CN117603306A

CN117603306A - Antibacterial peptide CAD-5 with candida biofilm removal activity and application thereof

Info

Publication number: CN117603306A
Application number: CN202311625761.1A
Authority: CN
Inventors: 李瑞芳; 吴嘉莎
Original assignee: Henan University Of Technology High Tech Industrial Technology Research Institute Co ltd; Henan University of Technology
Current assignee: Henan University Of Technology High Tech Industrial Technology Research Institute Co ltd; Henan University of Technology
Priority date: 2023-11-30
Filing date: 2023-11-30
Publication date: 2024-02-27

Abstract

The invention relates to the technical field of antibacterial peptides, in particular to an antibacterial peptide CAD-5 with candida biofilm removal activity and application thereof. The antibacterial peptide CAD-5 provided by the invention can target candida beta-1, 6-glucan synthase KRE9, has broad-spectrum antibacterial activity and low hemolytic activity; the antibacterial peptide CAD-5 provided by the invention has the functions of inhibiting formation of candida biofilm and removing candida formed biofilm activity, has the candida biofilm removing activity stronger than that of fluconazole, is expected to become a novel candidate medicament for treating candida persistent infection, and has good application prospect.

Description

Antibacterial peptide CAD-5 with candida biofilm removal activity and application thereof

Technical Field

The invention relates to the technical field of antibacterial peptides, in particular to an antibacterial peptide CAD-5 with candida biofilm removal activity and application thereof.

Background

Bacterial resistance due to antibiotic abuse and fungal resistance due to biofilm formation or stubborn bacteria formation, cause a global growing problem of antibiotic resistance.

Biofilm formation can help fungi avoid host cell immune system clearance and drug attack, and persistent infection is a serious threat to human health. Candida albicans is a common conditional pathogenic bacteria for clinical infection and is also a main research model for the research of fungus biofilm at present. The mechanisms of the extracellular matrix, the expression of drug resistance genes, and the like, have been found to be closely related to the development of biofilm resistance [ Li Ruilian, wang, du Yi Guang, candida albicans biofilm research progress, microbiology report, 2017, 57 (8): 1206-1218]. Thus, the study of antimicrobial envelope-active substances is of great importance for the treatment of persistent infections with candida.

Extracellular polysaccharides, including beta-1, 3-glucan and beta-1, 6-glucan, are the major components of biofilm structure and are also key factors in the development of biofilm resistance [ Li Ruilian, wang, du Yi Guang. Most antibiotics do not have the inhibitory formation and scavenging activity of candida biofilm due to the mechanism of action. Antibacterial peptides are an important component of the natural immune system of the animal body. Unlike traditional antibiotics which act primarily by inhibiting a certain biosynthetic pathway (e.g., cell wall, protein), most antimicrobial peptides inhibit or kill pathogenic bacteria by a multi-pathway, multi-target mechanism of action. The unique mechanism of action of the antimicrobial peptides makes it possible to combat candida biogenesis and clearance. Thus, antimicrobial peptides have great potential clinically for the treatment of chronic candida infections.

Disclosure of Invention

The invention provides an antibacterial peptide CAD-5 with the activity of eliminating candida biofilm and application thereof, aiming at solving the problem of chronic candida infection caused by candida biofilm formation.

In order to achieve the above purpose, the present invention provides the following technical solutions:

the invention provides an antibacterial peptide CAD-5, the amino acid sequence of which is NH2-HARKKKKRAH-COOH.

Preferably, the carbon end of the antibacterial peptide CAD-5 is subjected to amidation modification.

The invention also provides application of the antibacterial peptide CAD-5 in preparation of medicines for resisting pathogenic bacteria infection.

Preferably, the pathogenic bacteria include fungi and bacteria.

Preferably, the fungus is candida albicansCandida albicans) Tropical candida (Tropical candida)Candida tropicalis）。

Preferably, the bacteria is enterococcus faecalisEnterococcus faecalis) And staphylococcus aureus @ sStaphylococcus aureus）。

Preferably, the candida albicans include candida planktonic, candida albicans forming a biofilm, and candida albicans forming a biofilm. The candida tropicalis is candida planktonic and candida tropicalis.

The invention also provides an anti-infective medicament, the effective components of which comprise the antibacterial peptide CAD-5 of claim 1 or 2.

Preferably, the minimum antibacterial concentration of the antibacterial peptide CAD-5 is 3.92-6.82 mug/mL, the minimum antibacterial concentration of the antibacterial peptide CAD-5 on refractory candida tropicalis is 31.32 mug/mL, the minimum concentration of candida albicans inhibiting biofilm formation is 37.71 mug/mL, and the minimum concentration of candida albicans eliminating formed biofilm is 125 mug/mL.

Advantageous effects

The invention provides an antibacterial peptide CAD-5 with candida biofilm removal activity, wherein the amino acid sequence is NH2-HARKKKKRAH-COOH. The antibacterial peptide CAD-5 provided by the invention is a cationic antibacterial peptide with 8 positive charges, targets candida cell wall beta-1, 6-glucan synthase KRE9, and inhibits candida cell wall beta-1, 6-glucan synthesis by inhibiting the beta-1, 6-glucan synthase activity of the KRE9, so that candida cell wall integrity is damaged; the antibacterial peptide CAD-5 provided by the invention has low hemolysis; the antibacterial peptide CAD-5 provided by the invention contains 10 amino acids, the amino acids are L-shaped amino acids, the synthesis cost is low, the antibacterial peptide CAD-5 is expected to become a novel antibacterial candidate drug, has good application prospect in the aspect of treating chronic candida infection, and has great potential in the aspect of solving the drug resistance of pathogenic bacteria.

Drawings

In order to more clearly illustrate the embodiments of the present invention or prior art solutions, the drawings that are used in the embodiments will be briefly described below.

FIG. 1 shows the experimental results of the inhibition of beta-1, 6-glucan synthase KRE9 by antibacterial peptide CAD-5;

FIG. 2 shows the molecular docking results of the antibacterial peptide CAD-5 and beta-1, 6-glucan synthase KRE 9.

Description of the embodiments

The invention provides an antibacterial peptide CAD-5 with candida biofilm removal activity, wherein the amino acid sequence is NH2-HARKKKKRAH-COOH.

The antibacterial peptide CAD-5 provided by the invention contains 10 amino acids, and all the contained amino acids are L-type amino acids. Molecular weight 1258.55 daltons, isoelectric point 12.03, instability index 33.11, aliphatic amino acid index 20.00, total average hydrophilicity value-2.74, net charge = 7. In vitro antibacterial experiments show that the antibacterial peptide CAD-5 has broad-spectrum antibacterial activity, has better antibacterial effect on candida albicans, candida tropicalis, enterococcus and staphylococcus aureus clinical source strains, and has a Minimum Inhibitory Concentration (MIC) value of 3.92-6.82 mug/mL and a minimum concentration for inhibiting candida albicans biofilm formation of 37.71 mug/mL; of particular interest, the antimicrobial peptide CAD-5 had a minimum inhibitory concentration of 31.32 μg/mL for candida tropicalis recalcitrant bacteria and a minimum concentration of 125 μg/mL for removing biofilm formed by candida albicans. Both of these activities are better than fluconazole. The antibacterial mechanism experiment shows that CAD-5 targets candida cell wall beta-1, 6-glucan synthase KRE9, inhibits the beta-1, 6-glucan synthase activity of KRE9, blocks candida cell wall beta-1, 6-glucan synthesis, and damages candida cell wall integrity and biofilm. Meanwhile, the antibacterial peptide CAD-5 has no hemolysis.

In the present invention, the preparation method of the antibacterial peptide CAD-5 preferably comprises a polypeptide solid-phase synthesis method, and the method has no special requirement on the polypeptide solid-phase synthesis method, and adopts a method well known to a person skilled in the art.

In the present invention, amidation modification is preferably performed at the carbon end of the antibacterial peptide CAD-5. The modification method of the present invention is not particularly limited, and methods well known to those skilled in the art may be employed.

The invention provides application of the antibacterial peptide CAD-5 in preparation of medicines for resisting pathogenic bacteria infection.

In the present invention, the pathogenic bacteria preferably include fungi and/or bacteria; the fungi include one or more of candida albicans and candida tropicalis; the bacteria are one or more of enterococcus faecalis and staphylococcus aureus; the candida albicans comprises one or more of candida albicans planktonic, candida albicans forming a biological film and candida albicans forming a biological film; the candida tropicalis includes one or more of candida planktonic and candida tropicalis.

The invention provides an anti-infective medicament, and the effective components of the anti-infective medicament comprise the antibacterial peptide CAD-5 in the technical scheme.

In the invention, the minimum antibacterial concentration of the antibacterial peptide CAD-5 is 3.92-6.82 mug/mL, the minimum antibacterial concentration of the antibacterial peptide CAD-5 on intractable candida tropicalis is 31.32 mug/mL, the minimum concentration of candida albicans inhibiting biofilm formation is 37.71 mug/mL, and the minimum concentration of candida albicans eliminating biofilm formation is 125 mug/mL.

For further explanation of the present invention, the antibacterial peptide CAD-5 and its application provided in the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.

The antibacterial peptide CAD-5 sequence and the preparation thereof are as follows:

the target peptide is synthesized by adopting a solid-phase Fmoc method, and is synthesized from the C end to the N end.

(1) And (5) activating the resin. 0.5 g Rink Amide-AM resin was weighed with an analytical balance and added to a solid phase polypeptide synthesis tube, 5 mL Dichloromethane (DCM) was added thereto, the synthesis tube was sealed, shaken up and down, and deflated 3 times. The solid-phase polypeptide synthesis tube was spun on a spin shaker, spun 8 h, to allow full activation of the Rink Amide-AM resin to swell, and then the solvent and soluble reagents were removed under vacuum with a vacuum pump.

(2) Fmoc protecting group removal and Kaiser test detection: to the activated Resin-NH-Fmoc solid phase polypeptide synthesis tube was added 5 mL of 20% methylpiperidine/N, N-Dimethylformamide (DMF) deprotection solution, and the reaction was carried out for 30 min to remove Fmoc protection. Vacuum reduced pressure suction filtration is carried out to remove the deprotection liquid. The resin was cross-rinsed with N, N-Dimethylformamide (DMF) and Dichloromethane (DCM) solution, vacuum filtered under reduced pressure until no solution flowed out, three times each, the last time until colorless liquid flowed out, and the filtration was continued for 1 min to ensure adequate draining of the solution. A small amount of resin was added to a glass tube (10 mm. Times.100 mm), kaiser test 30. Mu.L was added, and the mixture was placed in a 110℃heating metal bath hole and heated for 1 min. A positive Kaiser test (dark blue) shows that free amino groups exist, and Fmoc protecting groups are successfully removed to obtain Resin-NH2.

(3) Resin-CAD-5 synthesis: 1-Hydroxybenzotriazole (HOBT) (2.33 mmol,3 eq) and Fmoc-L-histidine (Fmoc-His (Trt) -OH) (2.33 mmol,3 eq) were dissolved in 4 mL of N, N-Dimethylformamide (DMF), vortexed and thoroughly dissolved, then all added to a solid-phase polypeptide synthesis tube containing 0.5 g Resin-NH2, followed by 200. Mu. L N, N' -Diisopropylcarbodiimide (DIC) (2.33 mmol,3 eq). Sealing the synthetic tube, shaking up and down, and deflating for 3 times. And (3) carrying out Kaiser test on the solid-phase polypeptide synthesis tube in a rotary shaking table, and carrying out rotary reaction 3 h, wherein the detection of the Kaiser test is negative (colorless) to indicate that Fmoc-His (Trt) -OH has been successfully coupled, so as to obtain Resin-NH-His-Fmoc. The solvent such as couplant, uncoupling Fmoc-His (Trt) -OH, etc. was removed in vacuo. The resin was cross-washed with N, N-Dimethylformamide (DMF) and Dichloromethane (DCM), the residual material was eluted, vacuum filtered under reduced pressure until no solution was allowed to flow out, three times each, the last time until colorless liquid was allowed to flow out, and suction filtration was continued for 1 min to ensure adequate draining of the solution. 5 mL of 20% methylpiperidine/N, N-Dimethylformamide (DMF) deprotection solution was added to the polypeptide synthesis tube and reacted for 30 min to remove Fmoc protection. Vacuum reduced pressure suction filtration is carried out to remove the deprotection liquid. The resin was cross-rinsed with N, N-Dimethylformamide (DMF) and Dichloromethane (DCM) solution, vacuum filtered under reduced pressure until no solution flowed out, three times each, the last time until colorless liquid flowed out, and the filtration was continued for 1 min to ensure adequate draining of the solution. A small amount of resin was added to a glass tube (10 mm X10-100 mm), 30. Mu.L of Kaiser test was added, and the mixture was placed in a metal bath hole heated at 110℃for 1 min. A positive Kaiser test (dark blue) shows that free amino groups exist, and Fmoc protecting groups are successfully removed to obtain Resin-NH-His (Trt) -NH2.

The steps are repeated to couple amino acids such as Fmoc-Ala-OH, fmoc-Arg (Pbf) -OH, fmoc-Lys (Boc) -OH, fmoc-Arg (Pbf) -OH, fmoc-Ala-OH, fmoc-His (Trt) -OH and the like in sequence. Finally, the Fmoc group at the last amino acid end was removed with 20% piperidine to give Resin-NH-His (Trt) -Ala-Arg (Pbf) -Lys (Boc) -Lys (Boc) -Lys (Boc) -Arg (Pbf) -Ala-His (Trt) -NH2.

(4) Antibacterial peptide cleavage. To a solid-phase polypeptide synthesis tube containing Resin-NH-His (Trt) -Ala-Arg (Pbf) -Lys (Boc) -Arg (Pbf) -Ala-His (Trt) -NH2 was added a4 mL lysis solution (trifluoroacetic acid/dimethylresorcinol/triisopropylsilane/thioanisole/water/phenol/=35:1:1:2:2:3V/V), the synthesis tube was sealed, shaken up and down, and deflated 3 times, and the solid-phase polypeptide synthesis tube was placed in a heated shaker for reaction at 37 ℃ and 250 rpm 2 h. The cut solution was then collected in a 50 mL centrifuge tube, followed by washing the resin twice with 2 mL trifluoroacetic acid and collecting the wash solution to the centrifuge tube. 3 mL cleavage liquid was again added to the solid-phase polypeptide synthesis tube. The above operation is repeated. Adding the filtrate into glacial ethyl ether for precipitation to obtain crude antibacterial peptide, and shaking the filtrate and the glacial ethyl ether in a volume ratio of 1:5, so that floccules are separated out of the antibacterial peptide and the antibacterial peptide is shaking the filtrate and the glacial ethyl ether. And (3) placing the centrifuge tube at the temperature of-20 ℃ for 10 min, and fully separating out the antibacterial peptide crude product. And then centrifuged at 3500 rpm for 5min. Removing the supernatant, adding glacial ethyl ether, vortex shaking for full washing, and repeating the steps for two times. Concentrating under reduced pressure by using a rotary evaporator to obtain a white crude product of the CAD-5 antibacterial peptide. The crude product obtained was purified by HPLC.

The antibacterial peptide CAD-5 antibacterial spectrum is measured, and the specific steps are as follows:

(1) Weigh and solubilize the protein. CAD-5 phosphoric acid solution was prepared at a first column initial concentration of 500. Mu.g/mL and a final concentration of 0.975. Mu.g/mL. Then 2mg/mL of CAD-5 stock solution was prepared. CAD-5 was dissolved in 20 mmol/L sodium phosphate buffer pH6.0 and the solution was filter sterilized with a 0.22 μm sterile filter.

(2) mu.L of 20 mmol/L sodium phosphate buffer pH6.0 was added to each well of the 96-well plate using a pipette, respectively, for dilution of CAD-5.

(3) 100. Mu.L of 2mg/mL SAMP1-A4 stock was pipetted into each well of the first column of 96-well plates.

(4) And repeatedly blowing and sucking the solution in the first row of the plate for 6-8 times, uniformly mixing, and avoiding splashing.

(5) And sucking 100 mu L from the first row, adding the mixture into the second row, repeatedly blowing and sucking the mixture for 6-8 times, and then sucking 100 mu L to the third row. This step is repeated to the tenth column.

(6) The 100. Mu.L aspirated from the tenth column was discarded without adding column 11, and column 11 was the negative control well.

(7) And respectively adding 100 mu L of bacterial suspension into the 1 st column to the 11 th column of the 96-well plate in sequence, repeatedly blowing and sucking for 6-8 times, and uniformly mixing. Note that the bacterial suspension is not added to column 12. Column 12 is blank wells.

(8) And (5) standing and incubating the 96-well plates. 48 h in the case of fungi at 30 ℃; in the case of bacteria, the culture is carried out at 37℃under 16 h.

(9) mu.L of 5/mg/mL MTT solution was added to each well and the incubation was continued for 4 h. The supernatant was pipetted, 100. Mu.L of dimethyl sulfoxide (DMSO) was added, the mixture was left at 37℃for 20 min and shaken from time to time, and after the crystals were completely dissolved, the OD was measured at 490 nm by an ELISA reader.

Antibacterial ratio (%) = [ (OD 570 (sample) -OD570 (blank) ]/[ OD570 (negative) -OD570 (blank) ]x100.

MIC ₁₀₀ The definition is as follows: the minimum concentration of CAD-5 is reached when the bacteriostasis rate reaches 99.9 percent.

(10) Each experiment was repeated three times.

The results are shown in Table 1. As shown by the results, CAD-5 has the strongest activity on enterococcus faecalis and Staphylococcus aureus, and the 99.9% antibacterial concentration is 3.95 mug/mL; the 99.9% inhibition concentrations for candida albicans and candida tropicalis were 6.36 μg/mL and 6.82 μg/mL.

The candida tropicalis single colony is selected and cultured in an SD liquid culture medium in a constant temperature culture shaking table at 28 ℃ and 180 rpm until the candida tropicalis single colony reaches the logarithmic phase. The culture broth was diluted to 1X 108 CFU/mL with SD. 100. Mu.L of the bacterial suspension was transferred to a 96-well plate and incubated at 37℃for 24 h. 100. Mu.L of fluconazole (1 XMIC) was added to the bacterial suspension, and the mixture was incubated at 37℃for 24 h. Washing with PBS for 3 times, removing the attached adhesion, and performing ultrasound for 5min to obtain intractable candida tropicalis. According to example 2: the minimum inhibitory concentration of the antibacterial peptide CAD-5 on the candida tropicalis is measured by the antibacterial peptide CAD-5 antibacterial spectrum measuring method, and the result is shown in table 2. The results show that: the antibacterial peptide CAD-5 has the 99.9% inhibition concentration of 31.32 mug/mL on the intractable candida tropicalis and has stronger activity than fluconazole.

The effect of the antimicrobial peptide CAD-5 on candida albicans and candida tropicalis biofilm formation was evaluated using crystal violet staining and 96-well microtiter plate methods. The method comprises the following specific steps:

the candida single colony is selected in SD liquid culture medium, and is cultivated at constant temperature of 28 ℃ and 180 rpm until the logarithmic phase. Then diluted to 1X 10 with RPMI 1640 medium containing 10% fetal bovine serum ⁶ CFU/mL. Then, 100. Mu.L of the bacterial suspension was added to a 96-well microtiter plate containing serial double dilutions of the polypeptide solution and incubated at 28℃for 24 h. The cells were washed 3 times with PBS (20 mM, pH 6.0) to remove non-adherent cells. Then, 100. Mu.L of methanol was added to fix the sample. 15 After min, the methanol is discarded and dried at room temperature (i.e. methanol in the roomAfter complete evaporation at temperature), it was stained with 100. Mu.L of crystal violet (0.1%) for 5min and washed with sterile water. Then 120. Mu.L of 95% ethanol was added to dissolve the staining solution for 30 min. Absorbance (OD) at 590 nm wavelength was measured with a multi-function microplate fluorescence reader. Candida tropicalis fluid without polypeptide treatment was used as a negative control group, and each experiment was repeated three times. The clinical antifungal agent fluconazole is used as a positive control. Minimum antimicrobial peptide concentration inhibiting formation of 50% biofilm (minimum biofilm inhibitory concentration, MBIC ₅₀ ) To evaluate the ability of the antimicrobial peptides to inhibit biofilm formation. The results are shown in Table 2. The result shows that the antibacterial peptide CAD-5 can inhibit the formation of candida biofilm, wherein the antibacterial peptide CAD-5 has a strong inhibition effect on the formation of candida albicans biofilm.

The ability of the antimicrobial peptide CAD-5 to clear Candida biofilm was assessed using crystal violet staining and 96 well microtiter plate methods. Briefly, a single colony of Candida is selected, inoculated in SD liquid medium, incubated at 28℃and 180 rpm to logarithmic phase, and diluted to 1X 10 with RPMI 1640 medium containing 10% fetal bovine serum ⁶ CFU/mL was inoculated into 96-well plates at a volume of 100. Mu.L per well, and incubated at 28℃for 24 h. Thereafter, the cells were removed by washing 3 times with PBS buffer (20 mM, pH 6.0) to obtain biofilm-forming Candida. 100. Mu.L of the continuous double-diluted polypeptide solution was added to the biofilm-formed 96-well plate, and incubated 24. 24 h in a constant temperature incubator shaker (28 ℃,100 rpm/min). Then, the sample was washed three times with 200. Mu.L of PBS buffer (20 mM, pH 6.0), and stained with 100. Mu.L of 0.1% crystal violet for 15 min. Wash with sterile water, dissolve with 95% ethanol, and determine absorbance (OD) at 590 nm wavelength. Bacterial solutions without polypeptide treatment were used as negative control groups, and each experiment was repeated three times. The clinical antifungal agent fluconazole is used as a positive control. The minimum concentration (minimum biofilm reduction concentration, MBRC 50) that cleared 50% of formed biofilm was used to evaluate the antimicrobial peptides for clear biofilm activity. The results are shown in Table 3. The results show that: CAD-5 scavenging Candida has formed a biofilm with higher activity than fluconazole, with the strongest scavenging activity on Candida albicans biofilm.

100 μl of 2% human red blood cell suspension was pipetted into a 96-well plate. 100 μl of CAD-5 solution was added to each well to give final CAD-5 concentrations of 250, 125, 62.5, 31.25, 15.6, 7.8, 3.9, 1.9, 0.45 and 0.23 μg/mL, respectively, and mixed well, and each concentration was repeated three times. After incubation at 37 ℃ for 30 min, the supernatant from each well was removed and transferred to a new 96-well microtiter plate and absorbance a of the sample was measured using a microplate reader at 570 nm. A negative control (0% hemolysis) was made of a suspension of human erythrocytes without CAD-5 and a positive control (100% hemolysis) was made of a suspension of human erythrocytes with 1% Triton X-100. The haemolysis rate was calculated.

The calculation formula of the hemolysis rate: percent hemolysis (%) = [ (sample a 570-negative control a 570)/(positive control a 570-negative control a 570) ] ×100%

The results are shown in Table 4. As a result, the CAD-5 concentration was 1062.90. Mu.g/mL at a hemolysis rate of 5%, which was higher than that of fluconazole. The result shows that the antibacterial peptide CAD-5 has high biological safety.

The CtKRE9 glucan synthase activity was evaluated by measuring the consumption of glucose with glucose as a substrate and a DNS reagent as a color developing agent.

First, a glucose solution standard curve is established. Briefly, glucose solutions of different concentrations (80. Mu.g/mL, 160. Mu.g/mL, 240. Mu.g/mL, 320. Mu.g/mL, 400. Mu.g/mL, 480. Mu.g/mL) were prepared. And respectively taking 2. 2 mL to-be-detected liquids with different concentrations, adding 1.5 mL of DNS reagent, carrying out water bath at 100 ℃ for 10 min, cooling, adding deionized water to a volume of 25 mL, and measuring the absorbance (OD value) at the wavelength of 540 nm by using a multifunctional microplate fluorescence reader. And drawing a standard curve by taking the glucose concentration as an abscissa and the absorbance as an ordinate.

Candida tropicalis was then inoculated into YPD liquid medium and cultured at 32 ℃ for 18 h. The cells were collected, washed twice with Tris-HCl (50 mM, pH 7.5) and sonicated in an ice bath. Collecting supernatant, precipitating with ethanol to obtain precipitate as CtKRE9 crude product, and freezing at-80deg.C for storage. Then, an equal volume of the antimicrobial peptide was added to a 2 mL enzyme solution (10. Mu.g/mL) in which the concentration of the antimicrobial peptide CGA-N12 and its structural analogs were 1 XMIC and the control was 2 mL deionized water. Both the experimental and control groups were incubated at 40℃for 10 min. After the incubation, 1 mL mixture was added with 1 mL glucose solution (20 mg/mL) and incubation was continued for 30 min at 40 ℃. After the reaction was completed, 1.5 mL of DNS reagent was added, water was used for 10 min at 100 ℃, deionized water was added to fix the volume to 25 mL after cooling, and absorbance (i.e., OD value) at 540, 540 nm wavelength was measured with a multi-functional microplate fluorescence reader. The amount of glucose consumed per unit time was calculated from the glucose standard curve. Each experiment was repeated three times.

With the glucose concentration on the abscissa and the absorbance at 540, 540 nm on the ordinate, we plotted a standard curve of glucose solution concentration versus absorbance (fig. 1A). Obtaining a linear regression equation: y= 0.0006898 ×x+ 0.01690, and the correlation coefficient R2 is 0.9996.

CtKRE9 protein as cell wall beta-1, 6-glucan synthase can consume glucose. The effect of CAD-5 on CtKRE9 protein can be expressed on glucose consumption (FIG. 1B). The results indicate that CAD-5 inhibited CtKRE9 protease activity and glucose consumption was reduced.

Using DS4.5 software, a rigid docking method was selected to molecularly dock CAD-5 with KRE9 protein.

In the DS4.5 software platform, CAD-5 molecular docking with KRE9 protein model was performed using the ZDOCK and RDOCK programs based on the fast Fourier transform technique, and the docking results were analyzed. RDOCK provides a set of optimization and scoring of docking protein poses generated by the ZDOCK algorithm, a rigid docking method using CHARMm minimization. In order to obtain the optimal binding posture of the polypeptide and the protein, ZRANK is also taken as a parameter, and the molecular docking results can be ordered according to detailed parameters such as electrostatic acting force, van der Waals acting force, desolventizing energy and the like. The interaction interface and potential binding sites in the optimal binding posture of the antibacterial peptide CAD-5 to the KRE9 protein were analyzed. The optimal docking posture of the antibacterial peptide CAD-5 and KRE9 protein is finally obtained, as shown in figure 2A. The type of force between CAD-5 and KRE9 binding sites is shown in FIG. 2B. The docking results show that CAD-5 interacts with KRE9 protein through hydrogen bonding, hydrophobic and Van der Waals forces, salt bridge forces, and other forces.

In conclusion, the antibacterial peptide CAD-5 provided by the invention has broad-spectrum antibacterial activity and low hemolysis, has a specific inhibition effect on the activity of beta-1, 6-glucan synthase KRE9, is expected to become a novel broad-spectrum antibacterial candidate drug, and has a good application prospect in the aspect of resisting candida chronic infection.

Although the foregoing embodiments have been described in some, but not all, embodiments of the invention, it should be understood that other embodiments may be devised in accordance with the present embodiments without departing from the spirit and scope of the invention.

Claims

1. An antibacterial peptide CAD-5 with candida biofilm removal activity and application thereof, which is characterized in that: the amino acid sequence is NH ₂ -HARKKKKRAH-COOH。

2. The antimicrobial peptide CAD-5 of claim 1, wherein the carboxy terminus of the antimicrobial peptide CAD-5 is amidated modified.

3. Use of the antibacterial peptide CAD-5 according to claim 1 or claim 2 for the preparation of an anti-infective medicament.

4. The use of claim 3, wherein the infection comprises a fungal infection and a bacterial infection.

5. According to claim 4The application is characterized in that: the fungus is candida albicansCandida albicans) And Tropical candidaCandida tropicalis）。

6. The use according to claim 4, characterized in that: the bacteria are enterococcus faecalisEnterococcus faecalis) And staphylococcus aureus @ sStaphylococcus aureus）。

7. The use according to claim 5, characterized in that: the candida albicans include candida planktonic, candida albicans forming a biofilm and candida albicans forming a biofilm; tropical candida include candida planktonic and candida intractable.

8. An anti-infective drug, characterized in that the active ingredient of the anti-infective drug comprises the antibacterial peptide CAD-5 of claim 1 or 2.

9. The anti-infective drug of claim 8, wherein the antimicrobial peptide CAD-5 has a minimum inhibitory concentration of 3.92-6.82 μg/mL, a minimum inhibitory concentration of 31.32 μg/mL for refractory candida tropicalis, a minimum concentration of 37.71 μg/mL for candida albicans inhibiting biofilm formation, and a minimum concentration of 125 μg/mL for candida albicans scavenging biofilm formation.