Arginine methylation modified antibacterial peptide for inhibiting multiple streptococci and application thereof

Abstract

The application belongs to the technical field of antibacterial peptides, and particularly relates to an arginine methylation modified antibacterial peptide for inhibiting multiple streptococci and application thereof. The modified antibacterial peptide is named as cd44-Cmethyl, the amino acid sequence of the modified antibacterial peptide is shown as SEQ ID NO:1, the N terminal is acetylated modification, the C terminal is amidation modification, the 17th arginine is Nω-monomethylated arginine, and the structural formula is Ac-MLTWLKKLQPYLISMMR(Me)-NH2. In vitro MIC determination shows that the MIC of cd44-Cmethyl for Streptococcus agalactiae ATCC 13813, Streptococcus pyogenes ATCC 19615 and Streptococcus dysgalactiae ATCC 27957 is 4 μg / mL, 4 μg / mL and 8 μg / mL respectively, and the antibacterial activity is increased by 4-8 times compared with the unmethylated control peptide.

Description

Technical Field

[0001] This invention belongs to the field of antimicrobial peptide technology, specifically relating to an arginine methylation modified antimicrobial peptide cd44-Cmethyl that inhibits various streptococci, and its application in the preparation of antimicrobial agents, anti-infective drugs or antimicrobial compositions that inhibit streptococci. Background Technology

[0002] Streptococci are an important group of Gram-positive pathogens that can cause a variety of infectious diseases in humans and animals. Among them, *Streptococcus agalactiae*, *Streptococcus pyogenes*, and *Streptococcus distilleriae* all possess strong pathogenic potential and are associated with clinical infections, animal infections, and livestock-related diseases. With the long-term use of antibiotics, bacterial resistance has become an increasingly serious problem, limiting the application of traditional antimicrobial drugs in the control of some bacterial infections. Therefore, developing novel antimicrobial molecules with new structural features and good antimicrobial activity is of great significance.

[0003] Antimicrobial peptides are a class of small polypeptide molecules typically composed of amino acids, characterized by their designable structure, ease of artificial synthesis, and diverse modes of action. Some antimicrobial peptides exert their antibacterial or bactericidal effects by interacting with bacterial cell membranes, cell walls, or other bacterial structures. However, natural or unoptimized antimicrobial peptides often suffer from insufficient antimicrobial activity, limited stability, or unclear target applications, which restricts their further development and application.

[0004] Terminal modification, amino acid substitution, or specific amino acid modification of antimicrobial peptides are important strategies for enhancing their antimicrobial activity and application potential. Arginine residues are generally associated with the cationic characteristics of antimicrobial peptides and their interaction with negatively charged structures on bacterial surfaces, significantly influencing antimicrobial peptide activity. However, existing technologies lack systematic research on how site-specific methylation modifications of arginine residues in specific antimicrobial peptide sequences affect their antimicrobial activity, especially their inhibitory effect on streptococci, and have not provided clear technical insights.

[0005] Therefore, there is still a need to develop a modified antimicrobial peptide with a well-defined structure, easy synthesis, and the ability to effectively inhibit various streptococci for use in the preparation of streptococcus-related antimicrobial agents. Summary of the Invention

[0006] To address the aforementioned technical problems, this invention provides an antimicrobial peptide cd44-Cmethyl obtained by N-terminal acetylation, C-terminal amidation, and arginine monomethylation at position 17, and its application in the preparation of products related to anti-streptococcal infection.

[0007] The basic amino acid sequence (SEQ ID NO:1) of the modified antimicrobial peptide cd44-Cmethyl of this invention was obtained through screening small open reading frames (sORFs) in the genomes of marine biofilm-bearing bacteria. Candidate sequences were then obtained through structural feature prediction and antimicrobial activity screening using a deep learning model. Based on these candidate sequences, specific terminal modifications and arginine methylation modifications were introduced via chemical synthesis. This antimicrobial peptide has a small molecular weight and a well-defined structure, making it easy to prepare using solid-phase peptide synthesis.

[0008] Specifically, the technical solution adopted in this invention is as follows: The first aspect of this invention provides an arginine methylation-modified antimicrobial peptide cd44-Cmethyl that inhibits various streptococci, the amino acid sequence of which is shown in SEQ ID NO:1: MLTWLKKLQPYLISMMR; wherein the modified antimicrobial peptide cd44-Cmethyl has an N-terminus acetylated modification and a C-terminus amidated modification, and its arginine residue is an Nω (ω position, i.e., the terminal guanidine nitrogen atom)-monomethylated arginine residue.

[0009] Furthermore, the modified antimicrobial peptide cd44-Cmethyl has the following structural formula: Ac-MLTWLKKLQPYLISMMR(Me)-NH2; where Ac represents N-terminal acetylation modification, R(Me) represents Nω-monomethylated arginine residue, and NH2 represents C-terminal amidation modification.

[0010] Furthermore, the molecular weight of the modified antimicrobial peptide cd44-Cmethyl is 2207.75 g / mol.

[0011] Furthermore, the modified antimicrobial peptide cd44-Cmethyl has an inhibitory effect on streptococci, including one or more of Streptococcus agalactiae, Streptococcus pyogenes, and Streptococcus faecium.

[0012] Specifically, the *Streptococcus agalactiae* is *Streptococcus agalactiae* ATCC 13813, the *Streptococcus pyogenes* is *Streptococcus pyogenes* ATCC 19615, and the *Streptococcus dysgalactiae* is *Streptococcus dysgalactiae* ATCC 27957. The minimum inhibitory concentrations (MICs) of the modified antimicrobial peptide cd44-Cmethyl against *Streptococcus agalactiae* ATCC 13813, *Streptococcus pyogenes* ATCC 19615, and *Streptococcus dysgalactiae* ATCC 27957 are 4 μg / mL, 4 μg / mL, and 8 μg / mL, respectively.

[0013] A second aspect of the present invention provides an anti-streptococcal composition comprising the modified antimicrobial peptide cd44-Cmethyl.

[0014] Furthermore, the anti-streptococcal composition further includes one or more of a pharmaceutically acceptable carrier, diluent, buffer, excipient, stabilizer, preservative, or solubilizer.

[0015] A third aspect of the present invention provides the use of the above-described modified antimicrobial peptide cd44-Cmethyl or the above-described antistreptococcal composition in the preparation of an antimicrobial agent that inhibits streptococci.

[0016] Furthermore, the antimicrobial agent for inhibiting streptococci uses the modified antimicrobial peptide cd44-Cmethyl as the effective active ingredient. In some embodiments, the anti-streptococcal product uses the modified antimicrobial peptide cd44-Cmethyl as the sole antimicrobial active ingredient.

[0017] Furthermore, the anti-streptococcal product is used to inhibit or kill one or more of Streptococcus agalactiae ATCC 13813, Streptococcus pyogenes ATCC 19615, and Streptococcus dysgalactiae ATCC 27957.

[0018] Furthermore, the antibacterial agent can be used to treat or inhibit diseases caused by streptococcal infection, and its application areas include medical and health care, animal husbandry, food preservation, personal care products and environmental disinfection.

[0019] Furthermore, the antibacterial agent is a pharmaceutical preparation, a veterinary preparation, a feed additive, or a disinfectant.

[0020] Compared with the prior art, the present invention has the following beneficial effects: 1. This invention provides a novel modified antimicrobial peptide cd44-Cmethyl, which has a well-defined amino acid sequence and a well-defined chemical modification method, facilitating artificial synthesis and quality control.

[0021] 2. The modified antimicrobial peptide cd44-Cmethyl of the present invention has a significant inhibitory effect on a variety of streptococci.

[0022] 3. Under BHI medium conditions, the MICs of cd44-Cmethyl against Streptococcus agalactiae ATCC 13813, Streptococcus pyogenes ATCC 19615, and Streptococcus dysgalactiae ATCC 27957 were 4 μg / mL, 4 μg / mL, and 8 μg / mL, respectively.

[0023] 4. Compared with the unmethylated homologue control peptide cd44-nc, the MIC of cd44-Cmethyl against Streptococcus agalactiae ATCC13813 decreased from 32 μg / mL to 4 μg / mL (an increase of approximately 8-fold), against Streptococcus pyogenes ATCC 19615 decreased from 16 μg / mL to 4 μg / mL (an increase of approximately 4-fold), and against Streptococcus dysgalactiae ATCC27957 decreased from 32 μg / mL to 8 μg / mL (an increase of approximately 4-fold).

[0024] 5. The modified antimicrobial peptide cd44-Cmethyl of the present invention can be used as an active ingredient in the preparation of antimicrobial agents, anti-infective drugs or antimicrobial compositions that inhibit streptococci, and has application value in medical and health and related fields. Attached Figure Description

[0025] The accompanying drawings, which form part of this specification, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0026] Figure 1 The diagrams show the structures of the control peptide cd44-nc and the modified antimicrobial peptide cd44-Cmethyl, where A is a planar structural diagram of the control peptide cd44-nc, B is a planar structural diagram of the modified antimicrobial peptide cd44-Cmethyl, and C is a three-dimensional structural model predicted based on the basic amino acid sequence shown in SEQ ID NO:1.

[0027] Figure 2 The inhibitory effects of control peptide cd44-nc and modified antimicrobial peptide cd44-Cmethyl on three streptococci are shown in Figure A, where MIC measurement results are shown in Figure B, and bacterial growth during the MIC measurement process is shown in Figure B.

[0028] Figure 3 The modified antimicrobial peptide cd44-Cmethyl exhibited a fold increase in antimicrobial activity compared to the control peptide cd44-nc. Detailed Implementation

[0029] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration of the invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0030] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0031] The pathogenic bacteria used in the following examples are from: agalactococcus ( Streptococcus agalactiae ATCC 13813, Streptococcus pyogenes ( Streptococcus pyogenes ATCC 19615 and Streptococcus lactis ( Streptococcus dysgalactiae ATCC 27957 was purchased from Beina Innovation Biotechnology Co., Ltd.

[0032] Example 1: Sequence screening and chemical synthesis of the modified antimicrobial peptide cd44-Cmethyl The basic amino acid sequence MLTWLKKLQPYLISMMR of the modified antimicrobial peptide cd44-Cmethyl of this invention was obtained from small open reading frame (sORF) information screening of marine biofilm bacteria genomes. The relevant biofilm samples were isolated from biofilm microbial communities on rocks and microplastic surfaces in the coastal waters of Qingdao, China. First, ribosomal sequencing (Ribo-seq) combined with genome small open reading frame prediction results was used to screen for sORF-encoded peptide sequences in an expression state. Subsequently, a CNN-BiLSTM-Attention deep learning antimicrobial peptide prediction model was used to predict the antimicrobial activity of candidate sORF-encoded peptide sequences, resulting in the candidate basic amino acid sequence MLTWLKKLQPYLISMMR (SEQ ID NO:1).

[0033] Based on this, N-terminal acetylation, C-terminal amidation, and arginine methylation at position 17 were introduced via solid-phase chemical synthesis to obtain the modified antimicrobial peptide cd44-Cmethyl, with the structural formula Ac-MLTWLKKLQPYLISMMR(Me)-NH2. This modified antimicrobial peptide contains 17 amino acid residues and has a molecular weight of 2207.75 g / mol.

[0034] The modified antimicrobial peptide cd44-Cmethyl was synthesized by Sangon Biotech (Shanghai) Co., Ltd. using a solid-phase chemical synthesis method, achieving a purity of >95%. The synthesized peptide was purified by reversed-phase high-performance liquid chromatography (RP-HPLC), and its molecular weight was confirmed to be consistent with the theoretical molecular weight by mass spectrometry. The purified peptide was then lyophilized and stored, and a stock solution of the required concentration was prepared using sterile water before use.

[0035] Example 2: Structural characteristics of control peptide cd44-nc and modified antimicrobial peptide cd44-Cmethyl The basic amino acid sequence of the polypeptide shown in SEQ ID NO:1 is MLTWLKKLQPYLISMMR.

[0036] Based on the above basic sequence, the following two polypeptides were prepared respectively: (1) Control peptide cd44-nc: Based on the basic sequence shown in SEQ ID NO:1, only N-terminal acetylation and C-terminal amidation are performed, and its structural formula is Ac-MLTWLKKLQPYLISMMR-NH2.

[0037] (2) Modified antimicrobial peptide cd44-Cmethyl: Based on the amino acid sequence of the control peptide, the arginine residue at position 17 was further methylated to obtain the modified antimicrobial peptide cd44-Cmethyl, with the structural formula Ac-MLTWLKKLQPYLISMMR(Me)-NH2. This modified antimicrobial peptide contains 17 amino acid residues and has a molecular weight of 2207.75 g / mol.

[0038] Therefore, the control peptide cd44-nc and the modified antimicrobial peptide cd44-Cmethyl have the same basic amino acid sequence. The difference lies in the fact that the arginine residue at position 17 of cd44-Cmethyl is further methylated. Therefore, in this embodiment, cd44-nc is used as an unmethylated homologous control to evaluate the effect of arginine methylation modification on antimicrobial activity.

[0039] Based on the amino acid sequences and modification methods of the control peptide cd44-nc and the modified antimicrobial peptide cd44-Cmethyl, their structural characteristics were analyzed, and the results are as follows: Figure 1 As shown. Figure 1 Figure A shows a schematic diagram of the planar structure of the control peptide cd44-nc and its N-terminal acetylation (Ac-) and C-terminal amidation (-NH2) modification sites; Figure 1 Figure B shows a schematic diagram of the planar structure of the modified antimicrobial peptide cd44-Cmethyl and its N-terminal acetylation, C-terminal amidation and arginine methylation (R(Me)) modification sites; Figure 1 C is a three-dimensional spatial structure model predicted based on the basic amino acid sequence MLTWLKKLQPYLISMMR shown in SEQ ID NO:1.

[0040] Since arginine methylation is a non-natural amino acid modification, to ensure consistency and comparability in structural modeling, this embodiment uses the basic amino acid sequence shown in SEQ ID NO:1 for modeling during the three-dimensional structure prediction process, and marks the methylation modification site R(Me) in the two-dimensional structure diagram and structural expression Ac-MLTWLKKLQPYLISMMR(Me)-NH2. The above structural analysis results are used to illustrate that, under the same basic amino acid sequence and the same N-terminal acetylation and C-terminal amidation modification background, the further introduction of arginine methylation modification at position 17 results in the modified antimicrobial peptide cd44-Cmethyl having a clear chemical modification site and possible spatial conformation characteristics, providing a structural basis for subsequent evaluation of its antimicrobial activity changes.

[0041] Preparation method: The control peptide cd44-nc was also synthesized by Sangon Biotech (Shanghai) Co., Ltd. using solid-phase chemical synthesis, achieving a purity of >95%. The synthesized peptide was purified by reversed-phase high-performance liquid chromatography, and its molecular weight was confirmed to be consistent with the theoretical molecular weight by mass spectrometry. The purified peptide was lyophilized and stored, and a stock solution of the required concentration was prepared with sterile water before use.

[0042] Example 3: Determination of the minimum inhibitory concentration (MIC) and evaluation of the antimicrobial activity of the modified antimicrobial peptide cd44-Cmethyl against three streptococci. The MIC of antimicrobial peptides was determined using the broth microdilution method, following the guidelines for antimicrobial susceptibility testing from the Clinical and Laboratory Standards Institute (CLSI). *Streptococcus agalactiae* (…) Streptococcus agalactiae ATCC 13813, Streptococcus pyogenes ( Streptococcus pyogenes ATCC 19615 and Streptococcus lactis ( Streptococcus dysgalactiae ATCC 27957 was inoculated into sterile BHI liquid medium and incubated overnight at 37°C. The above-mentioned streptococci were then inoculated into fresh BHI liquid medium at a 1 v / v inoculation rate and cultured until the logarithmic growth phase. The bacterial concentration was then adjusted to 1 × 10⁻⁶. 5CFU / mL of bacterial culture was obtained. Then, 180 μL of the bacterial culture was transferred to a 96-well plate. The modified antimicrobial peptide cd44-Cmethyl and the control peptide cd44-nc powder were dissolved in sterile water and diluted to sequentially 2-fold dilutions. 20 μL of each solution was added to the bacterial culture in the 96-well plate to maintain a peptide concentration range of 2–64 μg / mL. Solvent / growth control wells and culture medium blank wells were also prepared. The solvent / growth control wells contained bacterial culture and an equal volume of sterile water, but no peptide was added, to eliminate the influence of sterile water on bacterial growth and confirm the normal growth of the tested strain. The culture medium blank wells contained BHI liquid medium and an equal volume of sterile water, but no bacterial culture or peptide was added, to eliminate culture medium contamination and background interference. After incubating the 96-well plate at 37°C for 20 hours, the OD values ​​were measured using a microplate reader. 600 Bacterial growth was detected at the specified wavelength. Referring to the CLSI antimicrobial susceptibility testing standards, the MIC was defined as the lowest peptide concentration at which no visible bacterial precipitation was observed under the above testing conditions. The experiment was conducted in triplicate.

[0043] Test results are as follows Figure 2 As shown, A represents the MIC measurement results, and B represents bacterial growth during the MIC measurement process. The results show that the tested streptococci grew normally in both the solvent / growth control wells, with no inhibitory effect observed, indicating that sterile water itself does not affect the growth of the tested strains. No bacterial growth was observed in the culture medium blank wells, indicating that the experimental system was free of significant contamination. Therefore, the antibacterial effect observed in the experimental group originates from the added peptide itself.

[0044] The results showed that the minimum inhibitory concentrations (MICs) of the control peptide cd44-nc against Streptococcus agalactiae ATCC 13813, Streptococcus pyogenes ATCC 19615, and Streptococcus dysgalactiae ATCC 27957 were 32 μg / mL, 16 μg / mL, and 32 μg / mL, respectively; while the MICs of the modified antimicrobial peptide cd44-Cmethyl against Streptococcus agalactiae ATCC 13813, Streptococcus pyogenes ATCC 19615, and Streptococcus dysgalactiae ATCC 27957 were 4 μg / mL, 4 μg / mL, and 8 μg / mL, respectively (Table 1).

[0045] To further evaluate the antibacterial activity enhancement effect of the modified antimicrobial peptide cd44-Cmethyl compared with the control peptide cd44-nc, the activity enhancement factor was calculated based on the MIC results in Table 1.

[0046] The activity enhancement factor is calculated using the following formula: Activity enhancement factor = MIC of control peptide cd44-nc / MIC of modified antimicrobial peptide cd44-Cmethyl.

[0047] The results are shown in Table 1 and Figure 3 As shown in the figure, compared with the control peptide cd44-nc, the modified antimicrobial peptide cd44-Cmethyl exhibited approximately 8-fold increased antimicrobial activity against *Streptococcus agalactiae* ATCC 13813, and approximately 4-fold increased antimicrobial activity against *Streptococcus pyogenes* ATCC 19615 and *Streptococcus dysgalactiae* ATCC 27957. These results indicate that the modified antimicrobial peptide cd44-Cmethyl can effectively inhibit various streptococci, and its anti-streptococcal activity is significantly superior to that of the control peptide cd44-nc, suggesting that methylation modification of the arginine residue at position 17 can significantly enhance the antimicrobial activity of this antimicrobial peptide against streptococci.

[0048] Table 1. Comparison of antibacterial activity between control peptide cd44-nc and modified antimicrobial peptide cd44-Cmethyl cd44-nc <![CDATA[Ac-MLTWLKKLQPYLISMMR-NH2]]> 32 16 32 cd44-Cmethyl <![CDATA[Ac-MLTWLKKLQPYLISMMR(Me)-NH2]]> 4 4 8 Activity enhancement factor - 8 4 4 The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. An arginine-methylated modified antimicrobial peptide that inhibits various streptococci, characterized in that, The amino acid sequence of the modified antimicrobial peptide is shown in SEQ ID NO:1: MLTWLKKLQPYLISMMR; wherein the N-terminus of the modified antimicrobial peptide is acetylated, the C-terminus is amidated, and its arginine residue is an Nω-monomethylated arginine residue.

2. The modified antimicrobial peptide according to claim 1, characterized in that, The modified antimicrobial peptide has the following structural formula: Ac-MLTWLKKLQPYLISMMR(Me)-NH2; where Ac represents N-terminal acetylation modification, R(Me) represents Nω-monomethylated arginine residue, and NH2 represents C-terminal amidation modification.

3. The modified antimicrobial peptide according to claim 1, characterized in that, The modified antimicrobial peptide has an inhibitory effect on streptococci, including one or more of agalactolytic streptococci, pyogenic streptococci, and dysgalactolytic streptococci.

4. The modified antimicrobial peptide according to claim 3, characterized in that, The agalactolytic streptococcus is agalactolytic streptococcus ATCC13813, the pyogenic streptococcus is pyogenic streptococcus ATCC19615, and the galactolytic streptococcus is galactolytic streptococcus ATCC27957.

5. The modified antimicrobial peptide according to claim 4, characterized in that, The modified antimicrobial peptides have minimum inhibitory concentrations of 4 μg / mL, 4 μg / mL, and 8 μg / mL against Streptococcus agalactiae ATCC13813, Streptococcus pyogenes ATCC19615, and Streptococcus dysgalactiae ATCC27957, respectively.

6. An anti-streptococcal composition, characterized in that, The anti-streptococcal composition comprises the modified antimicrobial peptide according to any one of claims 1 to 5.

7. The anti-streptococcal composition according to claim 6, characterized in that, The anti-streptococcal composition further includes one or more of a pharmaceutically acceptable carrier, diluent, buffer, excipient, stabilizer, preservative, or solubilizer.

8. The use of the modified antimicrobial peptide according to any one of claims 1 to 5 or the antistreptococcal composition according to claim 6 or 7 in the preparation of an antimicrobial agent that inhibits streptococci.

9. The application according to claim 8, characterized in that, The streptococci include one or more of Streptococcus agalactiae ATCC 13813, Streptococcus pyogenes ATCC 19615, and Streptococcus dysgalactiae ATCC 27957.

10. The application according to claim 8 or 9, characterized in that, The antibacterial agent is a pharmaceutical preparation, a veterinary preparation, a feed additive, or a disinfectant.

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