A broad-spectrum antibacterial peptide escherichia coli-derived ESA_3 and application thereof

Abstract

The application discloses a broad-spectrum antibacterial peptide ESA_3 from Enterococcus faecalis and application thereof, the antibacterial peptide is composed of 15 amino acids and can be efficiently synthesized through an Fmoc solid-phase chemical method. The ESA_3 has significant bacteriostatic activity on various gram-positive and gram-negative bacteria such as Staphylococcus aureus ATCC 6538, methicillin-resistant Staphylococcus aureus USA300, Escherichia coli and Pseudomonas aeruginosa, and the inhibiting effect of the ESA_3 on the methicillin-resistant Staphylococcus aureus USA300 is better than that of human antibacterial peptide LL-37. Safety evaluation shows that the hemolysis rate of the ESA_3 is less than 5% under an effective antibacterial concentration, the survival rate of mammalian cells is kept above 80%, and the biological safety is excellent. In addition, the bacteriostatic activity of the peptide on the Staphylococcus aureus remains stable under a medium-low salt concentration. The antibacterial peptide can be widely applied to the preparation of drugs for resisting drug-resistant bacterial infection, antibacterial coatings of medical consumables, disinfectants, food preservatives and feed additives, and has a wide development prospect.

Description

Technical Field

[0001] This invention belongs to the field of antimicrobial peptide technology, specifically relating to a broad-spectrum antimicrobial peptide ESA_3 derived from Enterococcus faecalis and its applications. Background Technology

[0002] Bacterial infections are a significant threat to human health, animal health, and public health security. Pathogens such as Staphylococcus aureus, Pseudomonas aeruginosa, Acinetobacter baumannii, and Escherichia coli frequently cause skin and soft tissue infections, respiratory infections, sepsis, and intestinal diseases in livestock and poultry. Particularly concerning is the emergence of drug-resistant strains due to the long-term irrational use of antibiotics. Methicillin-resistant Staphylococcus aureus (MRSA), in particular, has become a globally concerning "superbug" due to its high resistance to various first-line clinical drugs, including β-lactams. Current treatments frequently fail, highlighting the urgent need to develop novel and highly effective antibacterial drugs.

[0003] Antimicrobial peptides (AMPs), as an important component of innate immunity, are considered ideal alternatives to traditional antibiotics due to their unique mechanism of action (primarily through physical disruption of bacterial membrane structure) and potential to reduce the likelihood of inducing drug resistance. However, existing antimicrobial peptides still face several bottlenecks in clinical translation and industrial application: First, it is difficult to balance activity and safety; many antimicrobial peptides with potent antibacterial activity are often accompanied by high hemolytic activity or toxicity to mammalian cells, limiting their drug development potential. Second, the poor stability of salt ions means that physiologically concentrated cations such as Na⁺ and Mg²⁺ compete with positively charged antimicrobial peptides for binding to bacterial membranes, leading to a significant decrease in antimicrobial activity, especially in high-salt environments (such as wound exudate, intestinal environment, and feed matrix). Third, their long sequences or complex structures result in high chemical synthesis costs, making large-scale production difficult.

[0004] Enterococci (Enterococcus spp.), as a normal flora in the intestines of humans and animals, are not only an important source of probiotics but also a treasure trove for discovering novel antimicrobial peptides. Some Enterococci (Enterococcus faecalis) can secrete bacteriocins or antimicrobial peptides with broad-spectrum antibacterial activity, showing great potential in food preservation, feed antibiotic alternatives, and anti-infection applications. Therefore, developing a novel antimicrobial peptide derived from Enterococci, possessing excellent broad-spectrum antimicrobial activity (especially highly effective inhibition of MRSA USA300), good safety, excellent salt stability, and ease of synthesis, is of significant scientific and clinical value in overcoming the limitations of existing antimicrobial agents and addressing the increasingly severe situation of bacterial resistance. Summary of the Invention

[0005] To address the above-mentioned problems, the present invention aims to provide a broad-spectrum antimicrobial peptide ESA_3 derived from Enterococcus faecalis and its applications.

[0006] The specific technical solution is as follows:

[0007] A broad-spectrum antimicrobial peptide ESA_3 derived from Enterococcus faecalis, the amino acid sequence of which is shown in SEQ ID NO.1.

[0008] The antimicrobial peptide ESA_3 contains multiple arginine (Arg) and tryptophan (Trp) residues, endowing it with strong cationic properties and moderate hydrophobicity. This structural feature facilitates its accumulation on the negatively charged bacterial cell membrane surface under electrostatic attraction, and its insertion into the cell membrane through hydrophobic interactions, inducing changes in membrane permeability or perforation, thereby exerting a rapid bactericidal effect.

[0009] The antimicrobial peptide ESA_3 consists of 15 L-amino acids, and its structural formula is as follows:

[0010]

[0011] The application of a broad-spectrum antimicrobial peptide ESA_3 derived from Enterococcus faecalis in the preparation of antimicrobial products, including antimicrobial agents and antimicrobial products, including personal care products, feed additives, antibiotic alternative feed additives, antimicrobial agents for medical consumables, antimicrobial coatings for medical consumables, cosmetics, disinfectants or detergents.

[0012] The bacteria are selected from at least one of Staphylococcus aureus (ATCC 6538), Methicillin-resistant Staphylococcus aureus (USA 300), Pseudomonas aeruginosa (ATCC 27853), Acinetobacter baumannii (ATCC 19606), Escherichia coli (ATCC 25922), and Salmonella typhimurium (14028S).

[0013] An antimicrobial composition comprising a broad-spectrum antimicrobial peptide ESA_3, and pharmaceutically, feed-grade, cosmetically, or materials-grade excipients, carriers, or matrices.

[0014] The beneficial effects of this invention are as follows:

[0015] 1) Strong anti-Staphylococcus aureus activity: The antibacterial activity against S. aureus ATCC 6538 and S. aureus USA300 is significantly better than that against the reference peptide LL-37;

[0016] 2) High safety: Hemolysis rate <5% and cell survival rate >80% at effective concentrations, providing a safety basis for clinical and oral (feed) applications;

[0017] 3) Excellent salt tolerance: It can withstand low to medium salt concentrations (Na+) + 0.125-2 mg / mL, Mg 2+ Stable antibacterial activity at concentrations of 25-200 μg / mL, suitable for wound exudate, intestinal environment and feed processing scenarios;

[0018] 4) Short sequence and easy to synthesize: It contains only 15 amino acids, has a clear structure, is easy to synthesize chemically, has low production cost, and is conducive to large-scale application. It contains multiple positively charged amino acids and has a certain degree of hydrophobicity, which is conducive to its interaction with bacterial cell membranes, thereby exerting antibacterial or bactericidal effects.

[0019] 5) Broad-spectrum antibacterial: It covers both Gram-positive bacteria (Staphylococcus aureus, Enterococcus) and Gram-negative bacteria (Escherichia coli, Pseudomonas aeruginosa, etc.), making it applicable to a wide range of conditions. Attached Figure Description

[0020] Figure 1The graph shows the results of determining the minimum inhibitory concentration (MIC) of the antimicrobial peptide ESA_3 of this invention against different pathogens.

[0021] Figure 2 This is a graph showing the hemolytic phenomenon of the antimicrobial peptide ESA_3 of this invention.

[0022] Figure 3 The graph shows the hemolysis rate of the antimicrobial peptide ESA_3 of this invention.

[0023] Figure 4 This is a graph showing the cytotoxicity evaluation results of the antimicrobial peptide ESA_3 of this invention on human bronchial epithelial cells (BEAS-2B).

[0024] Figure 5 This is a graph showing the cytotoxicity evaluation results of the antimicrobial peptide ESA_3 of this invention against porcine small intestinal epithelial cells (IPEC-J2).

[0025] Figure 6 The antimicrobial peptide ESA_3 of this invention is effective against different concentrations of Na + The results of MIC determination of MRSA USA 300 under the specified conditions are shown in the figure.

[0026] Figure 7 The antimicrobial peptide ESA_3 of this invention is effective against different concentrations of Mg. 2+ The results of MIC determination of MRSA USA 300 under the specified conditions are shown in the figure. Detailed Implementation

[0027] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of this invention, and not all of them. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0028] Example 1

[0029] Synthesis of antimicrobial peptides

[0030] The antimicrobial peptide ESA_3 was screened from the genome of Enterococcus faecalis and artificially synthesized by a professional peptide synthesis company using solid-phase peptide synthesis technology according to its sequence. It was then purified by reversed-phase high-performance liquid chromatography (RP-HPLC) to a purity of over 95%.

[0031] The amino acid sequence of the antimicrobial peptide ESA_3 is shown in SEQ ID NO.1: MWRRTWRNWRSAACR (Met-Trp-Arg-Arg-Thr-Trp-Arg-Asn-Trp-Arg-Ser-Ala-Ala-Cys-Arg).

[0032] Its structural formula is:

[0033]

[0034] The synthesized antimicrobial peptides were molecular weight identified by liquid chromatography-mass spectrometry (LC-MS), confirming that their molecular weight was consistent with the theoretical molecular weight. The purified antimicrobial peptides were lyophilized and stored. Before use, the antimicrobial peptides were dissolved in 1% DMSO prepared with sterile ultrapure water to the required concentration.

[0035] The antimicrobial peptide ESA_3 consists of only 15 L-amino acids, has a short sequence, high synthesis yield, and easy purity control, and has significant cost advantages and industrial production potential compared to long-chain peptides.

[0036] Example 2

[0037] Antimicrobial peptide MIC assay

[0038] This experiment aims to verify the broad-spectrum antibacterial activity of the antimicrobial peptide ESA_3 and compare it with that of the classic human antimicrobial peptide LL-37.

[0039] The in vitro antimicrobial activity of the antimicrobial peptide against the tested bacterial strains was initially tested using the micro-broth dilution method. The inhibition rate was calculated by measuring the absorbance at 600 nm, which allowed for a rapid determination of whether the peptide had the ability to inhibit bacterial growth.

[0040] 1) Preparation of bacterial culture: Six common clinical standard bacterial strains were selected: Gram-positive bacteria including *S. aureus* ATCC6538 and *Methicillin-resistant* *S. aureus* USA 300; and Gram-negative bacteria including *P. aeruginosa* ATCC 27853, *A. baumannii* ATCC 19606, *E. coli* ATCC 25922, and *S. typhimurium* 14028S. Human antimicrobial peptide LL-37 was used as a positive control peptide, and MHB liquid medium was used as a blank control.

[0041] The bacterial strains were removed from a -80℃ freezer and inoculated onto MHB solid medium using the streak plate method. The medium was then incubated upside down overnight in a 37℃ incubator. A single colony was picked and inoculated into 5 mL of MHB liquid medium. After mixing thoroughly by pipetting, the culture was incubated at 200 rpm for 12 h in a shaker at 37℃ to obtain the seed culture. 5 μL of the seed culture was transferred to 5 mL of fresh MHB liquid medium and cultured in a shaker until the bacterial concentration reached 10⁻⁶. 8 CFU / mL.

[0042] 2) Antibacterial experiment (peptide concentration gradients of 256 μmol / L, 128 μmol / L, 64 μmol / L, 32 μmol / L, 16 μmol / L, 8 μmol / L, 4 μmol / L, 2 μmol / L, 1 μmol / L, 0.5 μmol / L, and 0.25 μmol / L):

[0043] ① Peptide gradient dilution

[0044] Dissolve the peptide in 1% DMSO to a concentration of 10.24 mmol. Add 5 μL of peptide stock solution (10.24 mmol / L) to the first column of a 96-well plate, and add 100 μL of MHB liquid medium to each well. Add another 95 μL of MHB liquid medium to the first column, mix thoroughly by pipetting, and then transfer 100 μL of the mixture to the second column. Perform serial dilutions up to the tenth column. After mixing the tenth column, discard 100 μL of the diluted solution. Set up a control group: 100 μL of MHB liquid medium (the same volume of bacterial culture will be added subsequently).

[0045] ② Add bacteria

[0046] Dilute the bacterial culture to 1×10⁻⁶ 5 Add 100 μL of bacterial culture to each well at a concentration of CFU / mL.

[0047] ③Cultivation and Detection

[0048] The 96-well plate was incubated in a 37 ℃ incubator for 16 h, and the OD was measured. 600 .

[0049] ④ Calculate the antibacterial rate

[0050] Formula: Antibacterial rate (%) = (1 - (experimental group OD)) 600 - Blank group OD 600 ) / (Control group OD 600 - Blank group OD 600 ))×100%.

[0051] The experimental results are shown in Figure 1The results showed that ESA_3 exhibited significant antibacterial activity against the tested strains, with MIC values ​​ranging from 8 to 64 μmol / L for different strains. Notably, the MIC of ESA_3 was significantly lower than that of LL-37 in two Staphylococcus aureus strains (S. aureus ATCC6538 and MRSA USA 300), indicating that the antimicrobial peptide ESA_3 possesses stronger anti-Staphylococcus aureus activity.

[0052] Example 3

[0053] Hemolytic activity evaluation

[0054] This study aims to evaluate the blood compatibility of the antimicrobial peptide ESA_3, which is a key indicator for assessing its safety for injectable administration.

[0055] Mouse red blood cells were used. The cells were pre-cooled to 4°C in a centrifuge. Whole blood was centrifuged (3000 rpm / min, 10 min), the supernatant was discarded, and the bottom red blood cell pellet was collected. Approximately 5 volumes of sterile physiological saline were added, the red blood cells were gently resuspended, and centrifuged again. The supernatant was discarded, and the washing was repeated 4 times until the supernatant was colorless and transparent. Pre-cooled physiological saline was added to prepare a 4% (v / v) red blood cell suspension, and the mixture was thoroughly mixed. 1 ml of the red blood cell suspension was added to a 1.5 ml EP tube, centrifuged (3000 rpm / min, 10 min), and the supernatant was discarded. 1 ml of the antimicrobial peptide ESA_3 was added to the red blood cell pellet, and the mixture was gently mixed. After mixing, the concentrations were 4 μmol / L, 8 μmol / L, 16 μmol / L, 32 μmol / L, 64 μmol / L, 128 μmol / L, and 256 μmol / L, respectively. The pellets were incubated at 37°C for 3 h. Remove the EP tube, centrifuge, and visually inspect the supernatant for hemolysis. Take a photograph against a white background (e.g., ...). Figure 2 As shown, no obvious hemolysis was observed, indicating good blood compatibility and application safety. 200 μL of the supernatant from each tube was added to a 96-well plate, and the OD value was measured at 540 nm.

[0056] Sterile saline was used as a negative control, and 0.1% Triton X-100 was used as a positive control. The hemolysis rate was calculated using the following formula: Hemolysis rate = (OD) / (0.1% Triton X-100) 实验组 -OD 阴性组 ) / (OD 阳性组 -OD 阴性组 ) × 100%.

[0057] like Figure 3As shown, the antimicrobial peptide of this invention exhibits a hemolysis rate of less than 5% within the range of 4-256 μmol / L. This extremely low hemolytic activity means that the antimicrobial peptide ESA_3 has extremely high safety when administered systemically (e.g., intravenously), without causing serious red blood cell rupture side effects, making it superior to many cationic antimicrobial peptides with a high risk of hemolysis.

[0058] Example 4

[0059] Cytotoxicity evaluation

[0060] The toxicity of the antimicrobial peptides of this invention to mammalian cells was evaluated using the CCK-8 assay.

[0061] BEAS-2B and IPEC-J2 cells cultured to the 3rd generation were used for experiments. The old culture medium was discarded, cells were washed with PBS, digested with trypsin, and then counted. The cell suspension density was adjusted to 1×10⁻⁶. 5 Cells / mL. The adjusted cell suspension was evenly spread into 96-well plates at a volume of 100 μL per well. To avoid edge effects, the outermost wells of each 96-well plate were blocked with 150 μL of PBS solution and incubated overnight at 37 ℃ in a CO2 incubator. After incubation, 10 μL of different concentrations of the antimicrobial peptide ESA_3 solution (256 μmol / L, 128 μmol / L, 64 μmol / L, 32 μmol / L, 16 μmol / L, 8 μmol / L) were added to each well, with six replicates per group, including one blank control and one negative control. The blank control contained no cells but only culture medium, while the negative control contained both cells and culture medium. The 96-well plates were incubated for another 12 h. After incubation, 10 µL of CCK-8 solution was added to each well, and the plates were incubated for 3 h. The OD values ​​were then measured using a microplate reader. 450 The values ​​were calculated, and cell viability was determined.

[0062] The formula for calculating cell viability is: Cell viability = (OD) / (OD + ... 加药孔 -OD 空白孔 ) / (OD 阴性孔 -OD 空白孔 ) × 100%.

[0063] like Figure 4 and Figure 5 As shown, the antimicrobial peptide ESA_3 achieved a survival rate of over 80% in both cell types at low and medium concentrations (8 μmol / L-64 μmol / L) with no obvious toxicity, demonstrating good antimicrobial peptide safety.

[0064] Its excellent cell safety not only supports its application in clinical drugs (such as sprays and injections), but also provides a safety guarantee for its use in feed additives (which come into contact with intestinal epithelial cells).

[0065] Example 5

[0066] Salt ion stability experiment

[0067] 1) The preparation of bacterial suspension was the same as that for MIC determination. The bacterial suspension used in this experiment was MRSA USA 300 bacterial suspension.

[0068] 2) Dissolve and dilute NaCl with HEPES buffer, add 45 μl of salt solution to each well, so that the final NaCl concentrations per plate are 4 mg / ml, 2 mg / ml, 1 mg / ml, 0.5 mg / ml, 0.25 mg / ml, and 0.125 mg / ml, respectively, and the final MgCl2 concentrations are 800 μg / ml, 400 μg / ml, 200 μg / ml, 100 μg / ml, 50 μg / ml, and 25 μg / ml;

[0069] 3) Antibacterial assay: After fixing the salt concentration, the peptide concentration was diluted: the peptide was dissolved in 1% DMSO diluted with HEPES buffer to a concentration of 10.24 mmol, and serially diluted using 0.5 ml EP tubes. 5 μl of peptide solution was added to each well, resulting in final peptide concentrations of 256 μmol / L, 128 μmol / L, 64 μmol / L, 32 μmol / L, 16 μmol / L, 8 μmol / L, 4 μmol / L, and 2 μmol / L for each column. The MRSA USA 300 bacterial culture was diluted to 1×10⁻⁶. 5 CFU / mL, 50 μL of LMB HB liquid medium and 100 μL of bacterial suspension were added to each well; the blank group contained only the medium, and the control group contained salt solution, bacterial suspension and medium. The 96-well plate was incubated at 37 ℃ for 16 h, and the OD was measured. 600 ;

[0070] 4) Calculation of antibacterial rate:

[0071] Formula: Antibacterial rate (%) = (1 - (experimental group OD)) 600 - Blank OD 600 ) / (Control group OD 600 - Blank OD 600 ))×100%.

[0072] like Figure 6 and Figure 7 As shown, at low to medium salt concentrations (Na + : 0.125-2 mg / mL; Mg 2+Under conditions of 25-200 μg / mL, the MIC value of the antimicrobial peptide ESA_3 against MRSA USA 300 remained essentially unchanged. This characteristic is a significant advantage of ESA_3 over ordinary antimicrobial peptides, expanding its application scope in the pharmaceutical and animal husbandry fields.

Claims

1. A broad-spectrum antimicrobial peptide ESA_3 derived from Enterococcus faecalis, characterized in that, The amino acid sequence of the antimicrobial peptide ESA_3 is shown in SEQ ID NO.

1.

2. The broad-spectrum antimicrobial peptide ESA_3 derived from Enterococcus faecalis as described in claim 1, characterized in that, The antimicrobial peptide ESA_3 consists of 15 L-amino acids, and its structural formula is as follows: 。 3. The application of ESA_3, a broad-spectrum antimicrobial peptide derived from Enterococcus faecalis as described in claim 1 or 2, in the preparation of antimicrobial products, characterized in that, Antimicrobial products include antimicrobial agents and antimicrobial products. Antimicrobial products include personal care products, feed additives, antibiotic alternative feed additives, antimicrobial coatings for medical consumables, cosmetics, disinfectants, or detergents.

4. The application as described in claim 3, characterized in that, The bacteria are selected from at least one of the following: Staphylococcus aureus ATCC 6538, Methicillin-resistant Staphylococcus aureus USA 300, Pseudomonas aeruginosa ATCC 27853, Acinetobacter baumannii ATCC 19606, Escherichia coli ATCC 25922, and Salmonella typhimurium 14028S.

5. An antibacterial composition, characterized in that, The antimicrobial composition comprises the broad-spectrum antimicrobial peptide ESA_3 as described in claim 1 or 2, and pharmaceutically, feed-grade, cosmetically, or materials-grade excipients, carriers, or matrices.

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