CN116059311A - Application of antibacterial peptide in preparation of medicines for resisting staphylococcus aureus infection - Google Patents

Abstract

The invention provides application of an antibacterial peptide in preparation of a medicine for resisting staphylococcus aureus infection. In a pneumonitis mouse model infected by staphylococcus aureus, the treatment effect of GHb3K and GHbK4R is better than that of vancomycin, the inflammatory reaction can be obviously reduced, and the in vivo toxicity is lower than that of vancomycin. GHb3K and GHbR can remarkably promote healing of methicillin-resistant staphylococcus aureus MRSA chronic infectious wound surface, and the treatment effect is better than that of mupirocin ointment. The antibacterial peptide provided by the invention has the potential of being developed into a medicine for treating pneumonia caused by staphylococcus aureus infection and a medicine for treating chronic wound surface caused by MRSA infection.

Description

Application of antibacterial peptide in preparation of medicines for resisting staphylococcus aureus infection

Technical Field

The invention belongs to the field of biological medicine, and relates to application of an antibacterial peptide in preparation of a medicine for resisting staphylococcus aureus infection.

Background

With the continuous development of bacterial resistance to traditional antibiotics, the demand for novel antibacterial drugs is increasingly urgent. Antibacterial peptides are an important component of the natural immune system of organisms and often have broad-spectrum antibacterial effects. Most cationic antimicrobial peptides tend to form amphiphilic structures, selectively bind to microbial cell membranes by electrostatic adsorption, attach to or intercalate into cytoplasmic membranes, disrupt the membrane's intact structure, and cause leakage of the contents, thereby effectively killing pathogenic microorganisms. The specific mechanism of action of antimicrobial peptides, compared to traditional antibiotics, makes it difficult for bacteria to develop resistance to them.

Staphylococcus aureus (Staphylococcus aureus, s.aureus), also known as "staphylococcus aureus", is widely found in natural environments, and is distributed on the skin and mucosal surfaces of about 60% of adults, one of the most common pathogens from hospitals to communities, and can cause bacterial pneumonia, skin wound infections, etc. (Sakr, a.; bregeon, f.; mege, j.l.; et al, staphylococcus aureus Nasal Colonization: an Update on Mechanisms, epidemic, risk Factors, and subset information. Community-acquired staphylococcus aureus infection pneumonia is a major cause of death in children, and also commonly occurs in elderly people over 80 years old (Self, w.h.; wunderink, r.g.;

williams, d.j.; staphylococcus aureus Community-acquired Pneumonia:prevente, clinical Characteristics, and Outcomes Clin INfectdis, 2016,63 (3): 300-309.). Worse, the advent of methicillin-resistant staphylococcus aureus (MRSA) has further increased the difficulty of effective treatment of staphylococcus aureus infectious diseases. The staphylococcus aureus can be adhered to the surfaces of various medical instruments and implanted devices, such as catheters, artificial heart valves, central venous catheters, cardiac pacemakers and the like, and can form a biological film in body tissues (Otto, m.staphylococcus biofilms. Microbiol spectrum 2018,6 (4): 10.1128/microbiolspec. Gppp 3-0023-2018.). Bacteria in the biofilm are not metabolized actively to form antibiotic-resistant persisting bacteria, so that biofilm infection is extremely difficult to treat, chronic or recurrent infection is often caused, long-term antibiotic treatment is usually required or an implantation device is removed, a heavy burden is brought to patients, families and society, and the problem of bacterial drug resistance is further serious. In 2017, the World Health Organization (WHO) issued a list of bacteria that were in urgent need of new antibiotics, including MRSA, which was the leading cause of death in patients due to antibiotic resistant bacteria infection (tacsonelli, e.; carrara, e.; savoldi, a.; et al, WHO Pathogens Priority List Working group.discovery, research, and development of new antibiotics: the WHO priority list of antibiotic-resistant bacteria and, the laboratory diseases,2018,18 (3): 318-327).

Therefore, the development of a novel antibacterial agent which can rapidly sterilize and remove bacterial biofilm has important significance for coping with antibiotic drug resistance crisis.

Disclosure of Invention

Based on the technical defects, the invention provides application of an antibacterial peptide in preparing a medicine for resisting staphylococcus aureus infection.

In order to achieve the technical purpose of the invention, the invention adopts the following technical scheme:

the application of the antibacterial peptide in preparing a medicament for resisting staphylococcus aureus infection is provided, wherein the amino acid sequence of the antibacterial peptide is shown as SEQ ID NO.1, SEQ ID NO.2 or SEQ ID NO. 3.

Preferably, the staphylococcus aureus comprises methicillin-resistant staphylococcus aureus.

Preferably, the antimicrobial peptide is synthesized using N-9-fluorenylmethoxycarbonyl (Fmoc) solid phase synthesis.

More preferably, the C-terminal of the antibacterial peptide is subjected to amidation modification, and the purity of the antibacterial peptide is more than or equal to 95 percent by reversed-phase high performance liquid chromatography purification.

Preferably, the anti-staphylococcus aureus drug comprises a drug for treating staphylococcus aureus infection pneumonia and a drug for treating methicillin-resistant staphylococcus aureus infection chronic wound surfaces.

The invention has the beneficial effects that:

the invention designs and synthesizes a series of antibacterial peptides, including GHbR (the amino acid sequence is shown as SEQ ID NO. 1), GHb3K (the amino acid sequence is shown as SEQ ID NO. 2) and GHbK4R (the amino acid sequence is shown as SEQ ID NO. 3), and researches the anti-biofilm activity, the antibacterial activity and the mechanism of the antibacterial peptides on staphylococcus aureus and the in vivo anti-infection effect. In a pneumonitis mouse model infected by staphylococcus aureus, the treatment effect of GHb3K and GHbK4R is better than that of vancomycin, the inflammatory reaction can be obviously reduced, and the in vivo toxicity is lower than that of vancomycin. GHb3K and GHbR can remarkably promote healing of methicillin-resistant staphylococcus aureus MRSA chronic infectious wound surface, and the treatment effect is better than that of mupirocin ointment. Among other things, GHb3K also has the following outstanding advantages: the cytotoxicity is small; the molecule is small, the absorption is easy, and the anaphylaxis is low; high stability, long-term activity at normal temperature and easy storage; simple preparation process, low production cost, etc. Has the potential of being developed into a medicine for treating the pneumonia of the staphylococcus aureus infection and a medicine for treating the chronic wound surface of the MRSA infection.

Drawings

FIGS. 1 a-1 c show the bactericidal kinetics of the antibacterial peptides of the invention against Staphylococcus aureus.

FIG. 2 shows the induction of resistance of vancomycin to Staphylococcus aureus by the antibacterial peptide of the present invention.

FIG. 3a shows the ability of the antimicrobial peptides of the invention to inhibit biofilm formation; FIG. 3b shows the ability of the antimicrobial peptides of the invention to disrupt mature biofilms.

FIGS. 4 a-4 c show the effect of the antimicrobial peptides of the invention on the permeability of the membrane of the Staphylococcus aureus cell.

FIG. 5 shows a transmission electron micrograph of a treatment of Staphylococcus aureus with 12.5. Mu.M antimicrobial peptide for 1h.

FIGS. 6 a-6 c show cytotoxicity of the antimicrobial peptides of the invention.

FIGS. 7a and 7b show the bacterial load in organs of mice infected with Staphylococcus aureus after treatment with the antibacterial peptide of the present invention and vancomycin.

FIG. 8 shows IL-1 beta, TNF-alpha, MPO and MIP cytokine levels of pneumonitis mice lung and liver infected with Staphylococcus aureus after vancomycin treatment with the antibacterial peptides of the present invention.

Fig. 9a and 9b show changes in body weight of healthy mice injected with physiological saline, GHb3K, GHbK R and vancomycin.

Fig. 10a and 10b show that GHbR and GHb3K promote healing of MRSA infected wound in mice.

Fig. 11a and 11b show the bacterial load of wound mice infected with MRSA following GHbR and GHb3K treatment.

Fig. 12 shows the effect of GHbR and GHb3K treatment on inflammatory cytokine production from MRSA infected wound mice wounds.

Detailed Description

In order to more clearly illustrate the present invention, the present invention will be described in further detail below with reference to examples and with reference to the accompanying drawings. It is to be understood by persons skilled in the art that the following detailed description is illustrative and not restrictive, and that this invention is not limited to the details given herein.

Examples

Staphylococcus aureus (ATCC 25923) and methicillin-resistant staphylococcus aureus (ATCC 43300) used in the present invention were purchased from the guangdong collection of microorganism strains. Other clinically isolated methicillin-resistant staphylococcus aureus strains (MRSA 1-MRSA 4) were given the teaching of university of chinese medicine, guangxi Yang Ke, and have been identified by biochemical methods and 16s rDNA sequencing (GenBank database, accession No. OP 824645-824648).

Temporin-GHb (GHb) is an antibacterial peptide cloned from the skin of a biogas frog, but has low anti-staphylococcus activity. By comparing 110 identical family antibacterial peptide sequences, the frequency of occurrence of basic amino acid lysine (Lys, K) is found to be higher, 6.97%, arginine (Arg, R) and histidine (His, H) are relatively lower, 1.14% and 0.9%, respectively. Whereas in the GHb amino acid sequence, the basic amino acid is H. Different basic amino acids have different effects on the antibacterial activity and cytotoxicity of the antibacterial peptide. We have found that increasing R in antimicrobial peptides, while enhancing antimicrobial activity, tends to result in increased cytotoxicity. Therefore, in the modification of GHB, besides adopting R to replace H, K is also utilized to replace H, and R and K are simultaneously replaced, so that the antibacterial peptide with good activity and low toxicity is obtained.

Furthermore, the following 3 antibacterial peptides, GHbR, GHb3K and GHbK4R, were designed and synthesized by using GHb as a template and combining the analysis results of the antibacterial peptide big data. They consist of 13 amino acids, carrying 1-3 positive charges under physiological conditions, the amino acid sequences being respectively:

GHbR：FIRRIIGALGRLF-NH ₂ (SEQ ID NO.1)

GHb3K：FIKHIIGALGHLF-NH ₂ (SEQ ID NO.2)

GHbK4R：FIKRIIGALGKLF-NH ₂ (SEQ ID NO.3)

the antibacterial peptide is synthesized by adopting an N-9-fluorenylmethoxycarbonyl (Fmoc) solid-phase synthesis method. A liquid chromatograph (LC 6000) was prepared for the preparation and a high performance liquid chromatograph (LC 3000) was used for the analysis. Amidation modification of the C-terminal of the peptide, reversed phase high performance liquid chromatography purification, purity not less than 95%, and identification of the antibacterial peptide by electrospray mass spectrometry. The present invention is not further limited.

Next, the anti-biofilm activity, antibacterial activity and mechanism and in vivo anti-infective effect of these 3 antibacterial peptides on staphylococcus aureus were studied.

Test case

1. Antibacterial experiments

The minimum inhibitory concentration (Minimum inhibitory concentration, MIC) and minimum bactericidal concentration (Minimum bactericidal concentration, MBC) of the antimicrobial peptides were tested using a double broth microdilution method. Inoculating single colony into Tryptone Soybean Broth (TSB), culturing at 37deg.C for 8-12 hr, and diluting bacteria grown to logarithmic phase to 1×10 ⁶ CFU/mL. The antibacterial peptide is serially diluted into different concentrations in a 96-well plate, and an equal volume of bacterial suspension is added for incubation at 37 ℃ for 18-24h. Wells without antimicrobial peptide served as negative control wells and vancomycin (vancomycin) served as positive control. The concentration of the antimicrobial peptide corresponding to the well without bacterial growth observed with the naked eye was defined as MIC. When MBC was determined, 10. Mu.L of bacterial suspension was taken from the corresponding MIC wells on a 96-well plate for MIC or above and plated on TSB agar plates (TSA) and incubated at 37℃for 24h. The concentration of antimicrobial peptide that did not see bacterial growth was MBC.

As shown in Table 1, the antibacterial activity of GHB was weak, and the other 3 antibacterial peptides exhibited extremely strong antibacterial activity, and MIC was 3.1. Mu.M for both the staphylococcus aureus and MRSA standard strain. They also show activity against clinically isolated resistant bacteria MRSA1-MRSA4 with MIC of 1.6-6.2. Mu.M and MBC of 3.1-25. Mu.M.

TABLE 1 MIC of antibacterial peptides for Staphylococcus aureus and methicillin-resistant Staphylococcus aureus

Note that: SA is Staphylococcus aureus (ATCC 25923); MRSA is methicillin-resistant staphylococcus aureus (ATCC 43300); MRSA1-MRSA4 is a clinically isolated methicillin-resistant Staphylococcus aureus.

2. Determination of sterilization kinetics

Continuously diluting antibacterial peptide in EP tube in two-fold gradient, adding equal volume of diluted bacterial solution to make final bacterial concentration be 1×10 ⁶ CFU/mL, final peptide concentration was 1/2 XMIC, 1 XMIC, 2 XMIC, 4 XMIC, bacterial solution without peptide treatment served as negative Control (Control). Incubating at 37 ℃, taking out a proper amount of bacterial liquid after 15, 30, 60, 90, 120 and 180min respectively, diluting to a proper concentration by using TSB liquid culture medium, and coating 50 mu L of the bacterial liquid on a TSA plate. Incubation was carried out at 37℃for 24 hours, single colonies were counted, and a sterilization curve was drawn.

The results of the sterilization curves for staphylococcus aureus show (fig. 1 a-1 c), that the sterilization effect of GHbR and GHbK4R is most obvious, and that bacteria are killed at a concentration of 2×mic for 15min, and that GHb3K is killed at a concentration of 4×mic for 60 min.

3. In vitro drug resistance induction experiment

The staphylococcus aureus suspension is inoculated in fresh TSB liquid culture medium containing 1/2MIC antibacterial peptide, and cultured for 24 hours at 37 ℃. The bacterial suspension from the previous day was inoculated with an appropriate amount of fresh TSB liquid medium containing 1/2MIC antimicrobial peptide again daily for continued culture, with the TSB liquid medium treated Staphylococcus aureus as negative control and Vancomycin (Vancomycin) as positive control. The operation was repeated continuously for 50 days. The MIC of the antibacterial peptide against staphylococcus aureus was determined every 5 days.

The results showed that the MIC value of the antibacterial peptide against staphylococcus aureus remained stable at 3.1 μm after continuous passage for 50 passages with continuous exposure to the antibacterial peptide at sub-MIC concentration (fig. 2). However, the staphylococcus aureus can quickly generate drug resistance to vancomycin, and the MIC value of the staphylococcus aureus is increased to 32 times on the 45 th day. The results show that the staphylococcus aureus is difficult to generate drug resistance to GHbR and GHb3K, GHbK R.

4. Stability study

The effect of different salts, temperature, pH and storage time on the antimicrobial activity of the antimicrobial peptides was evaluated. The antibacterial peptide is prepared with salt (3.0 mM, 4.5mM, 6.0mM KCl,0.5mM, 1.0mM, 1.5mM MgCl) with different concentration ₂ 0.05M, 0.15M, 0.3M NaCl and 2.0mM, 2.5mM, 3.0mM CaCl ₂ ) Is dissolved in 2mM and incubated at 37℃for 24h.

The antibacterial peptide (2 mM) solutions were treated at different temperatures (25 ℃, 40 ℃, 70 ℃ and 100 ℃) for 30min, respectively.

The antimicrobial peptide solutions were incubated in solutions at pH 7 and 9, respectively, for 30min. The antibacterial peptide solution was stored at 25 ℃, 4 ℃ and-20 ℃ for 30 days, 60 days and 90 days, respectively. The MIC of the antimicrobial peptide after treatment was determined as described above.

The effect of storage temperature and time on antimicrobial activity of the antimicrobial peptides (Table 2) shows that the three antimicrobial peptides maintained good stability over 90 days with no change in MIC compared to the untreated group when stored at 25 ℃, 4 ℃ and-20 ℃ for 30 days, 60 days and 90 days, respectively. After 30min of treatment of the antimicrobial peptide at 25-100 ℃, the MIC was still 3.1. Mu.M. The antimicrobial peptides remained well stable at pH 7-9 (Table 3).

TABLE 2 influence of storage time and temperature on antibacterial activity of antibacterial peptides

TABLE 3 influence of temperature and pH on antibacterial Activity of antibacterial peptides

TABLE 4 influence of cations on the antibacterial activity of antibacterial peptides

In addition, the antibacterial activity of the antibacterial peptide was not adversely affected in the presence of the cation, and the antibacterial activity of GHbR was promoted (table 4).

The results show that 3 antibacterial peptides have excellent stability and can be kept stable under different storage conditions and body fluid environments. 5. Inhibition of Staphylococcus aureus biofilm formation by antibacterial peptide and action of destroying mature biofilm

The antibacterial peptide anti-Staphylococcus aureus biofilm effect was determined using 3- (4, 5-dimethylthiazole-2) -2, 5-diphenyltetrazolium ammonium bromide (MTT). The antibacterial peptide was serially diluted twice (final concentration 0.4-3.1. Mu.M) to 100. Mu.L in 96-well plates, and an equal volume of bacterial liquid (final concentration 1X 10) was added ⁶ CFU/mL), cultured at 37℃for 24 hours. The supernatant was discarded, washed 3 times with Phosphate Buffer (PBS), air-dried at room temperature, 95. Mu.L of PBS and 5. Mu.L of MTT (5 mg/mL) were added to each well, incubated at 37℃for 2-4h in the absence of light, and the supernatant was discarded. Blue-violet crystalline formazan was dissolved in 150. Mu.L of dimethyl sulfoxide (DMSO), incubated at room temperature in the dark for 20min, and absorbance at 570nm was measured (Multiskan Spectrum, bioTek, winooski, VT, USA). When the clearance of the antibacterial peptide to the mature biofilm of Staphylococcus aureus was measured, 200. Mu.L of bacteria was inoculated into a 96-well plate (1X 10) ⁶ CFU/mL), incubation at 37 ℃ for 24h, washing with pbs 3 times, air-drying at room temperature, adding 200 μl of 3.1-25 μΜ antimicrobial peptide, incubation at 37 ℃ for 24h, and the remaining steps were the same as above, with bacterial liquid not treated with antimicrobial peptide as negative Control (Control).

The results show that the antibacterial peptide has the capacity of inhibiting the formation of a biofilm by staphylococcus aureus and removing a mature biofilm, and has concentration dependence. GHbR has the ability to inhibit biofilm formation by 87% at 3.1 μm, and GHb3K and GHbK4R inhibit biofilm formation by about 65% at the same concentration (fig. 3 a). The antimicrobial peptides also have activity in clearing mature biofilms. GHbR and GHbK4R have the ability to clear 80% of the biofilm at 25. Mu.M and GHb3K has the ability to clear 70% of the biofilm at 25. Mu.M (FIG. 3 b). While GHb is 25 μm, the inhibition and clearance are only about 20%.

Thus, the other 3 antimicrobial peptides showed a stronger ability to inhibit the formation of the staphylococcus aureus biofilm and to clear the mature biofilm compared to GHb.

6. PI permeation experiments to detect the effect of antibacterial peptides on bacterial cell membranes

The membrane penetration capacity of the antibacterial peptide against staphylococcus aureus was evaluated using an Propidium Iodide (PI) uptake assay. When the cell membrane is damaged, PI permeates into the cell to combine with DNA, PI emits strong fluorescence, which indicates that the integrity of the bacterial cell membrane is destroyed and the permeability is increased.

Antibacterial peptide (100. Mu.L, 3.2-25. Mu.M) was double diluted in 96-well plates, and the staphylococcus aureus was washed 3 times with PBS and diluted, 92. Mu.L of bacterial liquid (final concentration 1X 10) was added ⁸ CFU/mL) was mixed with 8. Mu.L PI (final concentration 20. Mu.M). The measurement was performed using a multifunctional microplate reader at an excitation wavelength of 560nm and an emission wavelength of 620nm (Spark, tecan,

switzerland). The procedure was set, the temperature was 37℃and the measurement was carried out every 5 minutes for 2 hours, and the bacterial liquid which had not been treated with the antimicrobial peptide was used as a negative Control (Control).

As shown in fig. 4 a-4 c, the GHbR, GHb3K and GHbK4R fluorescence values increased in a concentration-dependent manner as the peptide concentration increased upon incubation of the staphylococcus aureus with the antimicrobial peptide. Maximum fluorescence values were reached at 5min when staphylococcus aureus was treated with 12.5 μm of GHbR, GHb3K and GHbK4R. The results show that GHbR, GHb3K and GHbK4R exert an antibacterial effect by increasing the permeability of the bacterial cell membrane and destroying the bacterial cell membrane.

7. Observation of bacterial morphology treated with antimicrobial peptides by transmission electron microscopy

Centrifuging the logarithmic phase of Staphylococcus aureus at 5000rpm for 5min, discarding supernatant, washing with PBS, and re-suspending, mixing antibacterial peptide (final concentration of 12.5 μm) with equal volume of bacterial liquid (final concentration of 1×10) ⁹ CFU/mL), with no peptide added as Control, incubated for 1h at 37 ℃. Centrifuging at 8000rpm for 5min, discarding supernatant, washing with PBS, fixing with 2.5% glutaraldehyde for 4h, centrifuging, washing with PBS for 3 times, sequentially gradient dehydrating with 30%, 50%, 70%, 100% ethanol for 10min, resuspending bacteria with 80 μl of absolute ethanol, dripping 10 μl of bacteria onto copper mesh, staining with 2% phosphomolybdic acid, and observing with transmission electron microscope.

As shown in FIG. 5, the Control bacterial cells were intact and the cells were in a regular circle. The staphylococcus aureus shapes treated with 12.5 μm GHbR, GHb3K and GHbK4R were significantly changed, cells shrunken, cell membranes ruptured, and contents leaked. It was further confirmed that the antibacterial peptide was dead by targeting the bacterial cell membrane, disrupting the cell membrane, and leaking the contents.

8. Cytotoxicity of cells

Human alveolar epithelial cells (A549), human breast cancer cells (MCF-7) and human keratinocytes (HaCaT) were cultured in high-sugar DMEM (containing 1% penicillin and streptomycin) with 10% Fetal Bovine Serum (FBS), at 37deg.C with 5% CO ₂ Culturing in an incubator. Cell coverage reaches 80% -90%, pancreatin digestion is used, and the cells are diluted to 1X 10 by using culture medium ⁵ cell/mL, 100. Mu.L per well in 96-well plates, was added and incubated for 24h. The original medium was discarded, 3.1-200. Mu.M of peptide diluted with DMEM containing 2% FBS was added to each well, cells not treated with peptide were used as Control wells, and blank wells without peptide and cells were used. The culture was continued for 24 hours, the supernatant was discarded, PBS was washed 3 times, 100. Mu.L of medium and 10. Mu.L of CCK8 solution were added, and the incubation was continued for 2-4 hours. Absorbance was measured at 450 nm.

The cytotoxicity of the antimicrobial peptides is shown in FIGS. 6 a-6 c. At 200. Mu.M concentration, GHB3K was not cytotoxic to HaCaT, MCF-7 and A549 cells. GHbR and GHbK4R have a certain toxicity to cells, half-maximal inhibitory concentrations (IC 50) for HaCaT cells of 15.4 and 18.5, respectively, and IC50 for A549 of 13.6 and 30.4. Mu.M, respectively. The result shows that GHb3K in the 3 antibacterial peptides has the characteristic of low toxicity.

9. Construction and treatment of staphylococcus aureus infected mouse pneumonia model

Six (6) to eight (8) week old, 72 healthy male BALB/cJ (20-24 g) mice were randomly divided into 9 groups of 8 mice each, with 1 group of healthy mice as a blank group (Control). After the rest 8 groups of mice were anesthetized with pentobarbital, they were intranasal inoculated with 30. Mu.L of 5X 10 ⁹ CFU/mL, mice were kept standing upside down for 30 seconds, and after 2h of infection, GHb3K or GHbK4R (5, 10, 15 mg/Kg) was intraperitoneally injected with vancomycin as a positive control (10 mg/Kg). Model and Control mice were intraperitoneally injected with 100 μl of saline, respectively. 1 time every 8h, 3 times after administration, the mice were sacrificed by anesthesia, blood collection from the eyeballs, and viscera (lung, heart, liver, kidney, spleen) were collected and recordedAnd (5) bacterial load.

After 2h of intranasal instillation of bacteria, model lung tissue became large and Control lung tissue did not change. HE staining results indicated that the pulmonary tissue of model mice had alveolar interstitial congestion and edema, pulmonary vasodilation, and a large number of inflammatory cell infiltrates. The number of bacteria in lung tissue of mice in model group was counted on mannitol sodium chloride agar plate to be more than 10 ⁶ CFU/g, indicated that the mice pneumonia model was successfully established.

The number of bacteria in the lung tissue of mice was counted after treatment (fig. 7a and 7 b), and the number of bacteria load in the lung tissue of the treated group was reduced compared to the Model group. Compared with Vancomycin (Vancomycin) treatment groups, the antibacterial peptide low-dose group is equivalent to Vancomycin, and the bacterial load of the medium-dose group and the high-dose group is obviously lower than that of the Vancomycin group. The staphylococcus aureus in heart, liver, spleen, lung and kidney is reduced by 16%, 21%, 32.6%, 23% and 15% respectively after vancomycin treatment. The number of staphylococcus aureus in the tissues after GHb3K and GHbK4R treatment was reduced in a concentration-dependent manner. In the GHb3K high dose group, the staphylococcus aureus was reduced by 48%, 20%, 50%, 49% and 15%, respectively; GHbK4R high dose groups were reduced by 31%, 20%, 43%, 46.5% and 46%, respectively. Vancomycin, GHb3K and GHbK4R high dose groups, the load of the lung bacteria of the mice is respectively reduced to 7.8X10 ⁷ 、5.3×10 ⁵ And 5.4X10 ⁵ CFU/g. Thus, the antimicrobial peptides GHb3K and GHbK4R are more effective than vancomycin in treating bacterial pneumonia in mice caused by staphylococcus aureus.

Lung and liver tissues were taken and assayed for effects on cytokines IL-1 beta, TNF-alpha, MIP-2, MPO using ELISA kit (fig. 8). The results show that the IL-1 beta, TNF-alpha, MIP-2 and MPO levels in lung and liver tissues are significantly increased after infection with the Staphylococcus aureus, and the levels of these inflammatory factors are significantly reduced after the antibacterial peptide treatment.

10. In vivo toxicity test of antibacterial peptide

To evaluate the in vivo toxicity of GHb3K and GHbK4R, healthy BALB/cJ mice were divided into 4 groups, and physiological saline (Control), 15mg/kg GHb3K,15mg/kg GHbK4R,10mg/kg Vancomycin (Vancomycin) were injected, respectively. The mice weights were recorded daily for 8 days of intraperitoneal injection of the drug, and no significant differences in weight were found between the 4 groups of mice (fig. 9a and 9 b). After 8 days of intraperitoneal injection of the drug, the blood index of mice, including alanine Aminotransferase (ALT), aspartate Aminotransferase (AST), alkaline phosphatase (ALP), was measured with no significant differences between the different groups. Indicating that GHb3K and GHbK4R have no significant hepatotoxicity. The pathological section of the mice was evaluated, and it was found that after vancomycin treatment, histopathological observation of liver and kidney was toxic, liver in liver was disturbed in its cable structure, and inflammatory cell infiltration was observed. The kidney has increased glomerular inflammatory cells and a narrowed renal cyst lumen. Mice treated with GHb3K and GHbK4R were not observed for necrosis, fibrosis and inflammatory lesions in heart, liver, spleen, lung, kidney, indicating that GHb3K and GHbK4R were not toxic in vivo at the doses used.

11. Construction and treatment of MRSA infected skin wound mouse model

Adult healthy female Kunming mice (18-22 g) were kept normally for 7 days, the hair on the back of the mice was removed by an electric shaver, washed with normal saline, dried, sterilized with 75% alcohol, and after anesthesia by intraperitoneal injection of pentobarbital, a circular full-thickness skin wound with a diameter of 10mm was cut on the epidermis of the mice with a skin punch, and iodophor was sterilized. 0.5mL of MRSA suspension (1X 10) was uniformly applied ⁸ CFU/mL), and after bacterial liquid absorption, mice were individually caged. 64 mice were randomized each time, 8 in each group, and divided into 8 groups, negative (Model), positive (Mupirocin), GHbR high, medium, low (12.5. Mu.M, 25. Mu.M, 50. Mu.M) treatment groups, GHb3K high, medium, low (12.5. Mu.M, 25. Mu.M, 50. Mu.M) treatment groups. A blank (Control) group, i.e., a group of mice with full-thickness skin wound that had not been infected with bacteria, was also provided. After 24h of infection, treatment was started, once daily, with 50 μl of saline applied to the negative group (Model), mupirocin ointment applied to the positive group (as indicated), 50 μl of different concentrations of the antimicrobial peptide solution applied to the treatment group, and 50 μl of saline applied to the blank group. On day 0, day 3, day 6, and day 10, photographs were taken once. Killing mice after treatment, sterilizing skin with alcohol cotton ball, collecting 0.05-0.1 g of skin at each wound, shearing, adding 1mL of sterile physiological saline into EP tube for each 0.1g of pathological tissue, homogenizing, diluting the homogenate to proper concentration, spreading on blood plate, and applying at 37deg.CThe incubator was incubated for 24 hours, and the colony count was recorded.

The high, medium and low dose groups treated with GHbR and GHb3K significantly accelerated repair of chronic infectious wound in mice (fig. 10a, fig. 10 b). The wound repair degree of the antibacterial peptide high-dose group is better than that of the positive control medicine mupirocin ointment. After 10 days of treatment, the bacterial count of the wound was significantly reduced, i.e. the low dose group of antimicrobial peptides was comparable to the positive control (Mupirocin) (fig. 11a and 11 b). On day 3 of treatment, the high dose groups of the antimicrobial peptides were significantly reduced in TNF- α, IL-1β and IL-6, with TNF- α substantially returning to Control levels and IL-1β and IL-6 levels even lower than the Control (FIG. 12). The positive control group had lower levels of reduction of the three inflammatory factors than the antibacterial peptide-treated group. After 10 days of treatment, the anti-bacterial peptides and inflammatory factors in the drug-treated group were substantially restored to the placebo level. The results show that the treatment effect of GHbR and GHb3K on the MRSA infection wound surface is better than that of a positive control medicine mupirocin ointment.

It should be understood that the foregoing examples of the present invention are merely illustrative of the present invention and not limiting of the embodiments of the present invention, and that various other changes and modifications can be made by those skilled in the art based on the above description, and it is not intended to be exhaustive of all the embodiments of the present invention, and all obvious changes and modifications that come within the scope of the invention are defined by the following claims.

Claims (5)

1. The application of the antibacterial peptide in preparing a medicament for resisting staphylococcus aureus infection is provided, wherein the amino acid sequence of the antibacterial peptide is shown as SEQ ID NO.1, SEQ ID NO.2 or SEQ ID NO. 3.

2. Use of an antimicrobial peptide according to claim 1 for the preparation of a medicament against staphylococcus aureus infections, wherein said staphylococcus aureus comprises methicillin resistant staphylococcus aureus.

3. The use of an antimicrobial peptide according to claim 1 for the preparation of a medicament against staphylococcus aureus infection, wherein the antimicrobial peptide is synthesized by an N-9-fluorenylmethoxycarbonyl solid phase synthesis method.

4. The use of an antimicrobial peptide according to claim 1 or 3 for preparing a medicament against staphylococcus aureus infection, wherein the C-terminal end of the antimicrobial peptide is amidated and purified by reverse phase high performance liquid chromatography with a purity of not less than 95%.

5. Use of an antimicrobial peptide according to claim 1 or 2 for the preparation of a medicament against staphylococcus aureus infections, wherein the medicament against staphylococcus aureus infections comprises a medicament for the treatment of pneumonia of staphylococcus aureus infections and a medicament for the treatment of chronic wounds resistant to methicillin staphylococcus aureus infections.

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