Use of a bacteriophage lytic enzyme in the preparation of an antibacterial agent

Abstract

The application discloses application of a bacteriophage lytic enzyme in preparation of an antibacterial agent. The bacteriophage lytic enzyme has a remarkable lytic effect on gram-negative bacteria, and thus can be used as a broad-spectrum antibacterial protein. For example, the antibacterial protein has a remarkable antibacterial effect on gram-negative bacteria such as Escherichia coli, Pseudomonas aeruginosa and Klebsiella pneumoniae. The antibacterial protein has high temperature tolerance and pH tolerance, can replace antibiotics, and is applied to prevention and treatment of bacterial diseases, sterilization of medical devices and medical sites as an external or internal medicine; and can be applied to feed processing, food industry, animal husbandry, beer industry, aquaculture, cosmetic production, fruit preservation and other fields as an additive.

Description

Technical Field

[0001] This invention relates to the application of a phage lysin in the preparation of antibacterial agents, and belongs to the field of phage lysins. Background Technology

[0002] With the widespread and prolonged use of antibiotics, the emergence of increasingly more drug-resistant strains has become a thorny medical problem, leading to the emergence of superbugs (bacteria resistant to almost all antibiotics). The terrifying aspect of these bacteria lies not in their lethality, but in their resistance to common antibiotics, leaving humans with virtually no effective treatments. The widespread use of antibiotics in food processing, livestock farming, agricultural production, and ecological pest control has also exacerbated the drug resistance problem. Globally, a strong effort to control antibiotic overuse is an inevitable trend. Therefore, there is an urgent need to find new antibacterial drugs to address the diseases, environmental, agricultural, and food safety issues caused by drug resistance. Bacteriophage lyases are highly specific bacterial cell wall hydrolases encoded by potent bacteriophage genes. They can effectively recognize and degrade peptidoglycan, a major component of bacterial cell walls, thereby specifically killing bacteria. Many studies have shown that bacteriophage lyases possess high antibacterial activity both in vitro and in vivo. In the context of multidrug-resistant pathogens, the exogenous sterilization of lyases undoubtedly represents a novel antibacterial development. Due to their high antibacterial activity, broad antibacterial spectrum, diverse types, and wide range of choices, they are considered the antibiotic alternatives with the best development prospects and application potential. The natural environment contains a large number of bacteriophages, which are a potential source of highly effective antibacterial proteins. They are a class of microbial antibacterial agents with great application potential. Summary of the Invention

[0003] Purpose of the invention: The technical problem to be solved by the present invention is to provide an application of bacteriophage lysin in the preparation of antibacterial agents.

[0004] Technical solution: To solve the above technical problems, the present invention provides the application of phage lysin or its active fragment or analogue with an amino acid sequence as shown in SEQ ID NO.1 in the preparation of antibacterial agents, wherein the active fragment or analogue has at least 85% sequence identity with the amino acid sequence shown in SEQ ID NO.1 or is an alternative amino acid sequence with the same functional group.

[0005] Preferably, the phage lysin or its binding domain is used in conjunction with a signaling molecule.

[0006] Preferably, the phage lysin or its binding domain forms a fusion product with the signaling molecule through gene fusion or chemical coupling.

[0007] The substituted amino acid sequence is obtained by conservative substitution of amino acids from the same amino acid group, which includes aliphatic amino acids, amino acids containing hydroxyl groups or sulfur / selenium, cyclic amino acids, aromatic amino acids or basic acidic amino acids and their amides.

[0008] The aliphatic amino acids include glycine, alanine, valine, leucine, or isoleucine; the amino acids containing hydroxyl groups or sulfur / selenium include serine, cysteine, threonine, or methionine; the cyclic amino acids include proline; the aromatic amino acids include phenylalanine, tyrosine, or tryptophan; the basic amino acids include histidine, lysine, or arginine; and the acidic amino acids and their amides include aspartic acid, glutamic acid, asparagine, or glutamine.

[0009] The antibacterial agent is used to inhibit and / or kill Gram-negative bacteria.

[0010] The Gram-negative bacteria include Escherichia coli, Klebsiella pneumoniae, or Pseudomonas aeruginosa.

[0011] The phage lysin is Klebsiella pneumoniae phage lysin, which is a bacterial cell wall hydrolytic enzyme.

[0012] The present invention also provides an antibacterial agent for inhibiting and / or killing Gram-negative bacteria, said antibacterial agent containing an antibacterial protein with an amino acid sequence as shown in SEQ ID NO.1. The amino acid sequence of the antibacterial protein is as shown in SEQ ID NO.1: MDIFGMLRIDEGYDSKIYKDTEGYWTIGIGHLLTKDPSKSLAISNLDKLVGRS TGGQITQAEAEVIFAKDVEKAIKGIVANATLNPVYNVLDDVRRAALINMVFQMGVSG VAGFPASMRLLLAKKWEAAAKELANSRWYRQTPNRARRVIETMRTGTWSAYQGK.

[0013] The nucleotides encoding the antimicrobial protein comprise the sequence shown in SEQ ID NO.2, or a synonymous codon sequence thereof. The nucleotide sequence of the antimicrobial protein is shown in SEQ ID NO.2: ATGGATATTTTTGGC ATGTTGCGTATCGATGAAGGATATGACAGCAAAATTTATAAAGATACCGAAGGGTATTGGACCATTGGTATTGGCCACTTACTGACTAAAGACCCGTCAAAATCTTTGGCTATTTCTAATCTGGACAAACTGGTAGGTCGTTCTACTGGTGGTCAAATTACTCAGGCTGAGGCAGAAGTAATTTTTGCCAAAGATGTTGAGAAGGCAATTAAAGGTATTGTTGCTAATGCTACATTAAA CCCGGTATAATGTATTAGATGATGTTCGTAGAGCTGCTCTGATTAACATGGTATTCCAAATGGGTGTGTCTGGTGTAGCCGGGTTCCCAGCTTCAATGAGGTTATTACTCGCTAAAAAGTGGGAAGCTGCTGCCAAGGAACTTGCAAATTCACGTTGGTATCGTCAGACACCTAATCGTGCTCGTCGTGTAATTGAAACAATGCGGACCGGAACTTGGTCTGCTTATCAAGGAAAA.

[0014] The phage lysin has a strong inhibitory effect on Gram-negative bacteria, has broad-spectrum antibacterial activity, and good thermal stability. It can effectively replace antibiotics and is widely used in the feed industry, medicine, food industry, animal husbandry, brewing industry, aquaculture and other fields.

[0015] The products mentioned include external or internal medicines, feed or food additives, disinfectants, cosmetic products, or medical devices.

[0016] Furthermore, the Gram-negative bacteria mentioned include Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, etc.

[0017] The concentration of the phage lysin is 0.1–1 mg / mL.

[0018] The antibacterial agent also contains EDTA.

[0019] The concentration of EDTA is 0.1–10 mM.

[0020] Furthermore, the concentration of EDTA is 0.5–10 mM.

[0021] Furthermore, the concentration of EDTA is 1–10 mM.

[0022] Furthermore, the concentration of EDTA is 5–10 mM.

[0023] The applicable temperature range for the antibacterial agent is 4–70°C.

[0024] Furthermore, the applicable temperature for the antibacterial agent is 4–50°C.

[0025] The applicable pH of the antibacterial agent is 3 to 11.

[0026] Furthermore, the applicable pH of the antibacterial agent is 3 to 10.

[0027] The present invention also provides a method for preparing the phage lysin, comprising the following steps: centrifugation after culture, collection of supernatant and separation and purification of phage lysin using a Ni-TED column, followed by dialysis displacement step, vacuum concentration and freeze drying to obtain the phage lysin; the molecular weight of the phage lysin is 18.9 kDa and the isoelectric point is 9.58.

[0028] The preferred culture conditions are: 16–30℃, 180–220 rpm, 12–18 h.

[0029] Specifically, the preparation method of the phage lysin is as follows:

[0030] S1: The culture medium was obtained at 30℃ and 180 rpm;

[0031] S2: Centrifuge the culture medium to collect the precipitate, centrifuge the lysed cells and take the supernatant, separate and purify it with a Ni-TED column, then perform a dialysis replacement step, and vacuum concentrate and freeze dry to obtain the phage lysin.

[0032] In the induction expression of Klebsiella pneumoniae phage lysin ATCCa, the final concentration of IPTG was 0.2–1 mM, and the temperature was 16–37 °C.

[0033] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages:

[0034] (1) The present invention isolates a novel antimicrobial protein that exhibits strong inhibitory effects against Gram-negative bacteria, particularly pathogenic Escherichia coli, Pseudomonas aeruginosa, and Klebsiella pneumoniae. It possesses broad-spectrum antimicrobial activity and good stability. This antimicrobial protein can replace antibiotics and can be used for the prevention and treatment of bacterial diseases, as well as for sterilization of medical devices and medical facilities. It can also be used as an additive in feed processing, food industry, animal husbandry, brewing industry, aquaculture, cosmetics production, and fruit preservation.

[0035] (2) The antimicrobial protein disclosed in this invention is a soluble protein, and the natural antimicrobial protein is produced from Escherichia coli. It has the characteristics of rapid reproduction, strong vitality, safety and non-toxicity, which facilitates the large-scale production of the natural antimicrobial protein and has great economic value. Attached Figure Description

[0036] Figure 1 Schematic diagram of the plasmid expressing Klebsiella pneumoniae phage lysin expression vector;

[0037] Figure 2 To optimize the concentration of isopropyl-BD-thiogalactoside (IPTG) during the expression of the lyase;

[0038] Figure 3 To optimize the induction temperature conditions during lysin expression;

[0039] Figure 4 SDS-PAGE electrophoresis images of the engineered bacteria expressing lysin and after lysis and purification; where: 1 is marker, 2 is total bacterial cells after lysis, 3 is precipitate after lysis, and 4 is supernatant after lysis.

[0040] Figure 5 Analysis of the optimal antibacterial concentration of the lysin;

[0041] Figure 6 Analysis of the optimal EDTA concentration for lysin;

[0042] Figure 7 This is a host spectrum of the lyase;

[0043] Figure 8 For the detection of the temperature tolerance of the lysin;

[0044] Figure 9 This is for the detection of pH tolerance of the lysin. Detailed Implementation

[0045] The technical solution of the present invention will be further described below with reference to the accompanying drawings.

[0046] For experiments not specifically described in the examples, the procedures or conditions should be followed according to the conventional experimental procedures described in the literature in this field. Reagents or instruments whose manufacturers are not specified are all commercially available conventional reagent products.

[0047] Experimental materials:

[0048] Enzymes and reagents: The enzymes used in the molecular biology operations in the examples were all purchased from TakaRa, and the corresponding operation steps were performed in strict accordance with the relevant product instructions.

[0049] Both liquid and solid culture media for Klebsiella pneumoniae were purchased from Solarbio, and their preparation methods were strictly in accordance with the relevant product instructions. The Klebsiella pneumoniae standard strain ATCC 13883 was purchased from Beijing Baosai Biotechnology Co., Ltd. Primer design and sequencing for the Klebsiella pneumoniae phage lysin and expression vectors involved in the examples were performed by Sangon Biotech (Shanghai) Co., Ltd. All other raw materials and excipients not otherwise specified were commercially available products.

[0050] Example 1: Construction of the expression vector for Klebsiella pneumoniae phage lysin ATCCa

[0051] Using the DNA of Klebsiella pneumoniae phage pATCCa obtained by screening sludge collected from the Xiqing District Wastewater Treatment Plant in Tianjin with ATCC13883 as the host bacterium, primer pairs ATCCa-F (5'-GGAATTCCATATGatggatatttttggcatgttgcgta-3') and ATCCa-R (5'-CCGCTCGAGttttccttgataagcagaccaagtt-3') were designed to amplify the lyase gene.

[0052] Table 1 PCR system

[0053]

[0054] Table 2 PCR reaction procedures

[0055]

[0056] The amplified lyase gene and vector pET30a were digested with restriction endonucleases NdeI and XhoI, respectively, and then ligated using T4 ligase. Figure 1 As shown, the recombinant plasmid pET30a-ATCCa was successfully constructed and then transformed into Escherichia coli BL21(DE3) to construct recombinant Escherichia coli BL21 / pET30a-ATCCa.

[0057] Example 2: Optimization of expression and purification conditions for Klebsiella pneumoniae phage lysin ATCCa

[0058] 1. Optimization of IPTG concentration-induced expression conditions: Single colonies containing recombinant plasmids were randomly picked using a sterile pipette tip and inoculated into LB liquid containing 50 μg / ml kanamycin, and cultured overnight at 37°C with shaking. 1% of the plasmid was transferred to 50 ml of LB liquid containing 50 μg / ml kanamycin and cultured at 37°C and 220 rpm with shaking until the logarithmic growth phase. IPTG was then added to final concentrations of 0.2 mM, 0.4 mM, 0.6 mM, 0.8 mM, and 1 mM, respectively. Another bottle was used as a non-induced control without IPTG. The cells were then cultured overnight at 25°C and 180 rpm with shaking. The cells were collected by centrifugation at 4°C. The cells were then sonicated (46% power; 8 min, 5 s sonication, 5 s interval) until the bacterial culture was clear. Total protein was collected for protein electrophoresis. Results are as follows: Figure 2 As shown, the protein expression level increased significantly under IPTG induction. The expression level of ATCCa showed little difference under different IPTG concentrations. The optimal IPTG concentration was 0.2 mM in the following experimental expression process.

[0059] 2. Optimization of temperature-induced expression conditions: Single colonies containing recombinant plasmids were randomly picked using a sterile pipette tip and inoculated into liquid LB containing 50 μg / ml Kanamycin, and cultured overnight at 37°C with shaking. 1% of the plasmid was then transferred to 50 ml of liquid LB containing 50 μg / ml Kanamycin and cultured at 37°C and 220 rpm with shaking until the logarithmic growth phase. The optimal IPTG concentration obtained in the previous experiment was added to the final concentration, and the cells were cultured overnight at shaking temperatures set to 16°C, 20°C, 25°C, 30°C, 37°C, and 180 rpm. The cells were collected by centrifugation at 4°C. The cells were then sonicated (sonication conditions: 46% power; 8 min, 5 s sonication, 5 s interval) until the bacterial solution was clear. Total protein was collected for protein electrophoresis. Results are as follows: Figure 3 As shown, the protein expression level varies under different temperatures, with the highest protein expression level observed at 30℃. The optimal temperature for induction is 30℃.

[0060] 3. Expression and purification of Klebsiella pneumoniae phage lysin ATCCa: Recombinant Escherichia coli was inoculated into LB liquid medium containing 50 μg / ml Kanamycin and cultured at 37°C. 1% was transferred to 50 ml of LB liquid medium containing 50 μg / ml Kanamycin and cultured at 37°C and 220 rpm in a constant temperature shaker until the exponential growth phase. IPT G was added to a final concentration of 0.2 mM, and the culture was incubated overnight at 30°C in a constant temperature shaker. The cells were collected by centrifugation at 4°C. The cells were then sonicated (46% power; 8 min, 5 s sonication, 5 s interval) until the bacterial solution was clear. After protein disruption, purification was performed using Ni-NTA columns (Qiagen, Dusseldorf, Germany). Imidazole elution was performed at concentrations of 2 ml 20 mM and 2 ml 30 mM to remove contaminating proteins, and 4 ml 100 mM to elute the target protein. The protein was then dialyzed into PBS at pH 7. Results are shown below. Figure 4 As shown, the antimicrobial protein ATCCa was expressed in the strain BL21 / pET30a-ATCCa by constructing the strain. The lysin ATC Ca was solublely expressed and purified in Escherichia coli, and its lysing activity was determined. The amino acid sequence of the antimicrobial protein ATCCa is shown in SEQ ID NO.1: MDIFGMLRIDEGYDSKIYKDTEGYWTIGIGHLLTKDPSKSLAISNLDKLVGRSTGGQITQAEAEVIFAKDVEKAIKGIVANATLNPVYNVLDDVRRAALINMVFQMGVSGVAGFPASMRLLLAKKWEAAAKELANSRWYRQTPNRARRVIETMRTGTWSAYQGK

[0061] Example 3: Exploring the optimal inhibitory concentration of Klebsiella pneumoniae phage lysin ATCCa

[0062] Lysozyme alone has weak bactericidal activity. Numerous studies have reported that low concentrations of EDTA (ethylenediaminetetraacetic acid) and lysozyme can produce a synergistic effect. To verify whether the expressed and purified lysozyme in this invention can also interact with low concentrations of EDTA, a low concentration of EDTA was added while treating bacteria with the lysozyme, and the differences in experimental results were observed.

[0063] One random monoclonal host bacterium was cultured in liquid LB medium until the logarithmic growth phase. The bacterial cells were collected, treated with 100 mM EDTA at pH 7 at 37°C for 10 min, and then resuspended in PBS. Four groups were set up: one group with both antimicrobial protein ATCCa and EDTA, one group with only ATCCa, one group with only EDTA, and one control group. The final concentrations of ATCCa were set at 0.1, 0.5, and 1 mg / ml, respectively, and the final concentration of EDTA in the EDTA-treated groups was 10 mM. Each group was incubated at 37°C for 6 h, and bacterial counts were performed using plate plating. All experiments were performed in triplicate. Results are shown below. Figure 5 As shown, when used alone at 0.5 mg / ml, the antimicrobial protein ATCCa reduced the survival rate of logarithmic Klebsiella pneumoniae ATCC 13883 to 26.49%, and when used in combination with EDTA, the survival rate of ATCC decreased to 0.09%, and the antimicrobial activity was further enhanced when used in combination with EDTA.

[0064] Example 4: Exploring the optimal EDTA concentration for Klebsiella pneumoniae phage lysin ATCCa

[0065] One monoclonal host bacterium was picked and cultured in liquid LB medium until the logarithmic growth phase. The bacterial cells were collected, treated with 100 mM EDTA (pH 7) at 37°C for 10 min, and then resuspended in PBS. Using the optimal protein concentration (0.5 mg / ml) obtained in Example 3, EDTA groups with final concentrations of 0, 0.1, 0.5, 1, 5, and 10 mM were established. Each group was incubated at 37°C for 6 h, and plate counts were performed every 2 hours. All experiments were performed in triplicate. The results are as follows: Figure 6 As shown, when the EDTA concentration is 5 mM, the concentration of TCC13883 bacteria increases from 2 × 10⁻⁶ mM. 8 cfu / mL decreased to 5 × 10 4 The optimal EDTA concentration for this protein to inhibit bacterial growth is 5 mM (cfu / mL).

[0066] Example 5: Determination of the host spectrum of Klebsiella pneumoniae phage lysin ATCCa

[0067] This invention determines the host spectrum of different test strains by treating them with the antimicrobial protein ATCCa. To test whether the antimicrobial protein has a broad host spectrum, six clinical Klebsiella pneumoniae strains, one Escherichia coli BL21(DE3) strain, and one clinical Pseudomonas aeruginosa strain were selected. One monoclonal host bacterium was picked and cultured in liquid LB medium until the logarithmic growth phase. The bacterial cells were collected, treated with 100 mM EDTA at pH 7 at 37°C for 10 min, and then resuspended in PBS. The optimal protein and EDTA concentrations from Examples 3 and 4 were selected. Each group was incubated at 37°C for 6 h. The colony count on the plates was observed, and all experiments were performed in triplicate.

[0068] Table 3. Determination of Lysin Host Profile

[0069] Klebsiella pneumoniae ATCC13883 + Clinical Klebsiella pneumoniae S15 + Clinical Klebsiella pneumoniae S21 + Clinical Klebsiella pneumoniae S24 + Clinical Klebsiella pneumoniae S34 + Clinical Klebsiella pneumoniae S55 + Clinical Pseudomonas aeruginosa P8W + Escherichia coli BL21(DE3) +

[0070] The results are shown in Table 3 and Figure 7 As shown, in addition to its inhibitory effect on its own host bacteria, this protein also has a significant inhibitory effect on other Gram-negative bacteria, such as Escherichia coli, Pseudomonas aeruginosa, and drug-resistant Klebsiella pneumoniae.

[0071] Example 6: Temperature tolerance test of Klebsiella pneumoniae phage lysin ATCCa

[0072] One monoclonal host bacterium was selected and cultured in liquid LB medium until the logarithmic growth phase. The bacterial cells were collected, treated with 100 mM EDTA (pH=7) at 37°C for 10 min, and then resuspended in PBS (pH=7.4). The optimal protein concentration from Example 3 was selected, and different temperature gradients (4, 16, 25, 37, 50, and 70°C) were set for treatment. The optimal EDTA concentration from Example 4 was selected. Each group was incubated at 37°C for 6 h and plated for counting. All experiments were performed in triplicate. Results are as follows: Figure 8 As shown, in terms of temperature tolerance, the antimicrobial protein ATCCa remained stable after treatment at 4, 16, 25, 37 and 50 °C, but its antimicrobial activity decreased to approximately 80% after exposure to 70 °C for the same duration. Therefore, this antimicrobial protein exhibits good temperature tolerance.

[0073] Example 7: Klebsiella pneumoniae phage lysin ATCCapH tolerance test

[0074] One monoclonal host bacterium was selected and cultured in liquid LB medium until the logarithmic growth phase. The bacterial cells were collected, treated with EDTA for 10 min, and then resuspended in PBS. The optimal protein concentration from Example 3 was selected, and different pH gradients (3, 4, 5, 6, 7, 8, 9, 10, 11) were used for treatment. The optimal EDTA concentration from Example 4 was selected. Each group was incubated at 37°C for 6 h and plated for counting. All experiments were performed in triplicate. Results are as follows: Figure 9 As shown, regarding pH tolerance, the protein activity remains essentially unchanged between pH 3 and 10, while the antibacterial activity decreases to approximately 90% at pH 11. Therefore, this antibacterial protein exhibits good pH tolerance.

Claims

1. The application of phage lysin and EDTA, with amino acid sequences as shown in SEQ ID NO.1, in the preparation of antibacterial agents, characterized in that, The antibacterial agent is used to inhibit and / or kill Gram-negative bacteria; the Gram-negative bacteria are Escherichia coli or Pseudomonas aeruginosa; the concentration of the bacteriophage lysin is 0.1~1 mg / mL; the concentration of the EDTA is 0.1~10 mM.

2. The application according to claim 1, characterized in that, The applicable temperature range for the antibacterial agent is 4~70℃.

3. The application according to claim 1, characterized in that, The applicable pH for the antibacterial agent is 3-11.

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