Antibacterial peptide pa9 for inhibiting oral periodontal pathogenic bacteria and application thereof

Abstract

The application discloses an antibacterial peptide PA9 for inhibiting oral periodontal pathogenic bacteria and application thereof. The amino acid sequence of the antibacterial peptide PA9 is shown as SEQ ID NO. 1. The antibacterial peptide PA9 provided by the application is composed of 36 amino acids, has a molecular weight of 4049.99, an isoelectric point of 10.4, a net charge of 12 at pH 7.0, an average hydrophilicity of 0.6, and a hydrophilic residue ratio of 47%. It is found for the first time that the antibacterial peptide shown as SEQ ID NO: 1 is simple to prepare, has low cytotoxicity, has bacteriostatic and bactericidal activities on oral periodontal pathogenic bacteria, can target to destroy a biological membrane of the oral periodontal pathogenic bacteria, has a bactericidal effect, and can be used for preparing oral products, inhibiting growth of oral periodontal pathogenic bacteria, regulating balance of a micro-ecosystem of the oral cavity, and further used for preventing and treating oral diseases, in particular, diseases such as dental caries, periodontitis, oral mucosa disease or oral cancer.

Description

Technical Field

[0001] This invention relates to an antimicrobial peptide PA9 that inhibits periodontal pathogens in the oral cavity and its application, belonging to the field of antimicrobial peptide technology. Background Technology

[0002] Periodontal disease is a common oral disease, and oral pathogens and dental plaque play a direct role in its occurrence and development. Adjunctive treatments for periodontal disease primarily rely on antibiotics, but long-term use can easily lead to bacterial resistance and oral flora imbalance. Therefore, there is an urgent need to develop new drugs to avoid the risk of bacterial resistance.

[0003] Antimicrobial peptides, as novel antimicrobial agents, are widely available and possess broad-spectrum antimicrobial activity. Compared to traditional antibiotics, most antimicrobial peptides kill bacteria primarily by disrupting their cell membranes, making them less likely to induce antibiotic resistance and thus considered a good alternative. In 1980, Boman et al. successfully isolated and purified cephalosporin from silkworm pupae, marking the first discovery of an antimicrobial peptide. Subsequently, antimicrobial peptides have been discovered in bacteria, fungi, amphibians, higher plants, mammals, and humans. Currently, more than 40 antimicrobial peptides have been isolated from human oral epithelial cells, saliva, and periodontal tissues. Some antimicrobial peptides have shown antimicrobial activity against periodontal pathogens. For example, LL-37 has a cytotoxic effect against *Actinobacillus actinomycetii* and *Prevotella intermedius*; β-defensin has a cytotoxic effect against *Actinobacillus actinomycetii*, *Fusobacterium nucleatum*, *Porphyromonas gingivalis*, and *Prevotella intermedius*; and lactoferrin has a cytotoxic effect against *Porphyromonas gingivalis* and *Prevotella intermedius*.

[0004] Natural antimicrobial peptides suffer from high production costs, low antimicrobial activity, and poor stability, failing to meet practical application needs. With ongoing in-depth research into the properties, structures, and mechanisms of action of existing natural antimicrobial peptides, increasing research focuses on developing synthetically produced antimicrobial peptides. Researchers have successfully utilized various modern biotechnologies to molecularly modify or synthesize novel antimicrobial peptides. Therefore, developing antimicrobial peptides with simple structures and strong antimicrobial activity is of great significance. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides an antimicrobial peptide PA9 that inhibits oral periodontal pathogens and its applications. This antimicrobial peptide exhibits good antibacterial and bactericidal activity against oral periodontal pathogens such as *Fusobacterium nucleatum*, *Porphyromonas gingivalis*, and *Prevotella intermedius*, and also possesses advantages such as simple structure, easy synthesis, low hemolytic activity, and high safety.

[0006] The technical solution of the present invention is as follows:

[0007] The first aspect of the present invention provides an antimicrobial peptide PA9 that inhibits periodontal pathogens in the oral cavity, wherein the amino acid sequence of the antimicrobial peptide PA9 is shown in SEQ ID NO.1.

[0008] This invention is the first to discover that the antimicrobial peptide shown in SEQ ID NO:1 is not only simple to prepare and has low cytotoxicity, but also possesses antibacterial and bactericidal activity against oral periodontal pathogens. It can target and disrupt the biofilm of oral periodontal pathogens, thereby exerting a bactericidal effect. More specifically, the antimicrobial peptide PA9 provided by this invention consists of 36 amino acids, has a molecular weight of 4049.99, an isoelectric point of 10.4, a net charge of 12 at pH 7.0, an average hydrophilicity of 0.6, and a hydrophilic residue ratio of 47%.

[0009] The antimicrobial peptide PA9 can be synthesized according to a specific amino acid sequence using a solid-phase synthesis method, and then purified by HPLC reverse-phase column chromatography after desalting.

[0010] According to a preferred embodiment of the present invention, the oral periodontal pathogens are one or more of Fusobacterium nucleatum, Porphyromonas gingivalis, and Prevotella intermedius.

[0011] A second aspect of the present invention provides the use of the above-mentioned antimicrobial peptide PA9 in the preparation of an antibacterial agent for oral periodontal pathogens.

[0012] According to a preferred embodiment of the present invention, the oral periodontal pathogens are one or more of Fusobacterium nucleatum, Porphyromonas gingivalis, and Prevotella intermedius.

[0013] A third aspect of the present invention provides the use of the above-mentioned antimicrobial peptide PA9 in the preparation of oral care products for the treatment and / or prevention of oral diseases.

[0014] According to a preferred embodiment of the present invention, the oral disease is caused by one or more of Fusobacterium nucleatum, Porphyromonas gingivalis, and Prevotella intermedius.

[0015] According to a preferred embodiment of the present invention, the oral care product is one or more of toothpaste, mouthwash, mouthwash effervescent tablets, or oral care solution.

[0016] In the oral cavity, if the microecology is imbalanced, the microbial community can easily transform into a pathogenic community, which has a negative impact on oral health. At the same time, the increase of pathogenic bacteria can easily cause bad breath, which can lead to diseases such as dental caries, periodontitis, oral mucosal diseases or oral cancer. The antimicrobial peptide PA9 of the present invention can effectively inhibit oral periodontal pathogenic bacteria, thereby effectively regulating the balance of the oral microecology. Therefore, oral care products containing the above-mentioned antimicrobial peptide PA9 should be within the protection scope of the present invention.

[0017] The beneficial effects of this invention are as follows:

[0018] 1. The antimicrobial peptide PA9 provided by this invention has antibacterial and bactericidal activity against oral periodontal pathogens. It can target and destroy the biofilm of oral periodontal pathogens, inhibit the formation of biofilms, and exert a bactericidal effect. In particular, the minimum inhibitory concentration (MIC) against *Fusobacterium nucleatum* is 500 μg / mL, and the minimum bactericidal concentration (MBC) against *Porphyromonas gingivalis* is 250 μg / mL, and the MIC against *Prevotella intermedius* is 125 μg / mL, and the MBC against *Prevotella intermedius* is 500 μg / mL. It can be used to prepare oral care products, inhibit the growth of oral periodontal pathogens, regulate the balance of the oral microecology, and thus be used to prevent and treat oral diseases, especially dental caries, periodontitis, oral mucosal diseases, or oral cancer.

[0019] 2. The antimicrobial peptide PA9 provided by this invention has a short synthetic sequence, small molecular weight, and is easy to synthesize chemically. It can be directly synthesized into a high-purity product. At the same time, it has low hemolytic activity, high safety, clear effect, and wide application. Attached Figure Description

[0020] Figure 1 This is a high-performance liquid chromatogram of the antimicrobial peptide PA9 of the present invention;

[0021] Figure 2 This is the mass spectrum of the antimicrobial peptide PA9 of the present invention;

[0022] Figure 3 The results of the minimum inhibitory concentration experiment of the antimicrobial peptide PA9 of this invention against oral periodontal pathogens;

[0023] Figure 4 This is the experimental result of the minimum bactericidal concentration of the antimicrobial peptide PA9 of the present invention against oral periodontal pathogens;

[0024] Figure 5 The results of the experiment on crystal violet staining of oral periodontal pathogenic bacteria biofilm by the antimicrobial peptide PA9 of the present invention are as follows;

[0025] Figure 6 These are the experimental results of the antimicrobial peptide PA9 of this invention on the biomass of oral periodontal pathogenic bacteria biofilm;

[0026] Figure 7 The results of the hemolytic toxicity experiment of the antimicrobial peptide PA9 of this invention on mammalian erythrocytes are presented. Detailed Implementation

[0027] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments and accompanying drawings. The described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0028] Unless otherwise specified, the experimental techniques and scientific terms used in the following examples have the same meanings as commonly understood by a person skilled in the art. Unless otherwise noted, all experimental consumables and reagents are commercially available.

[0029] Example 1: Synthesis of antimicrobial peptide PA9

[0030] The amino acid sequence of the antimicrobial peptide PA9 of this invention is as follows:

[0031] LGAAVLKMLKPGLQTNTLFKKKKKKKKKGEKFSGF (as shown in SEQ ID NO.1).

[0032] Then, Shanghai Nuoyou Biotechnology Co., Ltd. was commissioned to synthesize the antimicrobial peptide PA9 artificially using a solid-phase synthesis method according to the specific amino acid sequence mentioned above. The peptide was then purified by HPLC reverse-phase column chromatography to obtain the antimicrobial peptide PA9.

[0033] The high-performance liquid chromatogram of the antimicrobial peptide PA9 prepared in this embodiment is shown below. Figure 1 As shown, the mass spectrum is as follows Figure 2 As shown.

[0034] Depend on Figure 1 and Figure 2 It can be seen that the basic physicochemical properties of the antimicrobial peptide PA9 prepared in this embodiment are as follows: molecular weight is 4049.99, isoelectric point is 10.4, net charge at pH 7.0 is 12, average hydrophilicity is 0.6, and the proportion of hydrophilic residues is 47%.

[0035] Example 2: Determination of the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of antimicrobial peptide PA9

[0036] 1. The oral periodontal pathogenic strains involved in this embodiment are: Fusobacterium nucleatum (Fn) and Porphyromonas gingivalis (Pg), which are available from the American Type Culture Collection, and Prevotella intermedia (Pi), which is available from Beijing BioBio Biotechnology Co., Ltd.

[0037] 2. The methods for determining the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of the antimicrobial peptide PA9 are as follows:

[0038] (1) Activate the bacterial strains: streak the periodontal pathogenic bacteria on BHI blood agar solid medium and incubate them in a 37°C anaerobic incubator; when the colonies grow to a suitable size, randomly pick a single colony and incubate it in BHI liquid medium in a 37°C anaerobic incubator until the logarithmic phase.

[0039] (2) Collect bacterial cells by centrifuging the bacterial culture from step (1) to the logarithmic phase at 5000 rpm for 5 min, and then dilute with BHI liquid medium to a concentration of 1×10⁻⁶. 8 CFU / mL;

[0040] (3) The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of antimicrobial peptide PA9 were determined by the standard microbroth dilution method: double serial dilutions of antimicrobial peptide PA9 were prepared in 96-well plates at a volume of 100 μL / well in the culture medium, with the concentration range of antimicrobial peptide from 1 μg / mL to 500 μg / mL; positive control (oral periodontal pathogenic strain and culture medium) and negative control (culture medium only) were set up at the same time, and three replicates were set up for each group;

[0041] (4) Place the 96-well plate from step (3) in an anaerobic incubator at 37℃ for 24 hours. Visually observe the turbidity of the culture medium in each well. The concentration corresponding to the well with clear culture medium is the MIC value. Take 10 μL of bacterial culture from each well corresponding to the MIC and above concentrations and streak it on a BHI blood agar plate. Observe the concentration corresponding to the absence of colony growth on the plate surface as the MBC value. The results are shown in Table 1. Figure 3 and Figure 4 As shown.

[0042] Table 1. Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) of Antimicrobial Peptide PA9

[0043]

[0044] Through Table 1, Figure 3 and Figure 4 It can be seen that the minimum inhibitory concentration (MIC) of antimicrobial peptide PA9 against *Fusobacterium nucleatum* is 500 μg / mL, and the minimum bactericidal concentration (MBC) is 500 μg / mL; against *Porphyromonas gingivalis*, the MIC is 250 μg / mL, and the MBC is 250 μg / mL; and against *Prevotella intermedius*, the MIC is 125 μg / mL, and the MBC is 500 μg / mL. This fully demonstrates that the antimicrobial peptide PA9 provided by this invention exhibits significant antimicrobial and bactericidal activity against oral periodontal pathogens such as *Fusobacterium nucleatum*, *Porphyromonas gingivalis*, and *Prevotella intermedius*.

[0045] Example 3: Effects of antimicrobial peptide PA9 on bacterial biofilms

[0046] The effect of the antimicrobial peptide PA9 on the formation of biofilms by oral periodontal pathogens was assessed using crystal violet staining. The specific method is as follows:

[0047] (1) Prepare a bacterial suspension of periodontal pathogens according to the method in Example 2;

[0048] (2) Take a 96-well plate and add 50 μL of bacterial suspension containing periodontal pathogens and 50 μL of antimicrobial peptide PA9 solution of different concentrations to each well, so that the final concentration of antimicrobial peptide PA9 in the well plate is MIC, 1 / 2MIC, 1 / 4MIC, and 1 / 8MIC. At the same time, set up a positive control (periodontal pathogens and culture medium) and a negative control (culture medium only), and set up three replicates for each group;

[0049] (3) Place the 96-well plate in an anaerobic incubator at 37°C and incubate anaerobically for 24 hours;

[0050] (4) Discard the supernatant and slowly rinse three times with 200 μL of PBS buffer to remove airborne periodontal pathogens. Let it dry at room temperature.

[0051] (5) Add 100 μL of methanol to each well, fix for 15 min, remove the methanol, and air dry;

[0052] (6) Add 100 μL of 1% crystal violet staining agent to each well to stain the biofilm at room temperature for 20 min;

[0053] (7) Remove the crystal violet, rinse off the excess staining agent with PBS, and air dry;

[0054] (8) Subsequently, 100 μL of 30% acetic acid was added to each well, and the mixture was gently shaken at room temperature for about 10 min to decolorize. The OD value at a wavelength of 595 nm was recorded using a microplate reader. The results are as follows: Figure 5 and Figure 6 As shown.

[0055] Depend on Figure 5 It can be seen that the control groups of Fn, Pg, and Pi all formed complete biofilms; when the concentration of antimicrobial peptide PA9 was MIC, the biofilms were almost completely unstained; when the concentrations of antimicrobial peptide PA9 were 1 / 2MIC and 1 / 4MIC, only very thin biofilms were formed; when the concentration of antimicrobial peptide PA9 was 1 / 8MIC, a complete biofilm was formed with deep staining.

[0056] Depend on Figure 6 It can be seen that, compared with the control group, when the concentrations of antimicrobial peptide PA9 were MIC, 1 / 2 MIC, and 1 / 4 MIC, the biofilm OD of Fn and Pi were significantly higher. 595 Decreasing the concentration of antimicrobial peptide PA9 at MIC and 1 / 2MIC reduces the biofilm formation rate. The biofilm OD of Pg decreases as the concentration of PA9 decreases. 595The amount of biofilm formed decreased, and the difference was statistically significant (P < 0.05). This fully demonstrates that the antimicrobial peptide PA9 provided by this invention can target and destroy the biofilm of oral periodontal pathogens, inhibit the formation of biofilms of oral periodontal pathogens, and has significant antibacterial and bactericidal activity against oral periodontal pathogens.

[0057] Example 4: Hemolytic toxicity of antimicrobial peptide PA9 on mammalian erythrocytes

[0058] 1 mL of sterile anticoagulated sheep blood was centrifuged at 3000 rpm for 10 min, washed three times with sterile PBS buffer, and sheep red blood cells were collected. These cells were then incubated in PBS buffer containing different concentrations of the antimicrobial peptide PA9 (62.5 μg / mL-500 μg / mL) at 37°C for 30 min. 1% Triton X-100 was used as a positive control, and sterile PBS buffer was used as a negative control. After incubation, the cells were centrifuged at 3000 rpm for 10 min, and the supernatant was measured at 570 nm using a microplate reader. Three replicates were set up for each group. The results are shown in Table 2. Figure 7 As shown.

[0059] The formula for calculating the hemolysis rate is: Hemolysis rate = (AT-AO) / (AC-AO)×100%.

[0060] In the formula: AT is the absorbance value of the experimental group, AC is the absorbance value of the positive control group, and AO is the absorbance value of the negative control group.

[0061] Table 2. Hemolytic activity of antimicrobial peptide PA9

[0062]

[0063] From Table 2 and Figure 7 It is known that under the minimum inhibitory concentration (MIC) condition for inhibiting oral periodontal pathogens, the hemolysis rate of antimicrobial peptide PA9 is less than 1%. Even at the maximum bactericidal concentration of 500 μg / mL, the hemolysis rate is only 0.27%, with almost no hemolysis. This indicates that the antimicrobial peptide PA9 of the present invention has good safety and great application prospects, and can be further studied and developed.

[0064] In summary, the antimicrobial peptide PA9 provided by this invention has antibacterial and bactericidal activities against oral periodontal pathogens. It can target and destroy the biofilm of oral periodontal pathogens, inhibit the formation of biofilms, and exert a bactericidal effect. It can be used to prepare oral care products, inhibit the growth of oral periodontal pathogens, regulate the balance of oral microecology, and thus be used to prevent and treat oral diseases, especially dental caries, periodontitis, oral mucosal diseases, or oral cancer.

Claims

1. An antimicrobial peptide PA9 that inhibits pathogenic periodontal bacteria in the oral cavity, characterized in that, The amino acid sequence of the antimicrobial peptide PA9 is shown in SEQ ID NO.

1.

2. The application of the antimicrobial peptide PA9, which inhibits periodontal pathogens according to claim 1, in the preparation of an antimicrobial agent for periodontal pathogens; wherein the periodontal pathogens are one or more of Fusobacterium nucleatum, Porphyromonas gingivalis, and Prevotella intermedius.

3. The use of the antimicrobial peptide PA9, which inhibits periodontal pathogens as described in claim 1, in the preparation of oral products for the treatment and / or prevention of oral diseases; wherein the oral diseases are caused by one or more of Fusobacterium nucleatum, Porphyromonas gingivalis, and Prevotella intermedius.

4. The application as described in claim 3, characterized in that, The oral care products mentioned are toothpaste or oral care solution.

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