Human nasal fluid alpha-2-macroglobulin antibacterial peptide A2M3 and application thereof

Abstract

The application discloses human nasal fluid alpha-2-macroglobulin antibacterial peptide A2M3 and application thereof, the amino acid sequence of the antibacterial peptide A2M3 is SGFIPLKPTVK, and the molecular weight of the antibacterial peptide A2M3 is 1186.45 Da.The antibacterial peptide A2M3 can produce a strong inhibitory effect on staphylococcus aureus, can be used for preparing medicines for treating or preventing diseases caused by staphylococcus aureus, feed additives, and can also be used for preparing food preservatives.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a human nasal α-2-macroglobulin antimicrobial peptide A2M3 and its applications. Background Technology

[0002] Staphylococcus aureus ( Staphylococcus aureus Staphylococcus aureus (S. aureus) is a clinically important Gram-positive pathogen, primarily existing in a symbiotic form with humans, capable of colonizing the skin and mucous membranes of both humans and animals. Most S. aureus infections are associated with colonization by indwelling medical devices, triggering a series of immune responses. In the United States alone, there are approximately 300,000 hospital discharges due to S. aureus infection. S. aureus is also a foodborne pathogen; consuming food contaminated with S. aureus can cause gastrointestinal illnesses, and in severe cases, can lead to meningitis, intestinal bleeding, and other fatal consequences. S. aureus has the ability to produce various toxins that can directly interact with the host, damaging its biofilm and causing cell death, such as staphylococcal enterotoxins (SEA, SEB, SEC, SED, SEE, SEG, SHE, and SEI), toxic shock syndrome toxin-1 (TSST-1), and leukocidins. These toxins are heat-resistant, surviving even after being killed by heat treatment, and they are also resistant to other environmental conditions such as low pH, dryness, and freezing. Currently, the main treatment for Staphylococcus aureus infections is the use of antibiotics, but this often leads to antibiotic resistance and has certain limitations. Therefore, there is an urgent need to find a novel method that is highly effective in inhibiting bacteria and is safe and does not induce drug resistance.

[0003] Antimicrobial peptides (AMPs) are a class of small peptides widely found in organisms in nature and are an important component of the body's innate immune system. They are characterized by good water solubility, low toxicity to higher animals, and no drug resistance, and are considered potential alternatives to antibiotics, showing promising applications in the pharmaceutical industry and food additives. The nasal cavity is a major accumulation site for staphylococci, such as Staphylococcus aureus and Staphylococcus hemolyticus. However, under normal circumstances, they maintain a state of equilibrium with the body, mainly because the body can secrete defensive factors, such as lysozyme, lactoferrin, and secretory leukocyte protease inhibitors (SLPIs), which are antimicrobial peptides. Host defense is a prominent function of nasal secretions. In fact, the nasal mucosa can directly respond to bacterial attacks through the production of cationic peptides, selectively destroying bacterial cell walls and membranes. This may also be one of the reasons why the body remains healthy even when infected with pathogens in the nasal cavity.

[0004] Therefore, providing an antimicrobial peptide that can effectively inhibit Staphylococcus aureus infection while maintaining good stability is a technical problem that urgently needs to be solved in this field. Summary of the Invention The purpose of this invention is to provide a human nasal α-2-macroglobulin antimicrobial peptide A2M3 and its application. By isolating and identifying the antimicrobial activity of antimicrobial peptide A2M3 against Staphylococcus aureus from nasal fluid, the invention aims to reduce the drug resistance of Staphylococcus aureus to antimicrobial drugs and reduce the use of antibiotics.

[0005] One of the technical solutions adopted by this invention to solve its technical problem is to provide an antimicrobial peptide A2M3 in human nasal secretions. Its amino acid sequence is SGFIPLKPTVK, as shown in SEQ ID NO: 1.

[0006] We first performed mass spectrometry on human nasal fluid and obtained and identified 1859 peptide sequences. Then, we used the Swiss-model server to predict the peptide structure and discovered an antimicrobial peptide sequence SGFIPLKPTVK with strong antibacterial activity against Staphylococcus aureus, which we named A2M3.

[0007] The antimicrobial peptide A2M3 has a molecular weight of 1186.454 Da, a charge of +2, and a total hydrophobicity of 36%.

[0008] Antimicrobial peptide A2M3 can damage bacteria in the following ways: First, the positive charge of antimicrobial peptide A2M3 can interact with the bacterial cell membrane, adsorb onto the surface of the bacterial cell membrane, and damage the cell membrane, leading to bacterial death. Second, while damaging the cell membrane, it also changes the permeability of the bacterial cell membrane and inhibits cell membrane formation, leading to bacterial death. Third, it can bind to bacterial genomic DNA, inhibiting bacterial DNA synthesis, thereby leading to bacterial death.

[0009] The second technical solution adopted by the present invention to solve its technical problem is: to provide the application of antimicrobial peptide A2M3 in human nasal secretions in the preparation of antimicrobial drugs, which are used to inhibit and / or kill Staphylococcus aureus.

[0010] The third technical solution adopted by the present invention to solve its technical problem is: to provide an antibacterial drug, the effective component of which includes human nasal α-2-macroglobulin antimicrobial peptide A2M3, wherein the amino acid sequence of the antimicrobial peptide A2M3 is SEQ ID NO: 1.

[0011] In a preferred embodiment of the present invention, the active ingredient of the antibacterial drug is nasal α-2-macroglobulin antimicrobial peptide A2M3, and the amino acid sequence of the antimicrobial peptide A2M3 is SEQ ID NO: 1.

[0012] In a preferred embodiment of the present invention, the antibacterial drug is used to inhibit and / or kill Staphylococcus aureus.

[0013] The fourth technical solution adopted by the present invention to solve its technical problem is: to provide a food preservative, the effective ingredient of which includes human nasal α-2-macroglobulin antimicrobial peptide A2M3, the amino acid sequence of antimicrobial peptide A2M3 being SEQ ID NO: 1.

[0014] In a preferred embodiment of the present invention, the active ingredient of the food preservative is human nasal secretion α-2-macroglobulin antimicrobial peptide A2M3, and the amino acid sequence of antimicrobial peptide A2M3 is SEQ ID NO: 1.

[0015] In a preferred embodiment of the present invention, the food preservative is used to inhibit and / or kill Staphylococcus aureus.

[0016] The fifth technical solution adopted by the present invention to solve its technical problem is: to provide a feed additive whose effective ingredient includes human nasal α-2-macroglobulin antimicrobial peptide A2M3, the amino acid sequence of antimicrobial peptide A2M3 being SEQ ID NO: 1.

[0017] In a preferred embodiment of the present invention, the active ingredient of the feed additive is human nasal secretion α-2-macroglobulin antimicrobial peptide A2M3, and the amino acid sequence of the antimicrobial peptide A2M3 is SEQ ID NO: 1.

[0018] In a preferred embodiment of the present invention, the feed additive is used to inhibit and / or kill Staphylococcus aureus.

[0019] The antimicrobial peptides of the present invention can be synthesized using methods known to those skilled in the art, such as solid-phase synthesis, and purified using methods known to those skilled in the art, such as high-performance liquid chromatography.

[0020] Implementing this invention has the following beneficial effects: This invention uses human nasal secretions as the research object. Through mass spectrometry identification and screening, a novel amino acid sequence peptide, A2M3, was discovered. The antibacterial activity of peptide A2M3 against Staphylococcus aureus was studied, and its antibacterial mechanism was investigated. Experimental results show that this peptide has a strong inhibitory effect on Staphylococcus aureus. Its antibacterial mechanism involves first adsorbing onto the bacterial surface, causing damage to the bacterial cell membrane, and then crossing the cell membrane to bind to the bacterial genomic DNA, inhibiting bacterial DNA synthesis, thereby leading to bacterial death. The antimicrobial peptide A2M3 of this invention can be used to prepare drugs, feed additives, or food preservatives for the prevention or inhibition of Staphylococcus aureus infection. Attached Figure Description

[0021] Figure 1 This is a mass spectrometry analysis chromatogram of the antimicrobial peptide A2M3 of the present invention.

[0022] Figure 2 This is a structural view of the predicted model of the antimicrobial peptide A2M3 of the present invention.

[0023] Figure 3 This is a comparison chart showing the determination of the minimum inhibitory concentration (MIC) of the antimicrobial peptide A2M3 of the present invention against Staphylococcus aureus.

[0024] A: The concentration of the antimicrobial peptide is 0 μg / mL; B: The concentration of the antimicrobial peptide is 1000.0 μg / mL; C: The concentration of the antimicrobial peptide is 500.0 μg / mL; D: The concentration of the antimicrobial peptide is 250.0 μg / mL; E: The concentration of the antimicrobial peptide is 125.0 μg / mL; F: The concentration of the antimicrobial peptide is 62.5 μg / mL.

[0025] Figure 4 This is a time-kill curve of the antimicrobial peptide A2M3 of the present invention against Staphylococcus aureus.

[0026] Dilute to 10 in 0.01M pH 7.2 phosphate buffer. 6-7 CFU / mL. The concentration of the antimicrobial peptide was 125.0 μg / mL.

[0027] Figure 5 This is a transmission electron microscope image showing observations of bacterial cell membranes. A: Staphylococcus aureus blank control group; B: Staphylococcus aureus after treatment with antimicrobial peptide A2M3 for 2 hours.

[0028] Figure 6 The change in fluorescence intensity of the antimicrobial peptide A2M3 of this invention on the PI of Staphylococcus aureus is shown.

[0029] Figure 7 This is a flow cytometry diagram of the antimicrobial peptide A2M3 of the present invention acting on Staphylococcus aureus.

[0030] Figure 8 This is an electrophoresis diagram of the binding of the antimicrobial peptide A2M3 of this invention to Staphylococcus aureus DNA, wherein... Band 7: Blank control; Bands 1-6: These represent A2M3 to DNA mass ratios of 200 / 1, 100 / 1, 50 / 1, 25 / 1, 25 / 2, and 25 / 4, respectively. Figure 9 This is the fluorescence spectrum of the antimicrobial peptide A2M3 of this invention competitively binding to DNA with EB. The fluorescence spectrum of the sample in the range of excitation wavelength 535 nm and emission wavelength 600~750 nm was measured using a multifunctional microplate reader. Detailed Implementation

[0031] To better understand the present invention, the present invention will be further described in detail below with reference to the embodiments and accompanying drawings. However, those skilled in the art will understand that the following embodiments are not intended to limit the scope of protection of the present invention, and any changes and variations made on the basis of the present invention are within the scope of protection of the present invention.

[0032] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0033] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0034] Example 1: Screening of antimicrobial peptide A2M3 First, a sample was taken from human nasal fluid. After centrifugation at 1.5 mL for 15 min, the supernatant was collected and filtered through a 3000 Da filter membrane. The sample was stored at -20°C and then further analyzed using a Nano Aquity UPLC system (Waters Corp).

[0035] Chromatographic conditions: Injection volume: 5.0 μl Chromatographic column: C18 analytical column, 25 cm in length and 75 μm in inner diameter. Mobile phase: A: 0.1% methanol aqueous solution B: Acetonitrile Using the search software MAXQUANT v1.6.5.0 and the database uniprot Homo sapiens (Human) protein library, the obtained peptide mass spectra were differentiated and identified, yielding 1859 peptides, such as... Figure 1 As shown. Using two online servers, APD3 and CAMP, to screen its sequence and predict the peptide structure using the Swiss-model server, an antimicrobial peptide with strong antibacterial activity against Staphylococcus aureus was discovered. The sequence is SGFIPLKPTVK, named A2M3, and the molecular weight is 1186.45 Da.

[0036] Example 2: 3D structure prediction of antimicrobial peptide A2M3 The structure of the antimicrobial peptide A2M3 was predicted using the online structure prediction server Swiss-model, and then edited and modified using Pymol software to obtain the secondary structure of the antimicrobial peptide A2M3, such as... Figure 2 As shown.

[0037] Example 3: Determination of the minimum inhibitory concentration (MIC) of antimicrobial peptide A2M3 Staphylococcus aureus was cultured at 37°C for 12 hours to reach the logarithmic growth phase, and then diluted to a final concentration of 10 μL in 0.01 M pH 7.2 phosphate buffer.6-7 CFU / mL. Antimicrobial peptide A2M3 was dissolved in phosphate buffer and incubated with an equal volume of Staphylococcus aureus at 37°C for 2 hours. The minimum inhibitory concentration (MIC) was defined as the lowest peptide concentration at which no significant growth was observed on the plate after overnight incubation at 37°C. Figure 3 As shown, the minimum inhibitory concentration (MIC) of antimicrobial peptide A2M3 against Staphylococcus aureus is 125.0 μg / mL.

[0038] Example 4: Time-kill curve determination of antimicrobial peptide A2M3 The time-bacterial killing curve of the antimicrobial peptide A2M3 was determined using the plate count method. Staphylococcus aureus was cultured at 37°C for 12 h to the logarithmic growth phase, and then diluted to 10⁻¹⁰ in 0.01 M pH 7.2 phosphate buffer. 6-7 CFU / mL. Equal volumes of 1×MIC and 2×MIC peptides were mixed with Staphylococcus aureus at 37°C and incubated separately. Samples were taken every 30 minutes and plated. After overnight incubation at 37°C, the total colony count was recorded. Figure 4 As shown, the antimicrobial peptide A2M3 showed a significant effect on Staphylococcus aureus starting at 1 hour; subsequently, the effect continued to decrease, killing Staphylococcus aureus within 3 hours. This indicates that the antimicrobial peptide A2M3 has a significant inhibitory effect on Staphylococcus aureus with increasing exposure time.

[0039] Example 5: Investigating the effect of antimicrobial peptides on cell membrane integrity using transmission electron microscopy (TEM) Transmission electron microscopy (TEM) can be used to observe the effects of A2M3 on the cell membrane of hemolytic Staphylococcus aureus. Bacteria at concentrations of 4.07–4.40 log CFU / mL were treated with 2×MIC A2M3 at 37°C for 2 h, centrifuged at 2700 rpm for 10 min, washed twice with phosphate-buffered saline (pH 7.2), fixed with 1% osmium tetroxide, dehydrated with ethanol, treated with acetone for 20 min, baked at 70°C for 24 h, ultrasectioned, and stained. The ultrastructure was observed and captured using an H-7650 TEM (Hitachi Co., Ltd., Japan).

[0040] like Figure 5 As shown, untreated bacteria maintained good intracellular organization and structural integrity. After treatment with the antimicrobial peptide A2M3, the bacterial cell membrane structure began to blur due to cell vacuolation, the cell shape became irregular, the cell membrane completely collapsed, and cytoplasmic contents leaked out. Transmission electron microscopy results indicated that the antimicrobial peptide A2M3 had a disruptive effect on the cell membrane and internal structure of Staphylococcus aureus.

[0041] Example 6: Effect of antimicrobial peptides on bacterial PI uptake Take 50 μl 106-7 CFU suspension was added to a sterile 1.5 mL centrifuge tube, followed by an equal volume of antimicrobial peptide solution of different concentrations (sterile water served as a control). After incubation at 37°C for 2 hours, 100 μl of PI (final concentration 10 μM) was added and the tube was incubated in the dark for 15 minutes. PI fluorescence intensity was detected at an excitation wavelength of 550 nm and an emission wavelength of 570 nm.

[0042] Propidium iodide (PI) is a DNA-binding dye that cannot penetrate the intact cell membrane of normal cells or early apoptotic cells. However, in late apoptotic cells and dead cells, PI can penetrate the cell membrane and stain the cell nucleus red. The PI fluorescence signal in Staphylococcus aureus treated with the antimicrobial peptide A2M3 was significantly increased compared to the control group, indicating that the antimicrobial peptide A2M3 disrupted the integrity of the Staphylococcus aureus cell membrane, allowing PI to bind to intracellular nucleic acids.

[0043] Example 7: Flow Cytometry Staphylococcus aureus was cultured at 37°C for 12 h to reach the logarithmic growth phase. 1 mL of the bacterial suspension was centrifuged at 12000 r / min for 1 min in a 1.5 mL centrifuge tube, the supernatant was removed, and the suspension was resuspended in 1 mL of sterile 0.01 M phosphate buffer. This process was repeated three times. After preparing the bacterial suspension in 0.01 M phosphate buffer, peptides at concentrations of 1×MIC and 2×MIC were mixed with the bacterial suspension in equal proportions (greater than 200 µl) and incubated at 37°C for 1 h, 2 h, and 3 h, respectively. The 0.01 M phosphate buffer served as a blank control. After incubation, the cells were stained with fluorescent isothiocyanate (FITC)-annexin V and propidium iodide (PI) in the dark for 15 min at room temperature. The samples were then loaded into a flow cytometer (CytoFLEX Beckman Coulter, Inc., USA) for analysis.

[0044] Annexin V and Propidium double staining combined with flow cytometry were used to further characterize the effects of the antimicrobial peptide A2M3 on the Staphylococcus aureus cell membrane, such as... Figure 7 As shown, Annexin V binds to PS (phosphatidylserine) with high affinity. Annexin V was labeled with FITC (fluorescein fluorescein), and the occurrence of apoptosis could be detected by flow cytometry. After 1 hour of culture, the late apoptosis rate of the control group was only 0.38%, while the late apoptosis rates of 1×MIC and 2×MIC were 9.04% and 12.84%, respectively. After 3 hours of incubation, the late apoptosis rates of 1×MIC and 2×MIC increased to 36.91% and 83.22%, respectively. These results further confirm that the antimicrobial peptide A2M3 affects the cell membrane permeability of Staphylococcus aureus, leading to apoptosis and inducing programmed cell death in Staphylococcus aureus.

[0045] Example 8: Interaction between antimicrobial peptide A2M3 and bacterial DNA The interaction between the antimicrobial peptide A2M3 and the genomic DNA of Staphylococcus aureus was investigated using the DNA gel retardation method. Staphylococcus aureus was cultured in 50 mL of nutrient broth (NB) at 37 °C for 12 h, and bacterial genomic DNA was extracted using a bacterial genomic DNA extraction kit. The optical density ratio (OD) at 260 and 280 nm was compared with that at 280 nm. 260 / OD 280 The purity of the extracted genomic DNA was evaluated using a concentration ≥1.90. Next, 10 μl of DNA (110 ng / μL) was mixed with a continuous stream of the antimicrobial peptide A2M3 at 25°C for 90 min, and the mixture was electrophoresed on a 1% agarose gel. Gel retardation was observed under UV irradiation using a GelDocXR gel imaging system (Bio-Rad, USA). Figure 8 As shown, after different concentrations of antimicrobial peptide A2M3 were applied to Staphylococcus aureus, no significant migration of the bacterial DNA bands occurred. This binding effect could still inhibit DNA migration even when the ratio of antimicrobial peptide A2M3 to DNA was 50 / 1.

[0046] Example 9: Fluorescence spectroscopy experiment on the competitive binding of antimicrobial peptide A2M3 to DNA by ethidium bromide (EB) Fluorescence spectroscopy analysis of the competitive binding of antimicrobial peptide A2M3 to DNA with ethidium bromide (EB) to analyze the interaction mechanism of antimicrobial peptide with Staphylococcus aureus genomic DNA: Staphylococcus aureus genomic DNA was diluted to 50 μg / mL with TE buffer. The reaction was performed in 96-well plates. First, 50 μl of DNA solution and 0.75 μl of 100 μg / mL EB solution were added to each well, mixed, and incubated at 37°C in the dark for 10 min. Then, 50 μl of antimicrobial peptide solutions of different concentrations were added, with 50 μl of distilled water used as a blank control group. After mixing, the plates were incubated at 37°C in the dark for 30 min. After incubation, the fluorescence spectra of the samples were measured using a multi-mode microplate reader in the excitation wavelength range of 535 nm and the emission wavelength range of 600–750 nm. In aqueous solution, EB fluorescence was weak, but its fluorescence intensity increased significantly when it bound to double-stranded DNA with high affinity and intercalation. If a substance in the EB-DNA complex system that can interact with DNA in a similar way competes with EB bound to DNA, the fluorescence intensity of the system will decrease, indicating that the competitor binds to DNA through the same intercalation mechanism as EB.

[0047] Therefore, by measuring the changes in the fluorescence spectrum of the DNA-EB complex system interacting with the competitor, it can be determined whether the competitor also binds to DNA via intercalation, similar to EB. Figure 9 It can be seen that as the concentration of antimicrobial peptide A2M3 increases, the fluorescence intensity of the EB-DNA complex also decreases significantly (p<0.05), indicating that antimicrobial peptide A2M3 intercalates with Staphylococcus aureus DNA, competing with EB that was previously bound to the DNA base pairs, and replacing EB to bind to DNA in an intercalating manner, thus reducing the fluorescence intensity of the entire system.

[0048] In summary, this invention provides a novel antimicrobial peptide, A2M3, with a minimum bactericidal concentration (MBC) of 125.0 μg / mL against Staphylococcus aureus, exhibiting a strong inhibitory effect on the bacteria. Its antibacterial mechanism involves first adsorbing onto the bacterial surface, causing damage to the bacterial cell membrane, then crossing the cell membrane and binding to the bacterial genomic DNA, inhibiting bacterial DNA synthesis and leading to bacterial death. While specific embodiments of the present invention have been described above, those skilled in the art should understand that the specific embodiments described are merely illustrative and not intended to limit the scope of the present invention. Equivalent modifications and variations made by those skilled in the art in accordance with the spirit of the present invention should be covered within the scope of protection of the claims of the present invention.

Claims

1. A human nasal α-2-macroglobulin antimicrobial peptide A2M3, the amino acid sequence of which is shown in SEQ ID NO:

1.

2. The application of the human nasal α-2-macroglobulin antimicrobial peptide A2M3 as described in claim 1 in the preparation of antimicrobial drugs, characterized in that: The antibacterial drug is used to inhibit and / or kill Staphylococcus aureus.

3. An antibacterial drug, characterized in that: Its active ingredient is nasal α-2-macroglobulin antimicrobial peptide A2M3, and the amino acid sequence of the antimicrobial peptide A2M3 is SEQ ID NO:

1.

4. An antibacterial drug as described in claim 3, characterized in that: The antibacterial drug is used to inhibit and / or kill Staphylococcus aureus.

5. A food preservative, characterized in that: Its active ingredient is nasal α-2-macroglobulin antimicrobial peptide A2M3, and the amino acid sequence of the antimicrobial peptide A2M3 is SEQ ID NO:

1.

6. A feed additive, characterized in that: Its active ingredient is nasal α-2-macroglobulin antimicrobial peptide A2M3, and the amino acid sequence of the antimicrobial peptide A2M3 is SEQ ID NO:

1.

7. A feed additive as described in claim 6, characterized in that: The feed additive is used to inhibit and / or kill Staphylococcus aureus.

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