Recombinant protein and peptide with antimicrobial properties, compositions comprising them, uses and methods utilizing thereof

Abstract

The invention relates to a recombinant protein with antimicrobial properties, having an amino acid sequence with at least 80% identity with the sequence of the cell wall binding domain CBD_Sp_B from Staphylococcus pettenkoferi , as shown in SEQ. ID No. 1, a peptide with antimicrobial properties, compositions comprising them, methods of using thereof, and uses thereof as an antibacterial agent and / or antifungal agent, an antiseptic and disinfecting agent, an antimicrobial agent for disinfecting surfaces and rooms in food processing, an antimicrobial agent in the food industry, a disinfecting agent, an agent preventing and limiting the growth of microorganisms on surfaces, a plant protection agent, agent for preserving materials, an agent preventing biodeterioration by microorganisms, an in vitro antimicrobial agent with antimicrobial activity in the cosmetic industry, a veterinary and pharmaceutical composition for use as a drug in the prophylaxis, prevention and treatment of a disease and / or condition caused by microorganisms selected from bacteria and / or fungi.

Description

[0001] Recombinant protein and peptide with antimicrobial properties, compositions comprising them, uses and methods utilizing thereof

[0002] TECHNICAL FIELD

[0003] The object of the invention is a recombinant protein with antimicrobial properties, having an amino acid sequence with at least 80% identity with the sequence of the cell wall binding domain CBD_Sp_B from Staphylococcus pettenkoferi, as shown in SEQ. ID No. 1, a peptide having antimicrobial properties, compositions comprising them, methods of using thereof, and uses thereof as an antibacterial agent and / or antifungal agent, an antiseptic and disinfecting agent, an antimicrobial agent for disinfecting surfaces and rooms in food processing, an antimicrobial agent in the food industry, a disinfecting agent, an agent preventing and limiting the growth of microorganisms on surfaces, a plant protection agent, an agent for preserving materials, an agent preventing biodeterioration by microorganisms, an in vitro antimicrobial agent with antibacterial activity in cosmetic industry, as well as a veterinary and pharmaceutical composition for use as a drug in the prophylaxis, prevention and treatment of a disease and / or condition caused by microorganisms selected from bacteria and / or fungi.

[0004] A recombinant cell wall binding domain protein CBD_Sp_B with antimicrobial properties, constituting a fragment of the peptidoglycan hydrolase SpM23_B from Staphylococcus pettenkoferi is disclosed. To date, the only known function of CBD domains was binding to cell walls, due to which their accompanying catalytic domains can cleave bonds in peptidoglycans (PGs) more efficiently, thereby leading to bacterial cell lysis. Unexpectedly, it was found that the CBD domain from Staphylococcus pettenkoferi also showed strong antibacterial and antifungal properties. The contact of such a domain with bacterial cells, both Gram-positive and Gram-negative, causes their death. Similar effects are observed in relation to fungi. Moreover, an active fragment of the domain was identified, which, also as an isolated peptide, shows antimicrobial properties.

[0005] STATE OF ART

[0006] Due to the growing problem of antibiotic resistance and the lack of new antibiotics on the market, new ways of combating bacteria that would be effective against strains resistant to antibiotics, and which, at the same time, would not induce generation of resistance are being sought. A similar situation also takes place in the case of fungi, which can attack not only humans and animals, but also plants and even inanimate objects such as books, walls, sculptures or paintings. A variety of substances with strong biocidal properties are used to combat fungi responsible for infections in humans and animals, plant diseases, food spoilage and biodeterioration. The fungicides used are not always effective and, moreover, they can pose a threat to the environment. Unfortunately, their widespread use causes contamination of the environment and emergence of resistance, and thus leads to a reduction or even complete elimination of the effectiveness of their action. Along with the growth of the awareness of the side effects of using the existing antifungal agents, alternative, safer and more effective methods and replacement products are being sought.

[0007] The fight against infectious bacterial diseases is mainly based on the use of antibiotics in the treatment process and a number of different substances with antibacterial properties for preserving food or disinfecting surfaces. Antibiotics are very commonly used in agriculture, in animal breeding, also to prevent diseases and thus accelerate the growth of livestock. The widespread over-use of antibiotics and incorrect dosing thereof have led to the rapid spread of antibiotic resistance. According to WHO predictions, the number of deaths related to antibiotic resistance could increase to even 10 million per year.

[0008] In turn, the fight against fungal plant diseases is based on the use of fungicides, which can be very dangerous for animals and the environment. The introduction of the EC recommendation to reduce the amount of chemical pesticides by even 35-50% by 2030 makes it necessary to introduce safe, natural plant protection products. Products based on the activity of beneficial microorganisms (e.g. Serifel by BASF), which are active in the roots, soil and on the surface of plants, are already on the market, but their activity is mainly preventive. For example, Bacillus subtilis strains JB3, JB 3.6, R1 and their antibacterial activity are known from ATE71802T1.

[0009] Both bacteria and fungi can also lead to the so-called biodeterioration, or the destruction of various materials. Works of art are a special case of biodeterioration. Due to the value and sensitivity of the materials from which they were created, there is not a very wide choice of effective and, at the same time, safe methods to save valuable objects. The currently used agents are not always safe for the cultural property treated with them, nor for people and the environment. It is estimated that annual losses in all materials caused by fungi reach tens of billions of USD (Sterflinger, 2013). According to some Polish sources, losses due to biodeterioration can reach up to 10% of GDP.

[0010] Peptidoglycan hydrolases, including those from staphylococci, which consist of a catalytic domain and a cell wall binding domain (CBD) are known in the state of the art. CBD domains include various domain subtypes, including SH3b (Src Homology 3b) domains, which play a key role in the binding of enzymes to bacterial cell walls. Apart from the naturally occurring peptidoglycan hydrolases, in which a binding domain is present, many chimeras that contain this type of domain / domains have been generated. In some cases, these chimeras showed higher activity compared to the isolated catalytic domains, in others they were more tolerant to ionic conditions in the environment and the pH (Jagielska 2016). Such effects of the CBD domain in enzymes are explained by the increased affinity for the substrate (cell walls), which is the ligand of the binding domains. In the literature, the mechanism of action of the SH3b-binding domain of peptidoglycan hydrolase produced by Staphylococcus simulans has been described, which is the binding of bacterial cell walls, specifically pentaglycine cross-bridges present in peptidoglycans, what has been confirmed by detailed structural studies (Mitkowski 2019, Tossavainen 2018, Gonzales 2020).

[0011] Although the binding of CBD domains to bacterial cell walls has been demonstrated many times (Oingping, 2015; Ko On Lee, 2029), their antibacterial or antifungal activity has never been confirmed. In the publication by Loessner et al. (2002) an isolated SH3b domain was used in lytic activity assays as a control for the chimera comprising thereof. While a very high lytic activity was observed in the case of the chimera, the results for the SH3b domain were at the level of the control without the added protein.

[0012] In the publication by Wysocka et al. 2021, new peptidoglycan hydrolases SpM23_A and SpM3_B were characterized, however, this publication does not reveal antimicrobial properties of the cell wall binding domains.

[0013] Therefore, in the state of the art, there are no known binding domains occurring in peptidoglycan hydrolases that would exhibit antimicrobial properties, in particular, antibacterial and antifungal properties of the CBD cell wall binding domains were not known.

[0014] DISCLOSURE OF THE INVENTION

[0015] In light of the described state of the art, the aim of the present invention is to overcome the indicated disadvantages and to provide new substances with antimicrobial properties that will be useful for applications as antibacterial and antifungal agents.

[0016] The invention relates to a fragment of peptidoglycan hydrolase, in particular a recombinant protein that is the CBD_Sp_B domain binding to the cell walls of microorganisms, as well as the CBD_Sp_Bpep peptide isolated from it, having antimicrobial properties, that very effectively eliminates bacteria and fungi.

[0017] Moreover, the invention also relates to the diverse use of the recombinant cell wall binding domain protein CBD_Sp_B, as well as the CBD_Sp_Bpep peptide isolated from it, with antimicrobial properties, as an antibacterial and antifungal agent.

[0018] The essence of the invention is a recombinant protein with antimicrobial properties, which has an amino acid sequence of at least 80% identity with the sequence of the cell wall binding domain CBD_Sp_B from Staphylococcus pettenkoferi, as shown in SEQ. ID No. 1. Preferably, the recombinant protein has an amino acid sequence with at least 85%, more preferably 90%, more preferably 95%, more preferably 99% identity with the sequence shown in SEQ ID No. 1; most preferably, it is the CBD_Sp_B domain from Staphylococcus pettenkoferi having the amino acid sequence SEQ ID No. 1.

[0019] The recombinant protein with antimicrobial properties preferably shows antibacterial and / or antifungal activity.

[0020] The invention also relates to a peptide with antimicrobial properties, which has an amino acid sequence with at least 80% identity with the sequence of the active fragment of the cell wall binding domain of CBD_Sp_Bpep from Staphylococcus pettenkoferi, as shown in SEQ ID No. 9.

[0021] Preferably, the peptide with antimicrobial properties is a peptide with an amino acid sequence with at least 85%, more preferably 90%, more preferably 95%, more preferably 99% identity with the sequence shown in SEQ ID No. 9; most preferably, it is the CBD_Sp_Bpep peptide with antimicrobial properties with the amino acid sequence of SEQ ID No. 9.

[0022] Preferably, the peptide shows an antibacterial and / or antifungal activity.

[0023] The invention also relates to a genetic construct encoding a recombinant protein with antimicrobial properties according to the invention and / or a peptide with antimicrobial properties according to the invention.

[0024] The invention also relates to a host cell, which comprises a genetic construct according to the invention.

[0025] The invention also relates to a composition comprising a recombinant protein with antimicrobial properties according to the invention and / or a peptide with antimicrobial properties according to the invention or a mixture thereof and a carrier.

[0026] The composition is preferably in the form of a solution, tablet, powder, granulate, suspension, emulsion, aerosol, gel, suppositories, vaginal suppositories, ointment, cream, cosmetic powder.

[0027] The composition is preferably intended for use as an antibacterial agent and / or antifungal agent.

[0028] The composition is preferably intended for use as an antibacterial agent against Gram(+) and / or Gram(-) bacteria, preferably against bacteria of the genus Listeria spp., Escherichia spp., Pseudomonas spp., Yersinia spp., Staphylococcus spp., Enterococcus spp., Streptococcus spp., more preferably against the species Listeria monocytogenes, Escherichia coli, Pseudomonas aeruginosa, Yersinia ruckeri, Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus simulans, Enterococcus faecium, Enterococcus faecalis, Streptococcus agalactiae, Streptococcus dysgalactiae, Streptococcus uberis, Streptococcus canis, Streptococcus egui. The composition is preferably intended for use as an antifungal agent against fungi and lichens -more preferably against single- and / or multi-cellular fungi, preferably against fungi from yeasts and Leotiomycetes classes, more preferably against fungi from the genera Candida ssp., Saccharomyces ssp., Phytophthora spp. and Botritis spp., more preferably against the species Candida albicans, Saccharomyces cerevisiae, Botritis cinerea.

[0029] The invention also relates to the non-medical use of a recombinant protein with antimicrobial properties according to the invention and / or a peptide with antimicrobial properties according to the invention and / or a composition according to the invention as an antibacterial agent and / or an antifungal agent.

[0030] In a preferred non-medical use, the antibacterial agent is used against Gram (+) and / or Gram (-) bacteria, preferably bacteria of the genus Listeria spp., Escherichia spp., Pseudomonas spp., Yersinia spp., Staphylococcus spp., Enterococcus spp., Streptococcus spp., more preferably against the species Listeria monocytogenes, Escherichia coli, Pseudomonas aeruginosa, Yersinia ruckeri, Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus simulans, Enterococcus faecium, Enterococcus faecalis, Streptococcus agalactiae, Streptococcus dysgalactiae, Streptococcus uberis, Streptococcus canis, Streptococcus egui.

[0031] In a preferred non-medical use, the antifungal agent is used against fungi and lichens, more preferably against single- and / or multi-cellular fungi, preferably against fungi from the classes of yeasts and fungi, more preferably against fungi from the genera Candida ssp., Saccharomyces ssp., Phytophthora spp. and Botritis spp., more preferably against the species Candida albicans, Saccharomyces cerevisiae, Botritis cinerea.

[0032] The invention also relates to the non-medical use of a recombinant protein with antimicrobial properties according to the invention and / or a peptide with antimicrobial properties according to the invention and / or a composition according to the invention as an in vitro antimicrobial agent with antibacterial and / or antifungal activity for inhibiting, eliminating and preventing the growth of microorganisms, wherein the microorganisms are preferably selected from bacteria, fungi and lichens.

[0033] The invention also relates to the use of a recombinant protein with antimicrobial properties according to the invention and / or a peptide with antimicrobial properties according to the invention and / or a composition according to the invention as an antiseptic and disinfectant.

[0034] The invention also relates to the use of a recombinant protein with antimicrobial properties according to the invention and / or a peptide with antimicrobial properties according to the invention and / or a composition according to the invention as an antimicrobial agent for disinfecting surfaces and rooms in food processing, preferably for disinfecting surfaces and / or tools that come into contact with food or semi-finished food products, preferably as an antimicrobial agent for disinfecting surfaces and rooms with antibacterial and / or antifungal activity. The invention also relates to the use of a recombinant protein with antimicrobial properties according to the invention and / or a peptide with antimicrobial properties according to the invention and / or a composition according to the invention as an antimicrobial agent in the food industry as an additive to human and / or animal food, preferably as a food additive, preferably as an antimicrobial agent with antibacterial and / or antifungal activity.

[0035] In a preferred use as an antimicrobial agent in the food industry, the agent is used to prevent infections and diseases caused by microorganisms in food, preferably listeriosis caused by Listeria monocytogenes.

[0036] The invention also relates to the use of a recombinant protein with antimicrobial properties according to the invention and / or a peptide with antimicrobial properties according to the invention and / or a composition according to the invention as a disinfectant for surfaces in health care in medicine, veterinary medicine, diagnostics, preferably for disinfecting hospital surfaces, medical and veterinary offices, laboratory surfaces, tool surfaces, surfaces of devices used in medicine, veterinary medicine, diagnostics.

[0037] The invention also relates to the use of a recombinant protein with antimicrobial properties according to the invention and / or a peptide with antimicrobial properties according to the invention and / or a composition according to the invention as an agent preventing and limiting the growth of microorganisms on the surfaces of devices used in medicine, veterinary medicine and diagnostics, preferably by contacting the surface with the agent, by covering the surface with the agent, wherein, preferably, the surfaces covered are the surfaces of catheters, implants, cannulae, endotracheal tubes.

[0038] The invention also relates to the use of a recombinant protein with antimicrobial properties according to the invention and / or a peptide with antimicrobial properties according to the invention and / or a composition according to the invention as a plant protection agent, wherein the microorganisms are preferably selected from bacteria, fungi and lichens

[0039] In the preferred use, the plant protection agent is applied to the plant material by immersing (dipping), spraying, pouring, dripping or misting (fogging) the plant material with the plant protection agent.

[0040] The invention also relates to the use of a recombinant protein with antimicrobial properties according to the invention and / or a peptide with antimicrobial properties according to the invention and / or a composition according to the invention as a preservative for materials for preserving the material by inhibiting and preventing the growth of microorganisms in / on the material, wherein the microorganisms are preferably selected from bacteria, fungi and lichens

[0041] In the preferred use, the material preservative is in the form of a paint, a liquid for application, a spray, a gel, a layer applied to the material, a liquid for immersing the material therein, an admixture to the material, a primer for the material, an adhesive for the material, a resin, a filler, a stain, a wax, a binder connecting the material. In a preferred use, the preserved material is selected from fabric, non-woven fabric, paper, canvas, painting support, painting loom, tissue paper, rocks, leather, wood, ceramics, metal, metal alloy, glass, plastic, cement, concrete.

[0042] The invention also relates to the use of a recombinant protein with antimicrobial properties according to the invention and / or a peptide with antimicrobial properties according to the invention and / or a composition according to the invention as an agent preventing biodeterioration by microorganisms, wherein the microorganisms are preferably selected from bacteria, fungi and lichens

[0043] In the preferred use, the agent preventing biodeterioration is used to prevent biodeterioration of fabrics, non-woven fabrics, paper, canvas, painting support, painting loom, tissue paper, rock, leather, wood, ceramics, metal, metal alloys, glass, plastic, cement, concrete, in particular books, old prints, paintings, sculptures, frescoes, tapestries.

[0044] The invention also relates to the use of a recombinant protein with antimicrobial properties according to the invention and / or a peptide with antimicrobial properties according to the invention and / or a composition according to the invention as an in vitro antimicrobial agent with antibacterial activity in the cosmetics industry, as an additive to cosmetics improving their microbiological quality, preferably as a biopreservative, preferably as an additive to liquids, creams, milks, lotions, mists, gels.

[0045] The invention also relates to a veterinary composition, which comprises a protein with antimicrobial properties according to the invention and / or a peptide with antimicrobial properties according to the invention and / or a composition according to the invention and a veterinary acceptable carrier for use as a drug for the prophylaxis, prevention and treatment of a disease and / or condition caused by microorganisms selected from bacteria and / or fungi.

[0046] The veterinary composition for use is preferably used in the prophylaxis, prevention and treatment of bacterial and / or fungal infections, it is preferably used in the treatment of mixed bacterial and fungal infections, in particular superficial infections, preferably skin infections.

[0047] The veterinary composition for use is preferably used against Gram(+) and / or Gram(-) bacteria, preferably against bacteria of the genus Listeria spp., Escherichia spp., Pseudomonas spp., Yersinia spp., Staphylococcus spp., Enterococcus spp., Streptococcus spp., more preferably against the species Listeria monocytogenes, Escherichia coli, Pseudomonas aeruginosa, Yersinia ruckeri, Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus simulans, Enterococcus faecium, Enterococcus faecalis, Streptococcus agalactiae, Streptococcus dysgalactiae, Streptococcus uberis, Streptococcus canis, Streptococcus egui.

[0048] The veterinary composition for use is preferably used against single- and / or multi-cellular fungi, preferably fungi from yeasts and Leotiomycetes classes, more preferably fungi from the genera Candida ssp., Saccharomyces ssp., Phytophthora spp. and Botritis spp., more preferably species Candida albicans, Saccharomyces cerevisiae, Botritis cinerea.

[0049] The veterinary composition for use is preferably used in the prophylaxis, prevention, treatment of superficial skin and / or wound infections, infected wounds, pressure sores, dermatoses, diabetic foot infections, burns, mastitis, vaginal infections, external ear infections, nail fungus, skin fungus.

[0050] The invention also relates to a pharmaceutical composition, which comprises a protein with antimicrobial properties according to the invention and / or a peptide with antimicrobial properties according to the invention and / or a composition according to the invention and a pharmaceutically acceptable carrier for use as a drug for the prophylaxis, prevention and treatment of a disease and / or condition caused by microorganisms selected from bacteria and / or fungi.

[0051] The pharmaceutical composition for use is preferably used in the prophylaxis, prevention and treatment of bacterial and / or fungal infections, preferably in the treatment of mixed bacterial-fungal infections, in particular superficial infections, preferably skin infections.

[0052] The pharmaceutical composition for use is preferably used against Gram(+) and / or Gram(-) bacteria, preferably against bacteria of the genus Listeria spp., Escherichia spp., Pseudomonas spp., Yersinia spp., Staphylococcus spp., Enterococcus spp., Streptococcus spp., more preferably against the species Listeria monocytogenes, Escherichia coli, Pseudomonas aeruginosa, Yersinia ruckeri, Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus simulans, Enterococcus faecium, Enterococcus faecalis, Streptococcus agalactiae, Streptococcus dysgalactiae, Streptococcus uberis, Streptococcus canis, Streptococcus egui.

[0053] The pharmaceutical composition for use is preferably used against single- and / or multi-cellular fungi, preferably fungi from yeasts and Leotiomycetes classes, more preferably fungi from the genera Candida ssp., Saccharomyces ssp., Phytophthora spp. and Botritis spp., more preferably species Candida albicans, Saccharomyces cerevisiae, Botritis cinerea.

[0054] The pharmaceutical composition for use is preferably used for the prophylaxis, prevention, treatment of superficial skin and / or wound infections, infected wounds, pressure sores, dermatoses, diabetic foot infections, burns, mastitis, vaginal infections, external ear infections, nail fungus, skin fungus.

[0055] The invention also relates to a cosmetic or care composition for cosmetic, care or hygiene uses for human or animal, which comprises a protein with antimicrobial properties according to the invention and / or a peptide with antimicrobial properties according to the invention and / or a composition according to the invention, wherein the composition is intended for external use, and wherein the recombinant protein with antimicrobial properties, the peptide with antimicrobial properties is used in the cosmetic or care composition to limit the occurrence of or eliminate microorganisms from the composition, wherein the microorganisms are preferably selected from bacteria and / or fungi. The invention also relates to a method for inhibiting or reducing or preventing the growth of microorganisms in vitro, wherein the method comprises the step of contacting the microorganism with a protein with antimicrobial properties according to the invention and / or a peptide with antimicrobial properties according to the invention and / or a composition according to the invention, wherein the microorganisms are preferably selected from bacteria, fungi and lichens.

[0056] In the method, the bacteria are preferably selected from Gram(+) and / or Gram(-) bacteria, preferably from bacteria of the genus Listeria spp., Escherichia spp., Pseudomonas spp., Yersinia spp., Staphylococcus spp., Enterococcus spp., Streptococcus spp., more preferably from bacteria of the species Listeria monocytogenes, Escherichia coli, Pseudomonas aeruginosa, Yersinia ruckeri, Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus simulans, Enterococcus faecium, Enterococcus faecalis, Streptococcus agalactiae, Streptococcus dysgalactiae, Streptococcus uberis, Streptococcus canis, Streptococcus egui.

[0057] In the method, the fungi are preferably selected from single- and / or multi-cellular fungi, preferably fungi from yeasts and Leotiomycetes classes, more preferably fungi from the genera Candida ssp., Saccharomyces ssp., Phytophthora spp. and Botritis spp., more preferably species Candida albicans, Saccharomyces cerevisiae, Botritis cinerea.

[0058] The invention therefore relates to solutions based on the CBD cell wall binding domain from Staphylococcus pettenkoferi, and more specifically to a recombinant protein that is a functional microbial cell wall binding domain CBD_Sp_B and its derivative, as well as the CBD_Sp_Bpep peptide and its derivative isolated from it, with antimicrobial properties, that very effectively eliminate bacteria and fungi.

[0059] Additionally, the invention also relates to the diverse use of the recombinant cell wall binding domain protein CBD_Sp_B and the peptide CBD_Sp_Bpep isolated from it, with antimicrobial properties, as an antibacterial and antifungal agent in their practical applications.

[0060] DETAILED DESCRIPTION OF THE INVENTION

[0061] The cell wall binding domain CBD_Sp_B from peptidoglycan hydrolase produced by Staphylococcus pettenkoferi, with antimicrobial properties, has been disclosed. This domain accompanies the M23 catalytic domain, which is assumed to be responsible for the lytic activity of bacteria. Interestingly, the CBD_Sp_B domain exhibits antibacterial properties not by lysing bacteria, but probably by destabilizing the bacterial cell membrane, which results in death of cells. A similar effect is observed in the case of its antifungal activity. Unexpectedly, it was found that as little as nanomolar amounts of the CBD_Sp_B binding domain eliminate millions of microbial cells within several dozen minutes. The CBD_Sp_B domain is effective against both Gram(+) and Gram(-) bacteria. The recombinant protein according to the invention also acts against all the fungi tested so far: Candida albicans and Saccharomyces cerevisiae (representatives of the fungi class of yeasts) and Phytophthora and Botritis cinerea (fungi from the Leotiomycetes class), which suggests a broad spectrum of antifungal activity. This is a very unexpected and at the same time extremely desirable property that can find various applications: for example, to protect humans, animals and plants from diseases caused by bacteria and fungi, and inanimate objects from biocorrosion.

[0062] To verify whether these properties are unique to the CBD_Sp_B domain or also apply to other binding domains found in peptidoglycan hydrolases, three other CBD domains from different peptidoglycan hydrolases were tested. It turned out that only the CBD_Sp_B domain showed strong antimicrobial properties (against both bacteria and fungi), while the other domains did not show such properties.

[0063] Therefore, the broad spectrum of antimicrobial activity of the recombinant cell wall binding domain protein CBD_Sp_B from Staphylococcus pettenkoferi, and in particular its active fragment in the form of the antimicrobial peptide CBD_Sp_Bpep, is an unknown and surprising property, which provides new possibilities for their wide application, either alone or in the form of compositions comprising them as antimicrobial agents acting against both bacteria and fungi. Their use in medicine in the treatment or prevention of mixed bacterial and fungal infections may be particularly important.

[0064] It was also confirmed that the use of preparations comprising the CBD_Sp_B domains and the CBD_Sp_Bpep peptide will be safe for plants, and therefore it was shown that it will be possible to use them as potential universal antimicrobial agents for plant protection with antibacterial and antifungal activity.

[0065] Whenever the term "comprising" or "including" is used in this description and claims, it does not exclude other elements. For the purposes of the present invention, the term "consisting of" is considered to be a preferred embodiment of the term "comprising" and "including". If a group is defined below that comprises at least a certain number of embodiments, this is also to be understood as disclosing a group that preferably consists of only those embodiments.

[0066] The terms "about" or "approximately" in the context of the present invention indicate a range of precision that a person skilled in the art would understand to be sufficient to still provide the technical effect of a given feature. The term "typically" indicates a deviation from the indicated numerical value of ±10%, and preferably ±5%.

[0067] BRIEF DESCRIPTION OF THE FIGURES OF DRAWING

[0068] The publications cited in this description and the references given therein are hereby incorporated by reference in their entirety. For a better understanding of the invention, it is illustrated in the embodiments and in the attached figures, in which:

[0069] Fig. 1. presents a picture of SDS-PAGE separation of the highly purified CBD_Sp_B domain protein. Protein sizes are presented in kDa.

[0070] Fig. 2. presents the results of the study of the antibacterial activity of the CBD_Sp_B domain depending on the concentration towards the selected bacterial strains (106CFU / ml). A- Escherichia coli DSM 1103, B- Listeria monocytogenes DSM 19094, C- Staphylococcus aureus NCTC 8325-4, D- Yersinia ruckeri CCM 4620, E- Klebsiella pneumoniae DSM 789, F- Pseudomonas aeruginosa DSM 939.

[0071] Fig. 3. presents a SDS-PAGE analysis of the purified CBD domains: CBD_Ss_L, CBD_Sp_A i CBD_Ss_R.

[0072] Fig. 4. presents the results of the study of the antibacterial activity of the CBD_Ss_L domain depending on the concentration towards the selected bacterial strains. (106CFU / ml). A- Escherichia coli DSM 1103, B- Listeria monocytogenes DSM 19094, C- Staphylococcus aureus NCTC 8325-4, D- Yersinia ruckeri CCM 4620, E- Klebsiella pneumoniae DSM 789, F- Pseudomonas aeruginosa DSM 939.

[0073] Fig. 5. presents the results of the reduction of the number of microorganisms expressed in orders of magnitude for proteins - different isolated and purified CBD domains at the concentration of 0.5 pM after 2-hour incubation the with selected bacterial strains (106CFU / ml).

[0074] Fig. 6. presents the results of the activity test of the different isolated and purified CBD domains on the Candida albicans ATCC 10231 fungal strain (105CFU / ml) used at the concentration of 0.5 pM and 5 pM after 2-hour incubation.

[0075] Fig. 7. presents the results of treatment of the fungus Hyphopichia burtonii with 5 pM solution of the CBD_Sp_B protein for 2 hours. CFU number- initial cell count, upper panel - control without the protein, lower panel - fungus treated with the CBD_Sp_B protein.

[0076] Fig. 8. presents: (A) a comparison of CBD_Sp_B and CBD_Ss_L sequences, the sequence of the CBD_Sp_Bpep peptide marked with a box; (B) the results of the antibacterial activity test of the CBD_Sp_Bpep peptide performed on Escherichia coli DSM 1103 (107CFU / ml) depending on the concentration.

[0077] Fig. 9. presents the results of the activity test of the recombinant peptide CBD_Sp_Bpep performed on Escherichia coli DSM 1103 (107CFU / ml) at different concentrations over time, after 1.5 and 3 hours of incubation.

[0078] Fig. 10. presents the results of safety tests conducted for the CBD_Sp_B domain. Germination test of (A) basil and (B) radish seeds. The number and quality of seeds germinated in the presence of 5 pM aqueous suspension of CBD_Sp_B did not differ from the control (germinated in the presence of water, dF O). (C) Toxicity test conducted on Galleria mellonella moth larvae. Injection of 20 pL of CBD_Sp_B with the concentration of 0.5 pM. Control larvae (c) were injected with 20 pL water. Developmental stages of G.mellonella: L - larvae, K - cocoon, P - pupae; nz - dead individuals. (D) Acute toxicity test performed on zebrafish embryos. CBD_Sp_B was added directly to water (immersion treatment). Normal morphology was observed 96 hours post-fertilization (hpf) in embryos treated with 500 nM CBD_Sp_B as compared to controls. The mock control received only the buffer in which CBD_Sp_B was stored. Images show lateral views; scale bar = 1 mm.

[0079] Fig. 11. presents the results of the test demonstrating the effectiveness of the selected proteins in decontamination of various surfaces contaminated with Listeria monocytogenes DSM 19094. Panel A - glass, panel B - silicone; C - control; 1 - CBD_Sp_Bpep, 2 - CBD_Sp_B.

[0080] EMBODIMENTS OF THE INVENTION

[0081] The following examples are provided for the purpose of illustrating the invention and explaining particular aspects thereof, and are not intended to be limiting and should not be construed with its entire scope, which is defined by the appended claims. Unless otherwise indicated, standard materials and methods have been used in accordance with the manufacturers' specifications.

[0082] Technical terms are used in accordance with their typical meaning. If a specific meaning is used, the meaning will be described in details with reference to the context, in which the term is used.

[0083] EXAMPLES

[0084] I.

[0085] Example 1. Cloning and production of the CBD Sp B domain from Staphylococcus pettenkoferi.

[0086] An expression vector containing the coding sequence of the cell wall binding domain of peptidoglycan hydrolase CBD_Sp_B (SEQ ID NO: 2) from Staphylococcus pettenkoferi was introduced into the E. coli bacterial strain BL21(DE3) and subjected to antibiotic selection. The production of the recombinant protein CBD_Sp_B domain (SEQ ID NO: 1) was carried out for about ~16 h at 18°C. After centrifugation and lysis of the cells, the supernatant comprising the produced protein was dialyzed and then the protein was purified using ion exchange chromatography on WB40S resin (Bio-Works, Sweden) and gel filtration on Superdex75 resin (GE Healthcare, USA) in 20 mM Tris-HCI, 200 mM NaCI, 10% glycerol pH 8.0 buffer. Purified CBD_Sp_B domain protein was separated by SDS-PAGE and visualized by Coomassie staining (Fig.l), which showed the appropriate purity and assumed size of the produced protein - CBD_Sp_B domain. The produced CBD_Sp_B domain protein was then used in further experiments and tests presented in the following examples. Example 2. Antibacterial activity studies of the CBD Sp B domain.

[0087] The antibacterial properties of the CBD_Sp_B domain were determined by the decrease in the initial number of bacterial cells (106CFU / ml) after treatment with the protein. 16 bacterial strains were used in the tests (Tab. 1). After adding the CBD_Sp_B solution to bacteria (106CFU / ml) to a final concentration of 0.0625 to 2 pM , the incubation was carried out at room temperature for 60 minutes. After this time, the number of living cells was determined by plating serial dilutions of the tested samples on TSB agar medium (Graso Biotech). Bacterial cells not treated with the protein was used as a control. The obtained results are illustrated in Fig. 2. As can be seen, the addition of the CBD_Sp_B domain at the concentration of 0.25 pM causes complete elimination of bacterial cells of all the tested strains.

[0088] Tab. 1. Summary of the antibacterial activity tests of the CBD_Sp_B domain on 16 Gram (+) / (-) bacterial strains. "+" indicates that the domain activity resulted in the elimination of all bacteria in the mixture (100% efficiency).

[0089] Example 3. Cloning and generation of other CBD-type binding domains.

[0090] To determine whether the properties observed for the CBD_Sp_B domain are unique to this domain or represent a general feature of this class of domains, three additional CBD domains were generated: CBD_Ss_L, CBD_Sp_A, and CBD_Ss_R. The corresponding proteins were expressed and purified for further analysis. The amino acid sequences of each domain are listed in Tab. 2. Nucleotide sequences encoding the CBD_Ss_L (SEQ. ID NO: 4), CBD_Sp_A (SEQ. ID NO: 6), and CBD_Ss_R (SEQ. ID NO: 8) domains were inserted into pET expression vectors. The correctness of the sequence was verified by sequencing. The obtained expression vectors were introduced into E. coli BL21(DE3) bacteria. The CBD_Ss_L, CBD_Sp_A and CBD_Ss_R proteins were first purified on ion exchange columns and then by gel filtration, similarly as described for the CBD_Sp_B domain protein in Example 1.

[0091] The obtained purified and concentrated protein preparations of CBD domains from different organisms were subjected to SDS-PAGE analysis (Fig. 3).

[0092] Tab. 2. The generated CBD domains.

[0093] Example 4. The test of antibacterial activity of the CBD Ss L domain.

[0094] To evaluate whether the properties of the CBD_Sp_B domain are unique, comparative tests were conducted using the CBD_Ss_L domain derived from lysostaphin produced by Staphylococcus simulans (SEQ ID NO: 3). This domain shares approximately 70% sequence identity with CBD_Sp_B (see Tab. 3). The assays were performed as described in Example 2, using the bacterial strains listed in Tab. 1. CBD_Ss_L was added to bacterial suspensions (106CFU / mL) at final concentrations ranging from 0.0625 to 2 pM, followed by a 1-hour incubation. After treatment, serial dilutions were plated on appropriate microbiological media to quantify viable cells. CBD_Ss_L domain did not exhibit significant bactericidal activity, even at the highest tested concentration (2 pM) (Fig. 4). In contrast, CBD_Sp_B reduced bacterial counts by 100% at 2 pM, demonstrating superior antibacterial efficacy.

[0095] Example 5. Antibacterial properties of CBD domains.

[0096] Antibacterial activity tests were also performed for other domains indicated in Tab. 2 on the following 10 strains of both Gram(-) and Gram(+) bacteria: Escherichia coll DSM 1103, Klebsiella pneumoniae DSM 789, Pseudomonas aeruginosa DSM 939, Yersinia ruckeri CCM 4620, Staphylococcus aureus NTCT 8325-4, Enterococcus faecium DSMZ2146, Enterococcus faecalis DSM 20376, Streptococcus canis DSM 20715, Listeria monocytoegnes DSM 19094, Bacillus subtilis DSM 10. The cell suspension (106CFU / ml) suspended in glycine buffer (50 mM glycine-NaOH, pH 8.0) was incubated in the presence of 0.5 pM protein for 2 hours at room temperature. Subsequently, 10-fold dilutions of the samples were prepared and plated on TSB-agar plates using the so-called drop dilution assay . Here, bacterial cells treated with glycine buffer were used as a control. The enzyme activity was determined based on the number of live cells (Fig. 5). Among the tested domains, only the CBD_Sp_B domain demonstrated potent antibacterial activity, resulting in at least a 4-log reduction in viable bacteria. No significant antimicrobial effects were observed for the other domains under the conditions tested.

[0097] Example 6. Antifungal properties of CBD domains

[0098] The antifungal properties of all the tested domains indicated in Tab. 2 were tested on the Candida albicans ATCC 10231 strain. The strain was cultivated on Sabouraud agar medium at 30°C for 48 h under aerobic conditions. Subsequently, a suspension of yeast colonies was prepared in water with a density of 0.5 McFarland, which corresponds to 105CFU / ml. The cells prepared in this way were incubated with individual domains at a concentration of 0.5 or 5 pM. After 2 h, the samples were serially diluted and 5pl of each suspension was plated on Sabouraud agar plate. In the control samples, cells were treated with glycine buffer alone (Fig. 6).

[0099] Moreover, the antifungal efficacy of the CBD_Sp_B domain was tested on filamentous fungi of the genera Phytophthora and Botritis.

[0100] The antifungal properties of the CBD_Sp_B domain were also tested on the yeast Hyphopichia burtonii. H. burtonii culture was maintained on solid Sabouraud-2% dextrose medium (VWR, BDH Chemicals), at room temperature, protected from light. The yeast suspension was prepared by pouring 2 ml of water over the plate and mixing gently by pipetting. For the experiment, the initial suspension with an optical density of 1.0 McFarland (corresponding to approx. 2x10scells in a mililiter of suspension) and its subsequent dilutions were prepared. The cells prepared in this way were mixed (1:1 v:v) with 10 pM CBD_Sp_B and then incubated (in Eppendorf tubes or multi-well plates) for two or 20-22 hours at room temperature, protected from light. Then, 50 pl of each suspension was plated and left for at least 2 days (under the conditions as above). The reading of the number of growing colonies taken after this time indicated a decrease in the number of yeast cells treated with CBD_Sp_B by four orders of magnitude (99.99% mortality), which confirms the antifungal properties of this protein. The obtained results are presented in Fig. 7.

[0101] Example 7. Preparation of the CBD Sp Bpep peptide having antibacterial properties.

[0102] As demonstrated in Example 5, the CBD_Sp_B domain exhibited significantly higher antibacterial activity compared to other tested domains, including the CBD_Ss_L domain derived from lysostaphin. This finding was particularly surprising given the high amino acid sequence identity (~70%) shared between these proteins (see Tab. 3). Comparative sequence analysis revealed a specific region at the C-terminus with lower similarity, enriched in basic (K) and aliphatic (A, P) residues (Fig. 8A).

[0103] To further investigate this region, the sequence of the CBD_Sp_B domain was analyzed using the ClassAmp algorithm (Joseph et al., 2012), which predicts antimicrobial properties of protein sequences and peptides. The analysis identified a specific fragment— peptide 61-GYGSKKNRKYVPIRTWNRNTNAMG- 84 (SEQ ID NO: 9), hereafter referred to as CBD_Sp_Bpep— as having the highest predicted antibacterial potential, including bactericidal activity.

[0104] This peptide (CBD_Sp_Bpep) was chemically synthesized (GenScript) and subjected to antibacterial activity testing. Escherichia coli DSM 1103 cells (107CFU / mL) were incubated with increasing concentrations of the peptide, followed by serial dilution and plating. The results (Fig. 8B) confirmed that CBD_Sp_Bpep exhibited strong antibacterial activity. However, approximately five times more peptide was required to achieve the same bactericidal effect as the full-length CBD_Sp_B domain (SEQ ID NO: 1).

[0105] Specifically, 2.5 pM of CBD_Sp_Bpep was sufficient to completely eradicate E. coli cells (107CFU / mL) within 1 hour of incubation.

[0106] Example 8. Production of the CBD Sp Bpep peptide in an expression system.

[0107] The expression construct pET28-SUMO-6xHis with an insert encoding CBD_Sp_Bpep (SEQ ID NO: 10) was transformed into E. coli BL21-Gold (DE3) bacteria. Cultures were grown until ODgoo = 0.8-1.0, and peptide overproduction was induced with 0.4 mM isopropyl-p-D-thiogalactopyranoside (IPTG) and grown overnight at 16°C. The cultures were centrifuged and the bacterial pellet was suspended in buffer A (300 mM NaCI, 25 mM Tris pH 8.0, 10% glycerol, 20 mM imidazole), sonicated and the obtained lysate was clarified by centrifugation. The supernatant was applied by gravity onto a column of complete His-Tag Purification Resin (Roche, Merck) equilibrated with buffer A. Nonspecifically bound proteins were washed from the column with buffer A, and proteins bound to the column were eluted with buffer B (300 mM NaCI, 25 mM Tris pH 8.0, 10% glycerol, 500 mM imidazole). To remove the SUMO-6xHis tag, SUMO protease was added to the protein solution. After overnight digestion at 4°C, the tag-free protein was concentrated on an Amicon Ultra 3K filter (Merck, Germany). The preparation was applied to a Superdex 75 16 / 600 column, equilibrated with S75 buffer (100 mM NaCI, 25 mM Tris pH 8.0, 10% glycerol) to remove remaining contaminants and exchange the buffer. Protein purity was analyzed on a tricine gel (SDS-PAGE) and analyzed using mass spectrometry. The purified fractions were concentrated, freezed using liquid nitrogen and stored at -80°C. The purified preparation of the recombinant CBD_Sp_Bpep peptide, produced as described in Example 8, was tested for antibacterial activity using the method outlined in Example 7. The results of these assays are presented in Fig. 9. The experiment confirmed the high antibacterial efficacy of the recombinant peptide. Notably, a concentration of 1.5 pM was sufficient to eliminate 99% of Escherichia coli DSM 1103 cells within 1.5 hours of incubation. These findings demonstrate that the recombinant CBD_Sp_Bpep peptide, produced in Escherichia coli BL21 (DE3), exhibits antibacterial activity comparable to that of the chemically synthesized peptide.

[0108] Example 9. Safety of using the CBD Sp B domain and the CBD Sp Bpep peptide.

[0109] To verify whether the produced antimicrobial protein (protein with antimicrobial properties) is safe for plants, 20 basil seeds or 16 radish seeds were placed in a Petri dish and treated with 300 pl of an aqueous solution of CBD_Sp_B at a concentration of 5 pM. Seeds treated with pure water were used as a control. No significant differences were observed in the rate and number of germinating seeds or seedling morphology - see Fig. 10A for basil and Fig. 10B for radish. It was therefore confirmed that the treatment of seeds with an aqueous solution of CBD_Sp_B has no negative effect on the germination and development of dicotyledonous seedlings.

[0110] Thus, it has been shown that preparations containing the recombinant protein domain CBD_Sp_B or peptides derived from it according to the invention will be safe for plants.

[0111] Another safety test was conducted using the Galleria mellonella moth model. The last larval stage of G. mellonella was used for the tests. 20 pL of an aqueous suspension of CBD_Sp_B at a concentration of 0.5 or 5 pM was injected into the abdominal segment at the last pair of non-segmented legs (posteriors) of G. mellonella larvae. Control larvae were injected with 20 pL of water. In larvae treated with 0.5 pM CBD_Sp_B, no negative effect of CBD_Sp_B on the development and survival of G. mellonella was observed (Fig. 10C).

[0112] The acute toxicity of CBD_Sp_B was also tested on zebrafish according to OECD test no. 246 using zebrafish (Danio rerio) embryos. Similarly as for G. mellonella, CBD_Sp_B at the concentration of 0.5 pM has no effect on the development and survival of D. rerio (Fig. 10D).

[0113] Example 10. Application of the CBD Sp B domain and / or the CBD Sp Bpep peptide in decontamination of surfaces from microorganisms.

[0114] Two different materials (glass and silicone) contaminated with Listeria monocytogenes DSM 19094 were used. The strain was grown in TSB overnight at 37°C with shaking at 80 rpm. Subsequently, the cells were centrifuged and suspended in water to a density of 1.8 McFarland, which corresponds to approximately 108CFU / ml. Sterile glass beads and silicone tubes (length 14 mm, internal diameter 6 mm) were immersed in the bacterial suspension and air-dried for 30 min. The prepared glass beads and silicone tubes were incubated for 60 minutes in a 2 pM solution of CBD_Sp_B, CBD_Sp_Bpep, or water (C-control), and then the collected fluid samples were serially diluted in water and plated on TSB agar. As can be seen in Fig. 11, the most effective bactericidal factor was the CBD_Sp_B protein (reduction in the number of bacteria by 2 to 3 orders of magnitude, 99-99.9% reduction in the number of bacteria). The CBD_Sp_Bpep peptide showed weaker bactericidal activity compared to the entire CBD_Sp_B domain.

[0115] Example 11. Sequence comparison

[0116] Comparison of amino acid sequences showed that the CBD_Sp_B domain has 70.3% identity with the CBD_Ss_L domain, 63.3% identity with the CBD_Sp_A domain and 50.5% identity with the CBD_Ss_R domain. The sequence identity expressed in [%] is presented in Tab.3. Tab. 3. Summary of amino acid sequence identity comparison between the selected CBD domains in [%].

[0117] A BLAST search of the NCBI sequence database was conducted to identify proteins with the highest sequence identity to the CBD_Sp_B domain. The analysis revealed that the peptidoglycan hydrolase SpM23_B, originally identified in Staphylococcus pettenkoferi, is also present in Staphylococcus argensis, sharing 97% overall sequence identity. Notably, the 3% sequence divergence is confined to the catalytic domain, while the cell wall-binding domain (CBD) exhibits 100% identity. In comparison, the CBD_Ss_L domain from lysostaphin shares 70.3% identity with CBD_Sp_B. No other CBD sequence in the NCBI database shows greater identity to CBD_Sp_B than the CBD_Ss_L domain.

[0118] IL

[0119] Summary of the properties and industrial applications of the recombinant protein with antimicrobial properties and peptide according to the invention derived from the cell wall binding domain (CBD_Sp_B) from Staphylococcus petenkoferi

[0120] Antibacterial activity.

[0121] The antibacterial activity of the produced proteins of the tested CBD domains and the CBD_Sp_Bpep peptide was tested on ten species of bacteria, four Gram-negative (Escherichia coli DSM 1103, Klebsiella pneumoniae DSM 789, Pseudomonas aeruginosa DSM 939 and Yersinia ruckeri CCM 4620) and six Grampositive (Staphylococcus aureus NTCT 8325-4, Enterococcus faecium DSMZ2146, Enterococcus faecalis DSM 20376, Streptococcus canis DSM 20715, Listeria monocytogenes DSM 19094, Bacillus subtilis DSM 10). The activities are summarized in Tab 4.

[0122] Tab. 4. Summary of the activities of the tested CBD domains and the CBD_Sp_Bpep peptide (nt - not tested). Among the tested proteins, the CBD_Sp_B domain demonstrated the highest antibacterial efficacy, completely eradicating all tested bacterial species at a low concentration (0.5 pM) within a short incubation period (2 hours). The CBD_Sp_Bpep peptide exhibited slightly lower activity compared to the full-length CBD_Sp_B domain. In contrast, the CBD_Ss_L, CBD_Sp_A, and CBD_Ss_R domains did not display any detectable antimicrobial activity under the conditions tested.

[0123] Antifungal activity.

[0124] The antifungal activity of all domains listed in Tab. 2 was evaluated using Candida albicans as a model organism. Additionally, the CBD_Sp_B domain was tested against a broader panel of fungal species, including Saccharomyces cerevisiae, Hyphopichia burtonii, Phytophthora spp., and Botrytis cinerea. The results (Example 6) demonstrated that only the CBD_Sp_B domain exhibited antifungal activity across all tested fungal strains. In contrast, the CBD_Ss_L, CBD_Sp_A, and CBD_Ss_R domains showed no detectable antifungal activity under the conditions tested.

[0125] CBD Sp Bpep Peptide.

[0126] Bioinformatic analysis of the CBD_Sp_B domain revealed the presence of specific sequence fragments with predicted antibacterial properties. Among them, a peptide corresponding to a selected region of the domain, designated CBD_Sp_Bpep, was identified as a potential contributor to its antimicrobial activity. Experimental validation confirmed that the isolated CBD_Sp_Bpep peptide possesses antibacterial properties, supporting its role as the active region within the CBD_Sp_B domain.

[0127] Method of producing the CBD_Sp_B domain and the CBD_Sp_Bpep peptide

[0128] DNA sequences and genetic constructs and methods of their production have been developed and presented, as well as methods of the efficient production and purification of CBD domains, including the recombinant CBD_Sp_B protein, and a method for producing the recombinant CBD_Sp_Bpep peptide with antimicrobial activity according to the invention (Examples 7, 8).

[0129] The proposed mechanism of action of the CBD_Sp_B domain and the CBD_Sp_Bpep peptide according to the invention

[0130] The conducted experiments show that the observed antimicrobial effects, i.e. antibacterial and antifungal effects, are not caused by direct cell lysis, but arise through another, still unknown mechanism. Without being bound by any theory, one possible explanation is that the killing properties of the recombinant protein with antimicrobial properties and peptide according to the invention demonstrated for the CBD_Sp_B domain may result from electrostatic interactions between the positively charged recombinant protein or peptide according to the invention and the cell wall of bacteria or fungi, which usually has a negative charge on its surface.

[0131] Safety of use as a biocidal agent. An important feature of antimicrobial substances is the safety of their use, therefore preliminary cytotoxicity tests of CBD_Sp_B were carried out on moth larvae and Danio rerio embryos and on basil and radish seedlings (Example 9). Recombinant CBD_Sp_B proteins at working concentrations (0.5 pM) did not show cytotoxicity in the models used even after continuous, four-day exposure. It is therefore possible to safely use them in preparations used in the environment, e.g. as plant protection products, agents for preserving materials, agents preventing biodeterioration.

[0132] Practical applications. To demonstrate the possibility of using protein for surface decontamination, tests were performed on various species of bacteria and on two different surfaces (glass and silicone, Example 10).

[0133] Due to the strong and universal antimicrobial properties demonstrated above and at the same time high biological safety, the CBD_Sp_B protein can find a number of practical and desirable applications. In medicine and veterinary medicine - for the treatment of bacterial, fungal, but also mixed infections, in particular surface infections. There is a serious problem of bacterial and fungal infections, e.g. on catheters and implants, which could be controlled using a protein with antimicrobial properties with such a broad spectrum of activity.

[0134] In agriculture, the CBD_Sp_B protein can be used as a modern, safe biofungicide, which, unlike the currently used chemicals, would not only be effective, but also safe, and its use would not require compliance with waiting periods. Similar benefits of using the protein are also found in food storage.

[0135] The CBD_Sp_B protein can be used to eliminate fungi and bacteria from various surfaces. Biodeterioration is a very common phenomenon and affects a wide variety of surfaces, walls, wood, paper, etc. Eliminating fungi and bacteria from such surfaces using strong chemicals can lead to their damage, which is extremely dangerous, especially in the case of valuable items such as books, old prints, paintings, sculptures, frescoes or fabrics.

[0136] Decontamination of various surfaces, especially those being in contact with food, is often ineffective, even with the use of strong chemicals, requires intensive rinsing and exclusion of these surfaces (e.g. production surfaces) from use, which generates very high costs. In addition, it creates the possibility of harmful substances getting into food and the environment.

[0137] The use of the recombinant protein and the peptide with antimicrobial properties according to the invention as antimicrobial agents for decontamination and disinfection and prevention of the growth of bacteria and fungi from various surfaces is extremely desirable, their use in the food processing and food preservation industry will play a particularly important role, especially in the prevention of infections and diseases caused by microorganisms occurring in food, e.g. listeriosis caused by Listeria monocytogenes. LITERATURE:

[0138] 1. Jagielska E, Chojnacka O, Sabala I. LytM Fusion with SH3b-Like Domain Expands Its Activity to Physiological Conditions. Microb Drug Resist. 2016 Sep;22(6):461-9. doi: 10.1089 / mdr.2016.0053

[0139] 2. Korndbrfer IP, Danzer J, Schmelcher M, Zimmer M, Skerra A, Loessner MJ. The crystal structure of the bacteriophage PSA endolysin reveals a unique fold responsible for specific recognition of Listeria cell walls. J Mol Biol. 2006 Dec 8;364(4):678-89. doi: 10.1016 / j.jmb.2006.08.069. Epub 2006 Aug 30. PMID: 17010991.

[0140] 3. Loessner MJ, Kramer K, Ebel F, Scherer S. C-terminal domains of Listeria monocytogenes bacteriophage murein hydrolases determine specific recognition and high-affinity binding to bacterial cell wall carbohydrates. Mol Microbiol. 2002 Apr;44(2):335-49. doi: 10.1046 / j.1365-2958.2002.02889.x.

[0141] 4. Loessner MJ, Wendlinger G, Scherer S. Heterogeneous endolysins in Listeria monocytogenes bacteriophages: a new class of enzymes and evidence for conserved holin genes within the siphoviral lysis cassettes. Mol Microbiol. 1995 Jun;16(6):1231-41. doi: 10.1111 / j.1365-2958.1995.tb02345.x.

[0142] 5. Mitkowski P, Jagielska E, Nowak E, Bujnicki JM, Stefaniak F, Niedzialek D, Bochtler M, Sabala I. Structural bases of peptidoglycan recognition by lysostaphin SH3b domain. Sci Rep. 2019 Apr 12;9(1):5965. doi: 10.1038 / s41598-019-42435-z

[0143] 6. Shen Y, Kalograiaki I, Prunotto A, Dunne M, Boulos S, Taylor NMI, Sumrall ET, Eugster MR, Martin R, Julian-Rodero A, Gerber B, Leiman PG, Menendez M, Peraro MD, Canada FJ, Loessner MJ. Structural basis for recognition of bacterial cell wall teichoic acid by pseudo-symmetric SH3b-like repeats of a viral peptidoglycan hydrolase. Chem Sci. 2020 Oct 23;12(2):576-589. doi: 10.1039 / d0sc04394j.

[0144] 7. Tossavainen H, Raulinaitis V, Kauppinen L, Pentikainen U, Maaheimo H, Permi P. Structural and Functional Insights Into Lysostaphin-Substrate Interactivity. Front Mol Biosci. 2018 Jul 3;5:60. doi: 10.3389 / fmolb.2018.00060

[0145] 8. Wysocka A, Jagielska E, t^zniak t, Sabala I. Two New M23 Peptidoglycan Hydrolases With Distinct Net Charge. Front Microbiol. 2021 Sep 24;12:719689. doi: 10.3389 / fmicb.2021.719689.

[0146] SEQUENCE LISTING

[0147] SEQ ID No. 1. Amino acid sequence of the CBD_Sp_B domain from Staphylococcus pettenkoferi, the underlined amino acids correspond to the amino acid sequence of the CBD_Sp_Bpep peptide MFKTNKYGTLYKSEKAS FTPNTNI ITRKTGPFRSMKQAGILKAGKKINYDEVMKQDGYVWLGYGSKKNRK YVPIRTWNRNTNAMGPIWGKI S

[0148] SEQ ID No. 2. Nucleotide sequence of the fragment of the gene encoding the Staphylococcus pettenkoferi CBD_Sp_B domain atgttcaaaactaataaatatggaacgctttataaatctgaaaaagcatcatttacaccaaatacaaata ttatcactagaaagacgggtccttttagaagtatgaaacaagcaggaattttaaaagctggtaagaaaat taactatgatgaagtaatgaaacaagatggttatgtttggttaggttatggaagtaaaaagaaccgtaaa tacgtaccaattagaacttggaatagaaatactaacgctatgggaccaatttgggggaaaattagttaa

[0149] SEQ ID No. 3. Amino acid sequence of the Staphylococcus simulans CBD Ss L

[0150] MGWKTNKYGTLYKSESASFTPNTDI ITRTTGPFRSMPQSGVLKAGQTIHYDEVMKQDGHVWVGYTGNSGQ RIYLPVRTWNKSTNTLGVLWGTIK

[0151] SEQ ID No. 4. Nucleotide sequence of the fragment of the gene encoding Staphylococcus simulans CBD_Ss_L atgggttggaaaacaaacaaatatggcacactatataaatcagagtcagctagcttcacacctaatacag atataataacaagaacgactggtccatttagaagcatgccgcagtcaggagtcttaaaagcaggtcaaac aattcattatgatgaagtgatgaaacaagacggtcatgtttgggtaggttatacaggtaacagtggccaa cgtatttacttgcctgtaagaacatggaataaatctactaatactttaggtgttctttggggaactataa ag SEQ ID No. 5. Amino acid sequence of the Staphylococcus pettenkoferi CBD_Sp_A

[0152] FQENQYGTLYKEEHASFTPNTSI ITRKTGPFINMPRGGTLEAGQTIHYDEVMKQDGYVWLGYDSNGGRRY

[0153] LPIRTWDRTTEQIGSMWGT IS

[0154] SEQ ID No. 6. Nucleotide sequence of the fragment of the gene encoding Staphylococcus pettenkoferi CBD_Sp_A atgttccaagaaaatcaatatggcacgctatacaaagaagaacatgcgtcatttaccccaaatacttcta ttattacacgtaaaacaggcccatttattaatatgccacgtggtggaactttagaagcaggtcaaacaat tcattacgatgaggttatgaaacaagatggttacgtatggctaggttacgattcaaatggtggtcgtcgc tacttaccaattcgcacatgggatcgcactactgaacaaataggaagtatgtggggaacaatttcttaa

[0155] SEQ ID No. 7. Amino acid sequence of the Staphylococcus simulans CBD Ss R domain

[0156] STNSTYKVDGKGTYYKAESAS FTANYDIKTRLNGPFRSNPQSGVLHPGQT IKYDTVMKQDGHVWVVYTGY SGKRI YLPVRTWDKNSNTLGPLWGI IN

[0157] SEQ ID No. 8. Nucleotide sequence of the fragment of the gene encoding Staphylococcus simulans CBD_Ss_R atgcaccatcatcatcatcattcttctggtgtagatctgggtaccgagaacctgtacttccaatccaatg ccacttataaagttgatggcaaaggtacttattacaaagcagaatcagcatcatttacagcaaactatga tattaaaactcgattgaatgggccatttagaagtaacccacaatcaggtgtgttacatcctggtcaaaca attaagtatgatacagtcatgaaacaagatggacatgtatgggttgtatatactggatattcaggaaaac gaatatatttaccagttagaacatgggataaaaattctaatacattaggcccgttatggggaattattaa ttaa

[0158] SEQ ID No. 9. Amino acid sequence of the Staphylococcus pettenkoferi CBD Sp Bpep peptide (a part of the active CBD_Sp_B domain). The sequence corresponds to SEQ ID No. 1 at positions 61-84 AA.

[0159] GYGSKKNRKYVPIRTWNRNTNAMG

[0160] SEQ ID No. 10. Nucleotide sequence of a fragment of the gene encoding the Staphylococcus pettenkoferi CBD_Sp_Bpep peptide. ggttatggaagtaaaaagaaccgtaaatacgtaccaattagaacttggaatagaaatactaacgctatgg ga

Claims

CLAIMS1. A recombinant protein with antimicrobial properties, characterized in that the recombinant protein has an amino acid sequence that with at least 80% identity with the sequence of the cell wall binding domain CBD_Sp_B of Staphylococcus pettenkoferi as shown in SEQ ID No. 1.

2. The recombinant protein with antimicrobial properties according to claim 1, characterized in that the recombinant protein has an amino acid sequence with least 85%, more preferably 90%, more preferably 95%, more preferably 99% identity with the sequence shown in SEQ ID No. 1; most preferably it is the CBD_Sp_B domain from Staphylococcus pettenkoferi having the amino acid sequence SEQ ID No. 1.

3. The recombinant antimicrobial protein according to claims 1-2, characterized in that it shows antibacterial and / or antifungal activity.

4. A peptide with antimicrobial properties, characterized in that the peptide has an amino acid sequence with at least 80% identity with the sequence of the active fragment of the cell wall binding domain of CBD_Sp_Bpep from Staphylococcus pettenkoferi, as shown in SEQ ID No. 9.

5. The peptide with antimicrobial properties according to claim 4, characterized in that the peptide has an amino acid sequence with at least 85%, more preferably 90%, more preferably 95%, more preferably 99% identity with the sequence shown in SEQ ID No. 9; most preferably, it is the antimicrobial peptide CBD_Sp_Bpep with the amino acid sequence of SEQ ID No. 9.

6. The peptide with antimicrobial properties according to the claims 4-5, characterized in that it shows antibacterial and / or antifungal activity.

7. A genetic construct characterized in that it encodes the recombinant protein with antimicrobial properties according to claims 1-3 and / or the peptide with antimicrobial properties according to claims 4- 6.

8. A host cell characterized in that it comprises a genetic construct according to the claim 7.

9. A composition comprising the recombinant protein with antimicrobial properties according to claims 1- 3 and / or the peptide with antimicrobial properties according to claims 4-6 or a mixture thereof, and a carrier.

10. The composition according to claim 9, characterized in that the composition is in the form of a solution, tablet, powder, granulate, suspension, emulsion, aerosol, gel, suppositories, vaginal suppositories, ointment, cream, cosmetic powder.

11. The composition according to claims 9-10, characterized in that it is intended for use as an antibacterial agent and / or an antifungal agent.

12. The composition according to claims 9-10, characterized in that it is intended for use as the antibacterial agent against Gram(+) and / or Gram(-) bacteria, preferably against bacteria of the genus Listeria spp., Escherichia spp., Pseudomonas spp., Yersinia spp., Staphylococcus spp., Enterococcus spp., Streptococcus spp., more preferably against the species Listeria monocytogenes, Escherichia coli, Pseudomonas aeruginosa, Yersinia ruckeri, Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus simulans, Enterococcus faecium, Enterococcus faecalis, Streptococcus agalactiae, Streptococcus dysgalactiae, Streptococcus uberis, Streptococcus canis, Streptococcus egui.

13. The composition according to claims 9-12, characterized in that it is intended for use as the antifungal agent against fungi and lichens, more preferably against single- and / or multi-cellular fungi, preferably against fungi from yeasts and Leotiomycetes classes, more preferably against fungi from the genera Candida ssp., Saccharomyces ssp., Phytophthora spp. and Botritis spp., more preferably against species Candida albicans, Saccharomyces cerevisiae, Botritis cinerea.

14. A non-medical use of the recombinant protein with antimicrobial properties according to claims 1-3 and / or the peptide with antimicrobial properties according to claims 4-6 and / or the composition according to claims 9-13 as an antibacterial agent and / or an antifungal agent.

15. The non-medical use according to claim 14, characterized in that the antibacterial agent is used against Gram(+) and / or Gram(-) bacteria, preferably against bacteria of the genus Listeria spp., Escherichia spp., Pseudomonas spp., Yersinia spp., Staphylococcus spp., Enterococcus spp., Streptococcus spp., more preferable against the species Listeria monocytogenes, Escherichia coli, Pseudomonas aeruginosa, Yersinia ruckeri, Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus simulans, Enterococcus faecium, Enterococcus faecalis, Streptococcus agalactiae, Streptococcus dysgalactiae, Streptococcus uberis, Streptococcus canis, Streptococcus egui.

16. The non-medical use according to claim 14, characterized in that the antifungal agent is used against fungi and lichens, more preferably against single- and / or multi-cellular fungi, preferably against fungi from the yeast and Leotiomycetes classes, more preferably against fungi from the genera Candida ssp., Saccharomycesssp., Phytophthora spp. and Botritis spp, more preferably against species Candida albicans, Saccharomyces cerevisiae, Botritis cinerea.

17. A non-medical use of the recombinant protein with antimicrobial properties according to claims 1-3 and / or the peptide with antimicrobial properties according to claims 4-6 and / or the composition according to claims 9-13 as an in vitro antimicrobial agent with antibacterial and / or antifungal activity for inhibiting, eliminating and preventing the growth of microorganisms, wherein the microorganisms are preferably selected from bacteria, fungi and lichens.

18. Use of the recombinant protein with antimicrobial properties according to claims 1-3 and / or the peptide with antimicrobial properties according to claims 4-6 and / or the composition according to claims 9-13 as an antiseptic and disinfectant.

19. Use of the recombinant protein with antimicrobial properties according to claims 1-3 and / or the peptide with antimicrobial properties according to claims 4-6 and / or the composition according to claims 9-13 as an antimicrobial agent for disinfecting surfaces and rooms in food processing, preferably for disinfecting surfaces and / or tools that come into contact with food or semi-finished food products, preferably as an antimicrobial agent for disinfecting surfaces and rooms with antibacterial and / or antifungal activity.

20. Use of the recombinant protein with antimicrobial properties according to claims 1-3 and / or the peptide with antimicrobial properties according to claims 4-6 and / or the composition according to claims 9-13 as an antimicrobial agent in the food industry as an additive to human and / or animal food, preferably as a food additive, preferably as an antimicrobial agent with antibacterial and / or antifungal activity.

21. The use as the antimicrobial agent in the food industry according to claim 20, characterized in that the agent is used to prevent infections and diseases caused by microorganisms present in food, preferably listeriosis caused by Listeria monocytogenes.

22. Use of the recombinant protein with antimicrobial properties according to claims 1-3 and / or the peptide with antimicrobial properties according to claims 4-6 and / or the composition according to claims 9-13 as a disinfectant for surfaces in health care in medicine, veterinary medicine, diagnostics, preferably for disinfecting hospital surfaces, medical and veterinary surgeries, laboratory surfaces, tool surfaces, surfaces of devices used in medicine, veterinary medicine, diagnostics.

23. Use of the recombinant protein with antimicrobial properties according to claims 1-3 and / or the peptide with antimicrobial properties according to claims 4-6 and / or the composition according to claims 9-13 as an agent preventing and limiting the growth of microorganisms on the surfaces of devices used in medicine, veterinary medicine and diagnostics, preferably by contacting the surface with the agent, by covering the surface with the agent, wherein the surfaces preferably covered are surfaces of catheters, implants, cannulae, endotracheal tubes.

24. Use of the recombinant protein with antimicrobial properties according to claims 1-3 and / or the peptide with antimicrobial properties according to claims 4-6 and / or the composition according to claims 9-13 as a plant protection agent, wherein the microorganisms are preferably selected from bacteria, fungi and lichens.

25. The use according to claim 24, characterized in that the plant protection agent is applied to the plant material by immersing, pouring, dripping, spraying or misting the plant material with the plant protection agent.with antimicrobial properties according to claims 4-6 and / or the composition according to claims 9-13 as a preservative for materials for preserving the material by inhibiting and preventing the growth of microorganisms in / on the material, wherein the microorganisms are preferably selected from bacteria, fungi and lichens.

27. The use according to claim 26, characterized in that the material preservative is in the form of a paint, a liquid for application, a spray, a gel, a layer applied to the material, a liquid for immersing the material therein, an admixture to the material, a primer for the material, an adhesive for the material, a resin, a filler, a stain, a wax, a binder connecting the material.

28. The use according to claims 26-27, characterized in that the preserved material is selected from fabric, non-woven fabric, paper, canvas, painting support, painting loom, tissue paper, rocks, leather, wood, ceramics, metal, metal alloy, glass, plastic, cement, concrete.

29. Use of the recombinant protein with antimicrobial properties according to claims 1-3 and / or the peptide with antimicrobial properties according to claims 4-6 and / or the composition according to claims 9-13 as an agent preventing biodeterioration by microorganisms, wherein the microorganisms are preferably selected from bacteria, fungi and lichens30. The use according to claim 29, characterized in that the agent preventing biodeterioration is used to prevent biodeterioration of fabrics, non-woven fabrics, paper, canvas, painting support, painting loom, tissue paper, rock, leather, wood, ceramics, metal, metal alloys, glass, plastic, cement, concrete, in particular books, old prints, paintings, sculptures, frescoes, tapestries.

31. Use of the antimicrobial protein according to claims 1-3 and / or the peptide with antimicrobial properties according to claims 4-6 and / or the composition according to claims 9-13 as an in vitro antimicrobial agent with antibacterial activity in the cosmetics industry, as an additive to cosmetics improving their microbiological quality, preferably as a biopreservative, preferably as an additive to liquids, creams, milks, lotions, mists, gels.

32. A veterinary composition characterized in that it comprises the protein with antimicrobial properties according to claims 1-3 and / or the peptide with antimicrobial properties according to claims 4-6 and / or the composition according to claims 9-13 and a veterinary acceptable carrier for use as a drug for the prophylaxis, prevention and treatment of a disease and / or condition caused by microorganisms selected from bacteria and / or fungi.

33. The veterinary composition for use according to claim 32, characterized in that it is used in the prophylaxis, prevention and treatment of bacterial and / or fungal infections, it is preferably used in the treatment of mixed bacterial and fungal infections, in particular superficial infections, preferably skin infections.2634. The veterinary composition for use according to claims 32-33, characterized in that it is used against Gram(+) and / or Gram(-) bacteria, preferably against bacteria of the genus Listeria spp., Escherichia spp., Pseudomonas spp., Yersinia spp., Staphylococcus spp., Enterococcus spp., Streptococcus spp., more preferably against the species Listeria monocytogenes, Escherichia coli, Pseudomonas aeruginosa, Yersinia ruckeri, Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus simulans, Enterococcus faecium, Enterococcus faecalis, Streptococcus agalactiae, Streptococcus dysgalactiae, Streptococcus uberis, Streptococcus canis, Streptococcus egui.

35. The veterinary composition for use according to claims 32-34, characterized in that it is used against single- and / or multi-cellular fungi, preferably fungi from yeasts and Leotiomycetes classes, more preferably fungi from the genera Candida ssp., Saccharomyces ssp., Phytophthora spp. and Botritis spp., more preferably species Candida albicans, Saccharomyces cerevisiae, Botritis cinerea.

36. The veterinary composition for use according to claims 32-35, characterized in that it is used in the prophylaxis, prevention, treatment of superficial skin and / or wound infections, infected wounds, pressure sores, dermatoses, diabetic foot infections, burns, mastitis, vaginal infections, external ear infections, nail fungus, skin fungus.

37. A pharmaceutical composition characterized in that it comprises the protein with antimicrobial properties according to claims 1-3 and / or the peptide with antimicrobial properties according to claims 4- 6 and / or the composition according to claims 9-13 and a pharmaceutically acceptable carrier for use as a drug in the prophylaxis, prevention and treatment of a disease and / or condition caused by microorganisms selected from bacteria and / or fungi.

38. The pharmaceutical composition for use according to claim 37, characterized in that it is used in the prophylaxis, prevention and treatment of bacterial and / or fungal infections, preferably in the treatment of mixed bacterial-fungal infections, in particular superficial infections, preferably skin infections.

39. The pharmaceutical composition for use according to claims 37-38, characterized in that it is used against Gram(+) and / or Gram(-) bacteria, preferably against bacteria of the genus Listeria spp., Escherichia spp., Pseudomonas spp., Yersinia spp., Staphylococcus spp., Enterococcus spp., Streptococcus spp., more preferably against the species Listeria monocytogenes, Escherichia coli, Pseudomonas aeruginosa, Yersinia ruckeri, Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus simulans, Enterococcus faecium, Enterococcus faecalis, Streptococcus agalactiae, Streptococcus dysgalactiae, Streptococcus uberis, Streptococcus canis, Streptococcus egui.

40. The pharmaceutical composition for use according to claims 37-39, characterized in that it is used against single- and / or multi-cellular fungi, preferably fungi from yeasts and Leotiomycetes classes, more preferably fungi from the genera Candida ssp., Saccharomyces ssp., Phytophthora spp. and Botritis spp, more preferably species Candida albicans, Saccharomyces cerevisiae, Botritis cinerea.T141. The pharmaceutical composition for use according to claims 37-40, characterized in that it is used for the prophylaxis, prevention, treatment of superficial skin and / or wound infections, infected wounds, pressure sores, dermatoses, diabetic foot infections, burns, mastitis, vaginal infections, external ear infections, nail fungus, skin fungus.

42. A cosmetic or care composition for cosmetic, care and hygiene uses for human or animal, characterized in that it comprises the protein with antimicrobial properties according to claims 1-3 and / or the peptide with antimicrobial properties according to claims 4-6 and / or the composition according to claims 9-13, wherein the composition is intended for external use, and wherein the recombinant protein with antimicrobial properties, the peptide with antimicrobial properties is used in the cosmetic or care composition to limit the occurrence of or eliminate microorganisms from the composition, wherein the microorganisms are preferably selected from bacteria and / or fungi.

43. A method for inhibiting or reducing or preventing the growth of microorganisms in vitro, characterized in that it comprises a step of contacting the microorganism with the protein with antimicrobial properties according to claims 1-3 and / or the peptide with antimicrobial properties according to claims 4-6 and / or the composition according to claims 9-13, wherein the microorganisms are preferably selected from bacteria, fungi and lichens.

44. The method according to claim 43, characterized in that the bacteria are selected from Gram(+) and / or Gram(-) bacteria, preferably from bacteria of the genus Listeria spp., Escherichia spp., Pseudomonas spp., Yersinia spp., Staphylococcus spp., Enterococcus spp., Streptococcus spp., more preferably from bacteria of the species Listeria monocytogenes, Escherichia coli, Pseudomonas aeruginosa, Yersinia ruckeri, Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus simulans, Enterococcus faecium, Enterococcus faecalis, Streptococcus agalactiae, Streptococcus dysgalactiae, Streptococcus uberis, Streptococcus canis, Streptococcus egui.

45. The method according to claims 43-44, characterized in that the fungi are selected from single- and / or multi-cellular fungi, preferably fungi from yeasts and Leotiomycetes classes, more preferably fungi from the genera Candida ssp., Saccharomyces ssp., Phytophthoraspp. and Botritisspp., more preferably species Candida albicans, Saccharomyces cerevisiae, Botritis cinerea.

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