Recombinant protein and polypeptide with antibacterial properties, compositions comprising them, uses and methods utilizing thereof

Abstract

The present invention relates to a recombinant protein and polypeptide with antibacterial properties derived from the gene YR7 from Yersinia ruckeri, compositions comprising them, uses and the method utilizing thereof. The recombinant proteins and the polypeptide with antibacterial properties will be used to prevent the development of bacteria in aquatic environments and will have uses in the field of zootechnics, fishery and fish farming, in particular - of salmonid fish in aquacultures, especially against Yersinia ruckeri causing fish yersiniosis.

Description

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

[0002] TECHNICAL FIELD

[0003] The present invention relates to a recombinant protein and polypeptide with antibacterial properties derived from the gene encoding the YR7 protein from Yersinia ruckeri, compositions comprising them, uses and a method utilizing thereof. The recombinant proteins and the polypeptide with antimicrobial properties will be used to prevent the development of bacteria in an aquatic environment and will have uses in the field of zootechnics, fishery and fish farming, in particular of salmonid fish in aquacultures, especially against Yersinia ruckeri causing fish yersiniosis.

[0004] STATE OF ART

[0005] Yersinia ruckeri is a Gram-negative bacterium, the etiological agent of yersiniosis (Enteric Redmouth, ERM), one of the most dangerous diseases of salmonid fish, causing significant losses in fish farming. Yersiniosis manifests itself as redness in the fins, mouth and internal organs of the fish caused by internal and subcutaneous hemorrhages, and if left untreated, it leads to death, causing huge losses in industrial farms [1], This disease is a global problem, especially in places where salmonid fish farms constitute a huge part of the industry, including Norway or Chile. In Norway alone, the production of Atlantic salmon in 2019 reached 1.06 million megatons, and in 2022 the value of fish exported by this country amounted to €9 billion [2], In the years 2015-2019, a steady increase in losses of salmon in farming has been observed. In 2019, about 60 millions fish were reported dead due to bacterial infections, which shows that the potential market for modern antibacterial agents will grow. Prevention of yersinosis is mainly based on the prophylactic use of single- and multicomponent vaccines [3], Fish can be carriers of bacteria for a long time, mainly in the intestines. The infection follows an occurrence of stress and the reduction of immune resistance of the animals. Yersinia ruckeri, excreted with feces into water reservoirs, can survive for a long time in the form of a biofilm, causing infections in subsequent individuals. When symptoms of yersiniosis are diagnosed, the therapy usually consists of administering antibiotics, mainly amoxicillin, oxolinic acid and florfenicol in the feed [4], Although the use of antibiotics for preventive purposes has been legally restricted in many countries, the amount of antibiotics used in fish farming, especially in Asia, is still huge and contributes to the increase in antibiotic resistance [5], In the state of the art, attempts are undertaken to take an approach other than antibiotics to combat diseases caused by Y. ruckeri bacteria in aquacultures, such as using bacteriophages [6], Unfortunately, the undertaken attempts with the use of bacteriophages are very expensive, not widely available and not very effective. Another way to combat Yersinia ruckeri infections in aquacultures is the use of probiotics

[0006] [7], such as the oral administration of Bacillus subtilis and Bacillus licheniformis in rainbow trout farming

[0007] [8].

[0008] There is an urgent need to provide antimicrobials, e.g., in the form of new antimicrobial proteins, that are safe alternatives to antibiotics for use in fish farming. Moreover, it is desirable to provide proteins enabling the reduction in the development of bacterial pathogens of fish in the water for breeding, which will translate into reducing the possibility of infecting fish in aquacultures.

[0009] DISCLOSURE OF THE INVENTION

[0010] The invention relates to a recombinant protein with antibacterial properties comprising an active peptidoglycan binding domain LysM with an amino acid sequence in at least 80% identical with the sequence presented in SEQ ID No. 7 from Yersinia ruckeri.

[0011] Preferably, the recombinant protein with antibacterial properties comprising an active peptidoglycan binding domain LysM, with an amino acid sequence in at least 85%, more preferably 90%, more preferably 95%, more preferably 99% identical with the sequence presented in SEQ ID No. 7; most preferably, it comprises an active catalytic domain LysM with the amino acid sequence of SEQ ID No. 7.

[0012] Preferably, the recombinant protein with antibacterial properties is an active peptidoglycan binding domain LysM, with the amino acid sequence presented in SEQ ID No. 7 from Yersinia ruckeri.

[0013] Preferably, the recombinant protein with antibacterial properties, moreover, comprises an amidase domain Ami with an amino acid sequence in at least 80% identical with the sequence presented in SEQ ID No. 6, more preferably in at least 85%, more preferably 90%, more preferably 95%, more preferably 99% identical with the sequence presented in SEQ ID No. 6, more preferably the amidase domain Ami is the amino acid sequence as presented in SEQ ID No. 6 from Yersinia ruckeri.

[0014] Preferably, in the recombinant protein, the amidase domain Ami is located in the direction of the N- terminus of the recombinant protein, with respect to the peptidoglycan binding domain LysM.

[0015] Preferably, the recombinant protein with antibacterial properties comprises a linker between the peptidoglycan binding domain LysM and the amidase domain Ami connecting them, wherein, preferably, the linker has a sequence in at least 80% identical with the sequence of the linker 2 presented in SEQ ID No. 10, more preferably the linker is the amino acid sequence presented as SEQ ID No. 10 from Yersinia ruckeri. Preferably, the recombinant protein with antibacterial properties, further comprises the peptidoglycan binding domain AMIN with an amino acid sequence in at least 80% identical with the sequence presented in SEQ ID No. 5, more preferably in at least 85%, more preferably 90%, more preferably 95%, more preferably 99% identical with the sequence presented in SEQ ID No. 5, more preferably the peptidoglycan binding domain AMIN is the amino acid sequence presented in SEQ ID No. 5 from Yersinia ruckeri.

[0016] Preferably, the recombinant protein, comprises the peptidoglycan binding domain AMIN located in the direction of the N-terminus of the recombinant protein, with respect to the peptidoglycan binding domain LysM.

[0017] Preferably, in the recombinant protein with antibacterial properties, there is a linker between the peptidoglycan binding domain LysM and the peptidoglycan binding domain AMIN, wherein, preferably, the linker has a sequence in at least 80% identical with the sequence of the linker 1 presented in SEQ ID No. 10, more preferably the linker is the amino acid sequence presented as SEQ ID No. 9 from Yersinia ruckeri.

[0018] Preferably, the recombinant protein with antibacterial properties is the amino acid sequence YR7_Sp_Del from Yersinia ruckeri presented in SEQ ID No. 2.

[0019] Preferably, the recombinant protein, further has an amino acid sequence useful for purification and / or isolation of protein attached to one of the termini, wherein, preferably, the amino acid sequence useful for purification and / or isolation is placed on C-terminus side of the protein, preferably the amino acid sequence useful for purification and / or isolation of the recombinant protein is the oligohistidine sequence presented in SEQ ID No. 8.

[0020] Preferably, the recombinant protein consists of the sequence SEQ ID No. 2 at the N-terminus of the recombinant protein and the oligohistidine sequence presented in SEQ ID No. 8 at the C-terminus of the recombinant protein.

[0021] The invention also relates to a genetic construct encoding the recombinant protein according to the invention.

[0022] The invention also relates to a host cell comprising the genetic construct according to the invention.

[0023] The invention relates to a composition comprising the recombinant protein according to the invention or a mixture of them and a carrier.

[0024] The invention also relates to a composition, moreover comprising an isolated amidase domain Ami protein, with an amino acid sequence having at least 80% identity with the sequence presented in SEQ ID No. 6, and / or an isolated peptidoglycan binding domain AMIN protein, with an amino acid sequence in at least 80% identical with the sequence presented in SEQ ID No. 5. The invention also relates to a non-medical use of the recombinant protein according to the invention and / or the composition according to the invention as an antibacterial agent.

[0025] In the preferred non-medical use, the antibacterial agent is used in an aqueous environment with a conductivity of 0 to 10 mS / cm.

[0026] In the preferred non-medical use, the antibacterial agent is used against bacteria of the genera Yersinia spp., Enterococcus spp., Listeria spp., Bacillus spp., Escherichia spp., Staphylococcus spp., Streptococcus spp., Micrococcus spp., more preferably, bacterial species selected from: Yersinia ruckeri, Enterococcus faecalis, Listeria monocytogenes, Escherichia coli, Staphylococcus aureus, Streptococcus uberis, Staphylococcus simulans, Staphylococcus epidermidis, Micrococcus luteus.

[0027] The invention also relates to the use of the recombinant protein as defined according to the invention and / or the composition as defined according to the invention as an antiseptic and disinfecting agent.

[0028] The invention also relates to the use of the recombinant protein according to the invention and / or the composition according to the invention as an antibacterial agent for the disinfecting surfaces and rooms in food processing, preferably for disinfecting surfaces that come into contact with food or with semifinished food products.

[0029] The invention also relates to the use of the recombinant protein as defined according to the invention and / or the composition according to the invention as an antibacterial agent in the food industry, as an additive to food for humans and / or animals, preferably as a feed additive, preferably as a fish feed additive.

[0030] The invention also relates to the use of the recombinant protein according to the invention and / or the composition according to the invention as a disinfecting agent for surfaces in health care in medicine, veterinary medicine, diagnostics, preferably for disinfecting hospital surfaces, medical and veterinary offices, laboratory surfaces, surfaces of tools, surfaces of instruments used in medicine, veterinary medicine, diagnostics.

[0031] The invention also relates the non-medical use of the recombinant protein as defined according to the invention and / or the composition according to the invention as an in vitro bactericidal agent to inhibit, eliminate bacteria, prevent the growth of Gram(+) and Gram(-) bacteria, preferably bacteria of the genus Yersinia spp., Enterococcus spp., Listeria spp., Bacillus spp., Escherichia spp., Staphylococcus spp., Streptococcus spp., Micrococcus spp.; more preferably the species of bacteria selected from Yersinia ruckeri, Enterococcus faecalis, Listeria monocytogenes, Escherichia coli, Staphylococcus aureus, Streptococcus uberis, Staphylococcus simulans, Staphylococcus epidermidis, Micrococcus luteus.

[0032] In the preferred non-medical use, the bactericidal agent is used in an aqueous environment with a conductivity of 0 to 10 mS / cm. The invention also relates the non-medical use of the recombinant protein according to the invention and / or the composition according to the invention as the bactericidal agent to inhibit, eliminate bacteria, prevent the growth of bacteria in water, preferably bacteria of the species Escherichia coli, Enterococcus feacalis, Yersinia ruckeri.

[0033] In the preferred use as the bactericidal agent to inhibit, eliminate bacteria, prevent the growth of bacteria in water, the bactericidal agent is used in aquacultures.

[0034] In the preferred use as the bactericidal agent to inhibit, eliminate bacteria, prevent the growth of bacteria in water, the bactericidal agent is used in fish hatcheries and rearing facilities, in recirculating water systems, in containers for the transport of fish and / or fry, in containers for the storage of fish and / or fry, in containers with eggs and / or larvae of aquatic animals, in containers with aquatic plants and for the equipment having contact with fish.

[0035] In the preferred use, the bactericidal agent to inhibit, eliminate bacteria, prevent the growth of bacteria in water, the bactericidal agent is used in aqueous environment with a conductivity of 0 to 10 mS / cm.

[0036] The invention also relates to the use of the recombinant protein as defined according to the invention and / or the composition according to the invention to eliminate and prevent biofilm formation on surfaces, preferably on the surfaces in contact with water in aquaculture reservoirs, preferably, to eliminate biofilm formed by Yersinia ruckeri.

[0037] The invention also relates to the use of the recombinant protein as defined according to the invention and / or the composition according to the invention as an cleaning agent for water reservoirs and devices, preferably for cleaning water reservoirs and devices used in fish farming.

[0038] The invention also relates to the use of the recombinant protein according to the invention and / or the composition as defined according to the invention as an antibacterial agent 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.

[0039] The invention also relates to a cosmetic or care composition for cosmetic, care and hygiene uses in humans or animals, which contains the recombinant protein as described according to the invention and / or the composition according to the invention, the composition is intended for external use, and wherein the recombinant protein is used in the cosmetic or care composition to limit the occurrence or eliminate bacteria from the composition.

[0040] The invention also relates to a veterinary and / or pharmaceutical composition that comprises the recombinant protein according to the invention and / or the composition according to the invention and a veterinarily and / or pharmaceutically acceptable carrier for use in the treatment of a disease and / or condition caused by bacteria, preferably bacteria of the genus Yersinia spp., Enterococcus spp., Listeria spp., Bacillus spp., Escherichia spp., Staphylococcus spp., Streptococcus spp., Micrococcus spp.; more preferably bacterial species selected from Yersinia ruckeri, Enterococcus faecalis, Listeria monocytogenes, Escherichia coii, Staphylococcus aureus, Streptococcus uberis, Staphylococcus simulans, Staphylococcus epidermidis, Micrococcus luteus.

[0041] The invention also relates to a veterinary composition comprising the recombinant protein according to the invention and / or the composition according to the invention and a veterinarily acceptable carrier for use in the prevention and / or treatment of fish diseases caused by Yersinia ruckeri, preferably for use in the prevention and / or treatment of yersiniosis in fish.

[0042] The veterinary composition for use is preferably used in the treatment of yersiniosis in salmonid fish, preferably selected from rainbow trout (Oncorhynchus mykiss), brook trout (Salvelinus fontinalis), river trout (Salmo trutta morpha fario), sea trout (Salmo trutta morpha trutta), Atlantic salmon (Salmo salar), lake trout (Salmo trutta morpha lacustris).

[0043] The invention also relates to a method for inhibiting or limiting the growth of Gram(+) and / or Gram(-) bacteria which comprises a step in which the bacteria are contacted with the recombinant antibacterial protein according to the invention and / or the composition of the invention in an environment with conductivity ensuring the bactericidal activity of the recombinant protein.

[0044] The invention also relates to the method in which the contacting is carried out in an aqueous solution with the conductivity of 0 to 10 mS / cm.

[0045] In another embodiment in the method, the bacteria are selected from the genus Yersinia spp., Enterococcus spp., Listeria spp., Bacillus spp., Escherichia spp., Staphylococcus spp., Streptococcus spp., Micrococcus spp.; more preferably the species of bacteria selected from Yersinia ruckeri, Enterococcus faecalis, Listeria monocytogenes, Escherichia coii, Staphylococcus aureus, Streptococcus uberis, Staphylococcus simulans, Staphylococcus epidermidis, Micrococcus luteus.

[0046] The invention also relates to a recombinant polypeptide with antibacterial properties, which comprises the peptidoglycan binding domain AMIN with an amino acid sequence in at least 80% identical with the sequence presented in SEQ ID No. 5 from Yersinia ruckeri, more preferably in at least 85%, more preferably 90%, more preferably 95%, more preferably 99% identical with the sequence presented in SEQ ID No. 5, more preferably the peptidoglycan binding domain AMIN is the amino acid sequence presented in SEQ ID No. 5 from Yersinia ruckeri; and the linker-connected (wherein, preferably, the linker has a sequence that is at least 80% identical to the sequence of linker 1 shown in SEQ ID No. 8, more preferably the linker is the amino acid sequence shown as SEQ ID No. 8 from Yersinia ruckeri) amidase domain Ami with an amino acid sequence having at least 80% identity with the sequence presented in SEQ ID No. 6, more preferably in at least 85%, more preferably 90%, more preferably 95%, more preferably 99% identical with the sequence presented in SEQ ID No. 6, more preferably the amidase domain Ami is the amino acid sequence presented in SEQ ID No. 6 from Yersinia ruckeri; wherein preferably the linker has a sequence in at least 80% identical with the sequence of the linker 1 presented in SEQ ID No. 8, more preferably the linker is the amino acid sequence presented as SEQ ID No. 8 from Yersinia ruckeri

[0047] Preferably, the recombinant polypeptide with antibacterial properties is the amino acid sequence YR7_LysM_Del from Yersinia ruckeri presented in SEQ ID No. 4.

[0048] The invention also relates to a composition comprising a recombinant polypeptide according to the invention or a mixture thereof and a carrier.

[0049] The invention will overcome the inconveniences presented in the state of the art by providing recombinant proteins and polypeptides derived from fragments of the YR7 gene (NCBI, Protein Database, accession no. WP_049689580.1) from the genome of the bacterium Yersinia ruckeri.

[0050] The compositions comprising the recombinant protein according to the invention or a mixture thereof and a carrier are preferably in the form of a solution, tablet, powder, granule, suspension, emulsion, aerosol, gel, cream.

[0051] The use of bactericidal enzymes in the form of recombinant proteins and recombinant polypeptides according to the invention, such as, for example, YR_7_Sp_Del and its derivatives, makes it possible to reduce the use of antibiotics in fish farming, and thus to reduce the transmission of antibiotic resistance genes, and thus the spread of antibiotic resistance among animals and humans. It will improve animal welfare and reduce losses for farmers, at the same time.

[0052] The recombinant proteins and polypeptides according to the invention, compositions comprising them or their mixtures can be used as a bactericidal and antibacterial agent to inhibit, eliminate and prevent the growth of both Gram(+) and Gram(-) bacteria. The recombinant proteins according to the invention exhibit antibacterial properties and bactericidal activity against both Gram(+) and Gram(-) bacteria, which has been demonstrated for the recombinant proteins according to the invention on the example of Yersinia ruckeri, Enterococcus faecalis, Listeria monocytogenes, Staphylococcus aureus, Streptococcus uberis, Staphylococcus simulans, Staphylococcus epidermidis, Escherichia coli, Micrococcus luteus. Similar biological properties are exhibited by the recombinant YR7_LysM_Del polypeptide, lacking the LysM domain, for it was unexpectedly found out that the YR7_LysM_Del protein has a strong antibacterial and bactericidal effect, when the domains forming it: AMIN and Ami are connected by a linker.

[0053] The recombinant proteins according to the invention can be used to inhibit, eliminate and prevent the growth of Gram(-) and Gram(+) bacteria, preferably bacteria of the genera selected from: Yersinia spp., Enterococcus spp., Listeria spp., Bacillus spp., Escherichia spp., Staphylococcus spp., Streptococcus spp., Micrococcus spp.; more preferably, bacterial species selected from: Yersinia ruckeri, Enterococcus faecalis, Listeria monocytogenes, Escherichia coli, Staphylococcus aureus, Streptococcus uberis, Staphylococcus simulans, Staphylococcus epidermidis, Micrococcus luteus. The particularly high activity of recombinant proteins according to the invention against Yersinia ruckeri allows them to be used in fish farms, especially industrial farms for the treatment and prevention of fish diseases caused by Yersinia ruckeri such as fish yersiniosis, especially in industrial farms of salmonid fish such as rainbow trout (Oncorhynchus mykiss), brook trout (Salvelinus fontinalis), river trout (Salma trutta morpha / or / o), sea trout (Salma trutta morpha trutta) and Atlantic salmon (Salma salar), lake trout (Salma trutta morpha lacustris).

[0054] The recombinant proteins according to the invention can be formulated into a form that is convenient for administration, e.g,. in the form of a solution, tablet, powder, suspension, which can be applied to water, bodies of water, especially fish aquacultures, preferably salmonid fish aquacultures.

[0055] The recombinant proteins according to the invention will be used to eliminate and prevent the formation of bacterial biofilms, including biofilms from Yersinia ruckeri from surfaces, surfaces of tanks, containers, devices, tables, pipes and other surfaces for breeding, especially the surfaces of tanks used for keeping fish or transporting fish, where fish are often infected from bacteria deposited on such a biofilm.

[0056] The recombinant proteins according to the invention will be particularly beneficial for the removal of Yersinia ruckeri bacteria from water in aquacultures, in closed circuits, in fry transport containers, in fish / fry storage containers during vaccinations, routine inspections, removal of other parasites (e.g. salmon louse), disinfection of eggs in hatcheries and during other experiments on fish and aquatic animals and plants.

[0057] When the term "comprising" or "including " is used in this description and disclaimers, it does not exclude other elements. For the purposes of the present invention, the term "consisting of" is considered a preferred embodiment of the term "comprising" and "including". If a group is defined below that contains at least a number of embodiments, this is also to be understood as disclosing a group that preferably consists only of those embodiments.

[0058] The terms "about" or "approximately" in the context of this 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 ±10%, and preferably ±5%.

[0059] Technical terms are in accordance with their typical meaning. If a specific meaning is used, the meaning will be described in detail with reference to the context in which the term is used. BRIEF DESCRIPTION OF THE FIGURES

[0060] Examples of the invention are presented in the figures of the drawing, in which

[0061] Fig. 1. presents the schematic structure of proteins divided into domains with respect to the initial YR7 sequence and the constructs derived from it, including the recombinant proteins according to the invention. Particular domains of the initial protein and the recombinant proteins are presented as rectangles, linkers are marked in the form of lines, and within the particular elements the numbers of amino acid residues of sequences limiting the length of domains and linkers are placed.

[0062] Fig. 2. presents the number of bacteria recovered from the Yersinia ruckeri biofilm treated with the YR7_Sp_Del protein at the concentration of 2 and 4 pM. The number of bacteria was shown as logic of cfu / ml and determined based on the estimated order of magnitude of the cell number. NT - untreated biofilm, Control - biofilm treated with glycine buffer (50 mM glycine-NaOH, pH 8.0), 2 pM, 4 pM, enzyme concentrations used during the biofilm treatment. The experiment was performed three times in triplicate. *P<0.05 (Student's t-test, paired).

[0063] Fig. 3. diagram of the structure of peptidoglycan which is a component of the cell walls of bacteria Yersinia ruckeri, in which the bonds that are cut by the enzyme YR7_Sp_Del are indicated by arrows.

[0064] Fig. 4. presents fragments obtained after digestion of Y. ruckeri CCM 4620 cell walls with the enzyme YR7_Sp_Del, the letter designations and given masses are consistent with the data in Tab. 4. A - fragment 905.51 Da; B - fragment 1365.68 Da; C - fragment 1932,12 Da.

[0065] Fig. 5. presents the bacteriolytic activity of the YR7_Sp_Del protein against bacteria Y. ruckeri CCM 4620 at the concentration of 0, 0.5, 1 or 2 pM in the presence of the SYTOX™ green reagent (Invitrogen). The experiment was performed in three biological replications, a.u. - arbitrary units, * - P = 0.0170, **** - P < 0.0001 (one-way ANOVA with Dunnett test).

[0066] Fig. 6. presents a comparison of the activity of the YR7_Sp_Del protein at 12°C and 30°C used at the concentration of 4 pM for 18 hours. The number of bacteria Yersinia ruckeri was shown as logic of cfu / mL and determined based on the number of cells estimated to the order of magnitude.

[0067] Fig. 7. presents images showing the morphology of Danio rerio in lateral view, treated with 0.5 pM YR7_Sp_Del protein. A) control - two-day-old embryos, sample without the protein; B) embryos treated with 0.5 pM YR7_Sp_Del protein, 1-2 days after fertilization; C) embryos treated with 0.5 pM YR7_Sp_Del protein, 1-3 days after fertilization. Scale = 1 mm.

[0068] Fig. 8. presents the stability of the YR7_Sp_Del protein stored at different times and exposed to the effect of different temperatures for 2 weeks, 1 month, 2 months, 6 months, or 11 months, imaged by SDS-PAGE separation method. The scale describing the bands of the protein size standard is expressed in kDa. M - protein size standard, -80 - sample stored at -80°C, 4 - sample stored at 4°C, 22 - sample stored at 22°C, 37 - sample stored at 37°C. Fig. 9. presents the SDS-PAGE electrophoretic separation of samples of the protein YR7_Sp_Del treated with proteinase K, trypsin and the control protein (not treated with enzymes). M - protein size standard in kDa, K - control sample not subjected to proteolytic digestion, P - sample subjected to proteinase K digestion, T - sample subjected to trypsin digestion.

[0069] The publications cited in the description and the references given therein are hereby included in their entirety as references.

[0070] EMBODIMENTS OF THE INVENTION

[0071] The following examples are provided only 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.

[0072] EXAMPLES

[0073] In the following examples, unless otherwise indicated, standard materials and methods used in the field were used or manufacturers' recommendations for specific materials and methods were followed.

[0074] Example 1. Preparation of recombinant proteins derived from YR7 sequences: YR7_Sp_Del, YR7_AMIN_Del, YR7_LysM_Del, YR7_AMIN, YR7_Ami and YR7_LysM

[0075] A fragment of the YR7 gene from the genome of Yersinia ruckeri (SEQ ID No. 1, NCBI Protein Database accession no. WP_049689580.1) corresponding to amino acids from amino acid 28 to 588 was cloned into the pLATEll vector carrying ampicillin resistance, obtaining the gene encoding the YR7_Sp_Del protein (SEQ ID No. 2, Fig. 1) in the expression vector. The YR7_Sp_Del protein (SEQ ID No. 2) was produced in E. coli ER2566 cells and then purified by ion exchange chromatography on the WorkBeads 40S resin (BioWorks). The remaining fragments of the YR7 gene were cloned into the pLATE31 vector carrying ampicillin resistance and encoding the C-terminal oligohistidine sequence (HisTag) (SEQ ID No. 8), obtaining genes encoding recombinant proteins YR7_AMIN_Del (SEQ ID No. 3), YR7_LysM_Del (SEQ ID No. 4), YR7_AMIN (SEQ ID No. 5), YR7_Ami (SEQ ID No. 6) and YR7_LysM (SEQ ID No. 7) with an oligohistidine sequence at the C-terminus cloned into the expression vector (Fig. 1). The proteins YR7_AMIN_Del (SEQ ID No. 3), YR7_LysM_Del (SEQ ID No. 4), YR7_AMIN (SEQ ID No. 5) and YR7_LysM (SEQ ID No. 7) were obtained in E. coli ER256 cells, while the YR7_Ami protein (SEQ ID No. 6) was obtained in E. coli BL21 (DE3) cells. These proteins were purified by affinity chromatography on the WorkBeads 40 Ni-NTA resin. All preparations were purified to a homogeneity above 90% and suspended in a storage buffer: 20 mM Tris-HCI (with a pH of 7.0 for YR7_Ami, and for other proteins with a pH of 7.5), 500 mM NaCI, and 10% glycerol. All proteins were stored in portions at -80°C, at concentrations ranging from 4.87 to 28.67 mg / ml. The preparations stored in this way were then dialyzed for 18 hours in 2 liters of buffer (20 mM Tris-HCI, pH 7.5, 10% glycerol) at 16°C, and then used for activity and toxicity tests at the concentration of 500 nM (0.0295 mg / ml for YR7_Sp_Del, 0.0212 mg / ml for YR7_AMIN_Del, 0.0219 mg / ml for YR7_LysM_Del, 0.0063 mg / mL for YR7_AMIN, 0.0128 mg / mL for YR7_Ami, 0.0069 mg / mL for YR7_LysM) or higher. Preparations of proteins produced in this way were used in further studies presented in the examples below.

[0076] To carry out the experiments according to the invention, the YR7_Sp_Del protein without the HisTag tag at the C-terminus of the protein was used.

[0077] Example 2. Determination of the optimal in vitro reaction conditions for YR7_Sp_Del activity

[0078] In order to investigate the optimal conditions of the in vitro conductivity of the reaction environment for the bactericidal activity of the YR7_Sp_Del protein against Yersinia ruckeri, the enzyme activity was examined in a buffer containing increasing concentrations of NaCI, i.e. increasing conductivity of the environment. The protein YR7_Sp_Del (SEQ. ID No. 2) at the concentration of 500 nM (0.0295 mg / ml) was incubated with Y. ruckeri CCM 4620 bacteria and cell survival after incubation with the enzyme was studied. For this purpose, TSB (Graso Biotech) medium was inoculated with a single colony and incubated at 30°C, and the next day the cultures were rejuvenated in a fresh portion of media and incubated until the logarithmic growth phase was reached (OD6oo=0.6). Each culture was centrifuged and the bacteria were suspended in 50 mM glycine-NaOH buffer, pH 8.0 to obtain a cell suspension with a density of 107cfu / ml. The suspension was incubated with the addition of 500 nM YR7_Sp_Del protein for 2 hours at 30°C with stirring. After incubation, the suspension was diluted serially 10 times and plated on petri dishes on TSB-Agar medium in the form of droplets. The bacteriolytic activity of the protein is presented as a reduction in the initial number of bacteria expressed in orders of decrease in the number of bacteria (cfu / ml logic). The reaction buffer contained an increasing concentration of NaCI (from 0 to 100 mM) with a conductivity from 0.065 to 10.05 mS / cm. The results presented in Tab. 1 show that the protein shows the highest activity in the range of conductivity of the reaction environment from 0 to 1 mS / cm, which corresponds to the values of freshwater conductivity in salmonid fish aquaculture. YR7_Sp_Del loses some of its activity already in the presence of 2.5 mM NaCI, and above 10 mM NaCI (1 mS / cm) it becomes inactive. The results of the experiment clearly indicate the possibility of using the protein to remove Yersinia ruckeri bacteria from bodies of water, transport containers intended for breeding and transporting salmonid fish, as well as to eliminate the bacteria in egg hatcheries. Overall, the results of the experiment indicate the possibility of using the protein to remove bacteria in closed circuit bodies of water. The conductivity of water taken from Atlantic salmon aquaculture is 0.065 mS / cm. Tab. 1. Effect of the conductivity of the reaction environment on the activity of the YR7_Sp_Del protein presented as a reduction in the initial number of bacteria measured in orders of magnitude (cfu / ml logio) and in percentage compared to the initial number of bacteria in the assay. Example 3. In vitro bactericidal activity of the recombinant YR7_Sp_Del protein

[0079] To verify whether the YR7_Sp_Del protein exhibits bactericidal activity against different bacterial strains, including Yersinia ruckeri, it was incubated with suspensions of different bacterial species, and then the number of cells that survived incubation was determined (the protocol as in Example 2, although for E. coli LB medium was used, and for M. luteus nutrient broth was used). The results presented in Tab. 2 show that the YR7_Sp_Del protein exhibits bactericidal activity not only against Yersinia ruckeri, but also against many other species of Gram-negative and Gram-positive bacteria. Protein with the concentration of 500 nM (0.0295 mg / ml) is the most active against Yersinia ruckeri bacteria - it eliminates 100%* from 1 million bacterial cells within 2 hours. Therefore, the YR7_Sp_Del protein under optimal conditions (conductivity of the reaction environment up to 1 mS / cm) can also be used to remove other bacteria from surfaces, e.g., Listeria monocytogenes from surfaces and production devices during food processing, or Staphylococcus aureus living on the surfaces of hospital devices.

[0080] Tab. 2. The bactericidal activity of the YR7_Sp_Del protein against different bacterial strains presented as a reduction in the initial number of bacteria expressed in the orders of decrease (cfu / ml logio) and in percentage compared to the initial number of bacteria in the assay. * or below detection in the assay

[0081] Example 4. The removal of Yersinia ruckeri bacterial cells from biofilms

[0082] Yersinia ruckerii forms biofilms on the surfaces of water reservoirs and devices used for fish farming, which makes the biofilm a source of recurrent infections. To determine whether the YR7_Sp_Del protein also removes bacteria from the biofilm structure, an in vitro experiment was carried out, in which the cultured Yersinia ruckeri biofilm was treated with the enzyme YR7_Sp_Del. For this purpose, Yersinia ruckeri CCM 4620 bacteria were suspended in TSB medium with the addition of 0.26% NaCI and 0.33% glucose after night culture, so that the bacterial suspension had an optical density of ODsoo=2.0. Subsequently, a 10- fold diluted suspension with TSB, 0.26% NaCI, 0.33% glucose was applied to a 96-well plate and incubated for 48 hours at 28°C. The obtained biofilm was rinsed once with glycine buffer (50 mM glycine-NaOH, pH 8.0), then the enzyme YR7_Sp_Del at a concentration of 2 pM and 4 pM, also diluted in glycine buffer (50 mM glycine-NaOH, pH 8.0). The controls were treated with an enzyme-free buffer. The plate was incubated at room temperature for 2 hours. After removing the enzyme and flushing the biofilm with glycine buffer, the cells from the biofilm were suspended in the same buffer and serial dilutions were plated on TSB-agar plates in the form of 5 pl drops. The results presented in Fig. 2 indicate that the YR7_Sp_Del enzyme eliminates at least 90% to 99% of Yersinia ruckeri cells from the biofilm (1-2 logic cfu / ml), which enables its use for cleaning water reservoirs in fish farms where the Yersinia ruckeri biofilm is a serious threat and a reservoir of bacteria that constantly infect fish.

[0083] Example 5. Identification of the minimum sequence necessary to obtain a bactericidal effect against Yersinia ruckeri

[0084] The bactericidal activity of the particular derivatives of the YR7_Sp_Del protein obtained in Example 1, i.e. YR7_Sp_Del (SEQ ID No. 2), YR7_AMIN_Del (SEQ ID No. 3), YR7_LysM_Del (SEQ ID No. 4), YR7_AMIN (SEQ ID No. 5), YR7_Ami (SEQ ID No. 6) and YR7_LysM (SEQ ID No. 7) in relation to Yersinia ruckeri bacteria, was investigated. The bacteria for the experiment were prepared as in Example 2 and then incubated in the presence of 500 nM proteins for 2 hours at room temperature. After incubation, the bacteria were plated as in Example 2. The data presented in Tab. 3 shows that the sequence of the YR7_AMIN_Del protein (SEQ ID No. 3) retains high bactericidal activity against Yersinia ruckeri. It is a protein containing a single aminase domain YR7_Ami (SEQ ID No. 6) and the YR_7_LysM domain (SEQ ID No. 7) connected by a linker, whereby the addition of both domains as separate isolated recombinant proteins (YR7_Ami and YR7_LysM) to the reaction environment does not reproduce the activity of the YR7_AMIN_Del protein (SEQ ID No. 3). Protein variants have lower or no activity. Interestingly, the addition of three separate domains (YR7_AMIN, YR7_Ami, and YR7_LysM) to the reaction environment does not eliminate Yersinia ruckeri to the same extent as the YR7_Sp_Del protein, which consists of the same domains, but connected by linkers into a single protein molecule, although the percentage of bacterial reduction shown is at a high level of 90-99%. The results clearly indicate that bactericidal activity is provided by the isolated domain of YR7_LysM (SEQ ID No. 7) alone, all tested proteins comprising the domain, as well as mixtures comprising it showed bactericidal activity against Yersinia ruckeri. Unexpectedly, it was also found that the recombinant polypeptide YR7_LysM_Del, not comprising the LysM domain, but comprising the AMIN and Ami domains connected by a linker, also exhibits bactericidal activity, despite the fact that each of the domains that build it separately does not have such properties.

[0085] Table 3. Bactericidal activity of proteins against Yersinia ruckeri CCM 4620 presented as a reduction in the initial number of bacteria expressed in the orders of decrease (cfu / ml logio) and in percentage compared to the initial number of bacteria in the assay. Example 6. Confirmation of the activity of the peptidoglycan hydrolase of the recombinant YR7_Sp_Del protein and the bacteriolytic properties on the strain Yersinia ruckeri

[0086] The active YR7_Sp_Del protein contains, among others, the YR7_Ami domain (SEQ. ID No. 6), which is homologous to other known domains of peptidoglycan hydrolases with the catalytic activity of amidase, i.e., the enzyme that crosses amide bonds between the N-acetylmuramic acid residue in the sugar chain, and the first amino acid in the peptidoglycan cross-linking peptide, i.e., L-alanine in the peptidoglycan. In order to confirm that the protein is an amidase, the peptidoglycan isolated from the bacterial cells of Yersinia ruckeri was incubated in the presence of the YR7_Sp_Del protein, and then the obtained digestive products were separated by chromatographic methods and their masses were determined by mass spectrometry. Peptidoglycan was isolated from Yersinia ruckeri CCM 4620 according to the previously defined protocol [9], and then 500 pg of the preparation was incubated overnight in the presence of 10 pM of the YR7_Sp_Del protein at 30°C in a 50 mM glycine-NaOH buffer, pH 8.0. The peaks observed on the mass spectrum correspond to products formed as a result of the activity of the catalytic domain of the amidase nature. The site of cleavage of peptidoglycan by the YR7_Sp_Del enzyme is shown in the diagram (Fig. 3.), while the fragments formed after pruning are shown in Fig. 4. Tab. 4 presents the intensities of these peaks in the control sample (without the added protein) and in the incubated sample with the tested protein. The difference in the intensity of the peaks in both tests indicates the appearance of digestion products of the appropriate size peptidoglycan isolated from the cell walls of Yersinia ruckeri. This clearly indicates the amide bonds in peptidoglycan (peptidoglycan hydrolase) of the YR7_Ami domain, which is a component of the YR7_Sp_Del protein. It can be assumed that in vivo, under favorable conditions and upon allowing contact with the peptidoglycan layer in the bacterial cell wall, i.e. after crossing the outer membrane in the Y. ruckeri cell wall, this domain would hydrolyze amide bonds in the peptidoglycan.

[0087] Tab. 4. The amidase activity of the protein YR7_Sp_Del presented as an increase in the intensity of peaks with masses corresponding to the predicted products of digestion of isolated peptidoglycan from the walls of Yersinia ruckeri CCM 4620. The letter designations of the products of digestion correspond to the designations in Fig. 4. Example 7. Confirmation of the lytic activity of the recombinant YR7_Sp_Del protein towards Y. ruckeri CCM 4620 cells

[0088] In order to verify whether the YR7_Sp_Del protein kills Y. ruckeri CCM 4620 cells causing their lysis, an experiment was performed with the SYTOX™ green (Invitrogen) reagent binding to nucleic acids. This reagent does not penetrate the intact cell membrane of living cells, and during lysis, when the membrane permeabilizes and the cell contents are ejected to the outside, it can easily bind to nucleic acids by emitting green light with a wavelength of 523 nm when excited by a wavelength of 504 nm. Y. ruckeri CCM 4620 cells were grown as in Example 2 and suspended in a 50 mM glycine-NaOH buffer, pH 8.0. Cells were suspended to OD595 = 1.0 and then incubated with enzyme YR7_Sp_Del at 0 (non-enzyme control), 0.5, 1 or 2 pM in the presence of 5 pM of SYTOX™ green reagent. After one hour of incubation at 22°C, 100 pl from each sample was transferred to a 96-well plate. The fluorescence measurement was performed using the Infinite M1000 Pro spectrofluorometer (Tecan Trading AG) at an excitation wave of 504 nm and a fluorescence reading at 523 nm. The experiment was performed in three biological replications. The results obtained in this way (Fig. 5) indicate the lytic nature of cell death, as the fluorescence reading at the highest concentration of the enzyme is more than ten times higher than in the sample without the added protein. This effect can be indirectly related to the result of the experiment in Example 6, which demonstrated the digestion of isolated peptidoglycan, but this does not constitute direct evidence of the cause of cell lysis. The structure of the cell wall of Gram(-) bacteria does not reflect the conditions of the digestion reaction of the isolated peptidoglycans in vitro.

[0089] Example 8. The activity of the recombinant protein YR7_Sp_del at low temperature.

[0090] In order to investigate the effect of low temperature of the reaction environment on the bactericidal activity of the YR7_Sp_Del protein, an experiment was performed at temperatures of 12°C and 30°C. Temperatures of about 12°C prevail in closed salmonid fish farms and egg hatcheries. The test was performed as in Example 5. The results obtained (Tab. 5) prove that at a lower temperature the protein still retains its bactericidal activity and eliminates 90% of Yersinia ruckeri bacteria within 2 hours and indicate the possibility of its use in aquaculture conditions.

[0091] Table 5. The bactericidal activity of the YR7_Sp_Del protein at 12°C presented as a reduction in the initial number of bacteria expressed in orders of decrease (cfu / ml logio) and in percentage compared to the initial number of bacteria in the assay. Example 9. The activity of the recombinant protein YR7_Sp_Del in aquaculture water at low temperature

[0092] In order to investigate the application potential of the YR7_Sp_Del protein, a test of its bactericidal activity was performed under conditions similar to those found in Atlantic salmon (S. salar) farms. The test was performed as in Example 2 with modifications: the reaction was carried out at the temperature of 12 or 30°C in freshwater taken from Atlantic salmon aquaculture using 4 pM of protein for 18 hours. The obtained results (90% elimination of Y. ruckeri CCM 4620 cells, Fig. 6) confirm the activity of the protein under these conditions and the high potential of its use in salmonid fish breeding tanks.

[0093] Example 10. Safety of using the recombinant protein YR7_Sp_Del for eukaryotic organisms

[0094] It was investigated whether the YR7_Sp_Del protein is toxic to the model organism Zebrafish (Danio rerio). For this purpose, the OECD acute toxicity test no. 236 was used, in which groups (n=12) of the wild-type D. rerio embryos were placed in 3 cm diameter petri dishes filled with 2 ml of liquid, i.e. with a protein solution at a concentration of 0.5 pM in a 50 mM glycine buffer with pH 8.0, or glycine buffer alone (control). The embryos were incubated at 28.5°C for two days, exchanging fluids containing a fresh portion of protein every 24 hours. The YR7_Sp_Del protein was found to be non-toxic to D. rerio embryos (Fig. 7). The test result indicates that the protein is non-toxic to fish and can also be used in breeding of other fish species.

[0095] Example 11. The removal of Yersinia ruckeri bacteria from Danio rerio aquaculture by the recombinant protein YR7_Sp_Del.

[0096] As it was shown in Example 10 that the YR7_SP_Del protein is safe for use in Danio rerio fish and that the protein is bactericidal for Gram(-) Yersinia ruckeri bacteria, it was decided to verify whether in conditions similar to an aquaculture in the presence of 4-day-old Danio rerio fish, the YR7_Sp_Del enzyme would cause the elimination of Y. ruckeri bacteria.

[0097] For this purpose, Y. ruckeri CCM 4620 bacterial cells were prepared according to the protocol as in Example 2 and suspended in a 50 mM glycine-NaOH buffer, pH 8.0 to a final concentration of 107cfu / ml. The experiment was conducted on Danio rerio fish 24 hours after fertilization. In the petri dishes, there were 10 individuals in 2.5 ml of glycine buffer along with bacteria. A preparation of recombinant YR7_Sp_Del protein at the concentration of 0.5 pM, suspended in the above-mentioned glycine buffer, was added to the fish and incubated at 28°C for 2 hours. Subsequently, the bacteria were plated on TSB- Agar plates at 5 pl in the so-called droplet test and the reduction in the initial number of bacteria was estimated as an order of magnitude of login cfu / ml. The negative control consisted of Y. ruckeri bacteria incubated in the presence of fish without the added enzyme. The results shown in Tab. 6 indicate a high bactericidal efficiency of the tested YR7_Sp_Del protein, which eliminated Y. ruckeri bacteria in the presence of Danio rerio fish almost as effectively as in the optimal conditions (Tab. 6 "without fish" variant). Thus, no negative effect of the presence of fish on the bactericidal activity of the protein was observed, which indicates the potential effectiveness of the YR7_Sp_Del protein in aquaculture, and therefore the possibility of its use in fish farms for the treatment and prevention of fish diseases caused by Yersinia ruckeri, such as fish yersiniosis, especially in industrial farms of salmonid fish.

[0098] Table 6. Bactericidal activity of the YR7_Sp_Del protein in D. rerio aquaculture and conditions without fish, presented as a reduction in the initial number of bacteria expressed in orders of decrease (cfu / ml logio).

[0099] Example 12. The stability of the recombinant YR7_Sp_Del protein

[0100] To test the stability of YR7_Sp_Del protein stored in solution at different temperatures for several months, the protein obtained in Example 1 and suspended in a storage solution (20 mM Tris-HCI, pH 7.5, 500 mM NaCI, 10% glycerol) at the concentration of 12.19 mg / ml was stored in portions (25 pl) at -80°C, 4°C, 22°C and 37°C. Samples of protein in time intervals: 0.5 months, 1 month, 2 months, 6 months and 11 months were tested for bactericidal activity against Y. ruckeri CCM 4620 according to the protocol in Example 2 and the integrity of the proteins was assessed in the SDS-PAGE electrophoretic separation (Fig. 8). Both the results presented in Tab. 7 and the electrophoretic separation image in Fig. 8 indicate that the YR7_Sp_Del protein is very stable in solution, retains its integrity and biocidal activity. Only when stored at a higher temperature, 37°C, it undergoes faster proteolysis and its activity decreases slightly compared to that of a protein sample stored at a low temperature of -80°C. The thermal stability of YR7_Sp_Del protein was also verified by exposing it to high temperatures in a short time. The YR7_Sp_Del protein was incubated at 100°C for 0, 0.5, 1 or 5 hours and then cooled for 30 minutes at room temperature and tested as in Example 2. The results collected in Tab. 8 clearly indicate a slight decrease in the bactericidal activity of the YR7_Sp_Del protein.

[0101] Table 7. The activity of the YR7_Sp_Del protein stored from 2 weeks to 11 months in a solution at different temperatures presented as a reduction in the initial number of bacteria expressed in orders of decrease (cfu / ml logio) and in percentage compared to the initial number of bacteria in the assay.

[0102] Table 8. The activity of YR7_Sp_Del protein incubated for 0.5, 1 or 5 hours at 100°C in a solution presented as a reduction in the initial number of bacteria expressed in orders of decrease (cfu / ml logio) and in percentage compared to the initial number of bacteria in the assay.

[0103] Example 13. Inactivation of the enzyme - the recombinant protein YR7_Sp_Del

[0104] In order to test under what conditions the bactericidal activity of the YR7_Sp_Del protein can be inhibited, three methods of enzyme inactivation were tested: a) use of an EDTA solution, b) inactivation by increasing the conductivity of the reaction environment, and c) proteolysis. a) enzyme inactivation with an EDTA solution

[0105] A solution of EDTA(sodium edetate), chelates ions from the reaction environment, therefore it can remove zinc ions from the active site of the enzyme, i.e. the catalytic domain with amidase activity YR7_Sp_Del. A full-length active multidomain protein, containing the catalytic amidase domain (between 165 and 400 aa in YR7_Sp_Del protein, the isolated Ami domain has the sequence of SEQ. ID No. 6) was subjected to a bactericidal activity test against Y. ruckeri CCM 4620 according to the protocol from Example 2. Additionally, EDTA solution with concentrations ranging from 0 to 25 mM was added to the reaction buffer. The result of the experiment is presented in Tab. 9. and it indicates a low potential to inhibit the bactericidal activity of the enzyme. At 5 mM EDTA concentration in the reaction a slight decrease in the antibacterial properties of the protein is observed, intensifying at a concentration of 25 M. Above this EDTA concentration, the reaction solution was toxic to bacteria. This suggests that higher concentrations of EDTA are needed to completely inhibit the enzymatic activity of the amidase domain, indicating strong coordination of zinc ions at the active site of the enzyme, i.e. the amidase domain (YR7_Ami). b) deactivation by increasing the conductivity of the reaction environment

[0106] Taking into account the results obtained in Example 2, it is possible to inhibit the activity of the proteins obtained in Example 1 by increasing the conductivity of the reaction environment accordingly. The YR7_Sp_Del protein (SEQ. ID No. 2) is completely inhibited in the presence of 20 mM NaCI, corresponding to a conductivity of approximately 2 mS / cm (see Tab. 1). Therefore, it can be assumed that the protein is inhibited in a solution with a conductivity of at least 2 mS / cm. c) proteolysis

[0107] Proteolytic deactivation of an enzyme involves digesting the protein with an enzyme with protease activity that cuts the peptide bonds between the amino acids in the protein. In order to test whether this method of inactivation of the enzyme would be effective in the case of YR7_Sp_Del protein, the Milli-Q. enzyme YR7_Sp_Del with a concentration of 6.095 mg / ml in a buffer of 10 mM Tris-HCI, pH 7.5, 250 mM NaCI, 5% glycerol, 1 mM CaCL was subjected to proteolytic digestion using proteinase K and trypsin. The YR7_Sp_Del protein preparation (120 pg) was incubated in the presence of proteinase K (20 pg, A&A Biotechnology) or trypsin (10 pg, Merck) for 1 hour at 37°C. An image of the stained polyacrylamide gel is shown in Fig. 9. It shows that under both proteolytic enzymes, proteinase K and trypsin, treatment the protein is completely digested and has not retained its initial length. The digested protein preparations showed no bactericidal activity in the test carried out according to the same protocol as in Example 2.

[0108] SP - signal peptide AMIN -peptidoglycan binding domain AMIN

[0109] Ami - catalytic domain with amidase activity

[0110] LysM - peptidoglycan binding domain LysM

[0111] YR7 - full-length protein YR7 encoded by the gene WP_049689580.1 (SEQ ID No. 1)

[0112] YR7_Sp_Del - YR7_Sp_Del - protein with signal peptide (Sp) deletion at the N-terminus (SEQ ID No. 2) YR7_AMIN_Del -YR7_Sp_Del - protein with an additional deletion of the AMIN domain at the N-terminus of the protein (SEQ ID No. 3)

[0113] YR7_LysM_Del - YR7_Sp_Del - protein with deletion of the LysM domain at the C-terminus of the protein (SEQ ID No. 4)

[0114] YR7_AMIN - AMIN domain from the YR7_Sp_Del protein (SEQ ID No. 5)

[0115] YR7_Ami - amidase domain from the YR7_Sp_Del protein (SEQ ID No. 6)

[0116] YR7_LysM - LysM domain from the YR7_Sp_Del protein (SEQ ID No. 7)

[0117] NAM - N-acetylmuramic acid

[0118] NAG - N-acetylglucosamine

[0119] EDTA - ethylenediaminetetraacetic acid

[0120] TSB - bacterial culture medium, Tryptic Soy Broth

[0121] LB - bacterial culture medium, Lysogeny broth cfu - colony forming unit

[0122] LITERATURE:

[0123] 1. Ross AJ, Rucker RR, Ewing WH (1966) Description of a bacterium associated with redmouth disease of rainbow trout (Salmo gairdneri). Can J Microbiol 12:763-770

[0124] 2. Horne MT, Barnes AC (1999) Enteric redmouth disease (Yersinia ruckeri). In: Woo PTK, Bruno DW (eds) Fish diseases and disorders. Viral, bacterial and fungal infections. CABI Publishing, Wallingford, pp 445-477.

[0125] 3. Busch RA (1978) Protective vaccines for mass immunization of trout. Salmonide 1:10-22

[0126] 4. Michel C, Kerouault B, Martin C (2003) Chloramphenicol and florfenicol susceptibility of fish- pathogenic bacteria isolated in France: Comparison of minimum inhibitory concentration, using recommended provisory standards for fish bacteria. J Appl Microbiol 95:1008-1015

[0127] 5. Rodgers CJ (2001) Resistance of Yersinia ruckeri to antimicrobial agents in vitro. Aquaculture 196:325-345

[0128] 6. Ramos-Vivas J, Superio J, Galindo-Villegas J, Acosta F. Phage Therapy as a Focused Management Strategy in Aquaculture. Int J Mol Sci. 2021 Sep 28; 22(19):10436. DOI: 10.3390 / IJMS221910436. PMID: 34638776; PMCID: PMC8508683.

[0129] 7. Irianto A, Austin B (2002) Probiotics in aquaculture. J Fish Dis 25:633-642

[0130] 8. Raida MK, Larsen JL, Nielsen ME, Buchmann K (2003) Enhanced resistance of rainbow trout, Oncorhynchus mykiss (Walbaum), against Yersinia ruckeri challenge following oral administration of Bacillus subtilis and B. licheniformis (BioPlus2B). J Fish Dis 26:495-498

[0131] 9. de Jonge B. L., Chang Y. S., Gage D. and Tomasz A. (1992) Peptidoglycan composition of a highly methicillin-resistant Staphylococcus aureus strain. The role of penicillin binding protein 2A. Journal of Biological Chemistry 276 (16), 11248-11254. SEQUENCE LISTING

[0132] SEQ ID No. 1. Amino acid sequence of the protein encoded by the YR7 gene, identified in the NCBI database (Protein database, having the accession number WP_049689580.1) in the genome of the bacterium Yersinia ruckeri, from which the remaining derived proteins were cloned. The sequence contains a signal peptide (SP, amino acids indicated with a gray background and underlined) at the position 1-27 aa; the peptidoglycan binding domain (AMIN, amino acids in bold font) at the position 28- 133 aa; linker 1 (underlined amino acids) at the position 134-191 aa; the amidase domain (Ami, amino acids indicated with a grey background) at the position 192-427 aa; linker 2 (underlined amino acids) at the position 428-467 aa; the peptidoglycan binding domain (LysM, amino acids in italics) at position 468- 588 aa.

[0133] MMLSWRGFIATALLWSSVLTAPAAWAMKLTDIKVTNGSGESQITLSFDGKPIYAFFPLTGPDRVWDIRQSGNLSG LPLEFSGQNLLKRIRSSQPKDPQSTRLVFELTQKTKTRAVTQQVGGKYNWFTLSAVGATTRQTQMVATSVREAPKV AGSNPFNNKATWTSSPTITTSSNRPVKTANSSTSDRIWAIDAGHGGQDPGAIGQNGLKEKNVTIAI SRKLESLLN NDSMFKPVLTRNGDYFI SVMGRSDVARKQAANVLVSIHADAAPNRSANGASVWVLSNRRANSEMGNWLEQHEKQSEL LGGAGDVLANTSADPYLSQAVLDLQFGHSQRVGYDVAVKVLRELQTIGSIHKRRPEHASLGVLRSPDI PSLLVETGF I SNGTEERLLGSSAYQDKIAQAIYKGLRSYFLSHPLQADPKVENRPLGATAAVKRAAQQQRGINQPGPVMSTASSGG KTAASSKSQIHWKRGETLSGIASQYGVSMAAMRQNNTLRKDWWVGQRLRIPAAGTTVTAVPTPQKTVALKKSSPV KPIKHQVKRGDTLSAIAAKYGVSQSEIQRVNKLKSGSVQLGQTLTIPQS

[0134] SEQ ID No. 2. Amino acid sequence of the recombinant protein YR7_Sp_Del comprising the peptidoglycan binding domain (AMIN) (1-106 aa); linker 1 (107-164 aa); the amidase domain (Ami) (165-400 aa); linker 2 (401-440 aa); the peptidoglycan binding domain (LysM) (441-561 aa).

[0135] MKLTDIKVTNGSGESQITLSFDGKPIYAFFPLTGPDRVWDIRQSGNLSGLPLEFSGQNLLKRIRSSQPKDPQSTRL VFELTQKTKTRAVTQQVGGKYNWFTLSAVGATTRQTQMVAT S VREAPKVAGSNP FNNKATWT SSPTITTS SNRPV RTANSSTSDRIWAIDAGHGGQDPGAIGQNGLKEKNVTIAI SRKLESLLNNDSMFKPVLTRNGDYFI SVMGRSDVAR KQAANVLVSIHADAAPNRSANGASVWVLSNRRANSEMGNWLEQHEKQSELLGGAGDVLANTSADPYLSQAVLDLQFG HSQRVGYDVAVKVLRELQTIGSIHKRRPEHASLGVLRSPDI PSLLVETGFI SNGTEERLLGSSAYQDKIAQAIYKGL RS YFL S H P LQAD P KVENRPLGATAAVKRAAQQQRGINQPGPVMSTASSGGKTAAS SKSQIHWKR GE TLSGIASQ YG VSMAAMRQNNTLRKDWWVGQRLRIPAAGTTVTAVPTPQKTVALKKSSPVKPIKHQVKRGDTLSAIAAKYGVSQSEI QR VNKLKSGS VQLGQTLTIPQS

[0136] SEQ ID No. 3. Amino acid sequence of the recombinant protein YR7_AMIN_Del comprising the amidase domain (Ami) (1-236 aa); linker 2 (237-276 aa) and the peptidoglycan binding domain (LysM) (277-397 aa).

[0137] IWAIDAGHGGQDPGAIGQNGLKEKNVTIAI SRKLESLLNNDSMFKPVLTRNGDYFI SVMGRSDVARKQAANVLVSI HADAAPNRSANGASVWVLSNRRANSEMGNWLEQHEKQSELLGGAGDVLANTSADPYLSQAVLDLQFGHSQRVGYDVA VKVLRELQTIGSIHKRRPEHASLGVLRSPDI PSLLVETGFI SNGTEERLLGSSAYQDKIAQAIYKGLRSYFLSHPLQ ADPKVENRPLGATAAVKRAAQQQRGINQPGPVMSTASSGGKTAASSKSQJgWKRGETZSGTASQYGVSAaAMRQW TLRKDWWVGQRLRIPAAGTTVTAVPTPQKTVALKKSSPVKPIKHQVKRGDTLSAIAAKYGVSQSEIQRVNKLKSGS VQLGQTLTIPQS

[0138] SEQ ID No. 4. Amino acid sequence of the recombinant protein YR7_LysM_Del comprising the peptidoglycan binding domain (AMIN) (1-106 aa); linker 1 (107-164 aa) and the amidase domain (Ami) (165-400 aa).

[0139] MKLTDIKVTNGSGESQITLSFDGKPIYAFFPLTGPDRVWDIRQSGNLSGLPLEFSGQNLLKRIRSSQPKDPQSTRL VFELTQKTKTRAVTQQVGGKYNWFTLSAVGATTRQTQMVAT S VREAPKVAGSNP FNNKATWT SSPTITTS SNRPV RTANSSTSDRIWAIDAGHGGQDPGAIGQNGLKEKNVTIAI SRKLESLLNNDSMFKPVLTRNGDYFI SVMGRSDVAR KQAANVLVSIHADAAPNRSANGASVWVLSNRRANSEMGNWLEQHEKQSELLGGAGDVLANTSADPYLSQAVLDLQFG HSQRVGYDVAVKVLRELQTIGSIHKRRPEHASLGVLRSPDI PSLLVETGFI SNGTEERLLGSSAYQDKIAQAIYKGL RSYFLSHPLQADPKV

[0140] SEQ ID No. 5. Amino acid sequence of the recombinant protein YR7_AMIN (106 aa).

[0141] MKLTDIKVTNGSGESQITLSFDGKPIYAFFPLTGPDRVWDIRQSGNLSGLPLEFSGQNLLKRIRSSQPKDPQSTRL VFELTQKTKTRAVTQQVGGKYNWFTLSA

[0142] SEQ ID No. 6. Amino acid sequence of the recombinant protein YR7_Ami (236 aa).

[0143] IWAIDAGHGGQDPGAIGQNGLKEKNVTIAI SRKLESLLNNDSMFKPVLTRNGDYFI SVMGRSDVARKQAANVLVSIH ADAAPNRSANGASVWVLSNRRANSEMGNWLEQHEKQSELLGGAGDVLANTSADPYLSQAVLDLQFGHSQRVGYDVAVK VLRELQTIGSIHKRRPEHASLGVLRSPDI PSLLVETGFI SNGTEERLLGSSAYQDKIAQAIYKGLRSYFLSHPLQADP KV

[0144] SEQ ID No. 7. Amino acid sequence of the recombinant protein YR7_LysM (121 aa).

[0145] SKSQIHWKRGETLSGIASQYGVSMAAMRQNNTLRKDWWVGQRLRIPAAGTTVTAVPTPQKTVALKKSSPVKPIKHQ VKRGDTLSAIAAKYGVSQSEIQRVNKLKSGSVQLGQTLTIPQS

[0146] SEQ ID No. 8. Oligohistidine sequence (HisTag) derived from an expression vector (8 aa).

[0147] GHHHHHHG

[0148] SEQ ID No. 9. Sequence of linker 1 (58 aa).

[0149] VGATTRQTQMVATSVREAPKVAGSNPFNNKATWTSSPTITTSSNRPVKTANSSTSDR

[0150] SEQ ID No. 10. Sequence of linker 2 (40 aa).

[0151] ENRPLGATAAVKRAAQQQRGINQPGPVMSTASSGGKTAAS

Claims

CLAIMS1. A recombinant protein with antibacterial properties characterized in that it comprises an active peptidoglycan binding domain LysM, with an amino acid sequence in at least 80% identical with the sequence presented in SEQ ID No. 7 from Yersinia ruckeri.

2. The recombinant protein with antibacterial properties according to claim 1, characterized in that it comprises an active peptidoglycan binding domain LysM, with an amino acid sequence in at least 85%, more preferably 90%, more preferably 95%, more preferably 99% identical with the sequence presented in SEQ ID No. 7; most preferably, it comprises an active catalytic domain LysM with the amino acid sequence of SEQ ID No. 7.

3. The recombinant protein with antibacterial properties according to claims 1-2, characterized in that it is an active peptidoglycan binding domain LysM, with the amino acid sequence presented in SEQ ID No. 7 from Yersinia ruckeri.

4. The recombinant protein with antibacterial properties according to claims 1-3, characterized in that it, further comprises an amidase domain Ami with an amino acid sequence in at least 80% identical with the sequence presented in SEQ ID No. 6, more preferably in at least 85%, more preferably 90%, more preferably 95%, more preferably 99% identical with the sequence presented in SEQ ID No. 6, more preferably the amidase domain Ami is the amino acid sequence as presented in SEQ ID No. 6 from Yersinia ruckeri.

5. The recombinant protein according to claim 4, characterized in that the amidase domain Ami is located in the direction of the N-terminus of the recombinant protein, with respect to the peptidoglycan binding domain LysM.

6. The recombinant protein with antibacterial properties according to claims 4-5, characterized in that between the peptidoglycan binding domain LysM and the amidase domain Ami there is a linker connecting them, wherein, preferably, the linker has a sequence in at least 80% identical with the sequence of the linker 2 presented in SEQ ID No. 10, more preferably the linker is the amino acid sequence presented as SEQ ID No. 10 from Yersinia ruckeri.

7. The recombinant protein with antibacterial properties according to claims 1-7, characterized in that it further comprises the peptidoglycan binding domain AMIN with an amino acid sequence in at least 80% identical with the sequence presented in SEQ ID No. 5, more preferably in at least 85%, more preferably 90%, more preferably 95%, more preferably 99% identical with the sequence presented in SEQ ID No. 5, more preferably the peptidoglycan binding domain AMIN is the amino acid sequence presented in SEQ ID No. 5 from Yersinia ruckeri.

8. The recombinant protein according to claim 7 , characterized in that the peptidoglycan binding domain AMIN is located in the direction of the N-terminus of the recombinant protein, with respect to the peptidoglycan binding domain LysM.

9. The recombinant protein with antibacterial properties according to claims 7-8, characterized in that between the peptidoglycan binding domain LysM and the peptidoglycan binding domain AMIN there is a linker, wherein, preferably, the linker has a sequence in at least 80% identical with the sequence of the linker 1 presented in SEQ ID No. 10, more preferably the linker is the amino acid sequence presented as SEQ ID No. 9 from Yersinia ruckeri.

10. The recombinant protein with antibacterial properties according to claims 1-9, characterized in that it is the amino acid sequence YR7_Sp_Del from Yersinia ruckeri presented in SEQ ID No. 2.

11. The recombinant protein according to claims 1-10, characterized in that it further has an amino acid sequence useful for purification and / or isolation of protein attached to one of the termini, wherein, preferably, the amino acid sequence useful for purification and / or isolation is placed on C-terminus side of the protein, preferably the amino acid sequence useful for purification and / or isolation of the recombinant protein is the oligohistidine sequence presented in SEQ ID No. 8.

12. The recombinant protein according to claims 1-11, characterized in that it consists of the sequence SEQ ID No. 2 at the N-terminus of the recombinant protein and the oligohistidine sequence presented in SEQ ID No. 8 at the C-terminus of the recombinant protein.

13. A genetic construct characterized in that it encodes the recombinant protein as defined in claims 1- 12.

14. A host cell characterized in that it comprises the genetic construct as defined in claim 13.

15. A composition characterized in that it comprises the recombinant protein as defined in claims 1-12 or a mixture thereof and a carrier.

16. The composition according to claim 15, characterized in that it further comprises an isolated amidase domain Ami protein, with an amino acid sequence having at least 80% identity with the sequence presented in SEQ ID No. 6, and / or an isolated peptidoglycan binding domain AMIN protein, with an amino acid sequence in at least 80% identical with the sequence presented in SEQ ID No. 5.

17. A non-medical use of the recombinant protein as defined in claims 1-12 and / or the composition as defined in claims 15-16 as an antibacterial agent.

18. The non-medical use according to claim 17, characterized in that the antibacterial agent is used in an aqueous environment with a conductivity of 0 to 10 mS / cm.

19. The non-medical use according to claims 17-18, characterized in that the antibacterial agent is used against bacteria Yersinia spp., Enterococcus spp., Listeria spp., Bacillus spp., Escherichia spp., Staphylococcus spp., Streptococcus spp., Micrococcus spp., more preferably, bacterial species selected from Yersinia ruckeri, Enterococcus faecalis, Listeria monocytogenes, Escherichia coli, Staphylococcus aureus, Streptococcus uberis, Staphylococcus simulans, Staphylococcus epidermidis, Micrococcus luteus.

20. Use of the recombinant protein as defined in claims 1-12 and / or the composition as defined in claims 15-16 as an antiseptic and disinfecting agent.

21. Use of the recombinant protein as defined in claims 1-12 and / or the composition as defined in claims 15-16 as an antibacterial agent for the disinfecting surfaces and rooms in food processing, preferably for disinfecting surfaces that come into contact with food or with semi-finished food products.

22. Use of the recombinant protein as defined in claims 1-12 and / or the composition as defined in claim 16 as an antibacterial agent in the food industry as an additive to food for humans and / or animals, preferably as a feed additive, preferably as an additive to the feed for fish.

23. Use of the recombinant protein as defined in claims 1-12 and / or the composition as defined in claims 15-16 as a disinfecting agent for surfaces in health care in medicine, veterinary medicine, diagnostics, preferably for disinfecting hospital surfaces, medical and veterinary offices, laboratory surfaces, surfaces of tools, surfaces of instruments used in medicine, veterinary medicine, diagnostics.

24. A non-medical use of the recombinant protein as defined in claims 1-12 and / or the composition as defined in claims 15-16 as an in vitro bactericidal agent to inhibit, eliminate bacteria, prevent the growth of Gram(+) and Gram(-) bacteria, preferably bacteria of the genus Yersinia spp., Enterococcus spp., Listeria spp., Bacillus spp., Escherichia spp., Staphylococcus spp., Streptococcus spp., Micrococcus spp.; more preferably the species of bacteria selected from Yersinia ruckeri, Enterococcus faecalis, Listeria monocytogenes, Escherichia coli, Staphylococcus aureus, Streptococcus uberis, Staphylococcus simulans, Staphylococcus epidermidis, Micrococcus luteus.

25. The non-medical use according to claim 24, characterized in that the bactericidal agent is used in an aqueous environment with a conductivity of 0 to 10 mS / cm.

26. Use of the recombinant protein as defined in claims 1-12 and / or the composition as defined in claims 15-16 as a bactericidal agent to inhibit, eliminate bacteria, prevent the growth of bacteria in water, preferably bacteria of the species Escherichia coli, Enterococcus feacalis, Yersinia ruckeri.

27. The use according to claim 26, characterized in that the bactericidal agent is used in aquacultures.

28. The use according to claims 25-27, characterized in that the bactericidal agent is used in fish hatcheries and rearing facilities, in recirculating water systems, in containers for the transport of fishand / or fry, in containers for the storage of fish and / or fry, in containers with eggs and / or larvae of aquatic animals, in containers with aquatic plants and for the equipment having contact with fish.

29. The use according to claims 26-28, characterized in that the bactericidal agent is used in an aqueous environment with a conductivity of 0 to 10 mS / cm.

30. Use of the recombinant protein as defined in claims 1-12 and / or the composition as defined in claims 15-16 to eliminate and prevent biofilm formation on surfaces, preferably on the surfaces in contact with water reservoirs in aquaculture, preferably, to eliminate biofilm formed by Yersinia ruckeri.

31. Use of the recombinant protein as defined in claims 1-12 and / or the composition as defined in claims 15-16 as a cleaning agent for water reservoirs and devices, preferably for cleaning of water reservoirs and devices used in fish farming.

32. Use of the recombinant protein as defined in claims 1-12 and / or the composition as defined in claims 15-16 as an antibacterial agent 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.

33. A cosmetic or care composition for cosmetic, care and hygiene uses in humans or animals, characterized in that it contains the recombinant protein as defined in claims 1-12 and / or the composition as defined in claims 15-16, wherein the composition is intended for external use, and wherein the recombinant protein is used in the cosmetic or care composition to limit the occurrence or eliminate bacteria from the composition.

34. A veterinary and / or pharmaceutical composition characterized in that it comprises the recombinant protein as defined in claims 1-12 and / or the composition as defined in claims 15-16 and a veterinary and / or pharmaceutically acceptable carrier for use in the treatment of a disease and / or condition caused by bacteria, preferably bacteria of the genus Yersinia spp., Enterococcus spp., Listeria spp., Bacillus spp., Escherichia spp., Staphylococcus spp., Streptococcus spp., Micrococcus spp.; more preferably bacterial species selected from Yersinia ruckeri, Enterococcus faecalis, Listeria monocytogenes, Escherichia coli, Staphylococcus aureus, Streptococcus uberis, Staphylococcus simulans, Staphylococcus epidermidis, Micrococcus luteus.

35. A veterinary composition characterized in that it comprises the recombinant protein as defined in claims 1-12 and / or the composition as defined in claims 15-16 and a veterinarily acceptable carrier for use in the prevention and / or treatment of fish diseases caused by Yersinia ruckeri, preferably for use in the prevention and / or treatment of yersiniosis in fish.

36. The veterinary composition for use according to claim 35, characterized in that it is used in the treatment of yersiniosis in salmonid fish, preferably selected from rainbow trout (Oncorhynchus mykiss),T1brook trout (Salvelinus fontinalis), river trout (Salmo trutta morpha / brio), sea trout (Salmo trutta morpha trutta), Atlantic salmon (Salmo salar), lake trout (Salmo trutta morpha lacustris).

37. A method for inhibiting or limiting the growth of Gram(+) and / or Gram(-) bacteria characterized in that it comprises a step in which the bacteria are contacted with the recombinant antibacterial protein as defined in claims 1-12 and or the composition as defined in claims 15-16 in an environment with conductivity ensuring the bactericidal activity of the recombinant protein.

38. The method according to claim 37, characterized in that the contacting is carried out in an aqueous solution with the conductivity of 0 to 10 mS / cm.

39. The method according to claims 37-38, characterized in that the bacteria are selected from the genus Yersinia spp., Enterococcus spp., Listeria spp., Bacillus spp., Escherichia spp., Staphylococcus spp., Streptococcus spp., Micrococcus spp.; more preferably the species of bacteria selected from Yersinia ruckeri, Enterococcus faecalis, Listeria monocytogenes, Escherichia coll, Staphylococcus aureus, Streptococcus uberis, Staphylococcus simulans, Staphylococcus epidermidis, Micrococcus luteus.

40. A recombinant polypeptide with antibacterial properties characterized in that it comprises the peptidoglycan binding domain AMIN with an amino acid sequence in at least 80% identical with the sequence presented in SEQ ID No. 5 from Yersinia ruckeri, more preferably in at least 85%, more preferably 90%, more preferably 95%, more preferably 99% identical with the sequence presented in SEQ ID No. 5, more preferably the peptidoglycan binding domain AMIN is the amino acid sequence presented in SEQ ID No. 5 from Yersinia ruckeri; and the linker-connected amidase domain Ami with an amino acid sequence having at least 80% identity with the sequence presented in SEQ ID No. 6, more preferably in at least 85%, more preferably 90%, more preferably 95%, more preferably 99% identical with the sequence presented in SEQ ID No. 6, more preferably the amidase domain Ami is the amino acid sequence presented in SEQ ID No. 6 from Yersinia ruckeri, wherein preferably the linker has a sequence in at least 80% identical with the sequence of the linker 1 presented in SEQ ID No. 8, more preferably the linker is the amino acid sequence presented as SEQ ID No. 8 from Yersinia ruckeri.

41. The recombinant polypeptide according to claim 40, characterized in that it is the amino acid sequence YR7_LysM_Del from Yersinia ruckeri presented in SEQ ID No. 4.

42. A composition comprising the recombinant polypeptide as defined in claims 40-41 or a mixture thereof and a carrier.

43. A non-medical use of the recombinant polypeptide as defined in claims 40-41 and / or the composition as defined in claim 42 as an antibacterial and / or bactericidal agent, preferably against Yersinia ruckeri.

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