Antibacterial peptide AAP-2 and application thereof

Abstract

The present application relates to a kind of mylabris antifungal peptide and its application in antibacterial group bacteria.The new antibacterial peptide AAP-2 sequence is obtained by comparing the sequence measured from mylabris transcriptome, and the mature peptide is chemically synthesized in vitro, and the synthesized antibacterial peptide can effectively inhibit the proliferation of bacterial group bacteria, and the minimum inhibitory concentration is 25 μg / mL.The antibacterial peptide involved in the present application is a newly identified antibacterial peptide, which has the advantages of small toxicity, not easy to produce drug resistance, etc., and is expected to be developed into an antibiotic synergist or an alternative antibiotic treatment for walnut black spot, to reduce the harm caused by chemical antibacterial agent or antibiotic residue.

Description

Technical Field

[0001] This invention relates to the discovery of an antimicrobial peptide from armyworms and its application in combating pantothecin, particularly to a novel antimicrobial peptide AAP-2 derived from armyworms, and also to the in vitro synthesis of this antimicrobial peptide and its inhibitory effect and mechanism against pantothecin. This invention belongs to the field of antimicrobial peptide technology. Background Technology

[0002] In my country, bacterial black spot disease of walnuts is mainly caused by Pantotheca acuminata (Pantotheca cum Caulis). Pantoea agglomerans Caused by pathogens such as [unspecified pathogens], this disease leads to leaf perforation and shedding, and fruit rot and deformity, significantly reducing the commercial value and economic benefits of walnuts. It has become one of the main bottlenecks restricting the high-quality development of the walnut industry. Currently, the prevention and control of walnut black spot disease still mainly relies on chemical fungicides. However, long-term irrational use has led to increasing resistance year by year, and has also caused pollution of the soil, water bodies, and other ecological environments, threatening the quality and safety of agricultural products and human health. At the same time, it contradicts the concept of green development in modern agriculture, and there is an urgent need to explore safe, efficient, and environmentally friendly alternative prevention and control technologies.

[0003] Antimicrobial peptides, as a class of small molecule polypeptides with broad-spectrum antimicrobial activity, are widely distributed and possess significant advantages such as unique antimicrobial mechanisms, low susceptibility to drug resistance induction, and environmental friendliness, making them a hot topic in the research and development of novel green biological fungicides. In recent years, research on the application of antimicrobial peptides in the control of plant bacterial diseases has been continuously deepening. They exert their antimicrobial effects through multiple mechanisms, such as disrupting the integrity of pathogen cell membranes, inhibiting nucleic acid synthesis, or interfering with metabolic processes. Their multi-target action makes them less prone to drug resistance, providing new ideas and approaches for the green control of walnut black spot disease.

[0004] In recent years, research on the pathogens causing walnut black spot disease has largely focused on pathogen isolation and identification, pathogen genomics and proteomics studies, and fungicide screening. There has been little research on the pathogenic mechanisms, interaction mechanisms, and other molecular biological studies, as well as conventional research on green control methods. Based on this, this study screened a novel antimicrobial peptide, AAP-2, which exhibits good inhibitory activity against *Pantotheca acuminata*, and explored its antimicrobial mechanism, providing a new reference method for the green control of walnut black spot disease. Summary of the Invention

[0005] In response to the problems existing in the prior art, the inventors discovered a new antimicrobial peptide, AAP-2, in their research on armyworms and prepared it chemically in vitro, hoping that it could effectively inhibit the clustered pan-bacteria that cause black spot disease in walnuts.

[0006] The technical solution of the present invention is as follows: An antimicrobial peptide AAP-2, the amino acid sequence of which is shown in SEQ ID No. 3; the antimicrobial peptide AAP-2 is a mature antimicrobial peptide, the amino acid sequence of its precursor peptide before cleavage is shown in SEQ ID No. 2; the gene sequence encoding the precursor peptide is shown in SEQ ID No. 1.

[0007] The antimicrobial peptide AAP-2 can be prepared by in vitro chemical synthesis. The synthesized antimicrobial peptide mainly inhibits the formation of pantothenic bacteria by increasing the permeability of the cell membrane of the clustered pantothenic bacteria, causing leakage of intracellular macromolecules, and inhibiting their energy metabolism.

[0008] The antimicrobial peptide AAP-2 can effectively inhibit the proliferation of Pantotheca clumps, with a minimum inhibitory concentration of 25 µg / mL against Pantotheca clumps. It can be used for the prevention and control of walnut black spot disease caused by Pantotheca clumps.

[0009] Beneficial effects: 1. This invention is the first to identify the antimicrobial peptide AAP-2, providing a novel antimicrobial peptide; 2. This antimicrobial peptide can be prepared by in vitro chemical synthesis, which has the advantages of simple and rapid preparation, providing support for the in vitro chemical synthesis of antimicrobial peptides in the future; 3. The antimicrobial peptide involved in this invention can effectively inhibit the proliferation of clustered pantothecin, and is expected to replace the use of antibiotics in the prevention and control of walnut black spot disease, reducing the environmental pollution caused by antibiotics; 4. This invention explores the antimicrobial mechanism of action of this antimicrobial peptide, providing a reference for the field application of antimicrobial peptides. Attached Figure Description

[0010] Figure 1 The gene sequence, amino acid sequence, and cleavage site of the mature peptide precursor of the antimicrobial peptide of the present invention are shown below. The underlined part is the signal peptide sequence, the boxed part is the amino acid residues that are cleaved after translation, and the remaining 40 amino acids are the mature peptide sequence.

[0011] Figure 2 Mass spectrum of the chemically synthesized novel antimicrobial peptide after removing the nuclear localization signal region.

[0012] Figure 3 High-performance liquid chromatography (HPLC) chromatogram of the novel antimicrobial peptide after chemical synthesis with nuclear localization signal region removed.

[0013] Figure 4 The results of the minimum inhibitory concentration (MIC) of the new antimicrobial peptide against pantothecin (A) and the results of the inhibition curve determination (B) are shown.

[0014] Figure 5 The study investigated the effects of the new antimicrobial peptide on the leakage of alkaline phosphatase (A) and β-galactosidase (B) in clustered pantothenia, the results of PI staining (C), and the ultrastructure of clustered pantothenia (D).

[0015] Figure 6Results of leakage of the novel antimicrobial peptide into clustered pantothenic nucleic acid (A) and protein (B), and results of its interaction with clustered pantothenic DNA (C).

[0016] Figure 7 The results show the effects of the novel antimicrobial peptide on oxidative stress (A) and energy metabolism (B) in clustered pantothenic bacteria. Detailed Implementation

[0017] The present invention will now be described in detail with reference to the accompanying drawings.

[0018] The main raw materials used in the following embodiments are as follows: the chemical synthesis and detection of the antimicrobial peptides were completed by Jier Biochemical (Shanghai) Co., Ltd. Pantotheca agglomerata was donated by the Henan Academy of Forestry Sciences and is preserved in the inventor's laboratory. Other reagents and materials, such as resazurin, are all commercially available. Unless otherwise specified, the experimental methods described for other reagents and materials are standard experimental methods.

[0019] 1.1 Discovery of novel antimicrobial peptides The novel antimicrobial peptide provided by this invention is derived from the armyworm. The gene sequence of this antimicrobial peptide was discovered by comparing sequences obtained from armyworm transcriptome sequencing. The sequence was obtained through the following method: using the armyworm transcriptome as a database, the cDNA sequence of the ORF of the novel antimicrobial peptide involved in this invention was found using BLASTn screening based on identified antimicrobial peptide sequences, and the amino acid sequence was deduced. Based on a comprehensive analysis of literature reports, it was found that the antimicrobial peptide contains a conserved cleavage site AP, thus obtaining the mature antimicrobial peptide sequence. Figure 1 As shown, the gene encoding the novel antimicrobial peptide precursor is 195 bp in length (SEQ ID No. 1). The precursor contains 64 amino acid residues (SEQ ID No. 2). Compared with similar antimicrobial peptides, this antimicrobial peptide contains a conserved cleavage site. The mature antimicrobial peptide after cleavage has 40 amino acid residues (SEQ ID No. 3). This mature antimicrobial peptide is named AAP-2.

[0020] 1.2 Preparation of novel antimicrobial peptides The obtained mature antimicrobial peptide sequence was sent to Jier Biochemical (Shanghai) Co., Ltd. for synthesis. Mass spectrometry and high-performance liquid chromatography analysis revealed that the mature peptide could be chemically synthesized in vitro with a purity of up to 98%. Figure 2 and 3 As shown, based on the effective sequence of the cleaved antimicrobial peptide, the in vitro chemically synthesized peptide sequence, and the results of mass spectrometry and chromatography analysis, the chemically synthesized peptide is consistent with the mature peptide sequence containing 40 amino acid residues.

[0021] 1.3 Determination of minimum inhibitory concentration and inhibition curve The initial concentration of AAP-2 was 400 μg / mL, diluted two-fold with 0.9% NaCl. 50 μL was added to each well of a 96-well plate, followed by another 50 μL of approximately 2 × 10⁻⁶ NaCl. 6 CFU / mL of clumped pantothenic acid bacterial suspension was used, with NaCl + bacterial suspension as a negative control and ampicillin + bacterial suspension as a positive control. The experiment was repeated three times. Incubation was performed at 37°C for 16–18 h, with 10 μL of 0.1 mmol / L resazurine solution added to each well, followed by further incubation in the dark for 2–4 h. The minimum AAP-2 concentration at which no bacterial growth (the solution remains blue) was defined as the MIC. Figure 4 As shown in Figure A, the minimum inhibitory concentration (MIC) was determined using the serial dilution method, and the MIC was 25 μg / mL.

[0022] The antimicrobial curve was determined using the bacterial plate count method: Clumps of pantothenia in the logarithmic growth phase were collected by centrifugation and diluted with sterile LB liquid medium to a concentration of 2 × 10⁻⁶. 6 CFU / mL, 400 μL of bacterial culture was mixed with an equal volume of AAP-2 solution. The final concentrations of AAP-2 were 1 MIC (25 μg / mL), 2 MIC (50 μg / mL), and 4 MIC (100 μg / mL). 0.9% sterile NaCl was used as a blank control. The culture was incubated at 37℃ in a shaker. At 0, 2, 4, 6, 8, 10, 12, and 24 h, 100 μL of the culture was placed in each well of a 96-well plate, and the D600 value was measured using a multi-functional microplate reader. The results are as follows: Figure 4 As shown in B. The results indicate that AAP-2 has good antibacterial activity against Pantotheca clumps, and 1 MIC of AAP-2 can completely inhibit the growth of Pantotheca clumps within 24 hours.

[0023] 1.4 Effects of antimicrobial peptide AAP-2 on the cell membrane of clustered pantothenic bacteria Take 1 mL of *Pantheraea perfringens* cultured to the logarithmic growth phase, centrifuge at 12000 rpm for 1 min, discard the supernatant, wash twice with sterile PBS and resuspend, and divide into two groups: one group was treated with AAP-2 to a final concentration of 4 MIC, and the other group served as a control group treated with an equal volume of PBS. Place both groups of culture in a 37℃ water bath for 1 h. Centrifuge at 12000 rpm for 1 min, remove the supernatant, add 1 mL of electron microscopy fixative, and send to Wuhan Saiwei Biotechnology Co., Ltd. for sample preparation. Observe and photograph under a scanning electron microscope. The results are as follows: Figure 5 As shown in D.

[0024] Collect the logarithmic growth phase of *Pantheraea perlatum* bacterial suspension, centrifuge at 12000 rpm for 1 min, discard the supernatant, wash with sterile PBS and resuspend. Divide the resuspended bacterial suspension into 5 tubes, 1 mL in each tube, and add antimicrobial peptide AAP-2 sequentially to final concentrations of 0 MIC (blank control), 1 MIC, 2 MIC, and 4 MIC, respectively. Use Triton at a final concentration of 0.3% as a positive control. After mixing all samples, incubate at 37℃ for 1 h. Centrifuge to collect the supernatant, add it to a 96-well plate, add chromogenic buffer, mix well, and incubate at 37℃ for 0.5 h. Stop the reaction by adding stop solution. Measure the absorbance of each well at 405 nm using a microplate reader. The results are as follows: Figure 5 As shown in Figure A. The supernatant was added to a new 96-well plate, followed by 4.5 μL of ONPG at a final concentration of 3 mmol / L. The mixture was shaken and incubated at 37°C for 0.5 h. The D420nm value was then measured. The entire process was performed in the dark. The results are shown in Figure A. Figure 5 As shown in B.

[0025] Collect *Pantheraea perlatum* bacterial suspension in the logarithmic growth phase, centrifuge at 12000 rpm for 1 min, discard the supernatant, wash with sterile PBS and resuspend. Divide the resuspended bacterial suspension into 5 tubes, 1 mL in each tube, and add antimicrobial peptide AAP-2 sequentially to final concentrations of 0 MIC (blank control), 1 MIC, 2 MIC, and 4 MIC, respectively. Use Triton at a final concentration of 0.3% as a positive control. After mixing all samples, incubate at 37℃ and 220 rpm in a constant temperature shaking incubator. Take 100 μL samples at 0, 0.5, 1, 2, and 4 h of incubation, add 10 μL of PI staining solution at a final concentration of 5 μg / mL, and incubate at 37℃ for 30 min in the dark. Add 100 μL of each sample to a 96-well plate, and detect the fluorescence intensity at 615 nm emission wavelength and 535 nm excitation wavelength using an automated multi-functional microplate reader. The results are as follows: Figure 5 As shown in C.

[0026] Figure 5 In this study, transmission electron microscopy was used to observe and evaluate the overall effect of AAP-2 on *Pantotheca clumps*. A multi-functional microplate reader was used to detect the leakage of alkaline phosphatase and β-galactosidase within *Pantotheca clumps* cells, exploring the effect of AAP-2 on *Pantotheca clumps* cell membrane permeability. PI staining was used to evaluate *Pantotheca clumps* cell permeability. The results showed that 4MIC AAP-2 treatment for 1 hour caused significant swelling and deformation of *Pantotheca clumps* cells, cell membrane rupture and collapse, and a significant increase in cell permeability.

[0027] 1.5 Results of AAP-2 on leakage of nucleic acid and protein from pantothenic agglomerates and its interaction with pantothenic agglomerate DNA The overnight cultured *Pantotheca acuminata* bacterial suspension was centrifuged at 12000 rpm for 1 min, the supernatant was discarded, and the suspension was resuspended after washing with sterile PBS. The suspension was divided into four 1.5 mL tubes, and AAP-2 was added sequentially to achieve final concentrations of 0 MIC (control group), 1 MIC, 2 MIC, and 4 MIC. After thorough mixing, the samples were incubated in a 37°C water bath. At 0, 1, 2, 3, 4, 5, and 6 h, 100 μL of the supernatant was collected by centrifugation. The D260 nm and D280 nm values ​​in the supernatant were measured using a micro-ultraviolet spectrophotometer. The results are as follows: Figure 6 As shown in A and 6B.

[0028] Take the logarithmic growth phase of *Pantotheca acuminata* bacterial culture, centrifuge at 12000 rpm for 1 min, discard the supernatant, and extract *Pantotheca acuminata* DNA according to the genomic DNA extraction kit instructions. Measure the concentration using a micro-UV spectrophotometer and dilute 10-fold with TE-Buffer. Take five 1.5 mL centrifuge tubes, add 0.5 μg of DNA to each, and then add different amounts of AAP-2 to achieve DNA-to-DNA ratios of 0, 1, 2.5, 5, and 10, respectively. Mix well and incubate at 37 ℃ for 30 min. Perform 100 μL of the DNA solution on a 1.0% agarose gel electrophoresis and observe and photograph the results using a gel imaging system. The results are shown below. Figure 6 As shown in C.

[0029] Figure 6 In this study, the effects of AAP-2 on the leakage of nucleic acids and proteins from *Pantotheca agglomerata* were evaluated using a micro-spectrophotometer, and the effects of AAP-2 on intracellular DNA of *Pantotheca agglomerata* were evaluated by nucleic acid gel electrophoresis. The results showed that 1 MIC of AAP-2 significantly increased the permeability of *Pantotheca agglomerata* cells and caused significant leakage of intracellular macromolecules, but it did not bind to *Pantotheca agglomerata* DNA.

[0030] 1.6 Effects of AAP-2 on oxidative stress and energy metabolism in pantothenic agglomerates Clumps of pantothenic bacteria cultured to the logarithmic growth phase were centrifuged at 12,000 rpm for 1 min at room temperature. The bacterial pellet was collected, washed with sterile PBS, and resuspended. The pellet was divided into four groups, and AAP-2 was added to each group to achieve final concentrations of 0 MIC (blank control), 1 MIC, 2 MIC, and 4 MIC, respectively. The pellets were incubated in a 37°C water bath for 1 h, centrifuged at 4,000 rpm for 5 min, and the supernatant was discarded. The ATP concentration in the bacterial cells was determined according to the ATP assay kit instructions.

[0031] Clumps of *Pantotheca acuminata* cultured to the logarithmic growth phase were divided into four groups, with 1 mL of the solution in each group. A final concentration of 10 μmol / L of DCFH-DA fluorescent probe was added to the bacterial suspension in each group. After vortexing and mixing, the solution was transferred to a 37°C incubator and incubated in the dark for 1 h. Then, the same volume of AAP-2 was added to each group to achieve final concentrations of 0 MIC (blank control), 1 MIC, 2 MIC, and 4 MIC, respectively. The solutions were then incubated in a 37°C water bath for 1 h. 100 µL of the resulting bacterial suspension was then transferred to 96-well plates. Fluorescence intensity was measured using a multi-mode microplate reader at an excitation wavelength of 488 nm and an emission wavelength of 525 nm.

[0032] like Figure 7 As shown, the effects of AAP-2 on oxidative stress and energy metabolism in *Pantotheca cum Cubis* were detected using a multi-functional microplate reader. The results indicated that AAP-2 affects oxidative stress in *Pantotheca cum Cubis*, and 1 MIC of AAP-2 significantly reduced its ATP production.

Claims

1. An antimicrobial peptide AAP-2, characterized in that, The amino acid sequence of the antimicrobial peptide AAP-2 is shown in SEQ ID No.

3.

2. A precursor peptide, characterized in that, The amino acid sequence of the precursor peptide is shown in SEQ ID No.

2.

3. The precursor peptide as described in claim 2, characterized in that, The nucleotide sequence encoding this precursor peptide is shown in SEQ ID No.

1.

4. The application of the antimicrobial peptide AAP-2 as described in claim 1 in inhibiting clustered pantothenia.

5. The application of the antimicrobial peptide AAP-2 as described in claim 4 in inhibiting clustered pantothenia, characterized in that, The minimum inhibitory concentration of the antimicrobial peptide AAP-2 against pantothenia gravis is 25 µg / mL.

6. The application of the antimicrobial peptide AAP-2 as described in claim 1 in the prevention and control of walnut black spot disease.

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