Novel Coral Antimicrobial Peptides Targeting the 30S Small Subunit of Bacterial Ribosomes and Their Applications

Abstract

This invention discloses novel coral antimicrobial peptides targeting the 30S small subunit of bacterial ribosomes and their applications. The amino acid sequences of the novel coral-derived antimicrobial peptides PDAM-9 and PDAM-10 are GGHVSRIYAIRQAISKALVA and ATAVAFCKRGNGLIKINGCP, respectively. Both antimicrobial peptides possess typical physicochemical characteristics of antimicrobial peptides, such as positive net charge and amphiphilicity. They can specifically bind to the core functional proteins S9 and S16 of the bacterial 30S small subunit of ribosomes, competitively occupying the functional sites of S9 and S16 proteins, interfering with bacterial ribosome assembly and translation, blocking the synthesis of core bacterial proteins, and thus exerting antibacterial effects. This provides new targets and high-quality candidate molecules for the development of novel antimicrobial peptides. Through structure-activity relationship optimization and molecular modification, the antimicrobial peptides PDAM-9 and PDAM-10 can become novel marine-derived antimicrobial candidate drugs with high activity, high stability, and low toxicity, providing a new solution for the treatment of multidrug-resistant bacterial infections in clinical practice and a precise means for the prevention and control of coral pathogenic bacterial infections.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a novel coral antimicrobial peptide targeting the 30S small subunit of bacterial ribosomes and its applications. Background Technology

[0002] The global spread of antibiotic resistance has become a major challenge threatening public health. The continuous emergence of multidrug-resistant and extensively resistant strains has limited the clinical application of traditional antibiotics, making the development of antimicrobial drugs based on novel mechanisms of action a core research need. Antimicrobial peptides, as key effector molecules of the organism's innate immune system, have become an important direction for the development of novel antimicrobial drugs due to their broad-spectrum antimicrobial activity, low resistance-inducing properties, and strong targeting characteristics. However, currently developed antimicrobial peptides only target single or a few pathogenic bacteria, exhibiting bottlenecks such as narrow antimicrobial spectrum and concentration-dependent effects, severely restricting their clinical translation. Summary of the Invention

[0003] The purpose of this invention is to overcome the shortcomings of the prior art and to provide a novel coral antimicrobial peptide that targets the 30S small subunit of bacterial ribosomes and its applications.

[0004] To achieve the above objectives, the technical solution adopted by the present invention is as follows: One of the technical solutions adopted by this invention to solve its technical problem is: providing a novel coral antimicrobial peptide PDAM-9 that targets the 30S small subunit of bacterial ribosomes. The amino acid sequence of the antimicrobial peptide PDAM-9 is GGHVSRIYAIRQAISKALVA, as shown in SEQ ID NO: 1. The theoretical molecular weight of the antimicrobial peptide PDAM-9 is 2110.50 Da, the observed molecular weight is 2110.8 Da, the positive charge is +3.25, the isoelectric point (pI) is 11, it is readily soluble in water, and soluble in DMSO.

[0005] Preferably, the nucleic acid encoding the antimicrobial peptide PDAM-9 is used.

[0006] Preferably, the expression vector contains the above-mentioned nucleic acid.

[0007] Preferably, the recombinant cells contain the above-described expression vector.

[0008] The second technical solution adopted by this invention to solve its technical problem is: providing a novel coral antimicrobial peptide PDAM-10 targeting the 30S small subunit of bacterial ribosomes. The amino acid sequence of the antimicrobial peptide PDAM-10 is ATAVAFCKRGNGLIKINGCP, as shown in SEQ ID NO: 2. The theoretical molecular weight of the antimicrobial peptide PDAM-10 is 2033.45 Da, the observed molecular weight is 2033.4 Da, the positive charge is +3, the isoelectric point (pI) is 9.51, it is readily soluble in water, and soluble in DMSO.

[0009] Preferably, the nucleic acid encoding the above-mentioned antimicrobial peptide PDAM-10.

[0010] Preferably, the expression vector contains the above-mentioned nucleic acid.

[0011] Preferably, the recombinant cells contain the above-described expression vector.

[0012] Preferably, the above-mentioned antimicrobial peptide PDAM-9 or antimicrobial peptide PDAM-10 is used in the preparation of antimicrobial drugs.

[0013] The antimicrobial peptide PDAM-9 or antimicrobial peptide PDAM-10 of the present invention can be synthesized using methods known to those skilled in the art, such as solid-phase synthesis, and purified using methods known to those skilled in the art, such as high-performance liquid chromatography.

[0014] The third technical solution adopted by the present invention to solve its technical problem is: to provide an antibacterial drug, wherein the effective component of the antibacterial drug includes the above-mentioned antimicrobial peptide PDAM-9 or antimicrobial peptide PDAM-10.

[0015] Implementing this invention has the following beneficial effects: This invention successfully obtained coral-derived ribosome-targeting antimicrobial peptides PDAM-9 and PDAM-10. Both possess typical physicochemical characteristics of antimicrobial peptides, such as positive net charge and amphiphilicity. They can specifically bind to the core functional proteins S9 and S16 of the 30S small subunit of bacterial ribosomes, competitively occupying the functional sites of S9 and S16 proteins, interfering with bacterial ribosome assembly and translation processes, blocking the synthesis of bacterial core proteins, and thus exerting antibacterial effects. This provides new targets and high-quality candidate molecules for the development of novel antimicrobial peptides. In vitro antibacterial experiments confirmed that antimicrobial peptides PDAM-9 and PDAM-10 have clear antibacterial activity against E. coli MG1655, MRSA, and V. fortis S10-1. Both also exhibit a unique concentration-free characteristic—the bacterial ribosome translation process is completely blocked after reaching the minimum effective concentration, and further increases in concentration have no synergistic effect; the initial OD in the low concentration group... 600 The surge in values ​​is speculated to be related to non-specific interactions between peptides and bacterial cells or transient growth stimuli.

[0016] The coral-derived antimicrobial peptides PDAM-9 and PDAM-10 of this invention can be optimized through structure-activity relationship and molecular modification to become novel marine-derived antimicrobial candidate drugs with high activity, high stability and low toxicity, providing a new solution for the treatment of multidrug-resistant bacterial infections in clinical practice, and also providing a precise means for the prevention and control of coral pathogenic bacterial infections. Attached Figure Description

[0017] Figure 1Preliminary findings and predicted three-dimensional structures of antimicrobial peptides PDAM-9 and PDAM-10. A represents the KEGG functional enrichment analysis results of Blank26 with the LV26 and HV26 groups, respectively; B represents the predicted three-dimensional structures of antimicrobial peptides PDAM-9 and PDAM-10.

[0018] Figure 2 The results are HPLC detection results for antimicrobial peptides PDAM-9 and PDAM-10.

[0019] Figure 3 MS detection results for antimicrobial peptides PDAM-9 and PDAM-10.

[0020] Figure 4 This is a three-dimensional conformation diagram of the binding of antimicrobial peptides PDAM-9 and PDAM-10 to S16 and S9 proteins. Red represents the target proteins; cyan represents the antimicrobial peptides.

[0021] Figure 5 This is a two-dimensional interaction diagram of antimicrobial peptides PDAM-9 and PDAM-10 with S16 and S9 proteins. The green dashed lines represent hydrogen bonds, and the red arcs represent hydrophobic interactions.

[0022] Figure 6 This image shows the antibacterial effects of antimicrobial peptides PDAM-9 and PDAM-10. Among them, Group A represents a large proportion of low concentrations; Group B is a small-proportion, high-concentration group. Detailed Implementation

[0023] To better understand the present invention, the present invention will be further described in detail below with reference to the embodiments and accompanying drawings. However, those skilled in the art will understand that the following embodiments are not intended to limit the scope of protection of the present invention, and any changes and variations made on the basis of the present invention are within the scope of protection of the present invention.

[0024] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0025] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0026] The clustered helmet corals were provided by the Coral Conservation Center of the Third Institute of Oceanography, Ministry of Natural Resources. PDAM-9 and PDAM-10 were synthesized by Genscript using a chemical solid-phase peptide synthesis method.

[0027] Example 1 I. Discovery and Preliminary Molecular Docking of Coral Antimicrobial Peptides 1. Metagenomic sequencing and differential analysis This study used clustered helmet corals as the research object, and the experimental temperature was set at 26℃. The materials were divided into a blank control group (Blank26) and a low-concentration vibrio infection group (LV26, with a final concentration of vibrio 10). 3 CFU / mL) and the high-concentration Vibrio virulence group (HV26, final Vibrio virulence concentration 10 CFU / mL) compared to the high-concentration Vibrio virulence group (HV26, final Vibrio virulence concentration 10 CFU / mL). 5 (CFU / mL). After 24 hours of temporary rearing to acclimatize, corals were challenged with vibrio bacteria, while the control group received an equal volume of fresh seawater eluent. The experiment was terminated when obvious bleaching, ulceration, or other lesions appeared on the corals, and the coral tissues were flash-frozen in liquid nitrogen. Genomic DNA was extracted from the samples using the CTAB method, followed by library construction, PCR amplification, and metagenomic sequencing using sequencing-by-synthesis technology on the Illumina platform to obtain raw sequence data.

[0028] Bioinformatics analysis was performed on high-throughput sequencing of three coral groups and their symbiotic organisms using the MajorBio cloud platform (https: / / cloud.majorbio.com). The Blank26 group served as the control group, and genomic differences were compared with the LV26 and HV26 groups. Based on antimicrobial peptide-related pathways, the Blank26 group was compared with the LV26 and HV26 groups, and KEGG functional enrichment analysis was conducted to screen for functional information potentially associated with antimicrobial peptide genes. iPath pathway comparative analysis was used to identify pathways specific to the Blank26 group, obtaining KO numbers for pathways potentially related to antimicrobial function. Further annotation of the KO numbers yielded corresponding locus tags, and the amino acid sequences encoded by each functional gene were retrieved using the National Center for Biotechnology Information (NCBI) database.

[0029] Further functional and pathway analyses were conducted using typical known KEGG pathways directly related to antimicrobial peptides: ko00072 (ketone body synthesis and degradation), ko01051 (ansamycin biosynthesis), ko02024 (quorum sensing), and ko03010 (ribosomes). Compared to humans, marine invertebrates such as corals lack specific immunoglobulin-mediated adaptive immunity and primarily rely on innate immune molecules such as antimicrobial peptides to defend against pathogen invasion. Based on the needs of corals' own immune defense mechanisms, it was hypothesized that, compared to the Vibrio-infected corals LV26 and HV26, antimicrobial peptide-related functions showed a significant enrichment in healthy coral Blank26. KEGG functional enrichment comparisons yielded the following results: Figure 1As shown in Figure A, the pathway with pathway ID Ko03010 (ribosome) was significantly enriched in differentially expressed genes, suggesting that genes associated with this ribosome-enriched pathway are related to the mechanism of action of antimicrobial peptides. Simultaneously, the study identified a series of KO numbers associated with antimicrobial peptide functional genes through iPath pathway analysis, including K01635, K02896, K02908, K07344, K07711, K08321, K10556, K11531, K15670, K20330, K20527, and K20533. These KO numbers represent specific gene functional units, indicating functional genes playing roles in the biosynthesis, modification, and functional regulation of antimicrobial peptides. Furthermore, by linking pathway KO numbers with pathway IDs, K02908, K02896, and K02960 were identified. Under the condition of matching coral species, three functional gene numbers were obtained by searching and annotating them through NCBI: LOC113669380 (similar protein L30 of 60S ribosomal protein in *Coralia pulcherrima*), LOC113664512 (similar protein L24 of 60S ribosomal protein), and LOC113675475 (similar protein S16 of 40S ribosomal protein). Based on this, it is hypothesized that these three coral functional genes may achieve antibacterial effects by targeting and binding to bacterial ribosomes, interfering with their protein synthesis process. Most antimicrobial peptides are only about 10-50 amino acids long, suggesting that shorter amino acid sequences expressing antimicrobial peptides may be hidden within the amino acid sequences of these three functional genes.

[0030] 2. Antimicrobial peptide fragment prediction and screening Based on the amino acid sequences obtained above, the CAMPR3 online tool (http: / / www.camp3.bicnirrh.res.in / prediction.php) was used to generate peptide sequences in batches, with 20 amino acids set as the sliding window parameter, and the probability of each peptide being an antimicrobial peptide and the potential type of antimicrobial peptide were predicted.

[0031] The 10 most likely natural antimicrobial peptides (AMPs) were screened from the CAMPR3 prediction results. The net charge, GRAVY total average hydrophilicity, Wimley-White full residue hydrophobicity scale, and protein binding potential (Boman index) were predicted using APD3 (https: / / doi.org / 10.1093 / nar / gkv1278). The isoelectric point (pI) was predicted using ExPASy's ProtParam.

[0032] Further analysis of the amphiphilicity of candidate antimicrobial peptides was conducted using the ProtScale tool. The secondary structure of peptides was predicted using the SOPMA online tool (https: / / npsa-prabi.ibcp.fr / cgi-bin / npsa_automat.pl?page=npsa_sopma.html). The three-dimensional conformation, active site distribution, and potential molecular interaction regions of candidate peptides were analyzed using the PEP-FOLD4 online tool (https: / / mobyle2.rpbs.univ-paris-diderot.fr / cgi-bin / portal.py#forms:PEP-FOLD4). Sequence and domain annotation analysis of candidate antimicrobial peptides was performed using the NCBI database to detect the presence of conserved domains in known antimicrobial peptides, such as cysteine-rich domains.

[0033] To rapidly screen peptides with antibacterial activity, this study utilized bioinformatics techniques and the typical characteristics of antimicrobial peptides for secondary analysis. First, the amino acid sequence length of antimicrobial peptides was determined to be 10-50 amino acids. Therefore, a sliding window parameter of 20 amino acids was set in CAMPRO3, and the selection range was limited to native peptides. A higher probability score for the obtained peptide indicated a greater likelihood that it was an antimicrobial peptide. Regarding hydrophilicity and hydrophobicity, the amphiphilic structure of antimicrobial peptides facilitates insertion into the cell membrane. The hydrophilic region facilitates the interaction of the antimicrobial peptide with the aqueous environment, while the hydrophobic region can insert into the lipid bilayer of the bacterial cell membrane, disrupting the cell membrane's integrity and thus exerting an antibacterial effect. The Wimley-White relative hydrophobicity scale refers to the free energy of a peptide's transfer from the aqueous phase to the membrane interface; a negative value indicates that the antimicrobial peptide spontaneously interacts with the cell membrane. The Boman index is generally between 0 and 2 kcal / mol; a lower index value indicates less side effects from the antimicrobial peptide. The isoelectric point (pI) of antimicrobial peptides is usually alkaline. A positive average hydrophobicity index (GRAVY) value indicates that the protein is hydrophobic; peptides with stronger hydrophobicity are more likely to bind to the cell membrane, thus exerting an antibacterial effect against pathogenic bacteria. Using the above typical physicochemical properties of antimicrobial peptides as screening criteria, two peptides that met the criteria were screened and named PDAM-9 and PDAM-10, respectively. Their physicochemical properties are detailed in Table 1.

[0034] Table 1. Physicochemical properties of PDAM-9 and PDAM-10, which conform to the characteristics of classic antimicrobial peptides.

[0035] PDAM-9 has an α-helix proportion of 85%. Since bacterial cell membranes typically contain a large number of negatively charged components, positively charged α-helical antimicrobial peptides bind more easily to negatively charged bacterial cell membranes. Furthermore, when antimicrobial peptides come into contact with the amphiphilic environment of the plasma membrane, they adopt an α-helix conformation, promoting the insertion of hydrophobic peptide residues into the lipid bilayer. This allows them to replace polar head groups through the "ring mechanism" in pore formation, thereby disrupting the bacterial cell membrane. PDAM-10 has a random coil proportion of 35% and an extended chain proportion of 50%. The large proportion of extended chains and random coils is conducive to forming a "membrane-associated peptide structure" similar to that formed by indolecin in the presence of micelles. Three-dimensional predictions for PDAM-9 and PDAM-10 (e.g.) Figure 1 The B in the text corroborates the two-dimensional structure.

[0036] NCBI sequence alignment results showed that PDAM-9 had a 100% match with the 40S ribosomal protein S16 of *Hylocereus*, *Cyclophorus*, and *Francis*, while PDAM-10 also had a 100% match with the unnamed protein product of *Evermania*, *Leptochloa*, and the 40S ribosomal protein S16 of *Stachys staghorn*. Furthermore, the conserved domains of both antimicrobial peptides were highly homologous to the uS9 protein family. Based on the ribosomal pathway functional enrichment results, it can be inferred that PDAM-9 and PDAM-10 can mimic the structural features of eukaryotic ribosomal protein S16 and prokaryotic uS9 protein, targeting and binding to the 30S small subunit of the bacterial 70S ribosome, precisely recognizing and acting on the core functional regions of uS9 and uS16, thereby interfering with bacterial protein synthesis and exerting an antibacterial effect.

[0037] 3. Analysis of docking between antimicrobial peptides and target proteins The selected candidate antimicrobial peptides were subjected to molecular docking experiments with the S9 and S16 proteins of the 30S small subunit of E. coli ribosome to explore the peptide-target protein interaction mechanism. The crystal structure of the 30S small subunit of E. coli ribosome was obtained from the RCSB protein database (https: / / www.rcsb.org), with the corresponding PDB number 7OE1. In the docking pretreatment stage, PyMOL 3.0.3 software was used to uniformly process the 30S subunit of ribosome and the target peptide, removing redundant water molecules, adding polar hydrogen, and accurately calculating the longest axis parameter of each candidate antimicrobial peptide to provide data support for subsequent docking parameter settings.

[0038] Molecular docking experiments were conducted using AutoDock Vina 1.5.6 software, with strict control over docking parameters: the geometric center of the polypeptide chains containing S9 and S16 proteins was used as the center of the docking grid, and the side length of the docking grid for both target sites was uniformly set to 47.25 Å to ensure consistency and comparability of experimental conditions. After docking, PyMOL 3.0.3 and LigPlot+ v.2.3.1 software were used to visualize and deeply analyze the final binding mode and key functional residues of the candidate antimicrobial peptides and target proteins.

[0039] Molecular docking simulation results (e.g.) Figure 4 , Figure 5 Further verification (as shown) confirmed that both PDAM-9 and PDAM-10, two coral-derived antimicrobial peptides, can specifically bind to the functional domains of the S9 and S16 subunits of the 30S ribosomal small subunit in *E. coli*. The binding process is thermodynamically spontaneous. The binding energies and hydrogen bond numbers of each complex are as follows: S16-PDAM-9 (-9.1 kcal / mol, 12 hydrogen bonds), S16-PDAM-10 (-8.1 kcal / mol, 14 hydrogen bonds), S9-PDAM-9 (-9.6 kcal / mol, 18 hydrogen bonds), and S9-PDAM-10 (-8.2 kcal / mol, 6 hydrogen bonds). All complex binding energies are negative and have high absolute values, confirming that both antimicrobial peptides can form stable bindings with the target proteins, demonstrating excellent binding ability.

[0040] Three-dimensional combined conformational analysis (e.g.) Figure 4 As shown in the figure, both antimicrobial peptides can effectively embed into the hydrophobic binding pocket on the surface of the target protein, forming precise spatial complementarity with the protein backbone and amino acid side chains, thus meeting the spatial adaptation requirements for molecular targeted binding. Comparison of conformational characteristics reveals that PDAM-9 exhibits an extended state within the binding pocket, resulting in a larger contact area with the target protein surface and higher molecular adhesion; PDAM-10 has a relatively compact conformation, lower efficiency in filling the hydrophobic pocket, and slightly weaker spatial adaptation. This structural difference is the core reason for the difference in binding energy between the two. Two-dimensional interactions (such as...) Figure 5 The analysis further reveals the intermolecular interaction mechanism. Green hydrogen bonds are the core polar forces in the system. The S9-PDAM-9 complex has the most green hydrogen bonds, the highest total number of interactions, and the widest distribution. It involves the specific recognition and binding of polar residues such as Gln and Asn of the S9 protein. At the same time, hydrophobic residues such as Leu, Ile, and Val also form a large-scale hydrophobic stacking interaction with PDAM-9. The hydrogen bonds and hydrophobic interactions synergistically enhance the stability of the complex. S9 and PDAM-10 can also form green hydrogen bonds, which can form non-covalent interactions with target protein residues.

[0041] By combining the inherent physiological functions of S9 and S16, the antimicrobial pathways of the two antimicrobial peptides can be clarified. Both S9 and S16 are essential housekeeping proteins of the 30S small subunit of the E. coli ribosome, serving as core supporting elements in bacterial protein synthesis. S9 stabilizes the integrity of the 3' main domain of 16S rRNA and regulates translation initiation and peptide chain elongation; S16, as a key secondary binding protein of the 30S small subunit, directly mediates the correct assembly and maturation of the 30S small subunit. Therefore, it can be concluded that the antimicrobial peptides PDAM-9 and PDAM-10, through their binding properties, target and bind to the 30S small subunit and 16S rRNA, potentially blocking 30S small subunit assembly and inhibiting mature ribosome formation through steric hindrance, while simultaneously blocking the normal binding and codon decoding processes of mRNA and tRNA. In addition, as housekeeping gene proteins essential for bacterial survival, impaired function of S9 and S16 can directly lead to global inhibition of bacterial protein synthesis, blocking core life activities such as basal metabolism, growth and reproduction, and ultimately exerting a strong antibacterial and bactericidal effect.

[0042] In summary, both PDAM-9 and PDAM-10 can potently target and bind to the S9 and S16 housekeeping proteins of the bacterial 30S ribosomal small subunit, demonstrating good antibacterial target binding potential and antibacterial application value. Among them, PDAM-9 exhibits superior binding affinity, conformational stability, and specificity, making it a highly promising antibacterial candidate molecule. PDAM-10 can also be further explored and optimized as an alternative antibacterial peptide. The precise binding modes and potential antibacterial mechanisms of these two antibacterial peptides to their target proteins provide crucial theoretical support and experimental evidence for subsequent antibacterial peptide molecular modification, in vitro functional verification, and antibacterial application translation research.

[0043] II. Verification of the antibacterial activity of potential antimicrobial peptides 1. Growth curves were measured using a large-scale, low-concentration enzyme-linked immunosorbent assay (ELISA) reader. The screened antimicrobial peptides were synthesized by Genscript Biotech Co., Ltd. using a solid-phase peptide synthesis method. Samples were dissolved in analytical-grade DMSO, and the purity and molecular weight of the synthesized peptides were determined by mass spectrometry and HPLC. HPLC detection conditions were as follows: mobile phase A was deionized water containing 0.065% trifluoroacetic acid (v / v), mobile phase B was acetonitrile containing 0.05% trifluoroacetic acid (v / v), flow rate was 1 mL / min, and detection wavelength was 220 nm. The gradient program was: 0.01–25.00 min 5%–65% B, 25.01–27.00 min maintaining 95% B, 27.01–35.00 min recovering to 5% B and maintaining, and detection was stopped at 35.01 min. The mass spectrometry detection conditions were as follows: mobile phase 50% water / 50% methanol, nebulizer gas flow rate 1.5 L / min, drying gas flow rate 5 L / min, CDL temperature 250℃, block temperature 200℃, interface bias +4.5 kV, T. flow 0.2 mL / min, and positive ion mode.

[0044] The results are as follows Figure 2 As shown, HPLC analysis revealed that PDAM-9 had a purity of 98.009% and a main peak retention time of 15.242 min; PDAM-10 had a purity of 95.170% and a main peak retention time of 14.958 min. Figure 3 As shown, mass spectrometry analysis revealed that the theoretical molecular weight of PDAM-9 is 2110.50 Da, the observed molecular weight is 2110.8 Da, and the characteristic ion peak is [M+2H]. 2+ (1056.1) [M+3H] 3+ (704.6) [M+4H] 4+ (528.7); The theoretical molecular weight of PDAM-10 is 2033.45 Da, the observed molecular weight is 2033.4 Da, and the characteristic ion peak is [M+2H]. 2+ (1017.5) [M+3H] 3+ (678.8) [M+4H] 4+ (509.3) [M+5H] 5+ (407.5). The above results indicate that PDAM-9 and PDAM-10 have been successfully prepared and possess high purity, meeting the requirements for in vitro verification in subsequent experiments.

[0045] Activated OD 600 Inoculate with indicator bacterial suspension at a ratio of 0.1:1 at a ratio of 1:500. The indicator bacteria are Escherichia coli MG1655 ( Escherichia coli MG1655, E. coli MG1655, methicillin-resistant Staphylococcus aureus (MG1655, E. coli MG1655), ... Staphylococcus aureus MRSA), Vibrio virosa S10-1 ( Vibrio fortisS10-1 and V. fortis S10-1 were inoculated and then mixed 1:1 with PDAM-9 at concentrations of 9.476 μmol / L (0.02 mg / mL), 0.9476 μmol / L (0.002 mg / mL), and 0.09476 μmol / L (0.0002 mg / mL) and PDAM-10 at concentrations of 9.836 μmol / L (0.02 mg / mL), 0.9836 μmol / L (0.002 mg / mL), and 0.09836 μmol / L (0.0002 mg / mL). E. coli MG1655 and MRSA were cultured in LB medium, while V. fortis S10-1 was cultured in LB liquid medium containing 3% NaCl. A control group, namely the growth control (GC), was set up, inoculated with bacteria but without the addition of antimicrobial peptides, as a reference for normal bacterial growth. The indicator bacterial culture, after being mixed with PDAM-9 and PDAM-10, was cultured in a double-layer shaker incubator at 37°C and 220 rpm. Samples were taken from 96-well plates at regular intervals over several hours, and the spectrophotometry was measured at 600 nm using an ELISA reader to plot the growth curve.

[0046] E. coli MG1655 is a Gram-negative model strain used to elucidate the antimicrobial peptide mechanism; MRSA is a clinically resistant Gram-positive bacterium used to verify the antimicrobial effect of resistant bacteria; V. fortis S10-1 is an aquatic pathogen used to evaluate its broad-spectrum antimicrobial potential. The three strains complement each other, taking into account both mechanism exploration and application expansion.

[0047] First, large-scale, low-concentration groups were set up (1:500 inoculation ratio, peptide concentrations of 0.0001 mg / mL, 0.001 mg / mL, and 0.01 mg / mL). For example... Figure 6As shown in Figure A, in this experiment, PDAM-9 exhibited a time-dependent inhibitory effect on the in vitro growth of *E. coli* MG1655, MRSA, and *V. fortis* S10-1. In the early stage of culture (0-20 h), the growth curves of each treatment group and the growth control group (GC) highly overlapped, showing no significant inhibition. In the later stage of culture (20-35 h), the high-concentration group (0.01 mg / mL) gradually showed antibacterial activity against *E. coli* MG1655 and *V. fortis* S10-1, while antibacterial activity against MRSA was observed in the low-concentration group (0.001 mg / mL). PDAM-10 did not show significant antibacterial activity against the three indicator bacteria, possibly because it did not reach the inhibition concentration threshold. The growth control group showed a complete growth cycle, confirming the stability and reliability of the experimental system. These results suggest that PDAM-9 may exert its antibacterial effect by slowly interfering with ribosome function and blocking protein synthesis, with its effect gradually appearing with prolonged treatment time. Meanwhile, both showed no complete concentration dependence, suggesting that their mechanism of action, predicted by the domain, may be related to targeted or conformational binding of ribosomes.

[0048] 2. Growth curves were measured using an ELISA reader for small-scale, high-concentration groups. Three indicator bacteria were used as described above, but the conditions were changed by reducing the inoculation ratio of the indicator bacteria and increasing the concentrations of PDAM-9 and PDAM-10. Similarly, activated OD was used. 600 A 0.1 μL indicator bacterial suspension was prepared, and E. coli MG1655, MRSA, and V. fortis S10-1 bacterial suspensions inoculated at a 1:600 ​​ratio were mixed 1:1 with PDAM-9 at concentrations of 473.8 μmol / L (1 mg / mL), 94.76 μmol / L (0.2 mg / mL), and PDAM-10 at concentrations of 491.8 μmol / L (1 mg / mL) and 98.36 μmol / L (0.2 mg / mL), respectively. The mixed bacterial suspensions were cultured in the corresponding media as described above, with two control groups established: an uninoculated control (UC) containing only the corresponding culture medium without bacteria or antimicrobial peptides; and a growth control (GC) inoculated with bacteria but without the addition of antimicrobial peptides, serving as a reference for normal bacterial growth. The inoculated bacterial culture was also cultured in a double-layer shaker at 37°C and 220 rpm for several hours. Samples were taken from 96-well plates and their spectrophotometry was measured at 600 nm using an ELISA reader to plot the growth curve.

[0049] Based on the antibacterial results ( Figure 6Figure B) shows that the antimicrobial peptides PDAM-9 and PDAM-10 have significant antibacterial effects against E. coli MG1655 and MRSA, but there are no significant differences among the different concentration groups. The high concentration group (0.5 mg / mL) did not show superior inhibitory activity compared to the low concentration group. However, 0.5 mg / mL PDAM-9, 0.1 mg / mL PDAM-9, and 0.5 mg / mL PDAM-10 have significant antibacterial effects against V. fortis S10-1, with peak times of 32 h, 25 h, and 32 h, respectively. Some low concentration groups showed lower initial OD values. 600 The value has increased abnormally.

[0050] Antimicrobial peptides PDAM-9 and PDAM-10 both exhibited inhibitory effects against three indicator bacteria at specific concentrations, demonstrating a non-concentration-dependent effect. This also indicates that their antimicrobial effect is not dependent on increasing concentration. Combined with previous findings and molecular docking, it can be inferred that PDAM-9 and PDAM-10 may completely block the translation process after reaching a minimum effective concentration, and further increasing the concentration will not further enhance the inhibitory effect; some low-concentration groups showed initial OD... 600 The surge in values ​​is speculated to be related to non-specific interactions or stimulatory effects between the peptide and bacterial cells.

[0051] In summary, this invention successfully obtained coral-derived ribosome-targeting antimicrobial peptides PDAM-9 and PDAM-10. Both possess typical physicochemical characteristics of antimicrobial peptides, such as positive net charge and amphiphilicity. They can specifically bind to the core functional proteins S9 and S16 of the 30S small subunit of bacterial ribosomes, competitively occupying the functional sites of S9 and S16 proteins, interfering with bacterial ribosome assembly and translation processes, blocking the synthesis of bacterial core proteins, and thus exerting antibacterial effects. This provides new targets and high-quality candidate molecules for the development of novel antimicrobial peptides. In vitro antibacterial experiments confirmed that antimicrobial peptides PDAM-9 and PDAM-10 have clear antibacterial activity against E. coli MG1655, MRSA, and V. fortis S10-1. Both also exhibit a unique concentration-free characteristic—the bacterial ribosome translation process is completely blocked after reaching the minimum effective concentration, and further increases in concentration have no synergistic effect; the initial OD in the low concentration group... 600 The surge in values ​​is speculated to be related to non-specific interactions between peptides and bacterial cells or transient growth stimulation. Meanwhile, the efficient extraction technology system for marine invertebrate antimicrobial peptides established in this study provides a referable technical paradigm for the large-scale screening of marine-derived antimicrobial peptides, further enriches the theoretical research on coral innate immune mechanisms, and clarifies the core role of antimicrobial peptides in corals' defense against marine pathogen invasion.

[0052] The coral-derived antimicrobial peptides PDAM-9 and PDAM-10 of this invention can be optimized through structure-activity relationship and molecular modification to become novel marine-derived antimicrobial candidate drugs with high activity, high stability and low toxicity, providing a new solution for the treatment of multidrug-resistant bacterial infections in clinical practice, and also providing a precise means for the prevention and control of coral pathogenic bacterial infections.

[0053] While specific embodiments of the present invention have been described above, those skilled in the art should understand that the specific embodiments described are merely illustrative and not intended to limit the scope of the present invention. Equivalent modifications and variations made by those skilled in the art in accordance with the spirit of the present invention should be covered within the scope of protection of the claims of the present invention.

Claims

1. A novel coral antimicrobial peptide PDAM-9 targeting the 30S small subunit of bacterial ribosomes, characterized in that, The amino acid sequence of the antimicrobial peptide PDAM-9 is shown in SEQ ID NO:

1.

2. The nucleic acid encoding the antimicrobial peptide PDAM-9 as described in claim 1.

3. An expression vector containing the nucleic acid as described in claim 2.

4. Recombinant cells containing the expression vector as described in claim 3.

5. A novel coral antimicrobial peptide, PDAM-10, targeting the 30S small subunit of bacterial ribosomes, characterized in that... The amino acid sequence of the antimicrobial peptide PDAM-10 is shown in SEQ ID NO:

2.

6. The nucleic acid encoding the antimicrobial peptide PDAM-10 as described in claim 5.

7. An expression vector containing the nucleic acid as described in claim 6.

8. Recombinant cells containing the expression vector as described in claim 7.

9. The use of the antimicrobial peptide PDAM-9 of claim 1 or the antimicrobial peptide PDAM-10 of claim 2 in the preparation of antimicrobial drugs.

10. An antibacterial drug, characterized in that, The active ingredient of the antimicrobial drug shown includes the antimicrobial peptide PDAM-9 of claim 1 or the antimicrobial peptide PDAM-10 of claim 2.

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