Novel antibacterial peptide and application thereof

Abstract

The invention relates to the technical field of biology, in particular to novel antibacterial peptides (RW8, RI8 and Z2) and application thereof. The antibacterial peptide contains an anti-protease hydrolysis peptide structural unit, and the anti-protease hydrolysis peptide structural unit is composed of two polar positively charged amino acids and two hydrophobic amino acids. The research results of the antibacterial activity of the novel antibacterial peptide and the inhibition and elimination effects of fungal or bacterial biofilms provide an important scientific basis for the invention design of the antibacterial peptide.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to novel antimicrobial peptides (RW8, RI8 and Z2) and applications thereof. Background Art

[0002] Antibiotics can treat clinical infections caused by pathogenic microorganisms and are the most widely used antimicrobial drugs in the world. Antibiotics are substances derived from bacteria or fungi, or synthesized from scratch, with antimicrobial effects. They can inhibit or kill pathogenic microorganisms such as fungi, bacteria, spirochetes, and mycoplasmas by inhibiting pathogen cell wall synthesis, increasing cell membrane permeability, or interfering with protein synthesis, nucleic acid metabolism, and other metabolic processes.

[0003] The abuse of antibiotics causes pathogens to be constantly exposed to antibiotic environments, making them resistant. Antimicrobial peptides are regarded as the most promising new drugs to replace antibiotics because of their special antibacterial mechanism and are not easy to cause drug resistance. Some antimicrobial peptides not only show excellent antibacterial activity against free bacteria, but also have effects on bacterial biofilms. Biofilm is a self-protective bacterial group formed by bacterial aggregation, adhesion, maturation and aging. Bacteria are tightly grouped together through electrostatic force, van der Waals force, hydrophobic interaction and secreted extracellular polysaccharides, which can protect bacteria from tenacious survival even in extreme environments. Biofilms can attach to almost any surface of any material. In clinical practice, there has always been a stubborn problem of chronic infection caused by biofilm adhesion to medical devices. Antimicrobial peptides can interfere with the membrane potential of biofilms, affect the bacterial signal transduction system, degrade bacterial extracellular polymers, regulate the binding protein transport genes related to biofilm formation, synergistically inhibit the formation of biofilms from different angles, and even destroy and eliminate irreversible mature biofilms. Antimicrobial peptides from natural sources have certain limitations, including low antimicrobial activity, poor biocompatibility, and susceptibility to environmental factors. Therefore, circumventing the above defects through artificial de novo design has become a hot topic and difficulty in the development of antimicrobial peptide drugs. Summary of the invention

[0004] The purpose of the present invention is to provide a novel antimicrobial peptide (RW8, RI8 and Z2) and application thereof.

[0005] To achieve the above object, the object of the present invention is achieved through the following technologies:

[0006] A novel antimicrobial peptide, characterized in that the antimicrobial peptide contains a protease-resistant peptide structural unit, wherein the protease-resistant peptide structural unit is a structural unit composed of two polar positively charged amino acids and two hydrophobic amino acids.

[0007] The antimicrobial peptide is (XXYY)2P(YYXX)2 (X represents R; Y represents I or W).

[0008] The antimicrobial peptide is a sequence represented by the amino acids in any one of SEQ ID No. 1-3.

[0009] The antimicrobial peptides are specifically:

[0010] SEQ ID No.1 (RW8) is

[0011] Ac—RRWWRRWWPWWRRWWRR—NH2;

[0012] SEQ ID No.2(RI8) is

[0013] Ac'—RRIIRRIIPIIRRIIRR—NH2;

[0014] SEQ ID No.3(Z2) is

[0015] Ac—RKIIRRIIPIIRRIIKR—NH2.

[0016] An application of the novel antimicrobial peptide, and application of the antimicrobial peptide in fighting pathogenic bacteria.

[0017] An application of the novel antimicrobial peptide is the application of the antimicrobial peptide in anti-biofilm, wherein the biofilm is a biofilm of pathogenic bacteria; the pathogenic bacteria are bacteria or fungi.

[0018] The bacteria is Staphylococcus aureus and the fungus is Candida albicans.

[0019] An application of the novel antimicrobial peptide, and application of the novel antimicrobial peptide in anti-pepsin stability.

[0020] The present invention is beneficial in that:

[0021] The present invention designs and synthesizes a new type of antimicrobial peptide, and for the first time discovers the antimicrobial activity of RW8 / RI8 / Z2 against a variety of common pathogens and drug-resistant strains, for the first time discovers the inhibitory and elimination effects of RW8 / Z2 on Staphylococcus aureus and Candida albicans biofilms, and for the first time discovers the strong resistance of RW8 / RI8 / Z2 to pepsin. Currently, clinical chronic and recurrent postoperative bacterial infections are closely related to the spread of bacterial biofilms. Traditional antibiotics have limited effects in eradicating clinical biofilms, and it is difficult to eradicate biofilm infections in indwelling equipment. Therefore, the antimicrobial peptides of the present invention have very important scientific significance for the treatment of wound infections caused by clinical postoperative biofilms; specifically:

[0022] 1. The three novel antimicrobial peptides RW8, RI8 and Z2 described in the present invention contain selected positively charged amino acids (Arg) and 8 hydrophobic amino acids (Trp or Ile). Two polar positively charged amino acids and two hydrophobic amino acids constitute a unit, and a total of four units are distributed in pairs on both sides of proline (Pro).

[0023] 2. Application of two anti-biofilm peptides RW8 and Z2 in the antimicrobial peptides of the present invention in inhibiting biofilms of Staphylococcus aureus and Candida albicans; further, the obtained antimicrobial peptides were tested for antimicrobial and anti-biofilm activities, and it was found that RI8 and Z2 had significant antimicrobial effects on a variety of common pathogens and drug-resistant strains, and could effectively inhibit the formation of biofilms of Staphylococcus aureus ATCC6538 at a concentration of 32 μg / mL, with inhibition rates of up to 75.8% and 90.75%, respectively. In summary, RW8 and Z2 are two antimicrobial peptides with high application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is a reverse phase high performance liquid chromatogram of the antimicrobial peptide RW8 / RI8 / Z2 provided in an embodiment of the present invention.

[0025] Figure 2 This is the mass spectrum of the antimicrobial peptide RW8 / RI8 / Z2 provided in an embodiment of the present invention.

[0026] Figure 3 This is a spiral wheel projection diagram of the antimicrobial peptide RW8 / RI8 / Z2 provided in an embodiment of the present invention.

[0027] Figure 4 This is a spiral network projection diagram of the antimicrobial peptide RW8 / RI8 / Z2 provided in an embodiment of the present invention.

[0028] Figure 5 Tricine-SDS-PAGE electrophoresis diagram of the antimicrobial peptide RW8 / RI8 / Z2 provided in the embodiment of the present invention after being treated with pepsin for different time periods; Note: M: protein marker; 1-5: anti-protease antimicrobial peptide treated with 8 mg / mL pepsin for 30 min, 1 h, 2 h, 4 h, and 8 h; 6: anti-protease antimicrobial peptide control; 7: pepsin control. DETAILED DESCRIPTION

[0029] The present invention is further described in detail below in conjunction with embodiments and drawings, but the embodiments of the present invention are not limited thereto.

[0030] Example 1

[0031] Design of antimicrobial peptides

[0032] The design methods of three new antimicrobial peptides are as follows: 8 arginines (Arg) are selected as positively charged amino acids and 8 tryptophans (Trp) or isoleucines (Ile) are selected as hydrophobic amino acids. Two polar positively charged amino acids and two hydrophobic amino acids form a unit, and a total of four units are distributed on both sides of proline (Pro) in pairs to form the anti-protease hydrolysis peptide structural unit (XXYY)2P(YYXX)2 (X represents R; Y represents I and W), and RW8 and RI8 are obtained. The first and last arginines (Arg) in the RI8 sequence are replaced with lysine (Lys) to obtain Z2.

[0033] 1. Synthesis and purification of antimicrobial peptides

[0034] The antimicrobial peptide was commissioned to Jiangsu GenScript Biotechnology Co., Ltd. to complete the production using the Fmoc solid phase synthesis method. The synthesized peptide was purified to a purity of not less than 90% by reverse phase chromatography: eluent A was 0.065% TFA / water solution; eluent B was 0.05% TFA / acetonitrile solution, the elution concentration was 5% B to 95% B, the elution time was 25 minutes, and the flow rate was 1 mL / min.

[0035] 2. Identification of antimicrobial peptides

[0036] The antimicrobial peptides obtained above were analyzed by electrospray mass spectrometry, and the molecular weights (such as Figure 1 The molecular weight of the antimicrobial peptide is basically consistent with the theoretical molecular weight in Table 1, and the purity of the antimicrobial peptide is greater than 90%.

[0037] Figure 1 The sample information parameters are as follows:

[0038] Interface: Electrospray Spray gas flow rate: 1.5L / min Drying gas flow rate: 5L / min

[0039] RF temperature: 250℃ Blocking temperature: 200℃

[0040] Dissolution method: 85% water + 15% acetonitrile Ion source voltage: +4.5Kv Detector: -0.2Kv

[0041] Flow rate: 0.2ml / min Injection volume: 200μl RF voltage: 0v

[0042] Mobile phase ratio: 50% water / 50% methanol

[0043] RW8 sequence: Ac-RRWWRRWWPWWRRWWRR-NH2

[0044] RW8 theoretical molecular weight: 2854.31

[0045] RW8 measured molecular weight: 2854.2

[0046] RI8 sequence: Ac-RRIIRRRIIPIIRRIIRR-NH2

[0047] RI8 theoretical molecular weight: 2269.89

[0048] RI8 measured molecular weight: 2269.5

[0049] Z2 sequence: Ac-RKIIRRRIIPIIRRIIKR-NH2

[0050] Z2 theoretical molecular weight: 2213.86

[0051] Z2 measured molecular weight: 2213.5

[0052] The RW8 sequence is shown in SEQ ID No. 1, the RI8 sequence is shown in SEQ ID No. 2, and the Z2 sequence is shown in SEQ ID No. 3. The basic parameters of the three new antimicrobial peptides RW8, RI8 and Z2 are shown in Table 1.

[0053] The amino acid sequence of SEQ ID No.1 (RW8) is: Ac-Arg-Arg-Trp-Trp-Arg-Arg-Trp-Trp-Pro-Trp-Trp-Arg-Arg-Trp-Trp-Arg-Arg-NH2,

[0054] The amino acid sequence of SEQ ID No.2 (RI8) is: Ac-Arg-Arg-Ile-Ile-Arg-Arg--Ile-Ile-Pro--Ile-Ile-Arg-Arg--Ile-Ile-Arg-Arg-NH2,

[0055] The amino acid sequence of SEQ ID No.3(Z2) is: Ac-Arg-Lys-Ile-Ile-Arg-Arg--Ile-Ile-Pro--Ile-Ile-Arg-Arg--Ile-Ile-Lys-Arg-NH2.

[0056] Table 1 Basic parameters of three new antimicrobial peptides RW8 / RI8 / Z2

[0057]

[0058] Example 2

[0059] Antimicrobial peptide antibacterial activity assay

[0060] The strains frozen at -20℃ were streaked onto LB solid plate medium and cultured at 37℃ for 18-24h. Single colonies were picked with sterile bamboo sticks and inoculated into 100mL MH liquid medium respectively. They were cultured at 37℃ and 180rpm for 12h. After the culture reached the logarithmic growth phase, appropriate amount of each bacterial solution was centrifuged, the supernatant was discarded, and the bacteria were resuspended in MH liquid medium and diluted to 1-2×10 5 CFU / mL.

[0061] Among them, the strains include Escherichia coli (ATCC 9637), Klebsiella pneumoniae (CMCC46117), Salmonella paratyphi (ATCC 14028), Pseudomonas aeruginosa (ATCC9027), A. baumannii (ACCC11038), P. aeruginosa CMCC 46117, Multiple-resistant E. coli (SYP-B4288), Multiple-resistant E. coli SYP-B4289 and Staphylococcus aureus (ATCC6538).

[0062] The above-mentioned marker SYP belongs to a multidrug-resistant clinical isolate, which was donated by the Affiliated Hospital of China Medical University. All strains are preserved in this laboratory.

[0063] Discard the four circles of the 96-well plate, add 36μL of 640μg / mL polypeptide and 144μL of bacterial solution to each well of B2-D2 (E2-G2), add 180μL of bacterial solution to each well of B2-D11 (E2-G11), and dilute in sequence from the second column to the tenth column. The bacterial solution in the eleventh column is used as a negative control, and sterile MH liquid culture medium is added to the twelfth column as a blank control. The remaining empty wells are treated with sterile water. Incubate the 96-well plate at 37℃ for 24h. Observe whether the culture medium in the well is turbid, and measure the OD with an enzyme reader 600nm The absorbance was measured and the inhibition rate was calculated using the following formula. The peptide concentration at which no obvious bacterial growth was observed by naked eye and the inhibition rate was greater than 90% was the MIC (minimum inhibitory concentration). The inhibition rate was calculated using the following formula, and the geometric mean (Geometric Mean, ), and therapeutic index (TI=MHC / GM). The test results are shown in Table 2.

[0064] Inhibition rate (%) = [(OD 阴性对照 -OD 样品 ) / (OD 阴性对照-OD 空白对照 )]×100%.

[0065] Table 2 Determination of MIC values ​​of RW8 / RI8 / Z2

[0066]

[0067] As can be seen from Table 2, RI8 and Z2 have broad-spectrum antibacterial activity, and the geometric mean minimum inhibitory concentrations are 6.35 and 3.70 μg / mL, respectively, and have relatively good antibacterial activity against most of the tested bacteria.

[0068] Example 3

[0069] Determination of hemolytic activity of RW8 / RI8 / Z2

[0070] 1 mL of fresh human blood was collected, anticoagulated with heparin, dissolved in 2 mL of PBS solution, centrifuged at 1000 g for 5 min, and red blood cells were collected; washed with PBS 3 times, and then resuspended with 10 mL of PBS; 50 μL of red blood cell suspension was mixed with 50 μL of antimicrobial peptide solution of different concentrations dissolved in PBS, and incubated in a 37°C incubator for 1 hour; taken out after 1 hour, centrifuged at 4°C and 1000 g for 5 minutes; the supernatant was taken out and the light absorption value was measured at 570 nm with an enzyme marker; the average value of each group was taken and compared and analyzed. Among them, 50 μL of red blood cells plus 50 μL of PBS was used as a negative control; 50 μL of red blood cells plus 50 mL of 0.1% Tritonx-100 was used as a positive control. The minimum hemolytic concentration is the antimicrobial peptide concentration when the antimicrobial peptide causes a 5% hemolysis rate. The hemolysis rate was calculated using the following formula, and the test results are shown in Table 3.

[0071] Hemolysis rate (%) = [OD (样品 -OD 阴性对照 ) / (OD 阳性对照 -OD 阴性对照 )]×100%.

[0072] Table 3 Hemolytic activity of RW8 / RI8 / Z2

[0073]

[0074] It can be seen from Table 3 that the hemolysis rate of RI8 and Z2 at a concentration of 128 μg / mL is still less than 1%, that is, the minimum hemolysis concentration (MHC) is 128 μg / mL, and they have strong biocompatibility; the hemolysis rate of RW8 at a concentration of 16 μg / mL is greater than 5%, and MHC = 8 μg / mL.

[0075] Example 4

[0076] RW8 / RI8 / Z2 stability test

[0077] 1. Salt stability and serum stability: Each peptide was mixed with salt ions and fetal bovine serum of different concentrations, so that the final concentrations of salt ions were 150mM NaCl, 4.5mM KCl, 6mM NH4Cl, 8mM ZnCl2, 2.5mM CaCl2 and 4mMFeCl3; the final concentrations of fetal bovine serum were 12.5%, 25% and 50% respectively. The mixed peptides were incubated at 37°C for 2h, and the changes in the minimum inhibitory concentration of the incubated samples before and after peptide treatment were determined according to the antibacterial activity determination method.

[0078] Table 4 Salt ion stability of three new antimicrobial peptides RW8 / RI8 / Z2

[0079]

[0080] Table 5 Serum stability of three new antimicrobial peptides RW8 / RI8 / Z2

[0081]

[0082] It can be seen from Tables 4 and 5 that the antimicrobial activity of antimicrobial peptide RW8 / RI8 / Z2 was not significantly affected in different physiological salt solutions and different concentrations of serum solutions, and the minimum inhibitory concentration changed slightly. In general, the antimicrobial peptide RW8 / RI8 / Z2 has high salt stability and serum stability.

[0083] 2. Pepsin stability: 1M dilute hydrochloric acid solution was diluted with sterile water to pH 1.5, 10 mL was added with 0.08 g of pepsin powder, and mixed for use. After mixing equal volumes of polypeptide and pepsin solution, incubated at 37°C for 2 h, heated in a water bath at 80°C for 10 min, and then the changes in the antibacterial activity of the polypeptide after protease treatment were detected according to the antibacterial activity determination method.

[0084] Table 6 Protease stability of three new antimicrobial peptides RW8 / RI8 / Z2

[0085]

[0086] As can be seen from Table 6, after the antimicrobial peptide RW8 / RI8 / Z2 was incubated with 2 mg / mL pepsin for 2 h, the antibacterial activity did not change, and the minimum inhibitory concentration was the same as that of the control, indicating that the antimicrobial peptide RW8 / RI8 / Z2 has a high resistance to pepsin.

[0087] 3. Antimicrobial peptide Trince-SDS-PAGE protein electrophoresis: Mix equal volumes of 3.2 mg / mL peptide and 8 mg / mL pepsin and incubate at 37°C. Take out 100 μL at different time points, dilute and heat at 99°C for 10 min to inactivate pepsin, centrifuge at 13,000 rpm for 30 min, and retain the supernatant. Prepare a protein gel of 16.5% separation gel + 4% concentration gel. After the peptide sample is inactivated by the protein loading-buffer, load the sample, place the electrophoresis tank in an ice bath, and perform electrophoresis at a constant voltage of 30V for 1 hour. After the bromophenol blue is compressed into a straight line and reaches the separation gel, adjust the voltage to 180V and perform electrophoresis for 2 hours. After the electrophoresis is completed, take out the protein gel and place it in a 0.5% glutaraldehyde fixative for 20 minutes. Remove the fixative and transfer it to a Coomassie brilliant blue dye solution for overnight staining. Take out the protein gel, rinse with sterile water to remove the residual dye, and place it in a decolorizing solution for repeated decolorization until clear bands are visible. See attached for results. Figure 5 .like Figure 5 As shown in the figure, after being treated with pepsin, the samples of RI8 and RW8 collected at different time points all had bands similar to those of the control group, and there was almost no degradation phenomenon. The protein band of the sample at 8h was the same size as the band of the control group. On the other hand, the protein band of RI8 / RW8 showed a larger molecular weight than the actual one. This is because the amino acid sequence of RI8 / RW8 is short and rich in hydrophobic amino acids and polar positively charged amino acids. At high concentrations, the polypeptide is prone to intermolecular interactions to form oligomers, which affects the mobility of the polypeptide during electrophoresis, causing the apparent molecular weight of the protein band to differ by 2-4 times from the actual molecular weight. The above results show that RI8 / RW8 has a high resistance to pepsin, and no degradation phenomenon occurred after co-acting with 8mg / mL pepsin for 8h.

[0088] Example 5

[0089] Anti-biofilm activity of RW8 / Z2

[0090] 1. Crystal violet semi-quantitative staining was used to determine the inhibitory effect of RW8 / Z2 on biofilms of Staphylococcus aureus (ATCC 6538) and Candida albicans (ATCC 5314). Antimicrobial peptides were added to a polystyrene 96-well plate, and micro-gradient dilutions were performed using TSB-G or YPD medium. The bacterial solution was added to make the final concentrations of antimicrobial peptides reach 64, 32, 16, and 8 μg / mL, respectively; the concentration of Staphylococcus aureus in the wells reached 10 8 CFU / mL, the concentration of Candida albicans liquid reached 10 6CFU / mL, set up negative control (bacterial solution) and blank control (TSB-G or YPD medium) respectively. Culture at 37℃ for 24h. Discard the culture medium in the wells and wash twice with PBS. Add 0.1% crystal violet to stain for 10min, remove crystal violet, wash three times with PBS, add 33% glacial acetic acid to decolorize after drying, and measure the absorbance at 490nm wavelength with an enzyme reader. Calculate the biofilm inhibition rate according to the following formula, and the test results are shown in Table 7.

[0091] Inhibition rate (%) = [(OD 阴性对照 -OD 样品 ) / (OD 阴性对照 -OD 空白对照 )]×100%.

[0092] 2. Determine the elimination effect of RW8 / Z2 on mature biofilm. Add 200 μL of bacterial suspension to a 96-well plate and culture at 37°C for 24 hours to form a complete biofilm. Carefully aspirate the culture medium, add 200 μL of 10mM PBS to the wells and wash twice, add 200 μL of TSB-1% glucose liquid medium or YPD liquid medium containing different concentrations of polypeptides, and further culture at 37°C for 24 hours. Perform crystal violet quantitative staining according to the above method, read the crystal violet absorbance value, and calculate the biofilm elimination rate using the following formula. The test results are shown in Table 7.

[0093] Elimination rate (%) = [(OD 阴性对照 -OD 样品 ) / (OD 阴性对照 -OD 空白对照 )]×100%.

[0094] Table 7 Anti-biofilm effect of RW8 / Z2

[0095]

[0096] It can be seen from Table 4 that at a concentration of 64 μg / mL, the inhibition rates of RW8 and Z2 against Staphylococcus aureus ATCC 6538 biofilm were as high as 75.8% and 92.4%, respectively; the elimination rates were 30.5% and 42.1%, respectively. They can effectively inhibit biofilm formation and eliminate mature biofilms to a certain extent, and have the potential to become new anti-biofilm agents.

Claims

1. A novel antimicrobial peptide, characterized in that: The antimicrobial peptide contains a protease-resistant peptide structural unit, wherein the protease-resistant peptide structural unit is a structural unit composed of two polar positively charged amino acids and two hydrophobic amino acids.

2. The novel antimicrobial peptide according to claim 1, characterized in that: The antimicrobial peptide is (XXYY)2P(YYXX)2 (X represents R; Y represents I or W).

3. The novel antimicrobial peptide according to claim 2, characterized in that: The antimicrobial peptide is the sequence represented by the amino acids in any one of the sequences of SEQ ID No. 1-3.

4. A use of the novel antimicrobial peptide according to claim 1, characterized in that: Application of the antimicrobial peptide in resisting pathogenic bacteria.

5. A use of the novel antimicrobial peptide according to claim 1, characterized in that: Application of the antimicrobial peptide in anti-biofilm.

6. Use of the novel antimicrobial peptide according to claim 4 or 5, characterized in that: The biofilm is a biofilm of pathogenic bacteria; the pathogenic bacteria are fungi or bacteria.

7. A use of the novel antimicrobial peptide according to claim 1, characterized in that: Application of the novel antimicrobial peptide in anti-pepsin stability.

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