Polypeptide and application of the same

Abstract

To provide a new polypeptide.SOLUTION: A polypeptide has: a first domain; a linker; and a second domain. The first domain is a domain which has an amino acid sequence where, one or more and four or less amino acid residues may be substituted, depleted, inserted or added from any of multiple specific sequences, or is a domain which has an amino acid sequence which has at least 80% of homology to any of the multiple specific sequences and has enzymatic activity. The linker has a linker having 2 or more and 10 or less amino acid residue length. The second domain is a domain which has an amino acid sequence where, one or more and four or less amino acid residues may be substituted, depleted, inserted or added, from any of other specific sequences, or is a cell binding domain which has an amino acid sequence which has at least 80% of homology to any of other specific sequences.SELECTED DRAWING: Figure 2

Description

Technical Field

[0001] This invention relates to polypeptides and their uses. More specifically, this invention relates to endolysin having antibacterial activity against Staphylococcus and its uses.

Background Art

[0002] Patent No. 5383481 describes phage endolysin that lyses and kills Gram-positive bacteria. International Publication WO2012 / 145630 pamphlet describes PlySs2 as an endolysin against Staphylococcus aureus.

Prior Art Documents

Patent Documents

[0003]

Patent Document 1

Patent Document 2

Summary of the Invention

Problems to be Solved by the Invention

[0004] Since polypeptides have various uses, new polypeptides are desired. In particular, new polypeptides having bactericidal activity against Gram-positive bacteria (such as Staphylococcus) and their uses are desired.

Means for Solving the Problems

[0005] As shown by the examples, the novel polypeptide disclosed in this specification has antibacterial activity equivalent to or higher than that of PlySs2 against Gram-positive bacteria (such as Staphylococcus).

[0006] The first invention relates to a polypeptide. This polypeptide contains a first domain, a linker, and a second domain in this order. The first domain is (1) A domain having an amino acid sequence represented by any one of SEQ ID NOs: 9 to 13, (2) A domain having an amino acid sequence in which 1 to 10 amino acid residues are substituted, deleted, inserted or added to the amino acid sequence represented by any one of SEQ ID NOs: 9 to 13, (3) A domain having an amino acid sequence having 80% or more homology with the amino acid sequence represented by any one of SEQ ID NOs: 9 to 13 and having enzyme activity. The linker is a linker having a length of 1 to 20 amino acid residues. The second domain is (1) A domain having an amino acid sequence represented by any one of SEQ ID NOs: 14 to 19, (2) A domain having an amino acid sequence in which 1 to 10 amino acid residues are substituted, deleted, inserted or added to the amino acid sequence represented by any one of SEQ ID NOs: 14 to 19, (3) A domain having an amino acid sequence having 80% or more homology with the amino acid sequence represented by any one of SEQ ID NOs: 14 to 19 and being a cell-binding domain.

[0007] A preferred polypeptide is (1) A polypeptide consisting of a first domain, a linker, and a second domain, (2) A polypeptide consisting of a first domain, a linker, a second domain, and a tag sequence, and the tag sequence has an amino acid sequence having a length of 2 to 10 amino acid residues added to either or both of the first domain and the second domain.

[0008] In a preferred polypeptide, the first domain is a domain having an amino acid sequence represented by any one of SEQ ID NOs: 9 to 13, the linker is a linker having a length of 1 to 20 amino acid residues, and the second domain is a domain having an amino acid sequence represented by any one of SEQ ID NOs: 14 to 19.

[0009] A preferred polypeptide is (1) A polypeptide having an amino acid sequence represented by any one of SEQ ID NOs: 1 to 8, or (2) A polypeptide having an amino acid sequence in which one or more and ten or fewer amino acid residues are substituted, deleted, inserted or added in the amino acid sequence represented by any one of SEQ ID NOs: 1 to 8, or (3) A polypeptide having an amino acid sequence having 80% or more homology with the amino acid sequence represented by any one of SEQ ID NOs: 1 to 8 and having an antibacterial action against Staphylococcus bacteria.

[0010] A preferred polypeptide is a polypeptide having an amino acid sequence represented by any one of SEQ ID NOs: 1 to 8.

[0011] The following invention is an isolated polynucleotide containing a nucleic acid encoding any of the above polypeptides.

[0012] The following invention relates to a vector containing the above polynucleotide. An example of the vector is a plasmid.

[0013] The following invention relates to a host cell containing the above vector.

[0014] The following invention relates to an antimicrobial agent containing the above polypeptide, the above polynucleotide, the above vector, or the above host cell. Examples of the antimicrobial agent are (1) a food additive or a disinfectant, (2) a cosmetic raw material or a cosmetic additive, (3) an antifouling agent or a cleaning agent for a medical device, a medical manufacturing apparatus or a food manufacturing apparatus, (4) a therapeutic or prophylactic agent for a disease associated with Staphylococcus (Staphylococcus aureus) infection.

Effects of the Invention

[0015] This specification discloses a new polypeptide. Further, this specification discloses a new polypeptide having a bactericidal action against Gram-positive bacteria (e.g., Staphylococcus) and its uses.

Brief Description of the Drawings

[0016]

Figure 1

Figure 2

Figure 3

BEST MODE FOR CARRYING OUT THE INVENTION

[0017] Hereinafter, embodiments for carrying out the present invention will be described with reference to the drawings. The present invention is not limited to the embodiments described below, and also includes those appropriately modified by those skilled in the art within an obvious range from the following embodiments.

[0018] The first invention relates to a polypeptide. This polypeptide is a polypeptide containing a first domain, a linker, and a second domain in this order. The polypeptides, genes, etc. in this specification are preferably isolated and purified. This polypeptide may be in the form of a salt. Also, the polypeptide may be a solvate (for example, a hydrate) if it exists. This polypeptide may be (1) a polypeptide consisting only of the first domain, the linker, and the second domain, or (2) an arrangement consisting only of the first domain, the linker, the second domain, and a tag sequence. Also, this polypeptide is preferably endolysin.

[0019] The first domain preferably has an enzymatic activity. An example of the enzymatic activity is cell wall degrading activity. The cell wall degrading activity may be evaluated by the method shown in the examples. The first domain is any of the following domains. (1) The first domain is a domain having an amino acid sequence represented by any of SEQ ID NOs: 9 to 13. (2) The first domain is a domain having an amino acid sequence in which 1 to 10 (preferably 1 to 4, more preferably 1 or 2) amino acid residues are substituted, deleted, inserted or added from the amino acid sequence represented by any of SEQ ID NOs: 9 to 13. In this case, the first domain preferably has enzyme activity. The enzyme activity may be equivalent to that of a domain having an amino acid sequence represented by any of SEQ ID NOs: 9 to 13. The substituted, inserted and added amino acid residues may be modified amino acid residues (the same applies hereinafter). (3) The first domain is a domain having an amino acid sequence having 80% or more homology with the amino acid sequence represented by any of SEQ ID NOs: 9 to 13 and having enzyme activity. The enzyme activity may be equivalent to that of a domain having an amino acid sequence represented by any of SEQ ID NOs: 9 to 13. The enzyme activity may be equivalent to or higher than that of the corresponding domain of the polypeptide represented by SEQ ID NO: 30.

[0020] The linker is a region that links the first domain and the second domain. Specifically, the C-terminus of the first domain is adjacent to the N-terminus of the linker, and the C-terminus of the linker is adjacent to the N-terminus of the second domain. The linker is a portion having a length of 1 to 20 amino acid residues. The linker may have a length of 2 to 15 amino acid residues, may have a length of 2 to 10 amino acid residues, may have a length of 4 to 8 amino acid residues, or may have a length of 5 to 7 amino acid residues. A specific linker is a site having an amino acid residue sequence (GAGAGA) represented by SEQ ID NO: 20, or a site having an amino acid sequence in which 1 or 2 amino acid residues are substituted, deleted, inserted or added from the amino acid residue sequence represented by SEQ ID NO: 20.

[0021] The second domain is preferably a cell-binding domain (CBD). The CBD is, for example, a targeting motif that recognizes the outer surface of bacteria. The second domain is any of the following domains. (1) The second domain is a domain having an amino acid sequence represented by any of SEQ ID NOs: 14 to 19. (2) The second domain is a domain having an amino acid sequence in which 1 to 10 (preferably 1 to 4, more preferably 1 or 2) amino acid residues are substituted, deleted, inserted or added from the amino acid sequence represented by any of SEQ ID NOs: 14 to 19. (3) The second domain is a domain having an amino acid sequence having 80% or more homology with the amino acid sequence represented by any of SEQ ID NOs: 14 to 19, and is a cell-binding domain. As described above, the second domain preferably has cell-binding ability. The cell-binding ability may be evaluated based on the method in the examples described later. The cell-binding ability may be equal to or higher than the corresponding domain of the polypeptide represented by SEQ ID NO: 30.

[0022] The tag sequence is a site having a length of 2 to 10 amino acid residues added to either or both of the first domain and the second domain. A preferred tag sequence has a length of 4 to 8 amino acid residues. The tag sequence is preferably added to the second domain, and particularly preferably added to the C-terminus of the second domain. Examples of the tag sequence are His tag (polyHis tag), FLAG tag, HA tag and Myc tag. Among these, His tag (polyHis tag) is preferred. The specific tag sequence is a site having the amino acid residue sequence (HHHHHH) represented by SEQ ID NO: 21, or a site having an amino acid sequence in which 1 or 2 amino acid residues are substituted, deleted, inserted or added from the amino acid residue sequence represented by SEQ ID NO: 21.

[0023] The above-described polypeptide appropriately includes any of the above combinations. Among the above-described polypeptides, preferred ones are as follows. (1) A polypeptide having an amino acid sequence represented by any of SEQ ID NOs: 1 to 8, (2) A polypeptide having an amino acid sequence in which 1 to 10 (preferably 1 to 4, more preferably 1 or 2) amino acid residues are substituted, deleted, inserted or added from the amino acid sequence represented by any one of SEQ ID NOs: 1 to 8, or (3) A polypeptide having an amino acid sequence having 80% or more homology with the amino acid sequence represented by any one of SEQ ID NOs: 1 to 8 and having an antibacterial activity against staphylococci. The antibacterial activity against staphylococci may be at the same level as that of the polypeptide having the amino acid sequence represented by any one of SEQ ID NOs: 1 to 8. Alternatively, the above polypeptide may have an antibacterial activity equivalent to or higher than that of the polypeptide (PlySs2) having the amino acid sequence represented by SEQ ID NO: 30.

[0024] SEQ ID NO: 1 bbst1001 MQAKLTKKEFIEWLKTSEGKQYNADGWYGFQCFDYANAGWQVLFGYNLKGVGAKDIPSANDFNGLATVYQNTPDFLAQPGDMVVFGSNYGAGYGHVAWVIEATLDYIIVYEQNWLGGGWTDGVQQPGSGWEKVTRRQHAYDFPMWFIRPNFKSETAPRSVQSPTQASKKETGAGAGAEIAKQEVLPTGWKKNKHGTYYKAQKGSFINGNQPIQARYVGPFRLKNNAAGDLPANTKIEYDEIMLQDKHVWVGYDSFEGERIYLPVGTWNGKKPPKNKMKQVWGILHHHHHH

[0025] Sequence number 2 bbst1005 MQAKLTKKEFIEWLKTSEGKQYNADGWYGFQCFDYANAGWQVLFGYNLKGVGAKDIPSANDFNGLATVYQNTPDFLAQPGDMVVFGSNYGAGYGHVAWVIEATLDYIIVYEQNWLGGGWTDGVQQPGSGWEKVTRRQHAYDFPMWFIRPNFKSETAPRSVQSPTQASKKETGAGAGAVKNKPGSASTPANRRDMSGWKINKYGTYYKSEVAHFTPNTPIKTHYVGPFRSCPVSGVLQPGQIVRYDTVCKQDGHVWISYTAYNGKDVWLAVRTWDKNTDSLGKLWGTINHHHHHH

[0026] Sequence number 3 bbst1014 MQAKLTKKEFIEWLKTSEGKQYNADGWYGFQCFDYANAGWQVLFGYNLKGVGAKDIPSANDFNGLATVYQNTPDFLAQPGDMVVFGSNYGAGYGHVAWVIEATLDYIIVYEQNWLGGGWTDGVQQPGSGWEKVTRRQHAYDFPMWFIRPNFKSETAPRSVQSPTQASKKETGAGAGAVKNKPGSASTPANRRDMNGWKINKYGTYYKSEVARFTPNTPIKTHYVGPFRSCPVSGVLQPGQTIKYDTVCKQDGHVWVSYTAYNGKDVWLAVRTWNKTNDSLGKLWGTINHHHHHH

[0027] Sequence number 4: bbst1021 MKTQSQINARLNAYKNGTVDSPYRVKTWTSYDPAFGTMEPGCIDVDHAYHAQCADLPIDYILWLTDNQYRAWGNAKDFPNNKFPTGWKVIENLPSTVPQKGWIAVFSSGTYAQYGHIGLVYDGGNTNSFEILEQNWNGYANKKPTLRWDNYYGLTHFIVPPVAKEVHTLTTKVKEAPKQTGAGAGATKKTSTSNEKWNKNQYGILWRKEVGSFTCNVPQGIITRRIGPGRQYPIAGALKKAQTVNYTEIQKNDGYIWISWMTNSGYTVYMPVRQVKSDGSLGPLWGTIKHHHHHH

[0028] Sequence number 5: bbst1027 MAKTQTQINKLVDSYLGKYVDFDGYYAFQCMDLAVSYVYKLTDGSFRMYGNAKDAINNKFPSGWKVIRNQAATVPKKGWIAVYTTGVYQQYGHIGIVYNGGNTSQFQILEQNFDGLANSPAKLRWDNYSGLTHFIVPPTKSATTSSNGSAKTTTAKAKTSTPKTKKRKIMLVAGHGYNDPGAGAGASSNTVKPVASAWKRNKYGTYYMEESARFTNGNQPITVRKVGPFLSCPVGYQFQPGGYCDYTEVMLQDGHVWVGYTWVGQRYYLPIRTWNGSAPPNQILGDLWGEIHHHHHH

[0029] Sequence number 6: bbst1035 MQAKLTKKEFIEWLKTSEGKQFNVDLWYGFQCFDYANAGWKVLFGLLLKGLGAKDIPFANNFDGLATVYQNTPDFLAQPGDMVVFGSNYGAGYGHVAWVIEATLDYIIVYEQNWLGGGWTDRIEQPGWGWEKVTRRQHAYDFPMWFIRPNFKSETAPRSIQSPTQASKKETAKPQPKAVEGAGAGAKNQKNPPVPAGYTLDKNNVPYKKETGNYTVANVKGNNVRDGYSTNSRITGVLPNNATIKYDGAYCINGYRWITYIANSGQRRYIATGEVDKAGNRISSFGKFSTIHHHHHH

[0030] Sequence number 7: bbst1036 MQAKLTKKEFIEWLKTSEGKQFNVDLWYGFQCFDYANAGWKVLFGLLLKGLGAKDIPFANNFDGLATVYQNTPDFLAKPGDMVVFGSNYGAGYGHVAWVIEATLDYIIVYEQNWLGGGWTDGIEQPGWGWEKVTRRQHAYDFPMWFIRPNFKSETAPRSVQSPTQAPKKETAKPQPKAVEGAGAGAKNQKNPPVPAGYTLDKNNVPYKKETGNYTVANVKGNNVRDGYSTNSRITGVLPNNATIKYDGAYCINGYRWITYIANSGQRRYIATGEVDKAGNRISSFGKFSTIHHHHHH

[0031] SEQ ID NO:8 bbst1037 MQAKLTKKEFIEWLKTSEGKQFNVDLWYGFQCFDYANAGWKVLFGLLLKGLGAKDIPFANNFDGLATVYQNTPDFLAKPGDMVVFGSNYGAGYGHVAWVIEATLDYIIVYEQNWLGGGWTDGIEQPGWGWEKVTRRQHAYDFPMWFIRPNFKSETAPRSVQSPTQAPKKETAKPQPKAVEGAGAGATKKTSTSNEKWNKNQYGILWRKEVGSFTCNVPQGIITRRIGPGRQYPIAGALKKAQTVNYTEIQKNDGYIWISWMTNSGYTVYMPVRQVKSDGSLGPLWGTIKHHHHHH

[0032] The above-mentioned polypeptide is preferably an endolysin polypeptide specific to the genus Staphylococcus. This polypeptide is a polypeptide having lytic activity against the genus Staphylococcus. The lytic activity is preferably a peptidoglycan hydrolase activity against the peptidoglycan of the genus Staphylococcus. The genus Staphylococcus is preferably selected from the group consisting of Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus simulans and Staphylococcus carnosus. The lytic activity can be evaluated by any suitable method known to those skilled in the art. It is preferable to use one of the analytical methods described in the examples of this specification. The polypeptide may be modified by amino acids or substituents (for example, at the N-terminus or C-terminus) in the amino acid sequence in order to increase stability, solubility and activity.

[0033] The polypeptides of SEQ ID NOs: 1 to 8 include those having the amino acid residue sequences represented by SEQ ID NOs: 20 and 21 as tag sequences and linkers, respectively. The relationship between the polypeptides of SEQ ID NOs: 1 to 8 and the first domain and the second domain is as follows. Full length, First domain, Second domain SEQ ID NO:1 SEQ ID NO:9 SEQ ID NO:14 SEQ ID NO:2 SEQ ID NO:9 SEQ ID NO:15 Array number 3, Array number 9, Array number 16 Array number 4, Array number 10, Array number 17 Array number 5, Array number 11, Array number 18 Array number 6, Array number 12, Array number 19 Array number 7, Array number 13, Array number 19 Array number 8, Array number 13, Array number 17

[0034] As shown by the examples, the above polypeptide has the effect of killing staphylococci. Therefore, this specification also provides the following invention.

[0035] Another invention relates to a polynucleotide. This polynucleotide is an isolated polynucleotide containing a nucleic acid encoding any of the above polypeptides. This polynucleotide may be isolated and purified. The polynucleotide may be either RNA or DNA, with DNA being preferred. Examples of nucleic acids encoding any of the above polypeptides are nucleic acids having any of the base sequences of SEQ ID NOs: 22 to 29 (and their complementary base sequences). The nucleic acid encoding the polypeptide can be designed by analyzing the codons corresponding to the amino acid residues. And the method for obtaining such a nucleic acid is known. "Nucleic acid" may be a deoxyribonucleotide, ribonucleotide, or mixed polymer in single-stranded or double-stranded form.

[0036] Another invention relates to a vector. This vector is a vector containing the above polynucleotide. The vector may be any of a plasmid, cosmid, bacteriophage, and virus that is transformed by introducing the polynucleotide, with a plasmid vector being preferred.

[0037] Another invention relates to a host cell containing the above vector. Examples of host cells are Escherichia coli, Schizosaccharomyces pombe, and Saccharomyces cerevisiae.

[0038] Another invention relates to an antimicrobial agent. The antimicrobial agent is a composition having a bactericidal effect against Gram-positive bacteria (e.g., staphylococci including Staphylococcus aureus). This antimicrobial agent contains any of the above polypeptides, polynucleotides, vectors, or host cells. This antimicrobial agent may appropriately contain a carrier or diluent such as water, or may be a component of various products.

[0039] This antimicrobial agent can be used as a food additive or a disinfectant. A food additive is an agent added to various foods to prevent the foods from being contaminated by microorganisms. A disinfectant is a composition used to prevent contamination by microorganisms.

[0040] This antimicrobial agent can be used as a cosmetic raw material or an additive for cosmetics. A cosmetic raw material can be used as an active ingredient of a cosmetic having antibacterial properties against microorganisms. An additive for cosmetics can be utilized as an element added when manufacturing cosmetics.

[0041] This antimicrobial agent can be used as an antifouling agent or a cleaning agent for medical devices, medical manufacturing apparatuses, or food manufacturing apparatuses. In particular, as demonstrated by the examples described below, since this antimicrobial agent can effectively dissolve biofilms, it can be preferably used as an antifouling agent or a cleaning agent for medical devices, medical manufacturing apparatuses, or food manufacturing apparatuses.

[0042] This antimicrobial agent can be used as a therapeutic or prophylactic agent for diseases associated with staphylococcal infections (especially Staphylococcus aureus infections). This therapeutic or prophylactic agent contains an effective amount of any of the above polypeptides, polynucleotides, vectors, or host cells as an active ingredient. Examples of diseases associated with staphylococcal infections (especially Staphylococcus aureus infections) are skin infections, food poisoning, pneumonia, meningitis, and sepsis. This therapeutic or prophylactic agent can be produced by mixing known auxiliary materials (such as carriers like water) in addition to the above active ingredients. The effective amount may be appropriately adjusted according to the target species (human or non-human animal), sex, body weight, and age. An example of a specific effective amount is 1 to 1000 μg per kg of body weight, and the number of administrations may be appropriately adjusted according to the use. This specification also provides a method for killing microorganisms using the antimicrobial agent. This specification also provides a method for treating or preventing the above diseases, which includes the step of administering the above therapeutic or prophylactic agent to a subject (human or non-human animal).

[0043] [Evaluation Method] Cell wall degrading activity, cell binding ability, and antibacterial action can be indicated by the minimum growth inhibitory concentration and antibacterial activity described below.

Examples

[0044] Expression and Purification of Endolysin The purification of each endolysin was prepared by gravity flow or chromatography using nickel resin or cobalt resin. The specific procedure is described below.

[0045] Genes of each endolysin consisting of Array Nos. 22 to 29 were cloned into the E. coli expression vector pET17b vector (Novagen) so as to be expressed under the control of the T7 promoter, and this was transformed into the BL21(DE3)pLysS strain. The transformed bacteria were inoculated into 2 ml of LB medium containing ampicillin (ampicillin-containing LB medium) at a final concentration of 100 μg / ml and cultured with shaking overnight at 37°C. 1 ml of the overnight culture was subcultured into 100 ml of ampicillin-containing LB medium and cultured with shaking at 37°C. When the OD600 optical density of the culture reached 0.4 - 0.8, IPTG was added to a final concentration of 0.1 - 0.5 mM, and the culture was cultured with shaking overnight at 16°C. The culture after overnight culture was centrifuged to recover the bacterial cells.

[0046] To 100 mg of the recovered bacterial cells, 2 ml of xTractor TM buffer (Clontech) was added, and appropriate amounts of protease inhibitor and DNaseI were further added. This was incubated on ice or at room temperature until the bacteria lysed, and then centrifuged at 10000 g to recover the supernatant as the soluble fraction. The soluble fraction was added to a gravity flow column filled with 1 ml of cobalt resin to capture the endolysin on the cobalt resin. Then, the column was washed with 7 ml of washing solution (phosphate buffer containing 10 mM imidazole), and finally 5 ml of elution solution (phosphate buffer containing 150 mM imidazole) was added to the column, and the elution solution passing through the column was recovered as the purified endolysin fraction. If necessary, the purified endolysin fraction was dialyzed against PBS.

[0047] The results of SDS-polyacrylamide gel electrophoresis of the purified polypeptide of SEQ ID NO: 1 are shown in Figure 1.

[0048] In the above method, the culture scale is not limited to 100 mL and is variable. Depending on the amount of endolysin required for the test, culturing was carried out using 5 mL to 2 L of LB medium containing ampicillin. Also, the resin used for purification is not limited to cobalt resin, and nickel resin can also be used as an alternative. The purification method is not limited to gravity flow, and it is also possible to purify the polypeptides of SEQ ID NOs: 1 to 8 using chromatography. No significant difference was observed in the activity of the purified endolysin depending on the culture scale and purification conditions.

Example

[0049] Minimum inhibitory concentration (MIC) of endolysin against Staphylococcus aureus The antibacterial activities of each endolysin and antibiotic against Staphylococcus aureus were analyzed. For the analysis, methicillin-sensitive Staphylococcus aureus (Staphylococcus aureus NBRC 100910; obtained from the National Institute of Technology and Evaluation (NITE), Japan) and methicillin-resistant Staphylococcus aureus (Staphylococcus aureus clinical isolate No. 1658; provided by Gunma University, Japan) were used.

[0050] Staphylococcus aureus was grown in TSB medium containing 7.5% sodium chloride, and the culture solution was centrifuged at 6000 g for 5 minutes. The supernatant was removed and the cells were resuspended in 10 mM Tris-HCl (pH 8.0). The bacterial suspension thus prepared was diluted with 10 mM Tris-HCl (pH 8.0) to an optical density of 0.1 OD600, and this was further diluted 1:100 with 2x cation-containing Mueller-Hinton medium. Endolysin or antibiotic was injected into a 96-well plate, and various concentrations of protein and antibiotic were prepared in a two-fold dilution series with a final volume of 100 μl using 10 mM Tris-HCl (pH 8.0). 100 μl of the bacterial suspension was added to the 96-well plate and mixed. The plate was cultured at 37 °C for 18 hours, and the OD600 value of the well was measured to measure bacterial growth. The minimum inhibitory concentration (MIC), which is the minimum concentration of endolysin or antibiotic below which the OD600 value is less than 0.2, was determined.

[0051] The results of the minimum growth inhibitory concentration (μg / ml) are shown in Table 1 below. Table 1 Minimum growth inhibitory concentration (μg / ml)

Table 1

[0052] PlySs2 is an endolysin against Staphylococcus aureus reported by Fischetti Vincent et al. (WO2012 / 145630) and was used as a control. Vancomycin is a low-molecular-weight antibiotic and was used as a control.

[0053] MIC is a method widely used to evaluate the efficacy of antibacterial agents. Since the MIC value is the minimum antibacterial agent concentration required to inhibit the growth of bacteria, the lower the MIC value, the higher the antibacterial activity. The endolysins of SEQ ID NOs: 1-8 showed good antibacterial activity against methicillin-susceptible Staphylococcus aureus and methicillin-resistant Staphylococcus aureus as compared with the previously reported endolysin (PlySs2). Therefore, it was confirmed that the endolysins of SEQ ID NOs: 1-8 are useful as effective antimicrobial agents against Staphylococcus aureus.

Example

[0054] Minimum growth inhibitory concentration (MIC) of endolysin against Staphylococcus aureus in human serum The antibacterial activities of each endolysin and antibiotic in serum against Staphylococcus aureus were analyzed.

[0055] Staphylococcus aureus (Staphylococcus aureus NBRC 100910; obtained from the National Institute of Technology and Evaluation (NITE), Japan) was grown in TSB medium containing 7.5% sodium chloride, and the culture solution was centrifuged at 6000 g for 5 minutes. The supernatant was removed, and the cells were resuspended in 10 mM Tris-HCl (pH 8.0). The bacterial suspension thus prepared was diluted with 10 mM Tris-HCl (pH 8.0) to an optical density of 0.1 at OD600, and further diluted 1:100 with 2x cation-containing Mueller-Hinton medium. Endolysin or antibiotics at various concentrations that form a two-fold dilution series were prepared using human serum and added to a 96-well plate at a final volume of 100 μl. 100 μl of the bacterial suspension was added to the 96-well plate and mixed. The plate was incubated at 37°C for 18 hours, and the presence or absence of bacterial growth was visually determined. The minimum inhibitory concentration (MIC), which is the minimum concentration of endolysin or antibiotic at which no bacterial precipitate or turbidity of the culture solution due to bacteria was observed, was determined. The measurement was performed with n = 3, and the MIC values are shown as a range.

[0056] The results of the minimum inhibitory concentration (μg / ml) are shown in Table 2 below. Table 2 Minimum inhibitory concentration (μg / ml)

Table 2

[0057] The endolysins of SEQ ID NOs: 1-8 showed antibacterial activity even in serum. Also, it was shown that the MIC values of the endolysins of SEQ ID NOs: 1-8 were lower in the presence of serum compared to in the absence of serum (Table 1), and they were shown to have significantly higher antibacterial activity compared to the known endolysin (PlySs2). In infectious diseases, it is important for antibacterial agents to act in the blood, and from the above results, the usefulness of the endolysins of SEQ ID NOs: 1-8 against infections caused by Staphylococcus aureus was confirmed.

Example

[0058] Bactericidal activity of endolysin against Staphylococcus aureus The bactericidal activities of each endolysin and antibiotics against Staphylococcus aureus were analyzed.

[0059] Staphylococcus aureus (Staphylococcus aureus NBRC 100910; obtained from the National Institute of Technology and Evaluation (NITE), Japan) was grown in TSB medium containing 7.5% sodium chloride. The culture solution was centrifuged at 6000 g for 5 minutes, the supernatant was removed, and the cells were resuspended in a buffer for measuring bactericidal activity (20 mM Tris-HCl, pH 7.5, 150 mM sodium chloride, 0.9 mM magnesium chloride, 2.5 mM calcium chloride). The bacterial suspension thus prepared was diluted with the buffer for measuring bactericidal activity to an optical density of 0.8 at OD600, and 90 μl of the diluted suspension was added to each well of a 96-well plate. 10 μl of the buffer for measuring bactericidal activity, an endolysin solution prepared to 256 μg / ml in the same buffer, or an antibiotic was added, and immediately after mixing, the measurement of the OD600 value was started, and the OD600 value was recorded over 40 minutes.

[0060] The results of the time-course measurement of bactericidal activity are shown in Fig. 2.

[0061] Vancomycin, one of the antibiotics used for Staphylococcus aureus infections, did not show a clear bactericidal activity in the 40-minute measurement. On the other hand, the endolysins of SEQ ID NOs: 1-8 showed rapid bactericidal activity, which is characteristic of endolysins, similar to the previously reported endolysins.

Example

[0062] Biofilm-disrupting activity of endolysin against Staphylococcus aureus The biofilm-disrupting activities of each endolysin and antibiotic against the biofilm of Staphylococcus aureus were analyzed.

[0063] Staphylococcus aureus (Staphylococcus aureus NBRC 13276; obtained from the National Institute of Technology and Evaluation (NITE), Japan) was grown in TSB medium containing 7.5% sodium chloride and diluted with TSB medium containing 0.2% glucose (TSBg medium) to an optical density of 0.1 at OD600, and further diluted 1:100 with TSBg medium. The bacterial dilution thus prepared was suspended with a vortex mixer for 1 minute, and then 150 μl each was added to a 96-well plate made of polystyrene and cultured at 37°C for 24 hours to form a biofilm. The culture solution was removed, and the wells were washed by adding 300 μl each of PBS. This washing operation was performed two more times to completely remove the PBS. Endolysin or an antibiotic prepared at 25.6 μg / ml in PBS was added 150 μl each and incubated at room temperature for 24 hours. The supernatant was removed, and the wells were washed by adding 300 μl each of PBS. This washing operation was performed two more times to completely remove the PBS. A staining solution (0.05% crystal violet, 20% methanol solution) was added 150 μl each and incubated at room temperature for 15 minutes. The staining solution was removed, and the wells were washed by adding 300 μl each of PBS. The PBS was completely removed, 150 μl each of 33% acetic acid was added, and the stained matter was dissolved by pipetting, and the optical density at OD590 was measured.

[0064] The biofilm disruption activity was calculated by the following formula. Biofilm disruption rate (%) = (OD590 control - OD590 sample ) / (OD590 control - OD590 blank ) x 100 Here, OD590 control is the OD590 value of the biofilm without the addition of endolysin or an antibiotic, OD590 sample is the OD590 value of the biofilm with the addition of endolysin or an antibiotic, OD590 blank is the OD590 value of the well where there is no biofilm without the addition of Staphylococcus aureus.

[0065] The results of the biofilm disruption rate (%) are shown in Table 3 below. Table 3 Biofilm disruption rate (%) [Table 3]

[0066] Vancomycin, one of the antibiotics used for Staphylococcus aureus infections, did not show significant biofilm disruption activity. On the other hand, the endolysins of SEQ ID NOs: 1 to 8 showed high biofilm disruption activity, similar to the endolysins already reported. [Examples]

[0067] Bactericidal activity of endolysin against Staphylococcus epidermidis The bactericidal activities of each endolysin and antibiotic against Staphylococcus epidermidis were analyzed.

[0068] Staphylococcus epidermidis (Staphylococcus epidermidis, NBRC 113847; obtained from the National Institute of Technology and Evaluation (NITE), Japan) was grown in TSB medium containing 7.5% sodium chloride, and the bactericidal activities of the endolysins of SEQ ID NOs: 1 to 8 were measured by the same method as described in Example 4. The results of the bactericidal activity measurement over time are shown in Figure 3.

[0069] As shown in Figure 3, the endolysins of SEQ ID NOs: 1 to 8 have rapid bactericidal activity against not only Staphylococcus aureus but also Staphylococcus epidermidis, and it was confirmed that they are effective against Staphylococcus spp. across different species. [Industrial Applicability]

[0070] Since this invention relates to polypeptides having antibacterial effects against Staphylococcus, it can be used in the food additive, pharmaceutical, daily necessities, and cosmetic industries. [Sequence Listing Free-Text]

[0071] SEQ ID NOs: 1 to 8 Full-length polypeptides Array numbers 9 to 13, first domain Array numbers 14 to 19, second domain Array number 20, linker Array number 21, tag Array numbers 22 to 29, full-length nucleotide Array number 30, PlySs2

Claims

1. A polypeptide comprising a first domain, a linker, and a second domain in this order, wherein the first domain is (1) a domain having an amino acid sequence represented by any one of SEQ ID NOs: 9 to 13, or (2) a domain having an amino acid sequence in which 1 to 10 amino acid residues are substituted, deleted, inserted, or added from the amino acid sequence represented by any one of SEQ ID NOs: 9 to 13, or (3) a domain having an amino acid sequence having 80% or more homology with the amino acid sequence represented by any one of SEQ ID NOs: 9 to 13 and having enzyme activity, the linker is a linker having a length of 1 to 20 amino acid residues, the second domain is (1) a domain having an amino acid sequence represented by any one of SEQ ID NOs: 14 to 19, or (2) a domain having an amino acid sequence in which 1 to 10 amino acid residues are substituted, deleted, inserted, or added from the amino acid sequence represented by any one of SEQ ID NOs: 14 to 19, or (3) a domain having an amino acid sequence having 80% or more homology with the amino acid sequence represented by any one of SEQ ID NOs: 14 to 19 and being a cell-binding domain, a polypeptide.

2. The polypeptide according to claim 1, wherein it is a polypeptide consisting of the first domain, the linker, and the second domain, or it consists of the first domain, the linker, the second domain, and a tag sequence, and the tag sequence has an amino acid sequence having a length of 2 to 10 amino acid residues added to either or both of the first domain and the second domain, a polypeptide.

3. The polypeptide according to claim 2, wherein the first domain is a domain having an amino acid sequence represented by any one of SEQ ID NOs: 9 to 13, the linker is a linker having a length of 1 to 20 amino acid residues, and the second domain is a domain having an amino acid sequence represented by any one of SEQ ID NOs: 14 to 19, a polypeptide.

4. The polypeptide according to claim 1, wherein it is a polypeptide having an amino acid sequence represented by any one of SEQ ID NOs: 1 to 8, or it is a polypeptide having an amino acid sequence in which 1 to 4 amino acid residues are substituted, deleted, inserted, or added from the amino acid sequence represented by any one of SEQ ID NOs: 1 to 8, A polypeptide having an amino acid sequence having 80% or more homology with the amino acid sequence represented by any one of SEQ ID NOs: 1 to 8 and having an antibacterial action against staphylococci.

5. The polypeptide according to claim 1, which has an amino acid sequence represented by any one of SEQ ID NOs: 1 to 8.

6. An isolated polynucleotide comprising a nucleic acid encoding the polypeptide according to claim 1.

7. A vector comprising the polynucleotide according to claim 6.

8. The vector according to claim 7, which is a plasmid.

9. A host cell comprising the vector according to claim 7.

10. An antimicrobial agent comprising the polypeptide according to any one of claims 1 to 5, the polynucleotide according to claim 6, the vector according to claim 7 or 8, or the host cell according to claim 9.

11. The antimicrobial agent according to claim 10, which is a food additive or a disinfectant.

12. The antimicrobial agent according to claim 10, which is a cosmetic raw material or an additive for cosmetics.

13. The antimicrobial agent according to claim 10, which is an antifouling agent or a cleaning agent for medical devices, medical manufacturing devices or food manufacturing devices.

14. The antimicrobial agent according to claim 10, which is a therapeutic or prophylactic agent for diseases associated with staphylococcal infection.

15. The antimicrobial agent according to claim 10, which is a therapeutic or prophylactic agent for diseases associated with Staphylococcus aureus infection.

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