Lumbricin and application of lumbricin in broad-spectrum antibiosis

Abstract

The application provides an earthworm antibacterial peptide derived from a popular cavity earthworm and application thereof in broad-spectrum antibacterial aspects. The amino acid sequence of the antibacterial peptide is shown in SEQ ID NO. 1 or SEQ ID NO. 2, and both have 13 amino acid residues and isoelectric points of 10.31 or 10.06. The application adopts a bioinformatics method, finds a new earthworm antibacterial peptide through a machine learning algorithm, optimizes the amino acid sequence according to the prediction result, and finally obtains an antibacterial peptide with better antibacterial effect. The obtained antibacterial peptide has good antibacterial activity on gram-negative bacteria represented by escherichia coli and gram-positive bacteria represented by staphylococcus aureus, and various drug-resistant bacterial variants of the two kinds of bacteria, and can be applied to the preparation of antibacterial products, including antibacterial drugs, additives and preservatives and the like.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and relates to an earthworm antimicrobial peptide derived from common coelomic earthworms and its application in broad-spectrum antimicrobial activity. Background Technology

[0002] With the widespread use and even abuse of antibiotics, the problem of bacterial resistance is becoming increasingly serious. The emergence of various drug-resistant bacteria poses a huge challenge to public health, making the development of novel antimicrobial drugs an urgent priority. Antimicrobial peptides, as an important component of the body's innate immunity, possess broad-spectrum antimicrobial activity and favorable physicochemical properties. Their structures are mostly amphiphilic, primarily acting on the bacterial cell membrane through hydrophobic interactions and electrostatic adsorption, causing the outflow of bacterial contents to achieve an antimicrobial effect. This physical action is unlikely to induce bacterial resistance and there is no cross-tolerance with traditional antibiotics. Therefore, they can exert antimicrobial activity even against drug-resistant bacteria, making them a highly promising alternative to antibiotics.

[0003] Nature is a vast treasure trove for antimicrobial peptides, and earthworms, as annelids that have long lived in soil environments rich in humic acid and pathogenic microorganisms, have evolved an extremely powerful immune system. Studies have shown that earthworms contain a variety of polypeptides with antimicrobial activity. However, current development of earthworm antimicrobial peptides is mostly limited to biochemical extraction and separation. This traditional trial-and-error method is not only time-consuming and labor-intensive, with low yields, but also makes it difficult to discover novel antimicrobial peptides with low abundance.

[0004] In recent years, with the completion of genome sequencing for species such as *Eriocheir sinensis*, their genetic information has provided a data foundation for the discovery of novel antimicrobial peptides. The genome contains a large number of open reading frames (ORFs), many of which contain undiscovered bioactive peptide sequences. How to efficiently screen and discover novel, low-toxicity earthworm antimicrobial peptides with antimicrobial potential from massive amounts of genomic data using bioinformatics methods and machine learning algorithms is key to further utilizing biological resources.

[0005] Since the ban on electrofishing of earthworms in 2023, earthworm resources have become scarce, creating an urgent need for active ingredients derived from earthworms for drug production and clinical use. Chinese patent CN144773435B discloses an earthworm antimicrobial peptide, EWAMP-R, extracted and modified from the whole genome of *Eisenia fetida* and its antimicrobial applications. However, the earthworm used in this method is *Eisenia fetida*, belonging to the genus *Eisenia*, which is not listed in the *Chinese Pharmacopoeia*. Therefore, how to extract antimicrobial peptides from the earthworm genome specified in the *Chinese Pharmacopoeia* is a pressing technical problem that needs to be solved in this field. Summary of the Invention

[0006] To address the aforementioned technical problems, this invention provides an earthworm antimicrobial peptide derived from *Pheretima asiatica* and its application in broad-spectrum antibacterial activity. The earthworm used in this invention is *Pheretima asiatica*, belonging to the genus *Pheretima*, and specifically to *Pheretima spp.*, one of the four types of earthworms specified in the *Chinese Pharmacopoeia*. This invention employs a bioinformatics strategy combining machine learning algorithms (ANN, RF, SVM) with gene polymorphism analysis for efficient screening and optimization. From *Pheretima asiatica*, a novel earthworm antimicrobial peptide was obtained that exhibits activity against both standard Gram-positive and Gram-negative bacteria, as well as against various clinically resistant bacteria such as methicillin-resistant Staphylococcus aureus (MRSA) and extended-spectrum β-lactamase-resistant *Escherichia coli*. Furthermore, it exhibits low hemolytic activity and has development potential.

[0007] The objective of this invention can be achieved through the following methods:

[0008] <First Aspect> This invention provides an earthworm antimicrobial peptide derived from common coelomic earthworm, the earthworm antimicrobial peptide comprising MVAMP.1 and MVAMP.2, the amino acid sequences of which are shown in SEQ ID NO.1-2, respectively.

[0009] As one embodiment of the present invention, the MVAMP.1 has 13 amino acid residues and its isoelectric point is 10.31.

[0010] As one embodiment of the present invention, the MVAMP.2 has 13 amino acid residues and its isoelectric point is 10.06.

[0011] The novel earthworm antimicrobial peptide of this invention has 13 amino acid residues, exhibiting amphiphilic and cationic characteristics, with isoelectric points of 10.31 and 10.06, respectively. Based on this, the antimicrobial peptide can exert broad-spectrum antibacterial activity by disrupting bacterial cell membrane structure or interacting with intracellular molecules.

[0012] <Second aspect> This invention provides an application of the earthworm antimicrobial peptide derived from common coelomic earthworm in broad-spectrum antimicrobial activity.

[0013] As one embodiment of the present invention, the earthworm antimicrobial peptide is used for antimicrobial activity against Gram-negative and Gram-positive bacteria.

[0014] Furthermore, the MVAMP.1 is used for antibacterial activity against Gram-positive bacteria.

[0015] Furthermore, the MVAMP.2 is used for antibacterial activity against Gram-negative bacteria.

[0016] As one embodiment of the present invention, the Gram-positive bacteria include one of standard Staphylococcus aureus and methicillin-resistant Staphylococcus aureus.

[0017] As one embodiment of the present invention, the Gram-negative bacteria include one of standard Escherichia coli, extended-spectrum β-lactamase-resistant Escherichia coli, and carbapenem-resistant Escherichia coli.

[0018] Furthermore, the minimum inhibitory concentration of MVAMP.1 against standard Staphylococcus aureus is 128 μg / mL.

[0019] Furthermore, the minimum inhibitory concentration of MVAMP.2 against standard Escherichia coli is 128 μg / mL.

[0020] <Third aspect> The present invention also provides an antibacterial drug, additive, or preservative comprising the earthworm antimicrobial peptide.

[0021] Compared with the prior art, the present invention has the following beneficial effects: 1. This invention utilizes machine learning algorithms (ANN, RF, SVM) combined with bioinformatics strategies based on gene polymorphism analysis to efficiently screen and optimize novel antimicrobial peptides from the genome of *Eriocheir sinensis*. Compared to traditional experimental trial-and-error methods, this method can accurately identify active peptides with high stability and redundant sequences removed.

[0022] 2. The earthworm antimicrobial peptides MVAMP.1 and MVAMP.2 provided by this invention have short sequences, small molecular weight, are easy to synthesize, and have low cost.

[0023] 3. The antimicrobial peptides of the present invention are active against both standard Gram-positive and Gram-negative bacteria, and also exhibit inhibitory activity against a variety of clinically resistant bacteria (such as methicillin-resistant Staphylococcus aureus, extended-spectrum β-lactamase-resistant Escherichia coli, carbapenem-resistant Escherichia coli, etc.). Furthermore, at the effective antibacterial concentration, the hemolysis rate on mammalian erythrocytes is extremely low (<3%), demonstrating high biosafety and drug development potential. It can be applied to the preparation of antimicrobial drugs, feed additives, and preservatives. Detailed Implementation

[0024] The present invention will now be described in detail with reference to specific embodiments. The following examples are implemented under the premise of the technical solution of the present invention, providing detailed implementation methods and specific operating procedures, which will help those skilled in the art to further understand the present invention. It should be noted that the scope of protection of the present invention is not limited to the following embodiments; any adjustments and improvements made under the concept of the present invention are all within the scope of protection of the present invention.

[0025] Example 1: Screening and optimization of earthworm antimicrobial peptides Based on the whole genome of *Eupolyphaga sinensis* GCA_018105865.1, machine learning algorithms (ANN, RF, SVM) were used to predict ORFs (open reading frames), and long-chain polypeptides MVAMP-5 and MVAMP-6 with antibacterial potential were obtained. Their amino acid sequences are MCFSVCPCCCRGRFAWCKLKAYG (SEQ ID NO.3) and MGLRGCKKIAFINVHSKASKCAAQ (SEQ ID NO.4), respectively.

[0026] Primers were designed based on the predicted antimicrobial peptide sequences. Eight primer pairs and six DNA samples from *Eupolyphaga sinensis* (specific sequences of the eight primer pairs are shown in Table 1, and information on the six *Eupolyphaga sinensis* samples is shown in Table 2) were used for polymerase chain reaction (PCR) amplification. The PCR products were sent to Beijing Huada Genomics Co., Ltd. for gene sequencing. Based on the results, sequence differences were compared, single nucleotide polymorphisms were analyzed, and redundant sequences were removed to obtain optimized short-chain antimicrobial peptides MVAMP-5.1 (MVAMP.1) and MVAMP-6.1 (MVAMP.2), with amino acid sequences shown in SEQ ID NO.1 and SEQ ID NO.2, specifically RGFAWCKLKAYG and GLRGCKKIAFINV, respectively.

[0027] Table 1 Primer Design

[0028] Table 2 Information on common coelomic earthworm samples used

[0029] Experimental Example 2: Determination of the antibacterial activity of earthworm antimicrobial peptides MVAMP-5.1 and MVAMP-6.1 The short-chain antimicrobial peptides MVAMP-5.1 and MVAMP-6.1, which were screened and optimized in the previous step, were artificially synthesized using a solid-phase synthesis method with a purity greater than 95%. The antimicrobial activity of the artificially synthesized peptides was determined using the minimum inhibitory concentration (MIC) method. The specific steps are as follows: (1) Inoculate Escherichia coli ATCC25922 and Staphylococcus aureus ATCC29213 on TSA agar medium by streaking and incubate overnight at 37°C until single colonies grow. (2) Pick a single colony and inoculate it into fresh MH broth medium, and incubate at 37°C with shaking for one day; (3) Take 50 μL of the bacterial culture from step (2) overnight and add it to 5 mL of fresh MH broth medium. Incubate at 37°C with shaking for 2-3 hours until OD600≈0.5. (4) Inoculate the bacterial suspension from step (3) into fresh MH broth medium and shake to mix well, so that the bacterial density is approximately 1×10⁻⁶. 5 ~5×10 5 cfu / mL, to obtain the bacterial solution required for the assay; (5) Dissolve the earthworm antimicrobial peptides MVAMP-5.1 and MVAMP-6.1 described in this invention and several other candidate peptides that were predicted but not screened in the polymorphism optimization step in sterile water to prepare an antimicrobial peptide solution of 10 mg / mL. (6) Add 180 μL of the bacterial suspension obtained in step (4) to the first column of a sterile 96-well plate, and 100 μL of bacterial suspension to each of the other columns. Then add 20 μL of the antimicrobial peptide solution obtained in step (5) to the first column, mix well, and then transfer 100 μL to the second column. After mixing well, transfer 100 μL to the third column, and so on, to perform serial dilutions so that the final concentrations of the antimicrobial peptides in each column are approximately 1024, 512, 256, 128, 64, 32, 16, 8, 4, 2, 1, and 0.5 μg / mL. Each peptide is repeated three times. Incubate the 96-well plate at 37°C for 18–24 h. (7) Observe the bottom of each well for bacterial precipitation and turbidity caused by the light source. The minimum concentration without visible turbidity is the minimum inhibitory concentration of the antimicrobial peptide against this type of bacteria. Table 3 shows the sequences of the synthesized MVAMP-5.1, MVAMP-6.1 and other candidate peptides and the results of their minimum inhibitory concentration tests.

[0030] Table 3. Sequences and minimum inhibitory concentrations of MVAMP-5.1, MVAMP-6.1, and other candidate peptides.

[0031] As shown in the table above, the antimicrobial peptides MVAMP-5.1 (SEQ ID NO.1) and MVAMP-6.1 (SEQ ID NO.2), selected by polymorphism screening after prediction, have good antimicrobial activity. The minimum inhibitory concentrations against standard Staphylococcus aureus and Escherichia coli are MICS.aureus (5.1) = 128 μg / mL and MICE.coli (6.1) = 128 μg / mL, respectively.

[0032] Example 3: Determination of the antimicrobial activity of antimicrobial peptides MVAMP-5.1 and MVAMP-6.1 against multiple drug-resistant bacteria. The antimicrobial activities of the screened antimicrobial peptides MVAMP-5.1 and MVAMP-6.1 against various drug-resistant bacteria were determined using the minimum inhibitory concentration (MIC) method. Table 4 shows the results of the drug-resistant bacteria and their MIC tests.

[0033] Table 4. Minimum inhibitory concentrations of MVAMP-5.1 and MVAMP-6.1 against drug-resistant bacteria.

[0034] As shown in the table above, the minimum inhibitory concentrations (MICs) of MVAMP-5.1 and MVAMP-6.1 against a variety of common drug-resistant bacteria are mostly in the range of MIC = 128~256 μg / mL, indicating good antibacterial activity.

[0035] Experimental Example 4: Determination of the hemolytic activity of antimicrobial peptides MVAMP-5.1 and MVAMP-6.1 The hemolytic activity of the screened antimicrobial peptides MVAMP-5.1 and MVAMP-6.1 against mammalian (sheep) erythrocytes was determined, and their in vitro safety was evaluated. The specific steps are as follows: (1) Take 2 mL of defibrinated sheep blood, centrifuge at 3000 g for 10 min at 4℃, and retain the lower layer of cells; (2) Wash three times with PBS and add PBS to prepare an 8% red blood cell suspension; (3) Take 100 μL of the suspension from step (2) into a 96-well plate, and add 100 μL of antimicrobial peptide solution of different concentrations to make the final concentrations approximately 256, 128, 64, 32, 16, 8, 4, 2, 1, 0.5, 0.25, and 0.125 μg / mL, with a red blood cell suspension concentration of 4%. Use 100 μL of PBS as a negative control and 100 μL of 0.2% Triton X-100 as a positive control. Perform three replicates for each group. (4) After standing at 37℃ for more than 1 h, centrifuge at 3000g for 10 min at 4℃, transfer 100 μL of the supernatant to another 96-well plate, measure its absorbance at 576 nm, and calculate the hemolysis rate. The formula for calculating the hemolysis rate is: Table 5 shows the results of the hemolytic activity assays for MVAMP-5.1 and MVAMP-6.1.

[0036] Table 5. Results of the hemolysis rate assay of sheep erythrocytes using MVAMP-5.1 and MVAMP-6.1.

[0037] As shown in the table above, MVAMP-5.1 and MVAMP-6.1 exhibit hemolytic activity in mammalian (sheep) erythrocytes ranging from -0.94% to 2.77%. They are therefore renamed MVAMP.1 and MVAMP.2, respectively.

[0038] The experimental results show that the earthworm antimicrobial peptides MVAMP.1 and MVAMP.2 obtained in this invention have good antimicrobial activity against Escherichia coli, Staphylococcus aureus and various drug-resistant bacteria, and their hemolysis rate is low. They can be used for the development of antimicrobial drugs, additives and preservatives.

[0039] The specific embodiments of the present invention have been described above. It should be understood that the present invention is not limited to the specific embodiments described above, and those skilled in the art can make various modifications or variations within the scope of the claims, which do not affect the essence of the present invention.

Claims

1. An earthworm antimicrobial peptide derived from *Pheretima asiatica*, characterized in that, The earthworm antimicrobial peptides include MVAMP.1 and MVAMP.2, whose amino acid sequences are shown in SEQ ID NO.1-2, respectively.

2. The earthworm antimicrobial peptide according to claim 1, characterized in that, The MVAMP.1 has 13 amino acid residues and its isoelectric point is 10.31; the MVAMP.2 has 13 amino acid residues and its isoelectric point is 10.

06.

3. The application of an earthworm antimicrobial peptide derived from common coelomic earthworm as described in claim 1 or 2 in broad-spectrum antimicrobial activity.

4. The application according to claim 3, characterized in that, The earthworm antimicrobial peptide is used for antimicrobial activity against Gram-negative and Gram-positive bacteria.

5. The application according to claim 4, characterized in that, The MVAMP.1 is used for antibacterial activity against Gram-positive bacteria; the MVAMP.2 is used for antibacterial activity against Gram-negative bacteria.

6. The application according to claim 4, characterized in that, The Gram-positive bacteria include one of standard Staphylococcus aureus and methicillin-resistant Staphylococcus aureus.

7. The application according to claim 4, characterized in that, The Gram-negative bacteria include one of the following: standard Escherichia coli, extended-spectrum β-lactamase-resistant Escherichia coli, and carbapenem-resistant Escherichia coli.

8. The application according to claim 6, characterized in that, The minimum inhibitory concentration of MVAMP.1 against standard Staphylococcus aureus is 128 μg / mL.

9. The application according to claim 7, characterized in that, The minimum inhibitory concentration of MVAMP.2 against standard Escherichia coli is 128 μg / mL.

10. An antimicrobial drug, additive, or preservative comprising the earthworm antimicrobial peptide of claim 1 or 2.

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