PL229542B1 - Polypeptide, polynucleotide coding it, or expressive vector, Staphylococcus pseudintermedius strain and their application and method for receiving - Google Patents

Abstract

Polypeptide of antibacterial or lytic effect to bacterial cells or lytic or cytotoxic effect to eukaryotic cells, its encoding polynucleotide or expression vector, the new Staphylococcus pseudintermedius strain and their applications and production method of the said polypeptides were revealed.

Description

Description of the invention

The subject of the invention is a polypeptide having a bactericidal or lytic activity towards bacterial cells or a lytic or cytotoxic activity towards eukaryotic cells, a polynucleotide or expression vector encoding it, a new Staphylococcus pseudintermedius strain, and their uses and a method for obtaining said polypeptide.

Bacteria have the ability to produce many compounds that are capable of killing other microorganisms. One such group of biologically active molecules are bacteriocins - peptides or proteins produced by bacteria on the ribosomes, capable of killing strains closely related to the manufacturer's strain at low concentrations. Both bacterial strains producing bacteriocins and the bacteriocins themselves are nowadays widely used for the preservation of food and feed, as well as for selected medical, veterinary and life sciences applications.

The species of bacteria Staphylococcus pseudintermedius is a common opportunistic pathogen of domestic animals - especially dogs and horses, but also isolated from other species of higher animals. Recently, however, the first case of infection with this bacterial species was also found in humans (Bannoehr and Guardabassi, 2012, Garbacz et al., 2013, van Hoovels et al., 2006). A case of infection ended with the patient's death has also been described [Savini et al., 2013).

Bacteriocins are an important group of molecules that are produced and secreted by many bacteria. They are peptides or proteins synthesized on ribosomes, able at very low concentrations (nanomoles / liter) to kill bacteria closely related to the producer of these compounds. This activity makes bacteriocins one of the basic effectors thanks to which the producer strain gains an advantage over other microorganisms competing with it in the same ecological habitat or in the same physiological niche. Bacteriocins also serve as a kind of metabolic regulators, depending on the density of the producer's cell population, as well as the presence of other bacteria. Due to the lack or relative lack of toxic effects on higher organisms, they are safe to use and are now widely used as preservatives in many food products and fodder. The safety of the use of bacteriocins is related mainly to their universality, and more precisely to the prevalence of the bacteria that produce them, most often present in dairy products as well as in silage and meat products, which have been components of the human diet for many thousands of years. It should be remembered that bacteriocin particles are not antibiotics. Bacteriocins and antibiotics are two different groups of compounds. Antibiotics are products of the secondary metabolism of microorganisms, they are synthesized outside the ribosomes, they usually have a broad spectrum of activity, and their activity is generally lower than that of bacteriocins (an antibiotic, on average, requires concentrations of orders of magnitude higher than the analogous concentrations of bacteriocins to exert a lethal effect). Unlike bacteriocins, the use of antibiotics often causes many side effects, harmful to humans, animals and the environment, both in the short and long term. Hence, their use is mainly restricted to medical and veterinary purposes and must be strictly controlled (Riley and Chavan, 2007, Drider and Rebuffat, 2011).

Bacteriocins are extremely diverse molecules. They have different sizes, structures, mechanisms of action, and the organization of the genes that code for them. This differentiation makes their classification very difficult and complicated, which often differs between authors. They are produced by both gram-negative and gram-positive bacteria, while gram-negative bacteriocins are a much less diverse group of proteins and peptides than gram-positive bacteriocins, they usually have a narrower spectrum of activity, and are mostly associated with a specific group of proteins and peptides. receptor on sensitive cells, which most gram-positive bacteriocins do not have.

Gram-negative bacteriocins include 3 groups of proteins and peptides: colicins (produced by most of the so-called enterobacteria that dominate the human intestines), microcins, and phage tail-like bacteriocins.

On the other hand, the bacteriocins of gram-positive bacteria (to which the strain being the subject of the present invention belongs) are divided into four classes: class I contains post-translationally modified peptides, class II includes thermostable unmodified peptides with a mass below 10 kDa, class III contains large, temperature-sensitive proteins and class IV includes poorly understood, high molecular weight proteins covalently linked to carbohydrates or lipids. These classes are further divided into subclasses and groups depending on more specific criteria (Riley and Chavan, 2007).

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The bacteriocins produced by the genus Staphylococcus are referred to as staphylococcins, and are secreted both by the so-called coagulase-negative and coagulase-positive strains. Staphylococcins belong to four groups of bacteriocins: the la subclass, known as lantibiotics (nine of them have been known so far), the llb subclass and the lid (four known representatives), and class III (two representatives) (Bastos et al., 2009).

Two groups of staphylococcins should be noted in the context of the present invention. The first is a relatively recently described and still not very numerous group of gram-positive bacteria of gram-positive bacteria subclass IIb, which is aureocin A53, isolated from the Staphylococcus aureus strain A53, and epidermicin NI01, obtained from the Staphylococcus epidermidis 224 strain. Both peptides have 51 residues. They are linear, unmodified post-translationally, rich in tryptophan and cationic amino acids, have a high isoelectric point. They are plasmid-encoded, their operons also encode proteins involved in secretion and immunity to their action. Their mechanism of action is unknown, but it is postulated that they induce pore formation in bacterial cell membranes, causing the efflux of metabolites and cell death. However, so far there are no known examples of commercial use of the peptide staphylococcins (Netz et al., 2002, Sandiford and Upton, 2012).

The second group of staphylococcins worth emphasizing in the context of the present invention is lysostaphins. They belong to the lila bacteriocin subclass of gram-positive bacteria, containing muraminolytic enzymes that cause cell dissolution / lysis in bacteria, and consequently their death. Functionally, lysostaphins are similar to lysozymes - hydrolytic enzymes commonly found in the animal world, hydrolyzing peptidoglycans of bacterial cell walls. The lila staphylococcin subclass includes two similar enzymes, lysostafin, produced by Staphylococcus simulans biovar simulans, and endopeptidase ALE-1, produced by Staphylococcus capitis EPK1. The earliest discovered and best described is lysostafin, which is a mature 25 kDa protein with two domains: an N-terminal peptidase capable of digesting glycine-glycine peptide bonds and a C-terminal peptidase capable of binding to bacterial cell wall peptidoglycans. Lizostafin is a relatively rare example of a commercial bacteriocin used in life sciences. This enzyme in recombinant form is sold by many companies as an effective lysing / dissolving factor for bacteria of the Staphylococcus genus, very useful in molecular biology (Kumar, 1997, Sugai et al., 1997).

The functional potential of bacteriocins is related to the high safety of their use, and the classification of bakeriocins to the so-called "Natural" preservatives. According to various sources, it is estimated that in the next few years, the annual value of the global market for the sale of such natural preservatives will reach the value of USD 2.5-2.7 billion. In the food industry and feed processing, bacteriocins are used both in a purified form, directly as additives to products, as well as inoculation of products with strains of bacteria that these bacteriocins produce. An example of a bacteriocin directly added to products is mainly nisin, a peptide bacteriocin from the subclass of lantibiotics, mainly produced by strains of Lactococcus lactis and officially approved as so-called GRAS (generally recognized as safe) compound for use as a food preservative in over 50 countries. It kills microorganisms sensitive to MIC doses. minimal inhibitory concentration) in the range of nanomoles per liter, is a safe and, above all, stable preservative, resistant to high temperatures and low pH values, commonly used in the processing of food products (cheese, dairy products, canned food, meat, alcoholic beverages) and animal feed. To date, nisin and the bacterial strains producing it are the only examples of such widespread use on such a large commercial scale. Other bacteriocins are used much less frequently and their application mainly concerns the application of the bacteria that produce them. This applies in particular to microcin and colicin producing strains used as protective probiotics in poultry and cattle farming. Their administration with feed reduces the level of harmful pathogens in the digestive tracts of animals, significantly increasing the resistance to bacterial infections. On the other hand, an example of a bacteriocin used commercially in life sciences is the aforementioned lysostafin, sold by many companies as a lysing / dissolving factor for bacteria of the genus Staphylococcus, very useful in molecular biology. Lizostafin is also used in typing staphylococci, and is also in clinical trials as an anti-staphylococcal drug (Riley and Gillor, 2007).

It is therefore particularly desirable to provide novel bacteriocins and methods for their production, and bacterial strains suitable for use in such methods.

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Unexpectedly, the above stated object was achieved with the present invention.

The invention relates to a polypeptide having an amino acid sequence selected from SEQ. No. 1 or Seq. No. 3 or its fragment with bactericidal or lytic activity towards bacterial cells or lytic or cytotoxic activity towards eukaryotic cells.

Another object of the invention is a polynucleotide containing a sequence encoding a polypeptide as defined above selected from SEQ. No. 2 or Seq. No. 4.

Another object of the invention is a polypeptide according to the invention as defined above or a polynucleotide according to the invention as defined above for use in therapy.

Another object of the invention is a plasmid expression vector containing the polynucleotide according to the invention as defined above, containing the sequence shown in Fig. 4A.

Another object of the invention is the Staphylococcus pseudintermedius 222 strain deposited with the PCM under the accession number B / 00084.

A further object of the invention is the use of a polypeptide according to the invention or a polynucleotide according to the invention or a bacterial strain according to the invention for the preparation of a preparation having a bactericidal or lytic activity towards bacterial cells or a lytic or cytotoxic activity towards eukaryotic cells.

Another object of the invention is a method for the preparation of the polypeptide according to the invention as defined above, characterized in that a bacterial host containing the expression vector as defined above is cultivated, and then the obtained protein is isolated from the culture fluid.

Preferably, the isolation of the protein is carried out by a known method selected from: salting-out, dialysis, concentration on semipermeable membranes or reversed-phase chromatography.

Preferably, the Staphylococcus pseudintermedius 222 strain deposited with PCM under accession number B / 00084 is used as the bacterial host.

The new bacterial strain according to the invention, belonging to the species Staphylococcus pseudintermedius, has the ability to produce peptide and protein bacteriocins with a wide range of biological activity: bactericidal or lytic against gram-positive bacteria, as well as cytotoxic or lytic against eukaryotic cells. Additionally, significant features of one of the bacteriocins produced by the strain in question are the high resistance of its molecule to degradation by proteolytic enzymes, resistance to high temperatures, and the preservation of biological activity after partial fragmentation of the molecule with chemical agents.

A new strain of Staphylococcus pseudintermedius according to the invention, with the working name Sp222, was isolated from pathological skin lesions of the dog and deposited on January 14, 2015 in the patent deposit of the Polish Microbial Collection at the Institute of Immunology and Experimental Therapy of the Polish Academy of Sciences in Wrocław under the number PCM B / 00084.

The results of the research of the authors of the present invention showed that Sp222 produces and secretes into the environment, inter alia, two new bacteriocins. The first is a linear bactericidal peptide rich in tryptophan and cationic amino acids with the working name BacSp222. This peptide also has haemolytic and cytotoxic properties towards eukaryotic cells and demonstrates exceptional resistance to degradation by proteolytic enzymes. The second bacteriocin isolated from Sp222 is a new enzyme capable of lysing and, consequently, killing bacterial cells, with the working name of lysostafin Sp222 (Mak and Władyka, unpublished results).

A further aspect of the invention relates to a new, third representative of the staphylococcins described above, which is a peptide bacteriocin with the working name BacSp22, produced by the claimed bacterial strain Sp222. It has a unique amino acid sequence and no statistically significant similarities to any of the currently known bacteriocins, nevertheless its physicochemical properties (molecular mass, rich in tryptophan, lysine and arginine residues) and antibacterial activity allow it to be classified as a peptide similar to the above-mentioned. aureocin A53 and epidermicin NI01. BacSp222 bacteriocin is capable of killing gram-positive bacterial cells at micromolar and sub-micromolar concentrations, and demonstrates biological properties beyond those of typical bacteriocins. It is capable of haemolysis of red blood cells and also has cytotoxic activity against eukaryotic cells. This allows the presumption that this peptide has the ability to modify the immune response of the host organism. Regardless of these activities, the BacSp222 bacteriocin molecule distinguishes itself from many other bacteriocins by a high degree of resistance to degradation by proteolytic enzymes - both eukaryotic and prokaryotic. It is also stable at elevated temperatures and has been proven to be significant

PL 229 542 Β1 bactericidal activity after limited chemical fragmentation of the molecule. All these features - resistance to proteolysis, temperature stability, and preservation of biological activity after limited chemical decomposition - are very important from the point of view of application applications (Moreno et al. 2000).

In a further aspect, the invention relates to a novel member of lysostafins, having the working name lysostafin Sp222, produced by the bacterial strain Sp222 of the invention. The sequence of this enzyme is new, but it has statistically significant similarities to the already known lysostaphins from other bacteria, and the activity of the new enzyme is also similar to them.

The description of the invention is supplemented by the following tables and figures.

Table 1 shows the MIC doses of BacSp222 bacteriocin effective against various microorganisms. The doses determined are the average of three independent measurements.

Table 2 shows the residual activity of the BacSp222 peptide bacteriocin digested with various proteolytic enzymes. This activity was presented as the diameter of the zones of inhibition of bacterial growth (in mm) in the radial diffusion test. Test samples are bacteriocin incubated with peptidase, control samples are bacteriocin incubated without peptidase.

Fig. 1 shows the results of an experiment demonstrating the antibacterial activity of the Sp222 strain. A wide streak of Sp222 colonies was vertically streaked onto the left edge of the solid nutrient plate, while next to this streak, on the right side, four indicator bacteria were streaked. After incubating the plate for 24 hours at 37 ° C, it can be observed that the grown colonies of all four tested bacteria are asymmetrical - they have the shape of a crescent. Their growth was inhibited on the left side by antibacterial substances secreted into the medium by the Sp222 strain.

Fig. 2 shows the results of an experiment demonstrating the lytic activity of the Sp222 strain against bacterial cells. A suspension of Staphylococcus aureus RN4220 bacterial cells was placed in two wells of a 96-well microplate, one without additives (control) and the other with 10 μΙ of Sp222 culture medium. The plate was then incubated at 37 ° C while measuring the decrease in optical density due to the lysis of the bacteria. In a well containing Sp222 culture medium, this decrease is significantly greater than in a control well and demonstrates the presence of a bacterial cell lysis factor in the Sp222 culture medium.

Fig. 3 shows the results of the sequential steps in the purification of the BacSp222 peptide bacteriocin. In all panels, the bacteriocin peak or band is marked with an arrow. A) Reverse-phase high pressure liquid chromatography (RP-HPLC) on a Nucleosil C18 column. B) RP-HPLC chromatography on a Kromasil C4 column. C) SDS-PAGE denaturing electrophoresis of the fractions collected at each purification step (starting medium, after salting out, after C18 column and final preparation after C4 column). This image shows that the final purified bacteriocin solution contains a single and homogeneous peptide band with a molecular weight of approx. 5 kDa.

In Fig. 4 the sequence of the plasmid p222 is shown. Panel A shows the nucleotide sequence of the entire plasmid, with the fragment encoding the BacSp222 peptide bacteriocin being underlined. In panel B there is a fragment of the nucleotide sequence encoding the BacSp222 peptide bacteriocin, together with the corresponding amino acid residues.

Fig. 5 shows the results of an experiment demonstrating the haemolytic activity of the BacSp222 peptide bacteriocin. Activity was determined as the percentage of haemolysis of human erythrocyte suspension and calculated as the mean of three independent measurements.

Fig. 6 shows the results of an experiment demonstrating the cytotoxic activity of the BacSp222 peptide bacteriocin. The cytotoxic activity of bacteriocin was tested at concentrations ranging from 0.195 to 100 micromol / liter against human skin fibroblasts and against human lipogenic stem cells. The determination was performed using two different biological tests - LDH and MTT.

Fig. 7 shows the results of the study of conformational changes of the BacSp222 peptide bacteriocin molecule at different temperatures. Conformation changes were observed by examining the ellipticity at 220 nm in a circular dichroism spectrum of a peptide incubated at temperatures from 20 to 90 ° C.

Fig. 8 shows the results of an experiment demonstrating the bactericidal activity of a fragment of the BacSp222 peptide bacteriocin molecule lacking a formylated methionine at the N-terminus. The bactericidal activity of the starting bacteriocin solutions (marked in the figure as "+ fm") and truncated (marked as "- fm" in the figure), in the amounts of 190, 380 and 760 picomoles per well, was tested by radial diffusion in a solid nutrient medium with susceptible bacteria suspension.

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The obtained diameters of the zones of inhibition of bacterial growth indicate that bacteriocin in the form lacking the N-terminal formylated methionine requires, on average, two times higher concentrations to reach the diameter of the inhibition zone characteristic for the original bacteriocin.

Fig. 9 shows the results of the sequential steps in the purification of Sp222 lysostaphin. In all panels, the enzyme peak or band is indicated by an arrow. A) Reverse-phase high pressure liquid chromatography (RP-HPLC) on a Nucleosil C18 column. B) RP-HPLC chromatography on a Discovery C5 column. C) RP-HPLC chromatography on a Discovery C18 column. D) SDS-PAGE denaturing electrophoresis of the fractions collected at each purification step (starting medium, dialysate, after salting out, after Nucleosil C18 column, after Discovery C5 column and final preparation after Discovery C18 column). This image shows that the final purified lysostafin solution contains a single and homogeneous protein band with a molecular weight of approx. 30 kDa.

Fig. 10 shows the Sp222 lysostaphin coding sequence. Panel A has the nucleotide sequence, while panel B has the same nucleotide sequence with the appropriate amino acid residues of the Sp222 lysostafin molecule overlayed.

Fig. 11 shows the results of an experiment demonstrating the lytic activity of purified Sp222 lysostafin on bacterial cells and its comparison with that of commercial lysostafin. A suspension of Staphylococcus aureus RN4220 bacterial cells was placed in three wells of a 96-well microplate, one without additives (control), the other with 1 μο of Lysostafin Sp222 and the third with 1 μg of commercial lysostafin. The plate was then incubated at 37 ° C while measuring the decrease in optical density due to the lysis of the bacteria. In the well containing Sp222 lysostafin, the decrease is significantly greater than in the control well and in the well containing commercial lysostafin, demonstrating the high lytic activity of Sp222 lysostafin.

For a better understanding of the essence of the invention, it has been illustrated by the following examples. These examples are provided merely for the purpose of illustration and elucidation of the invention, not for limiting purposes, and should not be equated with the entire scope of the invention, as defined in the claims.

Example 1

Demonstration of the antibacterial activity of substances secreted into the environment by the Sp222 strain

In order to demonstrate the inhibitory effect of the Sp222 strain on the growth of other bacteria, the Sp222 strain was inoculated on solid medium alongside other bacteria, and the effect of the Sp222 strain on the growth of other bacteria was observed. For this purpose, a plate was prepared containing sterile solid bacterial nutrient medium (Tryptic Soy Agar, Sigma). Sp222 bacteria were vertically inoculated on one end side of this plate as a wide streak, then the plate was incubated for 18 hours at 37 ° C. After this time, next to this streak, colonies of four test bacteria were sown in spots: Staphylococcus aureus RN4220, Staphylococcus pseudintermedius 223 (from the collection of the Department of Microbiology of the Faculty of Biochemistry, Biophysics and Biotechnology of the Jagiellonian University), Bacillus subtilis LOCK 0816 (from the collection of the Institute of Microbiology and Fermentation Technology in Łódź). and Staphylococcus epidermidis ATCC 35547 (American Type Cell Culture Collection, USA). The plate was then incubated for 24 hours at 37 ° C. After incubation, the image shown in Fig. 1 was obtained. It can be seen that the grown colonies of all four tested bacteria are asymmetrical - they have the shape of a crescent. On the one hand, their growth was inhibited by antibacterial substances secreted into the medium by bacteria of the Sp222 strain.

Example 2

Demonstration of the lytic activity of substances secreted into the environment by the Sp222 strain against other bacterial cells

In order to demonstrate the lytic activity (i.e. the ability to liquefy the cell walls and thus to disintegrate the bacterial cell) of substances produced by the Sp222 strain against other bacterial cells, their suspension was incubated in the presence of the liquid Sp222 culture medium and the decrease in turbidity of this suspension was observed, which is proportional to the degree of bacterial lysis. For this purpose, an aliquot of sterile liquid growth medium for bacteria (Trypticase Soy Broth, Sigma) was inoculated with bacteria of the Sp222 strain and then cultured for 15 hours at 37 ° C on an orbital shaker at 180 rpm. The obtained bacterial suspension was centrifuged at 4 ° C at 4,000 g and the clear supernatant containing the culture medium was collected. This medium contained substances secreted into the environment by the Sp222 strain. A volume of 10 μΙ of this culture medium was added to the well of a 96-well polystyrene microplate containing 150 μΙ of the suspension.

PL 229 542 Β1 Staphylococcus aureus RN4220 bacteria (from the collection of the Department of Microbiology of the Faculty of Biochemistry, Biophysics and Biotechnology of the Jagiellonian University) in 20 mM Tris-HCI pH 7.5 buffer containing 150 mM NaCI. In an adjacent well there was 160 µΙ of a suspension of Staphylococcus aureus RN4220 in the above buffer. This suspension did not contain any other additives and served as the control well. The plate was then placed in a microplate reader (Synergy H1 Hybrid MultiMode Microplate Reader, BioTek, USA) and the drop in turbidity was measured at 600 nm every 2 min for 1 hour at 37 ° C. The decrease in this turbidity is proportional to the degree of lysis of the bacterial cells. The results of the measurement are presented in Fig. 2. It can be seen that in the well containing the culture medium the decrease in turbidity is much greater than in the control well, which proves the presence of the factor secreted by the Sp222 strain and causing the lysis of bacterial cells in the culture medium.

Example 3

Procedure for the purification of BacSp222 peptide bacteriocin

The purification procedure for BacSp222 peptide bacteriocin present in the Sp222 liquid culture medium involves salting out the peptide with high salt concentrations, followed by two reverse phase chromatography steps. A sterile liquid medium for bacteria (Trypticase Soy Broth, Sigma) was inoculated with bacteria of the Sp222 strain and then cultured for 15 hours at 37 ° C on an orbital shaker at 180 rpm. The obtained bacterial suspension was centrifuged for 20 min at 4 ° C at 5,000 g, after which the clear supernatant containing the culture medium was collected. This medium contained substances secreted into the environment by the Sp222 strain, including the substance with lytic activity as shown in Example 2. This medium was cooled to 4 ° C and then solid ammonium sulphate was added with constant stirring to precipitate the proteins and peptides. until the solution is 60% (by weight) saturated. After the salt was added, the medium was stirred for an additional 30 min and then left in the ice bath without stirring for 2 hours. The resulting protein pellet was centrifuged for 30 min at 4 ° C at 21,000 g. The supernatant was discarded and the pellet was dissolved in water containing 0.1% (v / v) trifluoroacetic acid (TFA) and 60% (v / v) acetonitrile. This solution was centrifuged for 5 min at 4 ° C at 16,000 g, the collected supernatant was filtered through a 0.45 µm filter and then separated by reversed-phase high pressure liquid chromatography (RP-HPLC). The separation was carried out on an Ultimate 3000 chromatography system (Dionex / Thermo, USA) and on a Nucleosil C18 300A 250 x 8 mm column (Macherey-Nagel, Germany). A gradient elution was used with two buffers, A: 0.1% (v / v) TFA in water, B: 80% (v / v) acetonitrile in water, containing 0.07% (v / v) TFA. A flow of 1.5 ml / min and spectrophotometric detection at 220 nm were used. Separation was started on a column equilibrated with 60% buffer B, after the sample was injected for 5 min, the column was still equilibrated with 60% buffer B, followed by linear gradient elution from 60 to 100% buffer B over 20 min. The bacteriocin fraction eluting on the chromatogram as a peak with a retention time within 18-20 min. collected in a tube, lyophilized, dissolved in water containing 0.1% (v / v) TFA and 30% (v / v) acetonitrile and resolved on a Kromasil C4 100A 250 x 4.6 mm column (Sigma, USA). A flow of 1 ml / min and spectrophotometric detection at 220 nm were used. Separation was started on a column equilibrated at 68% B buffer followed by linear gradient elution from 68 to 75% B buffer in 20 min. The bacteriocin fraction eluting as a peak with a retention time of 11-14.5 min. collected into a tube, lyophilized and then dissolved in water to give the final purified Sp222 bacteriocin solution. The concentration of this peptide in the solution was determined by amino acid composition analysis. The steps of the chromatographic purification are shown in Fig. 3. This figure also shows an electrophoretic image of the fractions collected at the individual purification steps. It can be seen in this picture that the final purified bacteriocin solution contains a single and homogeneous peptide band with a molecular weight of approx. 5 kDa.

Example 4

Determination of the amino acid sequence of the BacSp222 peptide bacteriocin

The amino acid sequence of the peptide purified according to the procedure described in Example 3 was determined on the Procise 491 automatic sequencer (Applied Biosystems, USA) realizing Edman degradation of the polypeptide chains. Prior to determining this sequence, the N-terminal formyl was chemically removed by incubating the peptide in 0.6 M hydrochloric acid for 24 hours. The resulting BacSp222 bacteriocin amino acid sequence has the following sequence of residues: fMAGLLRFLLSKGRALYNWAKSHVGKVWEWLKSGATYEQIKEWIENALGWR.

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This sequence is shown as single letter abbreviations of amino acid residues, with the lowercase "f" in front of the sequence indicating that the alpha-amino group of the N-terminal methionine residue is formylated. The molecular weight of BacSp222 bacteriocin was determined on a MALDI-ToF mass spectrometer, model ultrafleXtreme (Bruker, Germany). It was 5921.917 Da and corresponded to the theoretical peptide mass of 5921.888 Da, calculated based on the higher amino acid sequence. This consistency proves that the above peptide amino acid sequence was determined reliably.

Example 5

Determination of the coding sequence for the BacSp222 peptide bacteriocin.

The BacSp222 coding sequence was obtained by bioinformatic shot-gun sequence analysis of the Sp222 genome. Total DNA (deoxyribonucleic acid) isolated from Sp222 bacteria by standard techniques known to those skilled in the art was shot-gun sequenced using the MiSeq (Illumina) instrument using an instrument-specific kit of reagents and procedures. The obtained short sequence reads were assembled into longer whole (so-called contigs) using the MIRA (http://mira-assembler.sourceforge.net/) and CLC Main Workbench (CLC bio) programs. In one of the contigs containing the plasmid sequence (p222), the sequence encoding the BacSp222 peptide bacteriocin was identified. This was done by translating the plasmid DNA sequence based on the genetic code into sequences containing the so-called open reading frames, i.e. the sequences of amino acid residues found in proteins and peptides. One of the sequences obtained was identical to the BacSp222 sequence determined by sequencing using Edman degradation (see Example 4). The bacteriocin coding sequence is as follows:

ATGGCAGGATTACTACGTTTTCTTTTAAGTAAAGGTCGCGCCTTATACAATTGGGCAAAG 60 AGTCATGTTGGAAAAGTTTGGGAGTGGCTTAAATCAGGAGCTACATATGAACAAATTAAA 120 GAATGGATTG AAAACGCATTAGGTTGGAGATAA 153 and is located between the ATG codon (nucleotides 8590-8592) for a methionine codon, TAA (nucleotides 8740-8742), meaning a stop codon in the sequence of the entire plasmid P222, which is given in Fig. 4. The correctness of the coding sequence BacSp222 was confirmed by amplifying a DNA fragment encoding BacSp222 by PCR using oligonucleotide primers complementary to the fragments flanking the coding sequence followed by sequencing the reaction product using Sanger sequencing.

Example 6

Determination of the antibacterial activity of BacSp222 peptide bacteriocin

The antibacterial activity of the BacSp222 peptide bacteriocin purified according to the procedure of Example 3 was documented by determining the MIC doses (defined as the lowest concentration of bacteriocin causing growth inhibition of the tested microorganism) against representatives of Gram-positive bacteria, Gram-negative bacteria and fungi. MIC dose determination procedures were performed strictly according to the guidelines of the Clinical and Laboratory Standards Institute (CLSI, 2006), or, in the case of fungal cells, The European Committee on Antimicrobial Susceptibility Testing (EUCAST-AFST, 2012). The list of microorganisms used for the tests, together with the exact determinations of their strains, is presented in Table 1. The microdilution procedure was used for the determinations, which consisted in incubating in the wells of a microplate a 96-well portion of 100 μΙ suspensions of the tested microorganism containing 5 x 10 ⁴ colony forming units ( colony forming unit (CFU) with serial dilutions of bacteriocin. The suspensions of the tested microorganisms were made in sterile Mueller-Hinton Broth medium normalized for cations (Cation-adjusted MHB, Sigma) or, in the case of Streptococcus pyogenes and Streptococcus sanguinis, in sterile MHB medium enriched with 5% (by volume) horse blood lysate (Graso) . However, in the case of Candida albicans yeast-like fungi, cell suspensions were made in sterile RPMI 1640 medium (Sigma) enriched with 0.3 g / liter of L-glutamine, 34.53 g of morpholinopropane sulfate acid (MOPS) / liter, 18 g / liter of glucose, pH 7.0. After incubating the microplates for 24 hours at 37 ° C, a visual assessment of the degree of turbidity of the contents of the wells was made and the lowest concentration of bacteriocin which completely inhibited the growth of the tested microorganism (no turbidity) was taken as the MIC dose. The final MIC dose was calculated as the arithmetic mean of three independent measurements. The results of the determinations are shown in Table 1. These results indicate that bacteriocin

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BacSp222 kills many gram-positive bacteria at MIC doses of 0.1 to about a few micromol / liter. However, it is not able to kill the tested gram-negative bacteria and fungi in the tested concentrations (up to 100 micromoles per liter).

Example 7

Determination of the haemolytic activity of the BacSp222 peptide bacteriocin

The haemolytic activity (red blood cell lysis capacity) of the BacSp222 peptide bacteriocin purified according to the procedure of Example 3 was documented by incubating a 5% (v / v) suspension of human erythrocytes with serial dilutions of the bacteriocin. Incubation was in phosphate buffered saline for 1 hour at 37 ° C. After incubation, the erythrocytes were centrifuged (5 min at 2,000 g), the supernatant was transferred to the wells of a 96-well microplate, and the amount of released hemoglobin was measured by measuring absorbance at 540 nm. A suspension of erythrocytes lysed in the presence of 1% (w / w) sodium dodecyl sulfate detergent was used as a 100% cell lysis control. The results of the determinations are shown in Fig. 4 as the percentage of blood cell lysis for each tested bacteriocin concentration, averaged over three independent measurements. These results indicate that BacSp222 bacteriocin has a moderate haemolytic activity - in the highest peptide concentration tested, 100 μΜ, it lyses 40% of erythrocytes. On the other hand, at concentrations below 10 μΜ the haemolytic activity of the peptide is very low (less than 2% haemolysis).

Example 8

Determination of the cytotoxic activity of the BacSp222 peptide bacteriocin

The cytotoxic activity of the BacSp222 peptide bacteriocin, obtained as in Example 3, was tested on two types of human cells, in parallel and independently, by means of two different survival tests. Human skin fibroblast cells (HSF) and human fat-derived stem cells (ASC) were cultured at 37 ° C under 5% CO2 in standard T25 plastic bottles. Cells were grown in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with phenol red, 10% (v / v) calf serum (FBS), 0.1 mg / ml streptomycin and 0.15 U / ml penicillin. The medium was changed to fresh every three days. After the third passage, 7,000 cells were transferred to the wells of a 96-well microplate and incubated for 24 hours to be pelleted to the media. After this time, the medium was replaced with 50 μ aliquots of medium containing serial dilutions of bacteriocin in the range of 0.195 to 100 micromoles per liter. The negative control was cells in the medium without bacteriocin, while the positive control (100% mortality) were cells treated with 0.9% (v / v) Triton Χ-100 detergent. After 4 hours of incubation, the cell survival rate was determined separately and independently with the lactate dehydrogenase activity assay (LHD, CytoTox 96 kit from Promega) and with the MTT tetrazoline salt reduction assay (Sigma). The LDH assay was performed on 30 μ aliquots of medium taken from the cells with cells transferred to a new 96-well microplate and mixed with 30 μΙ aliquots of LDH substrate. The substrate reaction was stopped after 30 min by adding 30 µΙ 0.33 M acetic acid. Measurement of the absorbance of the resulting colored product was measured with a PowerWave X microplate reader (Bio-Tek) at 490 nm with subtraction of background absorbance at 690 nm. In contrast, the MTT test was performed on cells embedded in the wells, replacing the medium with bacteriocin for 55 µΙ aliquots of medium containing 0.5 mg / ml of MTT. After 3 hours of incubation, the reaction was stopped by the addition of 100 µl of isopropanol / HCl mixture and the absorbance of the resulting dye was measured with a PowerWave X microplate reader (Bio-Tek) at 540 nm with subtraction of background absorbance at 690 nm. The obtained absorbance values were averaged from three independent measurements and converted into percent cell death values, 0% taking the absorbance values for cells from the negative control wells and 100% mortality using the absorbance values for cells from the positive control wells (cells killed with detergent). The pooled results are shown in Fig. 6. These results show that the BacSp222 peptide bacteriocin has a real and measurable cytotoxic activity at concentrations above a few micromoles per liter for both cell types tested and documented with two different cytotoxic activity assays.

Example 9

Determination of the susceptibility of the BacSp222 peptide bacteriocin to degradation by proteolytic enzymes

The sensitivity of the BacSp222 peptide bacteriocin purified according to the procedure from example 3 to degradation by proteolytic enzymes was tested by incubating the peptide with various peptidases,

PL 229 542 Β1 and then by determining the residual antibacterial activity of bacteriocin by radial diffusion technique. The following peptidases produced by higher organisms were used: cathepsin G and elastase from human neutrophils (Biocentrum), trypsin from bovine pancreas (Sigma) and pork pepsin (Sigma), as well as the following bacterial peptidases: proteinase K (A&A Biotechnology), StpC proteinase (enzyme obtained in-house) and proteinase V8 (Sigma). For incubation with cathepsin G, 0.1 M Tris-HCl buffer, 0.5 M NaCl, pH 8.0 was used for incubation with elastase, trypsin and K proteinase 0.1 M Tris-HCl, pH 8.0, for incubation with pepsin 0.1 M ammonium acetate pH 4.0, for incubation with StpC proteinase 0.1 M Tris-HCl, 5 mM sodium ethylenediamine acetic acid (EDTA), 5 mM cysteine hydrochloride, pH 7.5, and for incubation with V8 proteinase 0.1 M phosphate buffer pH 7.8. Bacteriocin was incubated for 1 and 3 hours at 37 ° C in the above enzyme buffers at peptide: enzyme weight ratios of 50: 1 and 10: 1. After incubation, portions of the reaction mixtures were dispensed into wells cut in a sterile nutrient medium (Trypticase Soy Broth, Sigma) containing 1% (by weight) of agar and a bacterial suspension of the species Bacillus subtilis LOCK 0816 (from the collection of the Institute of Fermentation Technology and Microbiology of the Lodz University of Technology). Bacteriocins incubated in appropriate buffers without the addition of peptidases were dispensed into separate control wells. The well plate was then incubated at 37 ° C for 24 hours and the diameters of the zones of inhibition of bacterial growth that developed around each well were measured. The obtained results are presented in Table 2. The analysis of these results shows that regardless of the incubation time and the weight ratio of peptide to enzyme, the peptidases used do not reduce the bactericidal activity of bacteriocin.

Example 10

High temperature susceptibility determination of BacSp222 peptide bacteriocin

The sensitivity of the BacSp222 peptide bacteriocin purified according to the procedure in Example 3 to elevated temperature was tested by incubating the peptide at 100 ° C and then verifying the residual bactericidal activity by radial diffusion technique, and also, independently, by observing changes in the conformation of the molecule at elevated temperatures by means of spectral measurements circular dichroism. To determine the residual bactericidal activity, the BacSp222 peptide bacteriocin solution was incubated in phosphate buffered saline (PBS) at room temperature (control) and 100 ° C for 30 and 60 min. After incubation, portions of the incubated solutions were dispensed into wells cut in a sterile nutrient medium (Trypticase Soy Broth, Sigma) containing 1% (by weight) of agar and a bacterial suspension of the species Bacillus subtilis LOCK 0816 (from the collection of the Institute of Fermentation Technology and Microbiology of the Lodz University of Technology). The well plate was then incubated at 37 ° C for 24 hours and the diameters of the zones of inhibition of bacterial growth that developed around each well were measured. The obtained diameters of the zones of inhibition of bacterial growth were the same for the control samples as well as for the samples incubated at 100 ° C. To determine the conformation changes of the BacSp222 peptide bacteriocin molecule at elevated temperatures, a bacteriocin solution was prepared in 50 mM sodium phosphate pH 7.4 containing 100 mM NaF. This solution was placed in a thermostated quartz cuvette mounted in a J-715 spectropolarimeter (Jasco). Then, the ellipticity was continuously measured at a wavelength of 220 nm in the temperature range from 20 to 90 ° C. The temperature change rate used was 1 ° C / min. The obtained graph of changes in ellipticity is presented in Fig. 7. In this graph, no significant changes in ellipticity were observed, which proves that in the studied temperature range, the spatial conformation of bacteriocin did not change.

Example 11

Determination of the bactericidal activity of a fragment of the BacSp222 peptide bacteriocin molecule

In order to determine the bactericidal activity of a fragment of the BacSp222 peptide bacteriocin molecule purified according to the procedure of Example 3, it was subjected to cleavage of the N-terminal formylated methionine residue with a chemical agent and then measurement of the residual bactericidal activity by radial diffusion technique. Cleavage of the N-terminal formylated methionine residue from the BacSp222 bacteriocin molecule was accomplished by dissolving the peptide in 70% (v / v) trifluoroacetic acid (TFA) solution, adding one crystal of cyanogen bromide (CNBr) and incubating the mixture in the dark for 24 hours at room temperature. After incubation, the mixture was evaporated to dryness in a vacuum centrifuge, dissolved in water containing 0.1% (v / v)

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TFA and 65% (v / v) acetonitrile. This solution was separated by reversed-phase high pressure liquid chromatography (RP-HPLC). The separation was carried out on an Ultimate 3000 chromatography set (Dionex / Thermo, USA) and on a Kromasil C4 250 x 4.6 mm column (Sigma). A gradient elution was used with two buffers, A: 0.1% (v / v) TFA in water, B: 80% (v / v) acetonitrile in water, containing 0.07% (v / v) TFA. A flow of 1 ml / min was used. and spectrophotometric detection at 220 nm. Separation was started on a column equilibrated at 65% buffer B and elution was carried out with a linear gradient from 65 to 72% buffer B in 20 min. The fraction containing BacSp222 bacteriocin truncated, lacking the N-terminal formylated methionine, eluting as a peak with a retention time ranging from 9 to 10.5 min, was collected in a tube, lyophilized and dissolved in water. The antibacterial activity of the truncated bacteriocin and the starting bacteriocin was determined by radial diffusion in nutrient agar containing sensitive bacteria. For this purpose, portions of serially diluted trimmed bacteriocin and starting bacteriocin were dosed into wells cut in a sterile nutrient medium (Trypticase Soy Broth, Sigma) containing 1% (by weight) agar and a bacterial suspension of Bacillus subtilis LOCK 0816 (from the collection of the Institute of Fermentation Technology and Microbiology of the Polytechnic University of Technology). Łódzka). The well plate was then incubated at 37 ° C for 24 hours and the diameters of the zones of inhibition of bacterial growth that developed around each well were measured. The results of these measurements showed that the fragment of BacSp222 bacteriocin, devoid of the N-terminal formylated methionine, requires on average twice the concentration to reach the diameter of the zone of inhibition identical to that of the starting bacteriocin. It should therefore be concluded that the limited chemical fragmentation to some extent weakens the bactericidal activity of the peptide, but does not cancel it.

Example 12

Lysostafin Sp222 purification procedure

The purification procedure for the lytic enzyme present in the liquid Sp222 culture medium involves dialysis of the medium, plating the protein with high salt concentrations, followed by three steps of reverse phase chromatography. A sterile liquid medium for bacteria (Trypticase Soy Broth, Sigma) was inoculated with bacteria of the Sp222 strain and then cultured for 15 hours at 37 ° C on an orbital shaker at 180 rpm. The obtained bacterial suspension was centrifuged for 20 min at 4 ° C at 5,000 g, after which the clear supernatant containing the culture medium was collected. This medium contained substances secreted into the environment by the Sp222 strain. This medium was dialyzed against distilled water at 4 ° C using a dialysis bag with a cut-off of 14 kDa or concentrated on a semipermeable membrane with a cut-off of 10 kDa. The dialysate was frozen and then lyophilized. The obtained lyophilisate was dissolved in a little water and then the proteins present in it were salted out with ammonium sulphate to 60% (by weight) of saturation. After the salt was added, the solution was stirred for 30 min, then it was left in the ice bath without stirring for 2 hours. The resulting protein pellet was centrifuged for 30 min at 4 ° C at 21,000 g. The supernatant was discarded and the pellet was dissolved in water containing 0.1% (v / v) of trifluoroacetic acid (TFA). The solution thus obtained was centrifuged for 5 min at 4 ° C at 16,000 g, the collected supernatant was filtered through a 0.45 µm filter and then separated by reversed-phase high pressure liquid chromatography (RP-HPLC). The separation was carried out on an Ultimate 3000 chromatography system (Dionex / Thermo, USA) and on a Nucleosil C18 300A 250 x 8 mm column (Macherey-Nagel, Germany). A gradient elution was used with two buffers, A: 0.1% (v / v) TFA in water, B: 80% (v / v) acetonitrile in water, containing 0.07% (v / v) TFA. A flow of 1.5 ml / min and spectrophotometric detection at 220 nm were used. Separation was started on a column equilibrated at 38% B buffer followed by linear gradient elution from 38 to 53% B buffer in 15 min. The bacteriocin fraction eluting as a peak with a retention time ranging from 23.5 to 24.5 min was collected in a tube, lyophilized, dissolved in water containing 0.1% (v / v) TFA and 36% (v / v) acetonitrile, and re-separated on Discovery Bio Wide Pore C5 300A 250 x 4.6 mm column (Sigma, USA). A flow of 1 ml / min and spectrophotometric detection at 220 nm were used. Separation was started on a column equilibrated at 30% B buffer followed by linear gradient elution from 36 to 50% B buffer in 30 min. The bacteriocin fraction eluting as a peak with a retention time ranging from 17 to 18 min was collected in a test tube, lyophilized and then dissolved in water containing 0.1% (v / v) TFA and 37% (v / v) acetonitrile and re-separated on the Discovery Bio column Wide Pore C18 300A 250 x 4.6 mm (Sigma, USA). A flow of 1 ml / min and spectrophotometric detection at 220 nm were used.

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Separation was started on a column equilibrated at 37% B buffer followed by linear gradient elution from 37 to 45% B buffer in 20 min. The bacteriocin fraction eluting as pico with retention time ranging from 15 to 16.5 min. collected into a tube, 50 mM sodium acetate, pH 5.5, containing 1 M NaCl was added (to minimize loss during lyophilization), and then lyophilized. The final purified lysostaphin Sp222 solution contained, in addition to the protein, 10 mM sodium acetate pH 5.5 and 200 mM NaCl. The concentration of the enzyme in the final preparation was determined with a kit based on the reaction with bicinchoninic acid (BCA, Sigma). The described steps in the purification of Sp222 lysostaphin are illustrated in Fig. 9. The figure also shows an electrophoretic image of the fractions collected at the individual purification steps. It shows that the final solution of the purified enzyme contains a single and homogeneous protein band with a molecular weight of approx. 30 kDa.

Example 13

Determination of the amino acid sequence of the N-terminus of Lysostaphin Sp222

The amino acid sequence of Lysostafin Sp222 purified according to the procedure described in Example 12 was determined on the Procise 491 automatic sequencer (Applied Biosystems, USA) realizing Edman degradation of the polypeptide chains. 20 residues from the N-terminus were identified: AATSTSGSAAWLNOYPLNNG. The sequence is shown as single letter abbreviations of the amino acid residues.

Example 14

Determination of the Sp222 Lysostaphin Coding Sequence

The Sp222 lysostafin coding sequence was obtained by bioinformatic shot-gun sequence analysis of the Sp222 genome. Total DNA (deoxyribonucleic acid) isolated from Sp222 bacteria by standard techniques known to those skilled in the art was shot-gun sequenced using the MiSeq (Illumina) instrument using an instrument-specific reagent kit. The obtained short sequence readings were assembled into longer whole (so-called contigs) using MIRA (http://mira-assembler.sourceforge.net/) and CLC Main Workbench (CLC bio). In one of the contigs, the Sp222 lysostaphin coding sequence was identified. This was done by translating the plasmid DNA sequence based on the genetic code into sequences containing the so-called open reading frames, i.e. the sequences of amino acid residues found in proteins and peptides. One of the sequences obtained contained a sequence identical to the sequence of the Sp222 lysostaphine fragment determined by sequencing using Edman degradation (see example 13). The designated Sp222 lysostafin coding sequence and the sequence of the entire Sp222 lysostaphin protein are shown in Figure 10.

Example 15

Determination of Lysostaphin Sp222 Lytic Activity Against Bacterial Cells

In order to demonstrate the lytic activity (i.e. the ability to liquefy bacterial cell walls) of the lysostafin obtained according to the procedure described in Example 12, the suspension of the tested strain was incubated in the presence of Sp222 lysostafin and a decrease in the turbidity of this suspension was observed, proportional to the degree of bacterial lysis. For comparative purposes, the lytic activity of another commercially available lysostaphine was also determined. For this purpose, into the wells of a 96-well polystyrene microplate containing 150 μΙ of the suspension of Staphylococcus aureus RN4220 bacteria (from the collection of the Department of Microbiology of the Faculty of Biochemistry, Biophysics and Biotechnology of the Jagiellonian University) in 20 mM Tris-HCI buffer pH 7.5 containing 150 mM NaCI. Then 1 microgram of Lysostafin Sp222 obtained according to Example 12 was added to the first well, 1 microgram of commercial Lysostafin (A&A Biotechnology) was added to the second well, and the test bacteria suspension was in the third well, without additives. The plate was then placed in a microplate reader (Synergy H1 Hybrid MultiMode Microplate Reader, BioTek, USA) and the decrease in turbidity was measured at 600 nm every 2 min for 1 hour at 37 ° C. The decrease in turbidity is proportional to the degree of bacterial cell lysis. The results of the measurement are presented in Fig. 11. It can be seen that in the well containing the addition of purified Sp222 lysostaphin, the decrease in turbidity is much greater than in the control well, which proves the presence of the factor secreted by the Sp222 strain and causing the lysis of bacterial cells in the culture medium.

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Sequence Listing

Sequence No. 1 ibacteriocin) m'agllrfllskgralynwakshvgkvwewlksgatyeqikewienalgwr 50 ^ Methionine at the N-terminus is forrnylated

Sequence number 2

ATGGCAGGATTACTACGTTTTCTTTTAAGTAAAGGTCGCGCCTTATACAATTGGGCAAAG 60

AGT CAT G T T GGAAAAGT T T GG GAGTGGCT TAAATCAG GAGC TA.C AT AT GAAC AAAT T AAA

120

GAATGGATTGAAAACGCATTAGGTTGGAGATAA153

Sequence # 3 (Lysostaphine)

ΜΚΝ5ΚΚΙΟΙ5ΕΑΤΣΑΒ6.3Μ5νν30νΝΑΚΕ30νΚΡΑΡΚΤΥΤΣΑΑΤ3Τ3β3ΆΑΜΕΝΟΥΡΣΝΥ60

GFGSYNLPNNGGMHYGVDFGM.SVGTPVKAITGGTVLNTAWDPYGGGNTITIKETDGVHTQ120

WYMHL SKFNVQKGQKIKVGQVIGY SGNTGQS SGPHLH FQRMNGNP SNANAQDPLPFLKSI180

GYGKSSGGSSNTKPSTSGGFKVNQYGTLYKAESATFTANTTIITRYTGPFRSMPKAGTLK240

SGQSIKYDEVMKQDGHVWGYTNSAKKRVYVPVRTWNKNNNNSLGSLWGTIK2 91

Sequence No. 4

ATGAAAAATAGTAAAAAAATAGGTATCAGTTTAGCAACATTAGCTTTAGGTAGTATGTTA60

GTAGTAGGACAAGTCAATGCGAAAGAATTACAAGTGAAGCCCGCTCCTAAAACTTATACT120

CTTGCTGCTACTAGCACTAGTGGTTCTGCTGCTTGGTTAAATCAATATCCATTAAATTAT180

GGTTTCGGGAGTTATAACCTACCTAATAATGGTGGTATGCACTATGGGGTGGATTTTGGC240

ATGAGTGTCGGAACTCCCGTGAAAGCAATAACTGGTGGGACAGTTCTTAATACCGCTTGG300

GACCCTTATGGTGGTGGTAATACTATCACAATTAAAGAAACAGATGGCGTACATACACAA360

TGGTATATGCACTTGAGTAAATTTAATGTACAAAAAGGTCAAAAAATAAAGGTAGGACAA420

GTAATTGGATATTCAGGTAACACTGGTCAATCAAGTGGTCCACACCTTCA.TTTCCAAAGA480

ATGAATGGAAATCCTAGTAACGCAAATGCTCAAGATCCTCTACCATTCTTAAAATCTATT540

GGTTATGGAAAATCAAGTGGTGGTTCTTCAAATACAAAACCATCAACATCTGGTGGTTTT600

AAAGTAAACCAATATGGTACTTTATATAAAGCAGAATCTGCAACATTTACAGCAAATACT660

ACAATCATAACTCGATATACAGGCCCATTTAGATCAATGCCAAAAGCTGGTACGTTAAAA720

TCTGGTCAATCAATTAAATATGATGAAGTCATGAAACAAGATGGTCATGTTTGGGTTGGT780

TATACTAATTCAGCAAAGAAAAGAGTTTATGTTCCAGTTAGAACATGGAATAAGAATAAT840

AACTCCCTGGGAAGTTTATGGGGAACAATAAAATAG8 7 6

Literature

Bannoehr J., Guardabassi L. (2012) Staphylococcus pseudintermedius in the dog: taxonomy, diagnostics, ecology, epidemiology and pathogenicity. Vet Dermatol. 23, 253-66.

Bastos M.C., Ceotto H., Coelho M.L., Nascimento J.S. (2009) Staphylococcal antimicrobial peptides: relevant properties and potential biotechnological applications. Curr Pharm Biotechnol 10, 38-61.

CLSI (2006) Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically; approved standard-seventh edition, M7-A7. Clinical and Laboratory Standards Institute, Wayne, PA, USA.

Drider D., Rebuffat S. eds. (2011) Prokaryotic antimicrobial peptides. New York, Springer.

EUCAST-AFST (2012) EUCAST technical notę on the EUCAST definitive document EDef 7.2: method for the determination of broth dilution minimum inhibitors concentrations of antifungal agents for yeasts EDef 7.2. Clin. Microbiol. Inf. 18, E246-E247.

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Garbacz K., Żarnowska S., Piechowicz L., Haras K. (2013) Pathogenicity potential of Staphylococcus pseudintermedius strains isolated from canine carriers and from dogs with infection signs. Virulence 4, 255-259.

Kumar J.K. (1997) Lysostaphin: an antistaphylococcal agent. Appl Microbiol Biotechnol 80, 555-561.

Moreno I., Lerayer A.L.S., Baldini V.L.S., Leitao MFF (2000) Characterization of bacteriocins produced by Lactococcus lactis strains. Brown. J. Microbiol. 31, 183-191.

Netz D.J., Pohl R., Beck-Sickinger A.G., SelmerT., Pierik A.J., Bastos Mdo C. (2002) Biochemical characterization and genetic analysis of aureocin A53, a new, atypical bacteriocin from Staphylococcus aureus. J Mol Biol 319,745-756.

Riley M.A., Chavan M.A. ed. (2007) Bacteriocins ecology and evolution. Berlin, Springer.

Riley M.A., Gillor O. eds. (2007) Research and Applications in Bacteriocins. Norfolk, Horizon Scientific Press.

Sandiford S., Upton M. (2012) Identification, characterization, and recombinant expression of epidermicin NI01, a novel unmodified bacteriocin produced by Staphylococcus epidermidis that displays potent activity against staphylococci. Antimicrob Agents Chemother 56, 1539-1547.

Savini V., Barbarini D., Polakowska K., Gherardi G., Białecka A., Kasprowicz A., Polilli E., Marrollo R., DiBonaventura G., Fazii P., D'Antonio D., Miedzobrodzki J., Carretto E. (2013) Methicillin-resistant Staphylococcus pseudintermedius infection in a bonę marrow transplant recipient. J Clin Microbiol 51, 1636-1638.

Sugai M., Fujiwara T., Akiyama T., Ohara M., Komatsuzawa H., Inoue S. (1997) Purification and molecular characterization of glycylglycine endopeptidase produced by Staphylococcus capitis EPK1. J Bacteriol 179, 1193-1202.

Van Hoovels L., Vankeerberghen A., Boel A., Van Vaerenbergh K., De Beenhouwer H. (2006) First case of Staphylococcus pseudintermedius infection in a human. J Clin Microbiol 44, 4609-4612.

Table 1

BacSp222 bacteriocin MIC doses against various microorganisms. The doses determined are the average of three independent measurements

Microorganism Strain MIC fmicromol / iitri Gram-positive bacteria: .......................... Bacillus subtins ATCC 6633 * 0.16 Lactococcus lactis LOCK 0871 strain 239 ⁺ * 0.89 Micrococcus luteus ATCC 4698 = PCM 525 *** 0.11 Staphylococcus aureus CH91 **** 0.92 Staphylococcus aureus MRSA USA300 * 0.89 Staphylococcus a u reus KB / 8658 Rocky (dog} **** 1.30 Staphylococcus aureus RN 4220 **** 10.00 a.m. Staphylococcus epidermidis ATCC 35547 * 4.40 Staphylococcus intermedius ATCC29663 * 2.00 Staphylococcus intermedius R-2725 (dog) **** 1.20 Staphylococcus pseudintermedius 222 **** 2.10 Staphylococcus pseudin termedius LMG 22219 * **** 0.16 Staphylococcus saprophyticus ATCC 15305 * 0.93 Staphylococcus aureus ATCC25923 * 1.00 The scab is coccus pyogen es PCM 465 *** 7.80 Strup to coceus songuin is PCM 2335 *** 3.50 Gam-ujenme bacteria: Escherichia coli KI 2 * > 100 Klebsiella pneumoniae ATCC 13886 = PCM 1 *** > 100 Serratia marcescens ATCC 274 = PCM 499 *** > 100 Mushrooms: Candida albicans ATCC 10231 * 100

Strain Sources:

* ATCC * 'Collection of the Institute of Fermentation Technology and Microbiology of the Lodz University of Technology *** Polish Collection of Microorganisms of the Polish Academy of Sciences, Wrocław **** Collection of the Department of Microbiology of the Faculty of Biochemistry, Biophysics and Biotechnology of the Jagiellonian University

Beigjan Coordinated Collections of Microorganisms

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Table 2

Residual activity of the BacSp222 peptide bacteriocin digested with various proteolytic enzymes. This activity is presented as the diameter of the zones of inhibition of bacterial growth (in mm) in the radial diffusion test. Test samples mean bacteriocin incubated with peptidase, control samples mean bacteriocin incubated without peptidase

The ratio in ago ex e n zy m p ty d

| 50: 1 and 50: 1 10: 1 i Enzyme: : Attempt ; examined, t incubation 1 ! at ΐ Trial j control, i incubation 1 h. and Test sample, And incubation 3 ΐ ROOM. ί i 'i and Trial and control; incubation 3 and hours Test sample, incubation 1 hour. Tested sample, incubation 3 hours. Ecstasy ί 12..2 mm j 13.1 mm ΐ 11.2 mm j and 11.4 mm 13.9 mm 14.6 mm Ttypsin : 11.9 mm and 13 _; 1 mm ΐ 11.3 mm ί and 11.4 mm 14.2 mm 15.1 mm; Pepsin ; 12.5 mm ΐ 13.1 mm and 10.8 mm 11.3 mm 14.6 mm 14.1 mm; Cathepsyma G. £ 12.4 mm and 13.7 mm : 11.0 mm and 11.6 mm 14.8 mm 14.4 mm Proteinase V8 l 12.5 mm and 14.0 mm and 11.2 mm ί : 11.3 mm 15.0 mm 15.3 mm Protein, 3 for K. and 11.9 mm j 13.1 mm ί 10.8 mm and 11.4 mm 14.4 mm 14.8 mm Proteinase j & ę ....................... : 12.4 mm and 13.1 mm : 10.8 mm and 11.4 mm 14.2 mm 14.8 mm

Patent claims

Claims (9)

Patent claims A polypeptide comprising an amino acid sequence selected from Seq. No. 1 or Seq. No. 3 or its fragment with bactericidal or lytic activity towards bacterial cells or lytic or cytotoxic activity towards eukaryotic cells. 2. A polynucleotide comprising a nucleotide sequence encoding a polypeptide according to claim 1, 1 selected from Seq. No. 2 or Seq. No. 4. 3. The polypeptide of claim 1 Or the polynucleotide according to claim 1 2 for use in therapy. 4. A plasmid expression vector containing the polynucleotide according to claim 1. 2, including the sequence shown in Fig. 4 A. 5. Staphylococcus pseudintermedius 222 strain deposited with PCM under accession number B / 00084. 6. Use of a polypeptide according to claim 1 1 or a polynucleotide according to claim 1, 2 or the bacterial strain according to claim 2 For the production of a preparation having a bactericidal or lytic activity towards bacterial cells or a lytic or cytotoxic activity towards eukaryotic cells. A method of obtaining a polypeptide according to claim 1 A bacterial host comprising the expression vector according to claim 1, wherein the bacterial host is cultivated. 4, and then the obtained protein is isolated from the post-culture fluid. 8. The method according to p. The method of claim 7, characterized in that the isolation of the protein is carried out by a known method selected from: salting-out, dialysis, concentration on semipermeable membranes or reversed-phase chromatography. 9. The method according to p. The method of claim 7, wherein the bacterial host is the Staphylococcus pseudintermedius 222 strain deposited with the PCM under accession number B / 00084.