CN113461777A

CN113461777A - Antibacterial peptide with antifungal effect and preparation method and application thereof

Info

Publication number: CN113461777A
Application number: CN202110944196.XA
Authority: CN
Inventors: 党向利; 王丽芳; 丁康
Original assignee: Anhui Agricultural University AHAU
Current assignee: Anhui Agricultural University AHAU
Priority date: 2021-08-17
Filing date: 2021-08-17
Publication date: 2021-10-01
Anticipated expiration: 2041-08-17
Also published as: CN113461777B

Abstract

The invention discloses an antibacterial peptide with antifungal effect, a preparation method and application thereof, and relates to the technical field of biology. The amino acid sequence of the antibacterial peptideIs Ac-WKKWFR-NH₂The antibacterial peptide has antibacterial activity on candida albicans, and the minimum inhibitory concentration on the candida albicans is 50 mug/mL. It can destroy the integrity of Candida albicans cell wall, and cause the increase of cell membrane permeability in a short time. The antibacterial peptide treated Candida albicans has abnormal and irregular cell structure, causes cytoplasmic protoplasm disorder, and has multiple vacuoles inside the cell. The antimicrobial peptides can bind to Candida albicans genomic DNA, inducing an increase in intracellular ROS. The prepared antibacterial peptide can be widely applied to the fields of medicine raw materials, food, fruit and vegetable preservation and preservation, plant fungal disease control, feed additives and the like as an antibacterial agent.

Description

Antibacterial peptide with antifungal effect and preparation method and application thereof

Technical Field

The invention belongs to the technical field of biology, and relates to an antibacterial peptide with an antifungal effect, and a preparation method and application thereof.

Background

Fungal diseases affect plants and animals, and also have a great impact on global food safety and human health. Fungal infections can cause humans to suffer from serious and even life-threatening diseases. Patients taking potent antibiotics for extended periods of time may increase the risk of fungal infections because the antibiotics kill both harmful and beneficial microorganisms, resulting in a change in the microbial balance in certain parts of the body, such as the intestinal tract and the oral cavity. However, fungal infections are less of an external concern than viral and bacterial infections, as the latter spread rapidly and have a large impact on human health. However, some fungal pathogens cause serious diseases, and with medical advances in the treatment of aids, cancer and organ transplantation, the number of people with compromised immune systems increases, and these people are susceptible to fungal infections. In nosocomial infections, Candida albicans (Candida albicans) is one of the most common fungal pathogens, and its incidence in humans is high, with mortality rates approaching 50% for infections associated with systemic fungi, and in some developing countries, mortality rates of up to 100% for some fungal pathogens. There are problems with current treatment of fungal infections with antifungal drugs, the first few classes of antifungal drugs available for treatment of human fungal pathogens, as fungi have evolved close to humans, the differential targets for development of antifungal drugs are relatively few, and some antifungal drugs have a tendency to produce side effects. At present, only five types of antifungal drugs are clinically used, namely polyene, allylamine, azole, pyrimidine analogue and echinocandin, and the antifungal drugs are mainly used for treating fungal infection of human beings through oral administration, local or intravenous infusion. Secondly, many fungal species have intrinsic resistance to certain antifungal drugs, and the mechanism of this resistance has not yet been fully elucidated.

Antimicrobial peptides (AMPs) have been considered as one of the drugs that address the crisis of Antimicrobial resistance since the discovery of AMPs in the end of the 80's 20 th century. AMPs is a low molecular weight protein with broad spectrum antibacterial and immunomodulatory effects, active against infectious bacteria, viruses, fungi, etc., and has been defined as an effective candidate for new therapeutic strategies against pathogenic microorganisms. AMPs are between 12 and 50 amino acids in length, are generally positively charged and amphiphilic, and can interact with cell membranes. AMPs vary in sequence and structure and can be generally classified according to their conformation as α -helices, β -sheets, loops or peptides with extended structures. Wang (2015) classifies AMPs according to the mode of linkage of polypeptide chains, which are the linear polypeptide chain UCLL (e.g., LL-37 and bombesin), the side chain linked peptide UCSS (e.g., defensins and lantibiotics), the side chain linked to backbone polypeptide chain UCSB (e.g., Lassos), and the cyclic peptide UCBB (e.g., cyclic peptides) with seamless backbone. Current antifungal drugs are mostly membrane bound by pathogenic microorganisms or target ergosterol on membranes by inhibiting certain biosynthesis. AMPs, in turn, kill pathogenic microorganisms primarily through different mechanisms that disrupt cell membranes. At the same time, it has also been discovered that AMPs target processes of key cells of pathogenic microorganisms. Obviously, compared with the traditional antifungal drugs, AMPs have nonspecific action mechanism, and the characteristic is expected to limit the drug resistance of pathogenic microorganisms.

Some AMPs have been reported in the literature to have good antibacterial activity against candida albicans, for example, human lactoferrin (hLF) is a peptide that binds iron and has a protease action, hLF is present in saliva and other human secretions, and has been found to be active against candida albicans, hLF1-11 inhibits candida albicans biofilm formation in the early phase, interferes with biofilm cell density and metabolic activity, and induces down-regulated expression of biofilm and hypha associated genes; experiments show that LL-37 and RK-31 influence the cell membrane permeability of Candida and have high bactericidal activity on Candida albicans, human LL-37 can inhibit the adhesion of Candida albicans to tissues through the interaction with cell wall carbohydrates of yeast, and LL-37 can also be cracked into short peptides with high fungicidal activity on Candida albicans; histatin-5 is a fragment of the salivary protein Histatin-3, and comprises 24 amino acids. The peptide has strong antifungal activity, and can kill yeast and Candida filamentous. Histatin-5 can exert its bactericidal activity by binding to a candidate 67kDa protein, and then interfering with the efflux of non-soluble ATP, even at low concentrations (15-30. mu.M). In addition, Histatin-5 can significantly interfere with Candida albicans biofilm formation.

Based on the important role of AMPs, many researchers have developed the search and preparation of new AMPs. There are three ways to find and prepare AMPs: chemical synthesis, bioengineering (recombinant technology) and screening of natural AMPs from plants, animals, microorganisms and other organisms. Although bioengineering can produce AMPs on a large scale, bioengineering still has the problems of incorrect transcription, inaccurate folding of proteins, low or even no activity of expressed proteins, mastery of core technology by a few scientists and companies, and the like. Although there are a large number of naturally bioactive AMPs and they have great potential as antifungal agents, the isolation of natural AMPs is often cumbersome, a cumbersome process, low in content, expensive and not suitable for large-scale production. AMPs molecules still present some problems in clinical applications. A major drawback may be the potential toxicity to mammalian cells at therapeutic concentrations. In addition, some AMPs are inactive in the presence of physiological ions and salt concentrations in biological fluids. Increased knowledge of the mechanism of action and structure-activity relationships of AMPs may identify specific features and key modifications of the peptides that need to be improved to obtain AMPs suitable for clinical use. Thus, in many cases, more efficient AMPs molecules can be economically produced using chemical synthesis methods of polypeptides based on known or designed AMPs sequence information. In addition, the chemical synthesis method also has the advantages of simple preparation, difficult degradation, easy structure modification, easy optimization of antibacterial activity and the like.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provides an antibacterial peptide with antifungal effect and a preparation method and application thereof.

The invention is realized by the following technical scheme:

the invention provides an antibacterial peptide with antifungal effect, and the amino acid sequence of the antibacterial peptide is Ac-WKKWFR-NH₂According to the amino acid sequence of the antibacterial peptide, the antibacterial peptide is artificially synthesized from the C end to the N end of the antibacterial peptide by using a solid-phase synthesis method. The molecular weight of the compound is 991.1Da, the isoelectric point is 12.4, the compound has antibacterial activity on candida albicans, and the minimum inhibitory concentration on candida albicans is 50 mug/mL.

The invention also provides a preparation method of the antibacterial peptide, which comprises the following steps:

(1) placing an appropriate amount of resin in a solid phase reactor, adding DMF (dimethylformamide) to wash for 1 time, swelling with DCM (dichloromethane) for 10-15min, and removing DCM;

(2) adding DBLK solution (piperidine + DMF) to remove protection 2 times (time 10min and 15min respectively). The intermediate was washed 1 time with DMF. After the protection is removed, washing with DMF for 6 times;

(3) and (3) dropwise adding a Kaiser reagent for indene detection on a small amount of resin, and continuing the amino acid coupling reaction if the resin shows blue.

(4) Putting 4eq (material ratio, material to resin ratio) of amino acid, 4eq of HBTU (benzotriazole-N, N, N ', N' -tetramethyluronium hexafluorophosphate) and 4eq of HOBt (1-hydroxybenzotriazole) in a reactor, adding DMF for dissolving, dropwise adding 8eq of DIEA, reacting for 2 hours at room temperature, and washing with DMF for 3 times;

(5) and (3) dropwise adding a small amount of resin into the Cather reagent for indene detection, and repeating the steps from (2) to (4) to perform the next amino acid coupling reaction until the reaction is finished if the resin is transparent and colorless.

(6) And (3) cutting the synthesized polypeptide from the resin by using a cutting fluid, and then drying by blowing, washing and cold drying to obtain the antibacterial peptide.

The purity and the molecular weight of the prepared antibacterial peptide are detected by using a conventional HPLC method and a mass spectrometry method; measuring the antibacterial activity of the obtained antibacterial peptide on candida albicans by adopting a conventional microdilution measurement method; detecting the influence of the antibacterial peptide on the integrity of the cell wall of the candida albicans by adopting a crystal violet staining method; detecting the influence of the antibacterial peptide on the permeability of the candida albicans cell membrane by using an ultraviolet spectrophotometer method; detecting the change of the microstructure of the cell after the candida albicans is treated by the antibacterial peptide by adopting an electron microscope; detecting the effect of the antibacterial peptide on inhibiting the formation of the candida albicans biofilm by using a crystal violet method; detecting the effect of antimicrobial peptide treatment on intracellular ROS production of Candida albicans by measuring the fluorescence intensity of 2 ', 7' -Dichlorofluorescein (2 ', 7' -Dichlorofluorescein, DCF); detecting the binding capacity of the antibacterial peptide and Candida albicans genome DNA by electrophoretic mobility variation analysis; detecting the hemolytic activity of the antimicrobial peptide by measuring hemoglobin release; evaluating the resistance of candida albicans to the antimicrobial peptide by determining a minimum inhibitory concentration; the therapeutic effect of the antibacterial peptide on the larvae of the greater wax moth infected with candida albicans is evaluated by detecting the in vivo antibacterial activity of the antibacterial peptide on the larvae of the greater wax moth infected with the antibacterial peptide.

The antibacterial peptide prepared by the invention has antibacterial activity on Candida albicans, the minimum inhibitory concentration on the Candida albicans is 50 mug/mL, and the minimum bactericidal concentration is 125 mug/mL. The antibacterial peptide can destroy the integrity of the cell wall of the candida albicans, and the permeability of a cell membrane is increased in a short time. The candida albicans treated by the antibacterial peptide has abnormal and irregular cell structure, so that cytoplasm disorder of cells, nonuniform electron density and a plurality of vacuoles inside the cells are caused. Can be combined with Candida albicans genome DNA to induce the increase of intracellular ROS. The hemolytic activity to rabbit erythrocyte is low, and the antibacterial peptide is continuously treated for 14 days without the generation of candida albicans drug resistance. The antibacterial peptide treatment can obviously improve the survival rate of the larvae of the galleria mellonella infected with candida albicans, and can completely eliminate the fungus infected in the larvae after 72 hours of treatment.

The invention has the following beneficial effects: the invention has the advantages of simple preparation, short production period, difficult degradation of products, easy modification of structure, easy optimization of antibacterial activity and the like. The antibacterial peptide prepared by the invention has the characteristics of high purity, good antibacterial activity, unique action mechanism, low hemolytic activity, no induction of drug resistance generation and the like. The antibacterial peptide product is white powder, has molecular weight of 991.1Da and amino acid sequence of Ac-WKKWFR-NH₂And the amino acid group has a typical amphiphilic structure, and basic amino acids are gathered on the same side. Has antibacterial activity against Candida albicansThe minimum inhibitory concentration of the bacteria is 50 mug/mL. Has synergistic effect with antibiotic amphotericin B. Compared with traditional antibiotics, the product mainly inhibits and kills fungi through acting on cell walls, cell membranes and intracellular action mechanisms without specific receptors, so that the product is not easy to induce the resistance of microorganisms to the product. On the treatment effect of the candida albicans infected galleria mellonella, the survival rate of larvae infected with the candida albicans can be obviously improved by the antibacterial peptide with different dosages. After 72 hours of treatment, the infected candida albicans in the larvae can be completely eliminated. Histopathological analysis showed that the antibacterial peptide treated infected galleria mellonella showed fewer candida albicans cells. The preparation method of the invention can realize large-scale industrial production, has short production period and higher product yield and purity, and is not influenced by the external environment.

Drawings

FIG. 1 shows the purity test of the antibacterial peptide obtained in the embodiment.

FIG. 2 shows the molecular weight detection of the antibacterial peptide obtained in the example.

Figure 3 effect of antimicrobial peptides obtained in the example on c.

FIG. 4 shows the effect of the antibacterial peptide obtained in the example on the permeability of C.albicans cell membrane.

FIG. 5 scanning electron microscope image of C.albicans treated with the antibacterial peptide obtained in example 10 h.

FIG. 6 is a transmission electron microscope image of C.albicans treated with the antibacterial peptide obtained in example 10 h.

Inhibition of c.

Fig. 8 gel electrophoresis images of the antimicrobial peptides at different concentrations in the example after treatment of c.

Fig. 9 effect of antibacterial peptides obtained in example on c.

FIG. 10 is a diagram showing a hemolytic activity of the antibacterial peptide obtained in the example.

Fig. 11 colony count assay for greater wax moth in vivo infection c.

FIG. 12 effect of the antibacterial peptide treatment obtained in the example on the number of colonies infected with C.albicans in galleria mellonella.

Fig. 13 histopathological analysis of infected c. albicans wax moth larvae after treatment with the antimicrobial peptides obtained in the examples.

Detailed Description

The present invention will be described in more detail with reference to examples. It is to be understood that the practice of the invention is not limited to the following examples, and that any variations and/or modifications may be made thereto without departing from the scope of the invention.

In the present invention, unless otherwise specified, the methods used in the examples are those commonly used in the art, and all the instruments, raw materials and the like are commercially available or commonly used in the industry.

Example 1: preparation and detection of antibacterial peptide

The preparation of the antibacterial peptide is carried out according to the following steps:

the amino acid sequence of the antibacterial peptide is Ac-WKKWFR-NH₂The preparation of the polypeptide is carried out according to the amino acid sequence of the antibacterial peptide, starting from the C end of the polypeptide and going to the N end of the polypeptide. The method comprises the following steps:

(1) placing an appropriate amount of resin in a solid phase reactor, adding DMF (dimethylformamide) for washing once, then swelling with DCM (dichloromethane) for 10-15min, and removing DCM;

(6) The synthesized polypeptide is cleaved from the resin with a cleavage solution, and then blow-dried and washed.

The purity and molecular weight of the synthesized polypeptide are detected and analyzed by HPLC and a mass spectrometer. By HP LC detection, the prepared antibacterial peptide has peak-off time of 10.509min (figure 1), and has symmetrical peak pattern and high purity reaching 98.24%. The molecular weight of the antimicrobial peptide was 991.1 (FIG. 2) as determined by MS mass spectrometry.

Example 2: detection of antibacterial activity of antibacterial peptide on candida albicans

The Minimum Inhibitory Concentration (MIC) of the antimicrobial peptide against candida albicans (c.albicans) was determined by microdilution assay. Thawing the C.albicans glycerol strain stored at-80 deg.C on ice, adding 100 μ L glycerol strain into 2mL fresh SDB medium (glucose 40g, peptone 10g, and sterilized water to constant volume of 1L), and culturing at 30 deg.C and 160r/min overnight. The next day, 100. mu.L of C.albicans cells were inoculated from overnight cells into 2mL of fresh SDB medium, and cultured at 30 ℃ and 160r/min until logarithmic phase. Bacterial suspension (1.0X 10 concentration) was prepared using fresh SDB medium⁵cfu/mL). 10 mu L of the antibacterial peptide obtained in the example 1 diluted by sterile water and with different concentrations is added into a 96-hole enzyme label plate by a double dilution method, then 90 mu L of candida albicans suspension is added into holes corresponding to the antibacterial peptide with different concentrations, the bacteria liquid and the sterile water are used as positive controls, and the SDB culture medium and the sterile water are used as negative controls. Culturing at constant temperature in 30 deg.C incubator for 24h, and detecting OD of 96-well enzyme-labeled plate with microplate reader (Multiskan FC, China)₆₀₀The value is obtained.

Minimum Fungicidal Concentration (MFC) determination of antimicrobial peptides reference the MIC determination. After the 96-well enzyme label plate is cultured at the constant temperature of 30 ℃ for 24h, 50 mu L of bacterial liquid is taken from each well (uniformly mixed before taking) to the corresponding SDA culture medium (40 g of glucose, 10g of peptone and 20g of agar are added with sterile water to reach the constant volume of 1L) and is evenly smeared by coating beads. After being put into a 30 ℃ constant temperature incubator to be cultured for 24 hours, colonies of C.albicans on the SDA plate are counted, and the lowest concentration of the growth of the colonies is used as MFC of the antibacterial peptide pair C.albicans.

MIC and MFC of antimicrobial peptide to c.albicans were determined to be 50 and 125 μ g/mL, respectively.

Example 3: effect of antimicrobial peptides on cell wall integrity of Candida albicans

C.albicans suspension (concentration 2.0X 10) was prepared from Candida albicans in logarithmic growth phase in fresh SDB medium⁷cfu/mL), c.albicans suspension was treated with antimicrobial peptide at a final concentration of 2MIC, and sterilized water was treated as a Control (CK). Each group was cultured in an incubator at 30 ℃ for 10 hours, taken out, centrifuged at 3000r/min for 10min to collect the cells, and washed 3 times with Phosphate Buffer (PBS). Adding 5% of crystal violet for dyeing for 10min, adding PBS to suspend the bacterial liquid, centrifuging until the bacterial liquid is colorless, taking 5 mu L of bacterial liquid to precipitate on a glass slide, dropwise adding 10% of tannic acid for fixing, covering a cover glass, observing under an inverted microscope (MoticAE2000, China), randomly observing 100 cells and calculating the integrity rate of the cell wall of C.albic ans. Each set of experiments was repeated three times.

As shown in FIG. 3 (A: sterilized Water treatment (CK); B: antimicrobial peptide treatment), the cell wall integrity of CK group C.albicans was 95.67% (Table 1), the shape was circular or elliptical, and the cell wall structure was stained purple or colorless completely in the extracellular and cytoplasmic directions (FIG. 3A). The cell wall integrity of the antibacterial peptide treated group C.albicans was 40.27% and 32.86%, respectively, and was significantly lower than that of the CK group (P <0.05) (FIG. 3B), which indicates that part of the cell wall and cytoplasm of C.albicans was stained dark purple.

Effect of antimicrobial peptides on C.albicans cell wall integrity

TABLE 1

Different letters represent significant differences (P < 0.05).

Example 4: effect of antibacterial peptide on permeability of Candida albicans cell membrane

Taking out C.albicans from an ultra-low temperature refrigerator at-80 ℃, adding the C.albicans into a fresh SDB culture medium, and placing the mixture in a constant temperature shaking table for overnight culture at 30 ℃ and 160 r/min. Centrifuging at 3000r/min for 10min, collecting precipitate, and resuspending with PBS to obtain thallus with final concentration of 2.0 × 10⁷cfu/mL. Respectively, extracting with Candida albicans solutionTreatment with antimicrobial peptide, amphotericin B, at a final concentration of 1 MIC. Suspension treated with 10% Triton X-100 as a positive control and PBS as a negative control. The groups are respectively treated in a shaking table at 30 ℃ and 100r/min for 0h, 0.5h, 1h, 2h, 4h, 8h and 12 h. The cultured c.albicans was filtered using a 0.22 μm aqueous syringe filter, and the filtrate was collected. The OD of the filtrate was measured with a UV spectrophotometer (Soptop 752, China)₂₆₀The UV spectrophotometer was calibrated to zero with PBS filtered through a water system syringe filter. The cell membrane permeability after treatment of candida albicans with the antibacterial compound was calculated using the following formula:

wherein OD_260AMPUsing water-based syringe filter to filter the absorbance, OD of bacterial liquid after C.albicans treatment for antibacterial peptide_{260Triton X}C.albicans was treated with 10% Triton X-100 and the absorbance of the broth was filtered through an aqueous syringe filter.

As a result, as shown in fig. 4, it was found that the antimicrobial peptide-treated group resulted in an increase in the permeability of c. Treatment of c.albicans with antimicrobial peptide for only 0.5h increased the permeability from 9.90% to 19.4%, respectively, compared to CK. The permeability is increased gradually along with the increase of the action time, and the permeability reaches 27.77 percent at the treatment time of 12h and is obviously higher than CK.

Example 5: electron microscope observation after antibacterial peptide treatment of candida albicans

(1) Observation by scanning electron microscope

The C.albicans in logarithmic growth phase was collected and prepared in fresh SDB medium (concentration 2.0X 10)⁷cfu/mL), c. The groups are placed in a shaking table at 30 ℃ and 100r/min for culturing for 10h and then taken out, cells are collected by centrifuging at 3000r/min for 10min, PBS is used for washing, and 1mL of electron microscope fixing liquid is added into the precipitate and is fixed in a refrigerator at 4 ℃ overnight. Washed 3 times with PBS and dehydrated in graded ethanol series (50%, 70%, 80%, 95% and 100%). After freeze-drying and gold coating, scanning electron microscope at SU-8100(Hitachi, Japan). As can be seen from fig. 5, the control c. albicans cells appeared circular or oval, full, and smooth on the cell surface (fig. 5A). C. albicans had severe damage and morphological changes of the membrane after 10h of antimicrobial peptide treatment compared to the control group. The cell surface was mainly rippled (FIG. 5B), membrane rupture, perforation, leakage of contents (FIG. 5C), and cell membrane detachment (FIG. 5D).

(2) Observation by transmission electron microscope

The C.albicans in logarithmic growth phase was collected and prepared in fresh SDB medium (concentration 2.0X 10)⁷cfu/mL), c.albicans was treated with antimicrobial peptide at a final concentration of 1MIC, sterile water as a control. Culturing each group at 30 deg.C and 100r/min for 10h, taking out, centrifuging at 3000r/min for 10min to collect thallus, washing with PBS, adding 1mL of electron microscope fixing solution into precipitate, and fixing in refrigerator at 4 deg.C overnight. Wash 3 times with PBS and fix with 1% osmic acid in PBS for 2 h. The samples were washed 3 times with PBS, dehydrated in a graded ethanol series (50%, 70%, 80%, 95% and 100%) and then dehydrated with anhydrous acetone. The samples were transferred to a mixture of anhydrous acetone and epoxy resin (1:1 and 1:2) for 2h and overnight, respectively. Then transferred to epoxy resin and kept at 37 ℃ overnight. Ultrathin sections (60-80 nm) were stained with uranyl acetate and lead citrate and observed under HT-7700 transmission electron microscopy (Hitachi, Japan). The control group C.albicans cells were circular or elliptical in shape, the cell structure was intact (FIG. 6A), and most of the bacterial cells were significantly changed after the antibacterial peptide treatment for 10 h. The formation of cytoplasmic vacuoles, non-uniform electron density (fig. 6B), and membrane disruption (fig. 6C) were observed in C.

Example 6: effect of antimicrobial peptides on Candida albicans biofilm formation

Culturing C.albicans with fresh SDB culture medium overnight, transferring to fresh SDB culture medium the next day, culturing to logarithmic phase, and diluting with SDB culture medium to obtain bacterial liquid with concentration of 1.0 × 10⁵cfu/mL, adding 180 μ L of bacterial liquid into 96-well enzyme label plate containing 20 μ L of antibacterial peptide with different concentrations, adding sterilized water into the bacterial liquid as positive Control (Control), and adding sterilized water into SDB culture medium asAnd (5) negative control. Incubate at 30 ℃ for 24 h. Then, the bacterial liquid in the 96-well enzyme label plate is sucked out, 200 mu L PBS is added to remove the cells which are not adhered in the wells, the PBS is sucked out, 200 mu L99% methanol is added to fix for 15min, the methanol is sucked out and dried in a super clean bench, 200 mu L1% crystal violet is added to be placed in the super clean bench for 5min, and the wells are cleaned by streamline tap water and then are dried in the super clean bench. Finally, 200. mu.L of 33% glacial acetic acid was added to each well and OD was measured with a microplate reader (Multiskan FC, China)₆₀₀The value is obtained. Compared with a control, the antibacterial peptide has the inhibition rate of 98.28% at the concentration of 2MIC, and only 1.72% of C.albicans biofilm remains (figure 7); control in fig. 7: adding sterilized water into the bacterial liquid. Error bars represent standard error, different letters represent significant difference (P)<0.05)。

Example 7: binding analysis of antibacterial peptide and Candida albicans genome DNA

Genomic DNA of c.albicans was extracted using a fungal genomic DNA extraction kit (Solarbio, china). Determining the concentration (OD) of the extracted DNA₂₆₀/OD₂₈₀) And stored in a refrigerator at-20 ℃ for later use. To analyze whether the peptides were able to bind to the C.albicans DNA, 5. mu.L of varying concentration gradients of peptides were incubated with 5. mu.L of the extracted C.albicans DNA (40 ng/. mu.L diluted in sterile water) at room temperature for 1.5h, the C.albicans DNA treated with sterile water was used as a Control (CK), 2. mu.L of 6 XDNA Loading Buffer was added, electrophoresis was performed using 1% agarose gel (0.5 XTBE Buffer), ethidium bromide (10 mg/mL final concentration) was added to the gel, and electrophoresis was performed at 98V. After electrophoresis, pictures are taken under the UV irradiation of a gel imaging system to observe the DNA migration change of C.albicans, and the combination condition of the antibacterial peptide and Candida albicans genome DNA is analyzed by detecting the blocking condition of the DNA in the gel.

Electrophoresis results showed that the antimicrobial peptide was bound to the c.albicans genomic DNA (fig. 8). The distance of migration of the genomic DNA bound to the antimicrobial peptide was significantly different from the distance of migration of the control DNA. The change of the electrophoretic mobility of albicans DNA shows the concentration dependency relationship of the antibacterial peptide.

Example 8: intracellular ROS detection after antimicrobial peptide treatment of Candida albicans

Taking C.albicans at logarithmic growth phase, preparing the concentration of 2.0 × 10 with fresh SDB culture medium⁷cfu/mL of a suspension of C.albicans bacteria treated with the final concentrations of the antimicrobial peptides 1/2MIC, 1MIC and 2MIC, with sterile water as a Control (Control). Culturing in 30 deg.C constant temperature incubator for 10h, centrifuging at 3500r/min for 10min to collect cells, washing with extracellular fluid (Beyotime, China) for 2 times, and resuspending each group of samples with PBS to obtain 2.0 × 10 bacterial liquid⁷cfu/mL, and 100. mu.L of 2 ', 7' -dihydrodichlorofluorescein diethyl ester (2 ', 7' -Dichlorodi-hydrofluorescein diacetate, DCFH-DA) solution (Yingxin, China) at a final concentration of 10. mu.M was added and incubated at 30 ℃ for 30min in the absence of light. 200. mu.L of each of the bacterial solutions was put in a blackboard made of a 96-well plate, and the fluorescence intensity (excitation wavelength/emission wavelength: 488/525nm) of 2 ', 7' -Dichlorofluorescein (2 ', 7' -Dichlorofluorescein, DCF) was measured with a multifunction microplate reader (SpectraMax i3, USA).

The results show (FIG. 9, Control (CK): sterilized water treatment. error bars represent standard error, different letters represent significant difference (P <0.05)), and 1MIC and 2MIC showed significant increases of 236.64% and 328.24% in ROS compared to CK (control) group after 10h of antimicrobial peptide treatment, respectively. The antibacterial peptide can promote the accumulation of the C.albicans intracellular ROS and show a dose-dependent effect.

Example 9: hemolytic activity assay of antibacterial peptide

The hemolytic activity of the antimicrobial peptides was assessed by measuring the hemoglobin release at 414nm of a 4% fresh rabbit red blood cell suspension. Fresh rabbit erythrocytes are taken, centrifuged at 2000r/min for 5min, the supernatant is discarded, and the erythrocytes are washed 3 times with PBS and resuspended. Mixing the re-suspended rabbit erythrocyte and antibacterial peptide according to a ratio of 1:1, shaking and incubating for 1h at 37 ℃, centrifuging for 10min at 500 Xg, taking 80 mu L of obtained supernatant, detecting the absorption value at 414nm, wherein the positive control is 0.1% Triton X-100, and the negative control is PBS. The hemolytic activity of the antimicrobial peptide was calculated according to the following formula.

Wherein OD_peptideOn rabbit erythrocytes treated with antimicrobial peptideAbsorbance, OD, of supernatant at 414nm_PBSThe absorbance value, OD, of supernatant at 414nm after PBS treatment of rabbit red blood cells_{Triton X}The absorbance of the supernatant at 414nm after Triton X-100 treatment of rabbit erythrocytes was determined.

As shown in fig. 10, the hemolytic activity of the antimicrobial peptide on rabbit erythrocytes was low at all concentrations tested. The hemolytic activity of the antibacterial peptide to rabbit erythrocyte is only 2% -4% under the concentration of 7.8125 mug/mL-125 mug/mL, and even under the high concentration of 1000 mug/mL, the hemolytic activity of the antibacterial peptide to rabbit erythrocyte is lower than 10%.

Example 10: determination of resistance of Candida albicans to antibacterial peptide

Treatment with antimicrobial peptides at final concentrations of 0.75MIC, 1MIC and 1.25MIC, and amphotericin B at 1.0X 10⁵cfu/mL of c.albicans bacterial solution (peptide: bacteria: 1: 9). After 24h of each incubation, the MIC of the antimicrobial peptide-treated C.albicans was determined by the microdilution assay described above in example 2. Subculture and MIC determination were performed daily using the same method for a total of 14 d. Resistance to c.albicans was assessed by observing MIC values.

The results of the drug resistance measurement show that when the C.albicans cells are repeatedly treated with the antibacterial peptides with 0.75MIC, 1MIC and 1.25MIC, the MIC of the antibacterial peptide to the treated C.albicans is unchanged and is 50 mu g/mL, and the drug resistance of the C.albicans to the antibacterial peptide is not observed within the detection time range.

Example 11: evaluation of therapeutic effect of antibacterial peptide on infected galleria mellonella larvae

(1) Infection of greater wax moth larvae by C.albicans at various concentrations

The overnight cultured C.albicans suspension was centrifuged at 3000r/min for 10min, the supernatant was discarded, washed with PBS and resuspended to the desired concentration. Gently fixing the larvae on the injection platform, and respectively taking 5 μ L of C.albicans suspension (concentration 1.0 × 10) with different concentrations by using an injector⁵cfu/mL～2.0×10⁷cfu/mL), injected into the body via the left hind paw of the larva. The uninfected group and the PBS-injected group with the same volume were set as controls. Each group had 20 larvae. 7d was observed at 37 ℃. Survival of larvae was recorded daily.

Infection with C.albicans at various concentrationsThe survival rate of the galleria mellonella larvae is different. The survival rates of the uninfected group (Control) and the PBS-injected group (PBS)7d were 100%, 1.0X 10⁵cfu/mL、1.0×10⁶cfu/mL、1.0×10⁷cfu/mL and 1.5X 10⁷Survival rates of cfu/mL infected group infected with 7d were 90%, 70%, and 40%, respectively, 2.0X 10⁷The cfu/mL infected group died at 2d post-infection. Selecting 1.5 × 10 for use according to the survival rate of greater wax moth⁷The cfu/mL infected group was used for subsequent studies of antibacterial peptide therapy.

(2) In vivo colony assay of C.albicans greater wax moth larva

Injecting 5 μ L of C.albicans bacterial suspension (concentration is 1.0 × 10 in sequence) into larva of Helichrysum Macrocephalus⁷cfu/mL、1.5×10⁷cfu/mL and 2.0X 10⁷cfu/mL). The same volume of PBS was injected as a blank control. After 6h, 12h, 24h, 36h, 48h, 60h and 72h of infection, three larvae per group were randomly selected to extract hemolymph, their surfaces were disinfected with ethanol, larvae hemolymph was spilled and collected by gently scratching near their tails (away from the gut) with a scalpel. Draw 5. mu.L of hemolymph and place in 95. mu.L of PBS. After serial dilution with PBS at different fold, 50. mu.L of the suspension was dropped onto SDA solid plates containing 200. mu.g/mL chloramphenicol, the solution was mixed with coated beads, and colonies were counted after incubation at 30 ℃ for 48 h. Each set was coated with at least 3 SDA solid plates.

The results are shown in FIG. 11 (error bars represent standard error, different letters represent significant differences (P)<0.05)) is shown. It can be seen that 1.0X 10⁷The cfu/mL infected group showed an increasing trend in the number of colonies at 6h and 12h of infection, and a marked decrease after 12h of infection. 1.5X 10⁷The cfu/mL infected group showed an increasing trend in the number of colonies infected at 6h, 12h and 24h, and a marked decrease after 24h infection. 2.0X 10⁷The colony numbers of cfu/mL infected groups at 6h, 12h, 24h and 36h are obviously increased, and the larvae of the greater wax moth die after 36h infection. No c.albicans was detected in the white control (PBS injected) galleria mellonella larva hemolymph.

(3) Determination of toxicity of antibacterial peptide to galleria mellonella

The larvae were injected with 5. mu.L of antimicrobial peptides at different concentrations (10mg/kg, 20mg/kg and 40mg/kg) and the same volume of PBS was injected as a control group. 20 larvae were injected per concentration. 7d was observed at 37 ℃. Survival of larvae was recorded daily. Through determination, the antibacterial peptides injected into the body at different concentrations (10mg/kg, 20mg/kg and 40mg/kg) have no influence on the survival rate of the larvae of the galleria mellonella, which indicates that the antibacterial peptides have no toxicity to the galleria mellonella.

(4) Analysis of survival rate of galleria mellonella larvae infected with C.albicans antibacterial peptide after treatment

Selecting healthy larvae of Helichrysum Macrocephalus, injecting 5 μ L of the larvae with concentration of 1.5 × 10⁷cfu/mL of C.albicans bacterial solution, left at 37 ℃ for 2 h. The infected larvae were then injected with varying doses of antimicrobial peptides (10mg/kg, 20mg/kg and 40mg/kg) diluted in PBS using a microinjector. PBS and amphotericin B (2mg/kg) were injected in equal volumes as the untreated group and the positive control group, respectively, while the uninfected group was set. 20 larvae were injected per treatment. After injection, the mixture was observed at 37 ℃ for 7 days. Survival of larvae was recorded daily.

After 7 days of treatment, the survival rate of the large wax moth in the uninfected group (Control) is 100 percent; survival rate of greater wax moth in untreated group (infection + PBS) was 35%. Compared with untreated groups, the survival rate of the larvae of the greater wax moth can be improved by all doses of the antibacterial peptide, the survival rates of the greater wax moth are respectively 50%, 70% and 70% at the concentrations of 10mg/kg, 20mg/kg and 40mg/kg, and the survival rates of the antibacterial peptide treated by medium-high doses (20mg/kg and 40mg/kg) are the same as that of the greater wax moth treated by positive control amphotericin B (2 mg/kg).

(5) In vivo colony analysis of larval-infected wax moth after antibacterial peptide treatment

Healthy galleria mellonella larvae were selected and treated by injecting various doses (10mg/kg, 20mg/kg and 40mg/kg) of antimicrobial peptides into C.albicans infected galleria mellonella larvae as described above (example 11 (4)). Equal volumes of PBS and amphotericin B (2mg/kg) were injected as untreated and positive controls, respectively. Uninfected and uninfected PBS-injected larvae of galleria mellonella serve as blank controls. Groups of three larvae per group were collected at 6h, 12h, 24h, 36h, 48h, 60h and 72h of treatment. And disinfecting the surface with ethanol, and collecting hemolymph. mu.L of hemolymph was placed in 80. mu.L of PBS. After serial dilution with PBS at different fold, 50. mu.L of the suspension was dropped onto SDA solid plates containing 200. mu.g/mL chloramphenicol, the solution was mixed with coated beads, and the colonies were counted by incubation at 30 ℃ for 48 h. Each set was coated with at least 3 SDA solid plates.

The number of c.albicans colonies in the galleria mellonella larvae after treatment was lower than in the untreated group (infection + PBS) for each treatment group (figure 12, error bars represent standard error, different letters represent significant difference (P < 0.05)). Treatment with antimicrobial peptides (10mg/kg, 20mg/kg and 40mg/kg) significantly reduced the number of colonies in vivo infected with C.albicans wax moth larvae compared to the untreated group. When the treatment is carried out for 72 hours, the colony number of the larvae infected with C.albicans greater wax moth can be completely eliminated by treating the larvae with antibacterial peptide (20mg/kg) with lower concentration. Amphotericin B showed a decreasing trend in the number of colonies in vivo within 72h of treatment of galleria mellonella larvae. No c. albicans colonies were detected in the blank (no infection, PBS only injection) blood lymph.

(6) Histopathological analysis of infected galleria mellonella larva after antibacterial peptide treatment

The C.albicans greater wax moth larvae infected with 5. mu.L of the antimicrobial peptide (20mg/kg) were treated as described above (example 11 (4)). PBS and amphotericin B (2mg/kg) were injected in equal volumes as the untreated group and the positive control group, respectively, while the uninfected group was set. After incubating the larvae at 37 ℃ for 24h, 3 larvae per group were randomly selected, and the larvae were fixed with paraformaldehyde fixative (Servicebio, china) for 24h before cross-cutting and longitudinal-cutting. The sample is dehydrated by high-concentration ethanol, then dehydrated by xylene, and finally added with paraffin. Tissue sections were prepared for future use (4 μm) with a microtome, paraffin sections were deparaffinized to water, stained with hematoxylin and eosin in order, dehydrated and mounted for observation under an upright optical microscope (Nikon Eclipse E100, japan).

The results are shown in FIG. 13, where A and B: no infection. 24h post infection, C and D: no treatment; e and F: 20mg/kg antimicrobial peptide; g and H: amphotericin B2 mg/kg. FIG. B, D, F, H is an enlarged view of the area indicated in FIG. A, C, E, G, respectively. Squares in fig. C, E, G represent c.

The untreated group showed C. albicans cells spread throughout the organism at various sites (fig. 13C, D) compared to the uninfected group (fig. 13A, B); larvae treated with 20mg/kg antimicrobial peptide (fig. 13E, F) and 2mg/kg amphotericin B (fig. 13G, H) showed the presence of fewer c. The results show that compared with the untreated group, the antibacterial peptide and amphotericin B can obviously reduce the infection of C.albicans to the larvae of the galleria mellonella.

The above description is intended to describe in detail the preferred embodiments of the present invention, but the embodiments are not intended to limit the scope of the claims of the present invention, and all equivalent changes and modifications made within the technical spirit of the present invention should fall within the scope of the claims of the present invention.

Sequence listing

<110> agriculture university of Anhui

<120> antibacterial peptide with antifungal effect, preparation method and application thereof

<160> 1

<170> SIPOSequenceListing 1.0

<210> 2

<211> 6

<212> PRT

<213> 2 Ambystoma laterale x Ambystoma jeffersonianum

<400> 2

Trp Lys Lys Trp Phe Arg

1 5

Claims

1. An antibacterial peptide with antifungal effect is characterized in that the amino acid sequence of the antibacterial peptide is SEQ ID NO. 1.

2. A method for preparing the antibacterial peptide of claim 1, wherein the method for preparing the antibacterial peptide is characterized in that the antibacterial peptide is artificially synthesized from C end to N end of the polypeptide by a solid phase synthesis method.

3. The use of the antimicrobial peptide of claim 1 as an antimicrobial agent in the preservation and freshness of medical materials, foods, fruits and vegetables, the control of plant fungal diseases and feed additives.

4. The use of the antimicrobial peptide of claim 1 in the preparation of a medicament for combating fungal infections, wherein the fungus is candida albicans and the antimicrobial peptide has a minimum inhibitory concentration against candida albicans of 50 μ g/mL.