglycan hydrolase according to claim 1, characterized in that the active form of the AurR protein, is in the form of a two-domain active form of AurR, comprising the CD^AurR catalytic domain and the CWT^AurR bacterial cell wall binding domain, wherein the amino acid sequence of the CWT^AurR catalytic domain is in at least 80% identical with the amino acid sequence shown in SEQ ID NO: 2, more preferably the CWT^AurR sequence is in at least 85%, 90%, more preferably 95%, most preferably at the level of 98% or more identical with the amino acid sequence shown in SEQ ID NO: 2.

6. Peptidoglycan hydrolase according to claims 1 and 5, characterized in that the amino acid sequence of the CD^AurR catalytic domain is linked to the sequence of the CWT^AurR bacterial cell wall binding domain by the amino acid sequence of a linker, wherein the preferred sequence of the linker are the amino acids 139 -159 of the SEQ ID NO: 3.

7. Peptidoglycan hydrolase according to claims 1 and 5-6, characterized in that the active form of AurR in the two-domain form is AurR^158-410 having the SEQ ID NO: 3.

8. Peptidoglycan hydrolase according to claims 1 and 5-6, characterized in that the active form of AurR in the two-domain form is AurR+ having the SEQ ID NO: 5.

9. A genetic construct encoding a peptidoglycan hydrolase as defined in claims 1-8.

10. A composition for the proteolysis of cell walls of Gram(+) bacteria, characterized in that it comprises a peptidoglycan hydrolase as defined in claims 1-8 and a carrier, wherein the composition is intended for the proteolysis of cell walls of Gram(+) bacteria having glycine and/or glycine-serine interpeptide bridges within the peptidoglycan.

11. A pharmaceutical composition comprising a peptidoglycan hydrolase as defined in claims 1-8 and a pharmaceutically acceptable excipient for use as a medicament in the treatment of diseases and/or inflammations caused by Gram(+) bacteria having glycine and/or glycine-serine interpeptide bridges within the peptidoglycan, wherein, preferably, the Gram(+) bacteria are selected from the genus Staphylococcus, and preferably are S. aureus, S. simulans, S. epidermidis, S. intermedins, S. haemolyticus , S. warneri.

12. A pharmaceutical composition according to claim 11, characterized in that it is in the form of a liquid, emulsion, gel, spraying liquid, lotion, wet wipe or dressing, preferably the dressing is in the form of a plaster with a dressing, a plaster with a hydrogel, gauze with the composition applied, hydrocolloid dressing, hydrofibre dressing, an alginate dressing, a bandage with the composition applied.

13. A cosmetic or care composition for cosmetic, care and hygiene uses in humans and/or animals, characterized in that it comprises a peptidoglycan hydrolase as defined in claims 1-8, wherein the composition additionally comprises at least one carrier which is approved for cosmetic, care, and hygiene uses in humans and/or animals, and is intended for external use.

14. A composition comprising a peptidoglycan hydrolase as defined in claims 1-8 and a carrier for use as an antiseptic agent, antibacterial agent, an agent for disinfection of surfaces used against Gram(+) bacteria having glycine and/or glycine-serine interpeptide bridges within the peptidoglycan, wherein, preferably, the composition is used against Gram(+) bacteria of the genus Staphylococcus, preferably selected from S. aureus, S. simulans, S. epidermidis, S. intermedius, S. haemolyticus, S. warneri.

15. A composition according to claim 14 characterized in that it is the form of a liquid, emulsion, gel, spraying liquid, lotion, wet wipe.

16. The use of a peptidoglycan hydrolase as defined in claims 1-8 and/or a composition as defined in claim 10, for the proteolysis of cell walls of Gram(+) bacteria having glycine and/or glycine- serine interpeptide bridges within the peptidoglycan, wherein, preferably, the Gram(+) bacteria are bacteria of the genus Staphylococcus, preferably S. aureus, S. simulans, S. epidermidis, S. intermedius, S. haemolyticus, S. warneri.

17. Non-medical use of a peptidoglycan hydrolase as defined in claims 1-8 and/or a composition as defined in claim 10 as a bacteriostatic or bactericidal agent against Gram(+) bacteria having glycine and/or glycine-serine interpeptide bridges within the peptidoglycan, preferably of the genus Staphylococcus, preferably S. aureus, S. simulans, S. epidermidis, S. intermedius, S. haemolyticus, S. warneri.

18. The use according to claim 17, characterized in that the agent is used in the food industry, particularly as a food additive for humans and/or animals or for disinfecting surfaces and rooms that come into contact with food or food intermediates.

19. The use according to claims 17-18, characterized in that the agent is used in the dairy industry and milk products.

20. The use according to claim 17, characterized in that the agent is used in the cosmetics industiy, preferably as an additive to cosmetics improving their microbiological quality, preferably as an additive to liquids, creams, milks, lotions.

21. The use according to claim 17, characterized in that the agent is used in health care for decontamination of the surfaces of tools and instruments used in medicine and diagnostics, as well as other surfaces, in particular hospital and laboratory surfaces.

22. The use according to claim 17, characterized in that the agent is used as a bacteriostatic or bactericidal agent in the form of a liquid, emulsion, gel, spraying liquid, lotion, wet wipe.

23. A method of hydrolyzing peptidoglycans having glycine and/or glycine-serine interpeptide bridges, characterized in that it includes the following steps, in which a) the active form of AurR, as defined in claims 1-8, is contacted in an aqueous medium with a peptide substrate having glycine and/or glycine-serine interpeptide bridges within the peptidoglycan; b) the peptide substrate having glycine and/or glycine-serine interpeptide bridges within the peptidoglycan is being hydrolyzed.

24. A method of hydrolyzing peptidoglycans according to claim 23, characterized in that the peptidoglycans of the cell walls of Gram(+) bacteria, preferably of the genus Staphylococcus, preferably S. aureus, S. simulans, S. epidermidis, S. intermedius, S. haemolyticus, S. warneri, are hydrolyzed.

25. A method of hydrolyzing peptidoglycans according to claims 23-24, characterized in that the step b) is carried out at a temperature from 4 °C to 45 °C.