Method for identifying novel antibacterial peptide based on evolution selection driving strategy and application thereof

Abstract

The invention discloses a method for identifying a novel antibacterial peptide based on an evolution selection driving strategy and application thereof, and relates to the technical field of biology. Genes of 128 bacilli are mined through an evolutionary driving strategy and a chemical synthesis technology, eight lead compounds acting on drug-resistant pathogenic bacterium infection are obtained, namely, double-target antibacterial peptides Paenitracin, Bacillin A-C8, Bacillin B-C8, Bacillin C-C7, Bacillin A-L, Bacillin A-C3, Bacillin A-C5 and Bacillin B-C1 of a new structure, and the Bacillin A-L and the Bacillin A-C5 are lead compounds which are not prone to generating drug resistance.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a method for identifying novel antimicrobial peptides based on an evolutionary selection-driven strategy and its application. Background Technology

[0002] Antibiotic resistance is a global health challenge, impacting diverse populations across multiple geographical boundaries. The rapid spread of multidrug-resistant pathogens caused approximately 4.95 million deaths globally in 2019, and is projected to cause 10 million deaths by 2050. The six deadliest multidrug-resistant bacteria worldwide include Acinetobacter baumannii, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Staphylococcus aureus, and Streptococcus pneumoniae, yet highly effective antibiotics are lacking for clinical treatment. Clinical treatment urgently requires novel antibiotics with novel structural frameworks, novel mechanisms of action, and no cross-resistance with existing drugs. In recent years, large-scale microbial genome sequencing has revealed a wealth of untapped biosynthetic resources within biosynthetic gene clusters (BGCs). Some of these untapped biosynthetic gene clusters have the potential to become novel antibiotics for the treatment of drug-resistant bacteria. However, a priority evaluation system needs to be established to efficiently screen target BGCs and rapidly obtain their encoded compounds.

[0003] Most natural environments (such as soil, the deep sea, and the human gut) harbor a diverse array of microorganisms that must compete with other microorganisms producing in the same environment for resources such as space and nutrients. Microbial secondary metabolites are their weapons against other species and are crucial for their survival because compounds derived from these metabolites have the potential to inhibit the growth of other microorganisms. In particular, microorganisms also acquire specialized DNA (such as antibiotic biosynthetic gene clusters or resistance genes) from other microorganisms through mobile genetic elements, enabling them to adapt to highly variable environmental conditions. This horizontal gene transfer is a significant evolutionary driver of the complexity of microbial natural products. Naturally derived antibiotic analogs also prevent resistance from competing microorganisms through mutations in the core or accessory genes of their biosynthetic genes (BGCs). Therefore, naturally derived antibiotics often contain multiple structural analogs encoded by evolutionarily related synthetic gene clusters. These related but distinct antibiotic biosynthetic gene clusters ultimately form gene cluster families (GCFs).

[0004] Therefore, those skilled in the art are dedicated to developing a novel antimicrobial peptide identified based on an evolutionary selection-driven strategy, and applying it as a lead compound for the preparation of antibiotic drugs against multidrug-resistant bacteria. Summary of the Invention

[0005] In view of the above-mentioned deficiencies of the prior art, the technical problem to be solved by the present invention is how to develop a novel antimicrobial peptide identified based on an evolutionary selection-driven strategy, and use it as a lead compound for the preparation of antimicrobial drugs against drug-resistant bacterial infections.

[0006] To achieve the above objectives, this invention provides a method for identifying novel antimicrobial peptides based on an evolutionary selection-driven strategy, comprising the following steps: Step 1: Encode non-ribosomal antimicrobial peptide families from 128 genera in the order Bacillus with the suffix "-bacillus"; Step 2: The non-ribosomal antimicrobial peptide family obtained in Step 1 was screened using the synBNP method of structure prediction chemical synthesis to obtain a novel non-ribosomal antimicrobial peptide family. Step 3: The novel non-ribosomal antimicrobial peptide family obtained in Step 2 is investigated for its antimicrobial activity and mechanism, and novel antimicrobial peptides are obtained.

[0007] Furthermore, step 3 explores the antibacterial activity and mechanism, including: in vitro antibacterial activity, cytotoxicity, hemolytic activity, acute toxicity and antibacterial activity in mice, and analysis of the antibacterial mechanism.

[0008] This invention also provides three novel antimicrobial peptide families: the Bacitracin family, the Bacimycin family, and the Bacipeptin family.

[0009] Furthermore, the Bacitacin family includes the known Bacitracin and the newly discovered Peanitracin; the Bacimycin family includes Bacimycin A, Bacimycin B and Bacimycin C; the Bacipeptin family includes Bacipeptin AL, Bacipeptin A-C3, Bacipeptin A-C5 and Bacipeptin B-C1.

[0010] This invention also provides eight novel antimicrobial peptides: Peanitracin, Bacimycin A, Bacimycin B, Bacimycin C, Bacipeptin AL, Bacipeptin A-C3, Bacipeptin A-C5, and Bacipeptin B-C1.

[0011] Furthermore, the chemical structures of Peanitracin are shown in Formula 1, Bacimycin A in Formula 2, Bacimycin B in Formula 3, Bacimycin C in Formula 4, Bacipeptin A-L in Formula 5, Bacipeptin A-C3 in Formula 6, Bacipeptin A-C5 in Formula 7, and Bacipeptin B-C1 in Formula 8. Formula 1, Equation 2, Equation 3, Equation 4, Equation 5, Formula 6, Equation 7, Formula 8.

[0012] This invention also provides an application of a novel antimicrobial peptide in the preparation of antimicrobial drugs.

[0013] Furthermore, the antimicrobial drug is a drug that fights pathogenic bacteria, which are Gram-negative bacteria, Gram-positive bacteria, and / or pathogenic fungi.

[0014] Furthermore, the Gram-positive bacteria were vancomycin-resistant Enterococcus faecalis 35682 and penicillin-resistant Staphylococcus aureus ATCC 6538.

[0015] Furthermore, the pathogens also include Staphylococcus aureus ATCC 25923.

[0016] In a preferred embodiment 1 of the present invention, the process of mining the genomes of 128 Bacillus genera through an evolution-driven strategy and chemical synthesis technology is described in detail. In another preferred embodiment 2 of the present invention, the process of investigating the antibacterial activity and mechanism of Peanitracin is described in detail. In another preferred embodiment 3 of the present invention, the process of investigating the antibacterial activity and mechanism of Bacitracin and Bacimycin family is described in detail. In another preferred embodiment 4 of the present invention, the cytotoxicity and drug resistance experimental process is described in detail; In another preferred embodiment 5 of the present invention, the epidermal infection experimental process is described in detail.

[0017] The beneficial technical effects of this invention are as follows: This invention utilizes the hypothesis that "gene clusters forming families have a high probability of encoding active compounds" in highly competitive environments to successfully screen for novel structural natural products with significant antibacterial activity, providing an efficient discovery strategy for antibiotic development. By mining the genomes of 128 Bacillus genera using evolution-driven strategies and chemical synthesis techniques, eight lead compounds targeting drug-resistant bacterial infections were obtained: novel, dual-target antimicrobial peptides Paenitracin, Bacimycin A-C8, Bacimycin B-C8, Bacimycin C-C7, Bacipeptin AL, Bacipeptin A-C3, Bacipeptin A-C5, and Bacipeptin B-C1. Paenitracin possesses a relatively novel target, C55-PP. Bacimycin A-C8, Bacimycin B-C8, Bacimycin C-C7, Bacipeptin AL, Bacipeptin A-C3, Bacipeptin A-C5, and Bacipeptin B-C1 exhibit single or dual targets (targeting cell membrane components), demonstrating broad-spectrum activity against Gram-positive and Gram-negative bacteria. Among these, Bacipeptin AL and Bacipeptin B-C1 show particularly strong activity. A-C5 is not prone to developing drug resistance and has good antibacterial activity in mice.

[0018] Eight novel antibiotics with specific multi-targets targeting the cell membrane or cell wall were obtained. Peanitracin targets C55-PP; the Bacimycin family primarily targets phosphatidylethanolamine (PE) in Gram-negative bacteria, and phosphatidylglycerol (PG) and cardiolipin (CL) in Gram-positive bacteria; the Bacipeptin family primarily targets PE and lipopolysaccharide (LPS) in Gram-negative bacteria, and PG and CL in Gram-positive bacteria. These eight novel lead compounds with unique targets provide a basis for the treatment of drug-resistant pathogens.

[0019] Because Bacipeptin-L and Bacipeptin-C5 have dual targets on the bacterial cell membrane and strong binding affinity to their targets, they are unlikely to induce drug resistance. Using a 21-day continuous resistance induction experiment, we directly observed that Bacipeptin-L and Bacipeptin-C5 are two lead compounds that are unlikely to induce drug resistance in the treatment of infections caused by drug-resistant pathogens.

[0020] This invention utilizes an evolution-driven strategy to efficiently screen for promising microbial genetic compounds (BGCs) and combines this with the synBNP method to rapidly obtain target compounds. This provides a general strategy for the efficient mining of microbial natural products.

[0021] Bacipeptin AL and Bacipeptin A-C5 have essentially the same antibacterial activity as previously approved antimicrobial lipopeptides daptomycin and polymyxin, and are less likely to induce drug resistance, making them good substitutes. Bacipeptin AL and Bacipeptin A-C5 are obtained through conventional solid-phase peptide synthesis, with a large-scale production cost of 3,000-5,000 yuan / gram, making their production highly feasible.

[0022] The two antimicrobial lipopeptides Bacipeptin AL and Bacipeptin A-C5 obtained in this invention have good prospects for industrial application, while the remaining six antimicrobial peptides provide a new structural paradigm for the clinical development of antibiotics for multidrug-resistant bacteria.

[0023] The following will further explain the concept, specific structure, and technical effects of the present invention in conjunction with the accompanying drawings, so as to fully understand the purpose, features, and effects of the present invention. Attached Figure Description

[0024] Figure 1 This is a schematic diagram of a preferred embodiment of the present invention, showing how gene cluster families drive antibiotic structural diversification to circumvent drug resistance. Figure 2 This is a schematic diagram of the formation of a gene cluster family driven by gene mutation according to a preferred embodiment of the present invention. Figure 3 This is a graph showing the experimental results of the antibacterial activity of Peanitracin, a novel Bacitracin family compound, according to a preferred embodiment of the present invention. Figure 4 This is a graph showing the cytotoxicity test results of Peanitracin, a novel Bacitracin family compound, according to a preferred embodiment of the present invention. Figure 5 This is a diagram illustrating the analytical results of the antibacterial mechanism of the Bacimycin family in a preferred embodiment of the present invention. Figure 6 This is a graph showing the isothermal titration calorimetric analysis results of CL, PG, PE or LPS on Bacimycin A according to a preferred embodiment of the present invention. Figure 7 This is a diagram illustrating the analytical results of the antibacterial mechanism of the Bacipeptin family in a preferred embodiment of the present invention. Figure 8 This is a graph showing the isothermal titration calorimetric analysis results of CL, PG, PE or LPS on Bacipeptin AL according to a preferred embodiment of the present invention. Figure 9 This is a graph showing the results of safety, resistance, and serum activity analysis of the Bacimycin family in a preferred embodiment of the present invention. Figure 10 This is a graph showing the results of safety, resistance, and acute toxicity analysis of the Bacipeptin family in a preferred embodiment of the present invention. Figure 11 This is the experimental results of the antibacterial activity of Bacipeptin AL and Bacipeptin A-C5 in a Staphylococcus aureus USA300 mouse wound infection model according to a preferred embodiment of the present invention. Detailed Implementation

[0025] The following description, with reference to the accompanying drawings, illustrates several preferred embodiments of the present invention to make its technical content clearer and easier to understand. The present invention can be embodied in many different forms, and the scope of protection of the present invention is not limited to the embodiments mentioned herein.

[0026] Example 1: Mining the genomes of 128 Bacillus genera using evolution-driven strategies and chemical synthesis techniques.

[0027] This invention proposes the following hypothesis: if multiple uncharacterized biosynthetic gene clusters from microorganisms in a competitive natural environment tend to evolve into gene cluster families, the natural products they encode are likely to exhibit antibacterial activity.

[0028] We applied the above hypothesis to search for nonribosomal antimicrobial peptides in the genus *Bacillus*. Several genera in the order *Bacillus* ending in "-bacillus" (such as *Bacillus*, *Brevibacillus*, *Paenibacillus*, and *Virgibacillus*) have been reported as rich sources of nonribosomal antimicrobial peptides. These genera produce nonribosomal antimicrobial peptides with diverse chemical structures and mechanisms of action, including polymyxins (as a last line of defense against Gram-negative pathogens), bacitracin, cigligiline, laterosporin, octopeptin, decadecapeptin, and casein. Notably, except for bacitracin and casein, all of the above nonribosomal antimicrobial peptides have been reported to have at least one homologue, whose encoded synthetic gene clusters have evolved into gene cluster families. Furthermore, species within these genera can form widely distributed dormant spores in soil or other complex environments. These spores can remain viable for extended periods and resist extreme environmental factors (such as high temperatures and chemicals). These facts suggest that genera in the order Bacillus with the suffix "-bacillus" survive in a competitive natural environment, making them ideal subjects for verifying our hypothesis.

[0029] Gene cluster families drive antibiotic structural diversification to circumvent drug resistance. (See diagram below) Figure 1 As shown in the diagram, antibiotic biosynthetic gene clusters (BGCs) in bacteria living in competitive natural environments tend to evolve into gene cluster families (GCFs), thereby encoding a series of structurally similar antimicrobial compounds. Closely related strains acquire new DNA through horizontal gene transfer (HGT) via mobile genetic elements (MGEs), thus adapting to highly variable environments and circumventing environmental resistance mechanisms. A schematic diagram illustrating the formation of gene cluster families driven by gene mutations is shown below. Figure 2 As shown. Structurally similar antibiotic BGCs tend to evolve into GCFs through mutations in core or accessory genes during evolution.

[0030] Therefore, in this study, we combined the evolutionary selection-driven natural product discovery approach with the synBNP method for structure-predictive chemical synthesis to systematically study the non-ribosomal antimicrobial peptide families encoded by 128 genera in the order Bacillus with the suffix "-bacillus". This strategy successfully discovered three novel non-ribosomal antimicrobial peptide families—the Bacitracin family, the Bacimycin family, and the Bacipeptin family—containing a total of eight active linear or cyclic structures. These compounds showed good activity against multidrug-resistant Gram-positive and Gram-negative pathogens. Furthermore, the Bacipeptin family demonstrated significant efficacy against methicillin-resistant Staphylococcus aureus in a mouse wound infection model, and holds promise as a novel lead compound for treating severe infections caused by multidrug-resistant pathogens.

[0031] Example 2: Investigation of the antibacterial activity and mechanism of Peanitracin

[0032] The antibacterial activity and mechanism of Peanitracin were investigated. The results of the antibacterial activity experiment are as follows: Figure 3 Part a shows the chemical structural formula of Peanitracin; part b shows the antibacterial activity and cytotoxicity of Peanitracin; part c shows the accumulation of UDP-MurNAc-pentapeptide in Staphylococcus aureus cultures treated with Paenitracin (8× MIC) by LC-MS analysis; part d shows the antibacterial activity of Paenitracin against Staphylococcus aureus (n=2) in the presence of different concentrations of C55-PP; and part e shows the 21-day continuous passage resistance experiment of Staphylococcus aureus ATCC 6538 against Peanitracin. Peanitracin cytotoxicity assay results are also included. Figure 4 As shown, Figure 4Part a presents the hemolytic activity of Peanitracin against sterile defibrinated sheep blood; Part b presents the cytotoxicity of Peanitracin against human HeLa cells; and Part c presents the cytotoxicity of Peanitracin against human HEK293T cells. Parts a and b show that Paenitracin exhibits an antibacterial spectrum similar to Bacitracin, demonstrating significant activity against various Gram-positive pathogens, including vancomycin-resistant Enterococcus faecalis 35682 and penicillin-resistant Staphylococcus aureus ATCC 6538. Parts c and d show that mechanism-of-action studies indicate that, similar to Bacitracin, Paenitracin leads to the accumulation of UDP-MurNAc-pentapeptide, a major precursor in bacterial cell wall biosynthesis. Further analysis using Staphylococcus aureus ATCC 6538 as the test strain revealed that the addition of Bacitracin's target C55-PP significantly inhibited the antibacterial activity of Paenitracin in a dose-dependent manner, as shown in Part e. These results confirm that Paenitracin does indeed belong to the Bacitracin family. However, in a 21-day continuous resistance study, as shown in section f, Paenitracin caused Staphylococcus aureus ATCC 6538 to rapidly develop resistance, with the minimum inhibitory concentration increasing by up to 128-fold. Although Paenitracin, like Bacitracin, maintains antibacterial activity in the serum environment and did not exhibit significant hemolytic activity or cytotoxicity, the experimental results are as follows... Figure 4 As shown in the figure, but the present invention has not evaluated its in vivo activity in mouse models.

[0033] Example 3: Investigation of the antibacterial activity and mechanism of Bacitracin and Bacimycin family

[0034] A variety of Gram-negative bacteria, Gram-positive bacteria, and pathogenic fungi were selected for activity testing. The antibacterial activities of Bacimycin A, Bacimycin B, and Bacimycin C are shown in Table 1, and the antibacterial activities of Bacipeptin AL, Bacipeptin A-C3, Bacipeptin A-C5, and Bacipeptin B-C1 are shown in Table 2. The results showed that Bacimycin A, Bacimycin B, and Bacimycin C all had significant antibacterial activity against Gram-positive and Gram-negative bacteria, with Bacimycin A exhibiting the most prominent antibacterial activity, even against multidrug-resistant bacteria such as Acinetobacter baumannii and Staphylococcus aureus, with a MIC value reaching 4 μg / mL. Bacipeptin AL, Bacipeptin A-C3, Bacipeptin A-C5, and Bacipeptin B-C1 also showed broad-spectrum activity against Gram-positive and Gram-negative bacteria, and their activity was better than that of compounds in the Bacimycin family, with MIC values ​​reaching 1-2 μg / mL.

[0035] Table 1. Antibacterial activities of Bacimycin A, Bacimycin B and Bacimycin C

[0036] Table 2. Antibacterial activity of Bacipeptin AL, Bacipeptin A-C3, Bacipeptin A-C5 and Bacipeptin B-C1

[0037] The antibacterial mechanisms of compounds from these two families were elucidated. The elucidation process of the antibacterial mechanism of the Bacimycin family is as follows: Figure 5As shown, part a represents the bactericidal activity of Bacimycin A, Bacimycin B, and Bacimycin C against Staphylococcus aureus ATCC 25923; part b represents the depolarization results of the cell membrane potential of Bacimycin A, Bacimycin B, and Bacimycin C against Staphylococcus aureus ATCC 25923, indicating that Bacimycin A, Bacimycin B, and Bacimycin C can cause rapid lysis of Staphylococcus aureus ATCC 25923 cells; part c represents the antibacterial activity of Bacimycin A, Bacimycin B, and Bacimycin C in the presence of CL, PG, or PE. The in vitro small molecule addition experiment shows that Bacimycin A, Bacimycin B, and Bacimycin C can bind to PG and CL in the cell membrane of Gram-positive bacteria, and can also bind to PE in the cell membrane of Gram-negative bacteria. Next, an isothermal titration experiment will be conducted, using Bacimycin A as an example. The isothermal titration calorimetric analysis of Bacimycin A with CL, PG, PE, or LPS is as follows: Figure 6 As shown in the figure. The results show that Bacimycin A targets PG and CL in Gram-positive bacteria, and PE in Gram-negative bacteria.

[0038] The process of elucidating the antibacterial mechanism of the Bacipeptin family is as follows: Figure 7 As shown, part a represents the bactericidal activity of Bacipeptin AL, Bacipeptin A-C3, Bacipeptin A-C5, and Bacipeptin B-C1 against Staphylococcus aureus ATCC 25923; part b represents the cell membrane lysis results of Bacipeptin AL, Bacipeptin A-C3, Bacipeptin A-C5, and Bacipeptin B-C1 against Staphylococcus aureus ATCC 25923; and part c represents the cell membrane potential depolarization results of Bacipeptin AL, Bacipeptin A-C3, Bacipeptin A-C5, and Bacipeptin B-C1 against Staphylococcus aureus ATCC 25923. For the Bacipeptin family, Bacipeptin AL, Bacipeptin A-C3, Bacipeptin A-C5, and Bacipeptin B-C1 can induce rapid lysis of Staphylococcus aureus ATCC 25923 cells. Figure 7Part d represents the antibacterial activity (MIC: μg / mL) of Bacipeptin A-C3 or Bacipeptin B-C1 against Staphylococcus aureus ATCC 25923 in the presence of CL and PG; Part e represents the antibacterial activity of Bacipeptin AL and Bacipeptin A-C5 against Staphylococcus aureus ATCC 25923 in the presence of CL and PG. In vitro small molecule addition experiments show that Bacipeptin AL, Bacipeptin A-C3, Bacipeptin A-C5, and Bacipeptin B-C1 can bind to PG and CL in the cell membrane of Gram-positive bacteria, and simultaneously bind to PE and LPS in the cell membrane of Gram-negative bacteria. Isothermal titration experiments were performed using Bacipeptin A-L and Bacipeptin A-C5, which exhibit better activity. Using Bacipeptin AL as an example, the isothermal titration calorimetric analysis of CL, PG, PE, or LPS against Bacipeptin AL is shown below. Figure 8 As shown in the figure. The results show that Bacipeptin AL targets PG and CL in Gram-positive bacteria, and also has dual targets, PE and LPS, in Gram-negative bacteria. Furthermore, its binding affinity to both Gram-positive and Gram-negative bacteria is stronger than that of antimicrobial peptides in the Bacimycin family, explaining the superior antimicrobial activity of Bacipeptin AL and Bacipeptin A-C5 compared to Bacimycin A. Through the above experiments, this invention not only verifies the good broad-spectrum antimicrobial activity of Bacimycin and the Bacipeptin family, but also elucidates their antimicrobial mechanism.

[0039] Example 4 Cytotoxicity and Drug Resistance Experiments

[0040] The hemolytic activity of bacimycin A, bacimycin B, bacimycin C, bacipeptin A-L, bacipeptin A-C3, bacipeptin A-C5, and bacipeptin B-C1 was tested using sterile defibrinated sheep blood. It was found that none of them exhibited significant hemolytic activity. The experimental results for the hemolytic activity of bacimycin A, bacimycin B, and bacimycin C on sterile defibrinated sheep blood are as follows: Figure 9 As shown in section a (using Bacimycin A as an example only); the experimental results of the hemolytic activity of Bacipeptin AL, Bacipeptin A-C3, Bacipeptin A-C5, and Bacipeptin B-C1 on sterile defibrinated sheep blood are as follows. Figure 10The results are shown in section a (using Bacipeptin AL as an example only). Their cytotoxicity to human HeLa or HEK293T cells was also tested, and none showed significant cytotoxicity. The cytotoxicity results of Bacimycin A, Bacimycin B, and Bacimycin C against human HeLa cells are as follows: Figure 9 As shown in section b (using Bacimycin A as an example only), its cytotoxicity against human HEK293T cells is as follows: Figure 9 As shown in section c (using only Bacimycin A as an example); the cytotoxicity of Bacipeptin AL, Bacipeptin A-C3, Bacipeptin A-C5, and Bacipeptin B-C1 to human HeLa cells is as follows. Figure 10 As shown in section b (using Bacipeptin AL as an example only), its cytotoxicity to human HEK293T cells is as follows: Figure 10 As shown in section c (using Bacipeptin AL as an example only).

[0041] A 21-day resistance test was conducted. Results showed that Bacimycin A or Bacimycin B readily induced resistance, while Bacipeptin AL and Bacipeptin A-C5, due to their dual targets and stronger binding affinity, were less likely to induce resistance in multiple clinically resistant pathogens, thus demonstrating promising clinical development potential. The 21-day continuous testing results for Bacimycin A, Bacimycin B, and Bacimycin C were further detailed. Figure 9 As shown in section d; the results of the 21-day continuous resistance assay for Bacipeptin AL, Bacipeptin A-C3, Bacipeptin A-C5 and Bacipeptin B-C1 are as follows. Figure 10 As shown in section d. Furthermore, the antibacterial activity of Bacimycin A or Bacimycin B against Acinetobacter baumannii 1104008 and Staphylococcus aureus USA300 is significantly reduced in serum conditions. The minimum inhibitory concentrations (MICs) of Bacimycin A, Bacimycin B, and Bacimycin C against Acinetobacter baumannii 1104008 or Staphylococcus aureus USA300 in the presence or absence of serum are as follows: Figure 9As shown in section e, subsequent mouse experiments were not considered. Based on this, this invention investigated the acute toxicity of Bacipeptin AL and Bacipeptin A-C5 in mice using 6-week-old female ICR mice. The results showed that mice survived after intraperitoneal injection of a high concentration of 150 mg / kg Bacipeptin AL and Bacipeptin A-C5 for 1-5 days, indicating that Bacipeptin AL and Bacipeptin A-C5 had no significant acute toxicity. The results of the acute toxicity experiments of Bacipeptin AL, Bacipeptin A-C3, Bacipeptin A-C5, and Bacipeptin B-C1 in mice are as follows: Figure 10 As shown in part e.

[0042] Example 5: Epidermal Infection Experiment

[0043] In vivo antibacterial activity tests were conducted on Bacipeptin AL and Bacipeptin A-C5. The results of the antibacterial activity experiments of Bacipeptin AL and Bacipeptin A-C5 in a Staphylococcus aureus USA300 mouse wound infection model are as follows: Figure 11 As shown. First, a 1 cm [unclear text - possibly a typo, should be inserted here] is constructed on the mouse epidermis. 2 The wound was then inoculated with Staphylococcus aureus USA300, followed by six injections of Bacipeptin AL and Bacipeptin A-C5 at 12-hour intervals. The wound was observed for 11 days, and the epidermal wound healing was recorded. On day 11, skin samples were collected from the wound for CFU counting of pathogenic bacteria. A schematic diagram of the mouse wound infection experiment is shown below. Figure 11 As shown in section a. The results indicate that both Bacipeptin AL and Bacipeptin A-C5 exhibit excellent anti-Gram-positive bacterial activity in the wound infection model, with wound records as follows. Figure 11 As shown in section b, the statistical results of wound size are shown in section c, and the mouse weight change records are shown in section c. Figure 11 As shown in section d, the bacterial load count in the wound tissue 11 days after infection is illustrated in the figure. Figure 11 As shown in section e, after three consecutive days of injection, Bacipeptin AL and Bacipeptin A-C5 not only promoted wound healing but also significantly reduced bacterial load by an order of magnitude after day 11 of infection, with effects comparable to vancomycin. Their excellent resistance to drug resistance and significant in vivo efficacy suggest that Bacipeptin AL and Bacipeptin A-C5 hold promise as lead compounds for the treatment of multidrug-resistant bacteria.

[0044] The preferred embodiments of the present invention have been described in detail above. It should be understood that those skilled in the art can make numerous modifications and variations based on the concept of the present invention without creative effort. Therefore, all technical solutions that can be obtained by those skilled in the art based on the concept of the present invention through logical analysis, reasoning, or limited experimentation on the basis of existing technology should be within the scope of protection defined by the claims.

Claims

1. A method for identifying novel antibacterial peptides based on evolutionarily selected driving strategies, characterized in that, The method comprises the following steps: Step 1, screening a non-ribosomal antibacterial peptide family from 128 genera with "-bacillus" as a suffix in the order of Bacillus; Step 2, screening the non-ribosomal antibacterial peptide family obtained in step 1 by combining structure prediction with a chemically synthesized synBNP method to obtain a new non-ribosomal antibacterial peptide family; Step 3, exploring the antibacterial activity and mechanism of the new non-ribosomal antibacterial peptide family obtained in step 2 to obtain the new antibacterial peptide.

2. The method of claim 1, wherein, The antibacterial activity and mechanism exploration in step 3 are in vitro antibacterial activity, cytotoxicity, hemolytic activity, acute toxicity and antibacterial activity in mice, and antibacterial mechanism analysis.

3. A novel family of antibacterial peptides screened by the method of claim 1 or 2, characterized in that, The new antibacterial peptide family is a Bacitracin family, a Bacimycin family and a Bacipeptin family.

4. The novel family of antimicrobial peptides as claimed in claim 3, wherein, The Bacitacin family includes Peanitracin; the Bacimycin family includes Bacimycin A, Bacimycin B and Bacimycin C; and the Bacipeptin family includes Bacipeptin A-L, Bacipeptin A-C3, Bacipeptin A-C5 and Bacipeptin B-C1.

5. A novel antibacterial peptide screened by the method of claim 1 or 2, wherein the peptide is selected from the group consisting of SEQ ID NO: 1 to 20. The new antibacterial peptide is Peanitracin, Bacimycin A, Bacimycin B, Bacimycin C, Bacipeptin A-L, Bacipeptin A-C3, Bacipeptin A-C5 and / or Bacipeptin B-C1.

6. A novel antimicrobial peptide as claimed in claim 5, wherein, The chemical structure of Peanitracin is shown as formula 1, the chemical structure of Bacimycin A is shown as formula 2, the chemical structure of Bacimycin B is shown as formula 3, the chemical structure of Bacimycin C is shown as formula 4, the chemical structure of Bacipeptin A-L is shown as formula 5, the chemical structure of Bacipeptin A-C3 is shown as formula 6, the chemical structure of Bacipeptin A-C5 is shown as formula 7, and the chemical structure of Bacipeptin B-C1 is shown as formula 8. Formula 1, Formula 2, Formula 3, Formula 4, Formula 5, Formula 6, Formula 7, Formula 8.

7. Use of the new antibacterial peptide of claim 5 in the preparation of an antibacterial drug.

8. The application as described in claim 7, characterized in that, The antibacterial drug is an anti-pathogenic bacterial drug, and the pathogenic bacteria are gram-negative bacteria, gram-positive bacteria and / or pathogenic fungi.

9. Use according to claim 8, wherein The gram-positive bacteria are vancomycin-resistant Enterococcus faecalis 35682 and penicillin-resistant Staphylococcus aureus ATCC 6538.

10. Use according to claim 8, wherein the compound is ###0002### The pathogenic bacteria also include Staphylococcus aureus ATCC 25923.

Images