CN111675751B

CN111675751B - Antibacterial peptide and application thereof

Info

Publication number: CN111675751B
Application number: CN202010269627.2A
Authority: CN
Inventors: 张溪; 蔺晨; 杨艳艳; 弓磊; 张倩; 王永兰
Original assignee: STOMATOLOGICAL HOSPITAL TIANJIN MEDICAL UNIVERSITY
Current assignee: STOMATOLOGICAL HOSPITAL TIANJIN MEDICAL UNIVERSITY
Priority date: 2020-04-08
Filing date: 2020-04-08
Publication date: 2022-06-14
Anticipated expiration: 2040-04-08
Also published as: CN111675751A

Abstract

The invention provides an antibacterial peptide and application thereof, wherein the sequence of the antibacterial peptide is Gly-Leu-Leu-Trp-Arg-Lys-Cys-Cys-Arg-Arg-Lys-Lys. The antibacterial peptide uses natural antibacterial peptide which is not easy to cause drug resistance of human bodies, prevents and controls the bacteria attached to the oral cavity in the initial stage of biofilm formation, inhibits the formation of pathogenic microorganisms in the window period of tissue recovery after non-drug therapy by interfering the basic condition of biofilm formation, and achieves the purpose of preventing and controlling infectious diseases.

Description

Antibacterial peptide and application thereof

Technical Field

The invention belongs to the technical field of biomedical materials, and particularly relates to an antibacterial peptide and application thereof.

Background

Periodontitis is the sixth most common chronic disease in the world, and is also the most common disease causing missing teeth. Periodontitis is a common disease that is mediated by plaque bacteria and the host, causing damage to soft and hard tissues of the teeth. Currently, the global prevalence of periodontitis is about 10%, affecting 100 million people worldwide. The development of periodontitis can affect overall health in a number of ways, for example, periodontitis can cause tooth loss, discomfort and pain, which can cause difficulties in speaking, chewing and swallowing, severely reducing quality of life; even with a severe impact on a person's social and emotional well-being. For example, loss of personal self-esteem is associated with loss of teeth. In addition, there is increasing clinical and experimental evidence that there is a potential direct relationship between periodontitis and a variety of systemic diseases including diabetes, rheumatoid arthritis, atherosclerosis, alzheimer's disease and even cancer.

The traditional non-surgical treatment for periodontitis is to remove dental plaque by periodontal scaling and root angioplasty, followed by surgical treatment. However, it is important that neither non-surgical methods nor extensive periodontal surgical techniques completely eliminate periodontal microorganisms due to the powerful potential of pathogens to invade the gingival epithelium and connective tissue through the periodontal pocket epithelial cells. In addition, not all patients respond to non-drug treatment, and some patients have persistent infections and tissue destruction that ultimately leads to tooth loss. In this case, the combination with other drugs may have a better clinical efficacy than mere mechanical debridement.

The use of antibiotics is essential in view of the fact that periodontitis is a group of diseases caused by bacterial infections. However, the widespread use of antibiotics can lead to a number of side effects, such as allergic reactions, dysbacteriosis, and drug resistance. Due to the side effects of antibiotic use, there is an urgent need for a new biological agent to replace antibiotics in the treatment and prevention of disease. With the increasing understanding of the nature and mechanism of action of natural cationic peptides, the production and clinical trials of new synthetic peptides have shown great potential and antibacterial activity in animal models of infection by a variety of pathogens.

Disclosure of Invention

In view of this, the present invention aims to provide an antimicrobial peptide and its application to overcome the deficiencies of the prior art.

In order to achieve the purpose, the technical scheme of the invention is realized as follows:

an antimicrobial peptide, characterized by: the sequence of the antibacterial peptide is Gly-Leu-Leu-Trp-Arg-Lys-Cys-Cys-Arg-Arg-Lys-Lys.

The principle is as follows: glycine (Gly, G) is a good N-capping residue of the alpha-helix in the natural antimicrobial peptide, and therefore Gly is used as the N-terminal residue of the antimicrobial peptide. Tryptophan (Trp, W) has a great affinity for the interface between the lipid bilayer and the aqueous medium and helps anchor the peptide to the surface of the bacterial cell lipid bilayer, and studies have shown that it should be in the fourth position of the antimicrobial peptide sequence, an amphipathic interface that readily forms a helix; meanwhile, researches show that the change of the helicity of the alpha-helix of the polypeptide can influence the antibacterial activity of the antibacterial peptide. If an amino acid having a high helical tendency is used instead of a residue having a low helical tendency, the antibacterial activity is remarkably improved, and therefore leucine (Leu, L) and lysine (Lys, K) are used; amidation of the C-terminus is important for the antimicrobial activity of the antimicrobial peptide, and the amidated terminus increases the net charge of the antimicrobial peptide. Many natural antimicrobial peptides are amidated in the human body, so a positively charged amino acid residue is added at the C-terminus to achieve amidation to increase antimicrobial activity. While the ratio between the cationic lysine (Lys) and arginine (Arg) residues influences the membrane selectivity, since the guanidino function of arginine promotes a more efficient interaction with eukaryotic cell membranes compared to lysine. However, this usually comes at the expense of increased cytotoxicity. Thus, the present invention contemplates amino acid sequences wherein Lys: the molar ratio of Arg is 1: 1. The amino acid sequence is synthesized by Shanghai biological engineering Co., Ltd, and the purity of the amino acid sequence is more than 95% through high performance liquid chromatography detection and mass spectrometry. To test its antibacterial properties, it was tested for its resistance to the initial oral attachment of the bacterium streptococcus. Meanwhile, the efficiency of the streptococcus on resisting biofilm formation is realized by in vitro high-flux culture and visual observation of a microscope. Cytotoxicity experiments against human gingival fibroblasts clarified its biocompatibility. Its target genes for the formation of streptococcal biofilm are also elucidated.

The invention also provides the application of the antibacterial peptide in inhibiting streptococcus or inhibiting the formation of streptococcus biofilm.

Preferably, the streptococcus is one or more than two of streptococcus oralis, streptococcus gerdoniae and streptococcus sanguis.

Preferably, the Minimum Inhibitory Concentration (MIC) of the antimicrobial peptide is 156.25. mu.g/ml, the Minimum Bactericidal Concentration (MBC) against Streptococcus oralis and Streptococcus sanguis is 2500. mu.g/ml, the Minimum Bactericidal Concentration (MBC) against Streptococcus geldanae is 1250. mu.g/ml, and the upper concentration when used does not exceed 1/2 MBC.

Preferably, the concentration that inhibits the formation of streptococcal biofilms is 156.25. mu.g/ml.

The invention also provides application of the antibacterial peptide in medicines or care products for preventing and controlling chronic periodontitis.

The adhesion of pathogenic bacteria to the surface of the teeth is the first step in the formation of bacterial biofilms, an important biological factor in the development and progression of periodontitis. Thus, interfering with or inhibiting early bacterial adhesion and biofilm formation on the tooth surface is a key factor in the prevention of chronic periodontitis. Many studies have attempted to achieve the goal of controlling plaque formation with different approaches and types of antibacterial substances, the main objective being to kill the pathogenic bacteria, whereas antibacterial strategies directed only to pathogenic bacteria do not control biofilm formation, which can still lead to disease once dysbiosis of the flora is achieved. In the current strategy, the application of antibacterial peptide on the initial attached bacteria does not cause drug resistance of the organism to the maximum extent on the basis of interfering the biofilm formation, and is a rational choice for resisting the infectious diseases. Therefore, the antibacterial peptide which is not easy to generate drug resistance to human bodies is selected for interfering the initial attachment bacteria of the biofilm formation, so that the bacteria attachment is reduced, the initial attachment bacteria are killed, the formation of the oral biofilm can be effectively controlled, the development of the chronic periodontitis is controlled, and the incidence rate of periodontal diseases caused by bacterial plaque is reduced.

Compared with the prior art, the antibacterial peptide and the application thereof have the following advantages:

(1) the antibacterial peptide can be synthesized artificially, is convenient to popularize and use, can achieve 99.9% of bacterial inhibition, and has good biocompatibility.

(2) The antibacterial peptide can inhibit the formation of bacterial biofilms and still has the function of inhibiting the formation of biofilms after 48 hours.

(3) The invention uses natural antibacterial peptide which is not easy to cause drug resistance of human body, prevents and controls the initial attachment bacteria of oral biomembrane formation, inhibits the formation of pathogenic microorganism biomembrane in the window period of tissue recovery after non-drug therapy by interfering the basic condition of biomembrane formation, and achieves the purpose of preventing and controlling infectious diseases.

Drawings

FIG. 1 is a diagram showing the analysis of the physicochemical properties of the antimicrobial peptide GK12 according to the present invention;

FIG. 2 shows the results of the evaluation of the antibiotic membrane resistance of the antimicrobial peptide GK12 according to the present invention;

FIG. 3 shows the experimental results of the antibacterial activity of the antibacterial peptide GK12 according to the present invention;

FIG. 4 shows the CLSM results for the antimicrobial peptide GK12 according to the present invention;

FIG. 5 shows the results of time-lethal evaluation of the antimicrobial peptide GK12 according to the present invention;

FIG. 6 is SEM and TEM images of the effect of antimicrobial peptide GK12 on cells;

FIG. 7 shows the result of cytotoxicity test of antibacterial peptide GK12 on human gingival fibroblast;

FIG. 8 shows that the antibacterial peptide GK12 of the present invention interferes with the expression of key genes for the formation of streptococcal biofilms.

Detailed Description

Unless defined otherwise, technical terms used in the following examples have the same meanings as commonly understood by one of ordinary skill in the art to which the present invention belongs. The test reagents used in the following examples, unless otherwise specified, are all conventional biochemical reagents; the experimental methods are conventional methods unless otherwise specified.

The present invention will be described in detail with reference to examples.

Biofilm (Biofilm): refers to an entity beneficial to the survival of microorganisms, which is widely existed in the natural world, such as ocean, lake, pond, soil, sediment, etc. It is also present in the oral cavity, intestinal tract, bladder and skin of animals and humans, and usually occurs on the surface or interface of the membrane.

Dental Plaque Biofilm (Dental Plaque Biofilm): the bacterial plaque is bacterial plaque which can not be washed away or rinsed away by water in the oral cavity, is soft and unmineralized bacterial colony which is wrapped by matrix and mutually adhered or adhered to the surface of the tooth, the interdental space or the surface of a restoration body, forms more architectural ecological colonies which grow orderly, and is the basis of the survival, metabolism and pathogenesis of oral bacteria.

Antibacterial Peptide (antibacterial Peptide): the polypeptide is host defense polypeptide with broad-spectrum antibacterial property, is produced by different tissues and cells in all forms of life bodies, has the activities of broad-spectrum antibacterial property, antivirus property, antifungal property, antitumor property, promotion of body tissue healing, regulation of in-vivo immune system and the like, and plays an important role in defense barrier in the natural immune defense system of the host.

Designing and chemically synthesizing polypeptide, and synthesizing the required raw materials: c end to N end corresponding to single letter protection amino acid:

Rink-Amide-MBHA-Resin,Fmoc-Lys(Boc)-OH,Fmoc-Lys(Boc)-OH,Fmoc-Arg(pbf)-OH,Fmoc-Arg(pbf)-OH,Fmoc-Cys(trt)-OH,Fmoc-Cys(trt)-OH,Fmoc-Lys(Boc)-OH,Fmoc-Arg(pbf)-OH,Fmoc-Trp(Boc)-OH,Fmoc-Leu-OH,Fmoc-

Leu-OH,Fmoc-Gly-OH；

other reagents were: DMF (dimethylformamide), DCM (dichloromethane), (MeOH) methanol, 1-Hydroxybenzotriazole (HOBT), N-Diisopropylcarbodiimide (DIC), piperidine, ninhydrin detection reagent, TFA (trifluoroacetic acid), anhydrous ether.

The preparation method comprises the following steps:

1. weighing 1g Rink-Amide-MBHA-Resin with the degree of substitution of 0.3mmol/g, putting the Rink-Amide-MBHA-Resin into a reactor, adding DCM for swelling for half an hour, then pumping out DCM,

2. the first amino acid in the sequence, Fmoc-Lys (Boc) -OH (0.6mmol equiv.) HOBT, DIC (both 0.6mmol) in DMF (appropriate amounts are sufficient to allow the resin to fully swell) was added and the reaction was bubbled with nitrogen for one hour and washed with DMF, MeOH, DCM (three times each for 9 cycles). And (4) drying, detecting by using a ninhydrin color reagent, judging whether the reaction is complete, and indicating that the reaction is complete if the reaction is not blue.

3. The Fmoc (9-fluorenylmethyloxycarbonyl) protecting group was removed, 8-10ml of 20% by volume piperidine in DMF was added and the reaction mixture was reacted for 20 minutes, washed with DMF, MeOH, DCM (three times in 9 cycles each) and the ninhydrin detection should appear blue.

4. The second amino acid in the sequence, Fmoc-Lys (Boc) -OH (0.6mmol), 0.6mmol HOBT and DIC, were added to the reactor, and the reaction was bubbled with nitrogen for one hour, washed with DMF, MeOH and DCM (three times for 9 times), dried, and detected with ninhydrin reagent to determine if the reaction was complete, and no blue color appeared indicating complete reaction. The Fmoc (9-fluorenylmethyloxycarbonyl) protecting group was removed, 8-10ml of 20% piperidine in DMF was added and the reaction mixture was reacted for 20 minutes, washed with DMF, MeOH, DCM (three times in 9 cycles each) and the ninhydrin detection should be blue. (Pro takes off Fmoc to show reddish brown)

5. And (4) sequentially adding amino acids in the sequence in the modes of steps 3 and 4 until the reaction of the A (Fmoc-Gly-OH) is finished finally.

6. Adding 10ml of cracking reagent (V/V%, trifluoroacetic acid: p-cresol: water: Tis: mercaptan 82.5:5:5: 2.5) to crack at room temperature for 2-3 hours, pouring the cracking solution into pre-cooled 25ml of anhydrous ether, stirring, standing, centrifuging, washing with anhydrous ether for 4 times, and drying in vacuum to obtain crude polypeptide GLLWRKCCRRKK-NH 2. The purity of the product is more than 95% by high performance liquid chromatography detection and mass spectrometry.

7. Basic physicochemical properties of the polypeptides were analyzed using protein-related analysis software and peptide property calculator https:// pepcalc.com/ppc.php.

The polypeptide synthesized by the invention has the characteristics of positive charge, hydrophilicity, hydrophobicity and amphipathy, and has good solubility.

The following antibacterial experiments were performed on the synthesized polypeptide GLLWRKCCRRKK-NH 2:

first, Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) experiments

Streptococcus oralis is cultured in aerobic environment at 37 deg.C for 24 hr, and Streptococcus sanguis is cultured in facultative anaerobic environment at 37 deg.C for 48 hr on brain heart infusion agar plate containing 5 vol% sterile defibrinated sheep blood, 1 wt% hemagglutinin and 0.1 wt% menadione.

Three polypeptides were analyzed for initial attachment of Streptococcus oralis (S.oralis), Streptococcus gordonii (S.gordonii) and Streptococcus sanguis according to the American laboratory Standard (NCCLS M7-A3) broth microdilution methodMinimum inhibitory concentration of bacteria (s.sanguinis). The three streptococci were diluted to 5X 10 with the appropriate liquid medium⁵CFU/mL bacterial suspension, using PBS solution to dilute three polypeptides into polypeptide solutions with different concentrations, mixing 100 μ L of antibacterial peptide solutions with different concentrations (1/2MIC, MIC and 2MIC) with the same volume of bacterial suspension (experimental group), mixing the same volume of PBS solution with the same volume of bacterial suspension (blank control group), inoculating into 96-well plate, each group has three multiple wells, and culturing under the aerobic and facultative anaerobic conditions at 37 ℃ for 24 hours and 48 hours. The minimum inhibitory concentration of the chimeric peptide refers to the concentration of the chimeric peptide at which no bacterial growth and no change in Optical Density (OD) was observed in the right eye. All experiments were repeated three times.

The minimum inhibitory concentration of the chimeric peptide is taken as the basis, different concentrations of the chimeric peptide which is more than or equal to MIC and streptococcus are prepared for culturing for 24 hours, 20 mu L of empty sample liquid is taken to be coated on a BHI agar plate, and the sample liquid is cultured in an incubator at 37 ℃ for 5 days, and the number of colonies is counted. The minimum bactericidal concentration of the chimeric peptide is the concentration of the chimeric peptide at which 99.9% of the bacteria are inhibited. All experiments were repeated three times.

According to the experimental results of MIC and MBC, the antibacterial peptide synthesized by the invention has antibacterial property on all three streptococci.

TABLE 1 MIC and MBC of GK12 against Streptococcus

Second, biofilm susceptibility test

The biofilm susceptibility test is determined by crystal violet microtiter analysis, and the concentration of the antibacterial peptide used in the test is 312.5 mu g/mL. Bacterial suspension concentrations of s.oralis, s.gordonii and s.sanguinis were configured as above. Grouping experiments: (1) blank control group: bacterial suspension + PBS solution (2) experimental group: bacterial suspension + antimicrobial peptide. 100 μ L of bacterial suspensions of S.oralis, S.gordonii and S.sanguinis were inoculated into 96-well plates containing 100 μ L of PBS solution and 100 μ L of a chimeric peptide solution at a concentration of 312.5 μ g/mL, each set having three replicate wells; then incubated in an aerobic and anaerobic incubator at 37 ℃ for 24 hours and 72 hours, and the supernatant from each well was removed with a sterile pipette; washing each hole twice by using sterile deionized water, and washing off bacteria which are not adhered in each hole; drying for 20 minutes at room temperature; the biofilm formed in each well plate was fixed with 95% by volume methanol, stained with 0.5% by volume crystal violet for 15 minutes and destained with 95% by volume ethanol, and finally the Optical Density (OD) value at absorbance of 600nm was measured with a microplate reader. All experiments were repeated three times. The results are shown in FIG. 2.

The antibacterial peptide synthesized by the invention can inhibit the formation of bacterial biofilms and still has the function of inhibiting the formation of biofilms after 48 hours.

Third, antibacterial rate experiment

Diluting the bacterial culture solution to 5 × 10⁶CFU/mL. Placing the sterile coverslip in a sterile 24-well plate, mixing the bacterial suspension with GK12(MIC concentration), and incubating in an incubator for 24 hours; calculating the number of bacteria adhered to the surface of the sterile cover glass; the antibacterial rate is calculated by the following formula on the adhesion of bacteria: r ═ B-a)/B × 100%. Here, A is the average viable cell count on the treated group sterile cover glass, and B is the average viable cell count on the treated group sterile cover glass. All experiments were repeated three times. The results are shown in FIG. 3.

Four, laser confocal scanning microscope experiment

In a 24-well microtiter plate, a sterile glass cover slip was placed in each well, and 300. mu.L of bacteria and 900. mu.L of BHI broth were transferred to each well to form a biofilm. The culture was carried out at 37 ℃ for 24 hours. After incubation, the medium and unattached bacterial cells were washed out of the culture wells, added to saliva or serum GK12 (312.5. mu.g/mL), and incubated at 37 ℃ for 24 hours. Each group consisted of three parallel experimental samples, which were repeated three times. The AO/EB staining solution was mixed with the sample and allowed to stand at room temperature for 15 minutes. Green represents live bacteria, red represents dead bacteria, compared with a control group, the bacteria in the experimental group are obviously changed into red, and GK-12 plays an obvious bacteriostatic action.

Five, time-lethal experiment

GK12 was added to bacterial cultures with final peptide concentrations of 1-fold and 2-fold MBC, followed by anaerobic culture at 37 ℃. Sterile deionized water was used as a negative control. At 0min, 1min, 2min, 5min, 15min, 30min, 60min, 120min, 240min, 24 h, 48h, an aliquot of the suspension (10.0 μ L) was withdrawn and continuously diluted 10-fold. Aliquots of the dilutions (10.0. mu.L) were seeded on BHI agar and incubated anaerobically at 37 ℃ for 48 h. Each set consisted of three parallel experimental samples, and lg (CFU/mL) was plotted versus incubation time over 48 hours. The results of the experiment are shown in FIG. 5.

Sixthly, scanning electron microscope experiment

Established biofilms on sterile coverslips were placed in 12-well plates. Then fixed with glutaraldehyde at a volume concentration of 2.5%, washed with PBS, and gradually dehydrated with ethanol. The treated sample was subjected to gold sputtering, and an image was obtained using a scanning electron microscope (Jeol JSM-6310LV, Tokyo, Japan; Jeol Co., Ltd.). Each sample was scanned in three different regions at a time and the experiment was repeated three times.

Experiment of transmission electron microscope

After co-culturing or non-culturing GK12 with the bacteria (control group), the bacteria were fixed for 4 hours by using glutaraldehyde solution with a volume concentration of 2.5%; the samples were washed twice with sterile PBS solution and fixed for 2 hours with osmic acid at a concentration of 0.1% by volume; washing a sample with sterile PBS solution for three times, and dehydrating with ethanol solutions with volume concentrations of 50%, 70%, 85%, 90% and 100% for 15 minutes in sequence; the sample was cut into extremely thin slices, stained with uranyl acetate at a volume concentration of 2% and citrate at a volume concentration of 0.2%, and the cell structure was observed by Transmission Electron Microscopy (TEM).

The time results of SEM and TEM are shown in fig. 6.

Eighth, cytotoxicity test

In order to evaluate the biocompatibility of GK12, human gingival fibroblasts are inoculated on the surface of a sample modified by GK12, and a cell proliferation activity detection kit-CCK 8 is used for detecting the cell proliferation condition, so that the biocompatibility of the polypeptide is characterized. After the patient agrees to remove the gingiva, Human Gingival Fibroblast (HGF) cells were cultured by tissue block adherence. Fetal bovine serum (Thermo Fisher Scientific, Waltham, MA, USA) at a volume concentration of 10%, streptomycin (Gibco) at 100mg/mL and penicillin (Sigma) at 100U/mL were cultured in DMEM. MC3T3-E1 cells were mixed with GK12 at 37 ℃. Cytotoxicity assays were measured at different time points (1 day, 2 days, 3 days, 4 days) with cell count KIT-8(CCK 8; DojdIO laboratory, pandan, japan). Six replicates were obtained for each sample and the experiment was repeated three times. The results are shown in FIG. 7.

Nine, RT-PCR

Total RNA from s.gordonii cells was extracted with Trizol reagent and then reverse transcribed to 2 μ L RNA to synthesize first strand cDNA according to the instructions. The cDNA was amplified in a final volume of 20. mu.L, and 45 cycles of amplification were performed in a DNA thermal cycler. The pre-denaturation step was 95 ℃ for 15s, the reaction step was 95 ℃ for 10s, 60 ℃ for 20s, and 72 ℃ for 30s, and the sequences of the differentiation-labeled primers are shown in Table 2. After amplification, the CT value of each target gene in each sample was obtained using GAPDH as an internal reference gene and the sample No. 1 of the control group as a standard. Data were analyzed using relatively quantitative Δ CT. Relative quantitative values (RQ values) were obtained for all target genes according to the formula RQ ═ 2- Δ Δ CT, and the results for expression of key genes interfering with streptococcal biofilm formation for GK12 are shown in fig. 8.

TABLE 2

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.

Claims

1. An antimicrobial peptide, characterized by: the sequence of the antibacterial peptide is Gly-Leu-Leu-Trp-Arg-Lys-Cys-Cys-Arg-Arg-Lys-Lys.

2. Use of the antimicrobial peptide according to claim 1 for the manufacture of a medicament for inhibiting streptococcus or for inhibiting biofilm formation in streptococcus.

3. Use according to claim 2, characterized in that: the streptococcus is one or more than two of streptococcus oralis, streptococcus gerbera and streptococcus sanguis.

4. Use of the antimicrobial peptide of claim 1 for the preparation of a medicament or care product for the prevention and control of chronic periodontitis.