Hlhepcidin antibacterial peptide derived from leuciscus idus and application thereof in regulating intestinal flora of fish and preventing and controlling aeromonas hydrophila infection

Abstract

This invention discloses an antimicrobial peptide, HlHepcidin, derived from *Hemibarbus labeo*, and its application in regulating fish gut microbiota and preventing *Aeromonas hydrophila* infection. The antimicrobial peptide comprises the amino acid sequence QSHLSLCRYCCNCCRNKGCGYCCKF shown in SEQ ID NO:1, or a functional variant retaining antimicrobial activity and / or fish gut microbiota regulation activity. This invention further provides a nucleic acid molecule encoding the peptide, a recombinant expression vector, engineered bacteria or host cells, a preparation method, a composition containing the antimicrobial peptide, and its applications. The antimicrobial peptide exhibits inhibitory activity against various aquaculture-related bacteria and can be used to restore the diversity and community composition of fish gut microbiota after *Aeromonas hydrophila* infection. The antimicrobial peptide or composition can be prepared as a feed additive, bath agent, water treatment agent, spray, injection, or other aquaculture product, thereby providing a green technology solution for the prevention and control of bacterial diseases in fish and reducing antibiotic use.

Description

Technical Field

[0001] This invention belongs to the technical fields of aquatic animal disease prevention and control, antimicrobial peptides, intestinal microecological regulators, healthy fish farming, and veterinary biological agents. Specifically, it relates to an HlHepcidin antimicrobial peptide derived from Hemibarbus labeo, a nucleic acid molecule encoding the antimicrobial peptide, a composition containing the antimicrobial peptide, and its application in regulating the intestinal flora of fish, reducing the risk of bacterial infection in farmed fish, and preparing products for the prevention and treatment of Aeromonas hydrophila infection. Background Technology

[0002] Aquaculture plays a vital role in supplying high-quality protein and promoting rural industrial development. However, with increasing stocking densities and intensive farming of freshwater fish, bacterial diseases are becoming more frequent, posing a significant challenge to aquaculture profitability, survival rates, and the safety of aquatic products. Aeromonas hydrophila is a common Gram-negative opportunistic pathogen in aquaculture environments, which can cause septicemia, tissue damage, decreased feed intake, stunted growth, and even death in fish.

[0003] Currently, the control of bacterial diseases still relies to some extent on antibiotics, chemical disinfectants, and empirical medication. Long-term or inappropriate use of antibiotics may lead to the emergence of drug-resistant strains, increased risk of drug residues, disturbance of aquatic ecosystems, and consumer concerns about the safety of aquatic products. Therefore, it is necessary to develop bioactive molecules with clearly defined sources, diverse mechanisms of action, environmental friendliness, and suitability for aquaculture scenarios.

[0004] The gut microbiota of fish is a crucial component of host nutrient absorption, immune homeostasis, and resistance to infection. Pathogen invasion can not only directly cause tissue infection but also disrupt the richness, evenness, and composition of the gut microbiota, leading to an overgrowth of harmful or opportunistic pathogens, thereby exacerbating the host's inflammatory response and causing aquaculture losses. Techniques that only kill pathogens are often insufficient to address the post-infection gut microecological imbalance.

[0005] Hepcidin is a class of cysteine-rich antimicrobial peptides that play an important role in the regulation of innate immunity and iron metabolism in vertebrates. Fish hepcidin can directly inhibit various bacteria and may participate in the anti-infection process by limiting iron utilization by pathogens, influencing immune responses, and regulating the gut microbiota. The labeo mullet (Hemibarbus labeo) is an economically valuable freshwater fish, and hepcidin derived from this fish has the potential to be developed into an active molecule with both antibacterial and microecological regulatory functions.

[0006] In existing technologies, antimicrobial peptide products for bacterial diseases in fish mainly focus on in vitro antimicrobial activity or immune enhancement. Technical solutions for restoring gut microbiota diversity and community structure in fish after Aeromonas hydrophila infection using specific mature Hepcidin peptides remain insufficient. Therefore, it is necessary to provide an HlHepcidin antimicrobial peptide derived from the lip worm and its composition for the green prevention and control of bacterial diseases in fish and the restoration of gut microbiota after infection. Summary of the Invention

[0007] To address the aforementioned technical problems, this invention provides an antimicrobial peptide derived from the labium and its application in regulating the intestinal flora of fish and preventing Aeromonas hydrophila infection, thereby at least partially solving the problems of bacterial disease control relying on antibiotics, difficulty in restoring intestinal flora imbalance after infection, and lack of green prevention and control products in aquaculture.

[0008] The technical solution adopted in this invention is as follows: This invention provides an HlHepcidin antimicrobial peptide, which contains the amino acid sequence QSHLSLCRYCCNCCRNKGCGYCCKF, and this sequence is denoted as SEQ ID NO:1.

[0009] Preferably, the antimicrobial peptide is a mature peptide, a chemically synthesized peptide, a recombinant expressed peptide, a peptide obtained by enzymatic digestion of a fusion protein, an oxidized folded peptide forming a disulfide bond, a salt form peptide, a freeze-dried peptide, a purified peptide, or a derivative thereof that is acceptable for aquaculture, veterinary use, feed, or pharmaceutical use.

[0010] Preferably, the antimicrobial peptide contains eight cysteine ​​residues capable of forming four disulfide bonds. The antimicrobial peptide may include an N-terminal QSHLS motif, which is beneficial for maintaining the stable conformation and biological activity of Hepcidin-type antimicrobial peptides.

[0011] The present invention further provides functional variants of the antimicrobial peptide. These functional variants have at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with SEQ ID NO:1 and retain at least one of the following activities: inhibiting the growth of aquaculture-associated bacteria, inhibiting Aeromonas hydrophila infection, reducing bacterial load in fish tissues or the aquaculture environment, restoring the diversity of fish gut microbiota, improving the composition of fish gut microbiota, regulating post-infection inflammatory responses, or enhancing non-specific immune function in fish.

[0012] The present invention further provides a nucleic acid molecule encoding the antimicrobial peptide or a functional variant thereof, a recombinant expression vector containing the nucleic acid molecule, and an engineered bacterium or host cell containing the recombinant expression vector or integrating the nucleic acid molecule into the genome.

[0013] The present invention further provides a method for preparing the antimicrobial peptide, comprising: performing solid-phase chemical synthesis and oxidative folding according to the amino acid sequence shown in SEQ ID NO:1; inserting a nucleic acid molecule encoding the antimicrobial peptide into an expression vector, expressing it in a host cell, removing the fusion tag, purifying, refolding, and desalting it; or isolating and purifying the antimicrobial peptide from lip tissue, proteome samples, or peptide samples.

[0014] The present invention further provides a composition comprising HlHepcidin or a functional variant thereof, and an aquaculture-acceptable, veterinary-acceptable, feed-acceptable, water-treatment-acceptable, or pharmaceutically acceptable carrier, excipient, stabilizer, protectant, sustained-release material, or diluent.

[0015] Preferably, the composition is prepared as a powder, lyophilized powder, solution, granules, premix, feed additive, microcapsule formulation, liposome formulation, nanocarrier formulation, sustained-release formulation, bath agent, water treatment agent, environmental spray, injection, or external preparation for fish surface treatment.

[0016] The present invention further provides the use of the aforementioned antimicrobial peptide or composition in the preparation of products for regulating the gut microbiota of fish. The regulation includes restoring the Shannon and / or Simpson indices reduced due to Aeromonas hydrophila infection, improving infection-induced shifts in gut microbiota composition, reducing the risk of excessive expansion of pathogens or opportunistic pathogens, or restoring the post-infection gut microbiota to a state characteristic of healthy fish microbiota.

[0017] The present invention further provides the use of the aforementioned antimicrobial peptide or composition in the preparation of products for the prevention and treatment of bacterial diseases in fish. The bacterial diseases are preferably infections associated with Aeromonas hydrophila, but may also include diseases caused or associated with Vibrio anguillarum, Citrobacter flexneri, Escherichia coli, Shigella flexneri, Staphylococcus warwickii, Listeria monocytogenes, or other aquaculture-related pathogens.

[0018] Compared with existing technologies, this invention has the following advantages: First, HlHepcidin is a fish-derived endogenous antimicrobial peptide with a clear origin, making it suitable for development as a candidate molecule for green disease control in aquaculture. Second, HlHepcidin exhibits inhibitory activity against a variety of Gram-negative and Gram-positive bacteria, and can be used as an active ingredient in compound antimicrobial products or antibiotic alternatives. Third, HlHepcidin can be used to restore the diversity and composition of intestinal flora after Aeromonas hydrophila infection, thus possessing both antimicrobial and microecological regulation value. Fourth, HlHepcidin can be prepared as feed additives, bath agents, water treatment agents, sprays, injections, or other aquaculture products, which is beneficial for industrial application and reduces the use of conventional antibiotics. Attached Figure Description

[0019] Figure 1 This is a multiple sequence alignment diagram of HlHepcidin with other fish Hepcidins, showing the signal peptide, pre-domain, mature peptide region, and conserved cysteine ​​residues.

[0020] Figure 2 This is a phylogenetic tree showing the relationship between HlHepcidin and other Hepcidin in fish and mammals.

[0021] Figure 3 This figure shows the expression of pro-inflammatory cytokines IL-6, IL-8, and TNF-α in the body of a labialis after infection with Aeromonas hydrophila.

[0022] Figure 4 This diagram illustrates the tissue-specific expression of the Hepcidin gene in the labial region.

[0023] Figure 5 This diagram shows the changes in the expression of HlHepcidin in the liver, spleen, and head kidney after Aeromonas hydrophila infection.

[0024] Figure 6 Figure showing the β-diversity analysis of gut microbiota in the control group, infection group, and Hepcidin-treated infection group.

[0025] Figure 7 Diagram showing the gut bacterial composition at the phylum, genus, and species levels for the control group, infection group, and Hepcidin-treated infection group.

[0026] Figure 8 The LEfSe analysis plot shows the different bacterial groups of gut microbiota among the control group, the infection group, and the Hepcidin-treated infection group.

[0027] The accompanying drawings are provided to further illustrate the invention and form part of this specification. The drawings, together with embodiments of the invention, are used to explain the invention and do not constitute a limitation thereof. Detailed Implementation

[0028] The technical solutions in the embodiments of the present invention are clearly and completely described below. It should be understood that the embodiments described are only a part of the embodiments of the present invention and are not intended to limit the scope of protection of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort should fall within the scope of protection of the present invention.

[0029] Unless otherwise stated, the molecular biology, microbiology, peptide synthesis, fish infection models, and microbial sequencing methods used in this invention can all be employed using methods conventional in the art. The terms "comprising" and "including," and variations thereof, are intended to cover non-exclusive inclusion. Unless the context clearly indicates otherwise, the singular form also includes the plural form.

[0030] Example 1: Obtaining and structurally analyzing HlHepcidin

[0031] Based on liver transcriptome data from the lip and palpebrae, a Hepcidin homologous gene sequence was obtained. Open reading frame analysis revealed that this gene contains a 282-nucleotide open reading frame encoding a 93-amino acid precursor polypeptide. This precursor polypeptide includes a signal peptide, a pre-domain, and a mature peptide region. The mature peptide region is located at amino acids 69 to 93, with the sequence QSHLSLCRYCCNCCRNKGCGYCCKF, i.e., SEQ ID NO:1.

[0032] The mature peptide has a theoretical molecular weight of approximately 2.90 kDa and a theoretical isoelectric point of approximately 8.74. It contains eight conserved cysteine ​​residues that can form four disulfide bonds. The N-terminus of the mature peptide contains a QSHLS motif. These structural features indicate that this peptide belongs to the fish HAMP1 type of hepcidin, possessing the potential to be developed into an antibacterial and microecological regulatory molecule.

[0033] Example 2: Basic tissue expression and infection-induced expression of HlHepcidin

[0034] Liver, head kidney, spleen, gills, heart, muscle, skin, and midgut tissues were collected from healthy lip mites. Total RNA was extracted and reverse transcribed into cDNA, and HlHepcidin expression was detected using real-time quantitative PCR. The results showed that HlHepcidin was expressed in all examined tissues, with the highest expression in the liver, relatively high expression in the spleen and heart, and the lowest expression in muscle.

[0035] After establishing an Aeromonas hydrophila infection model, liver, spleen, and head kidney tissues were collected at 4 h, 8 h, 12 h, and 24 h post-infection. Results showed that HlHepcidin was significantly upregulated in the early stages of infection. At 4 h post-infection, expression levels in the liver, spleen, and head kidney increased by approximately 14.2-fold, 19.6-fold, and 12.7-fold, respectively. These results indicate that HlHepcidin is involved in the early innate immune response of the labial lip to Aeromonas hydrophila infection.

[0036] Example 3: Chemical synthesis and folding of mature HlHepcidin peptide

[0037] The mature peptide of HlHepcidin was prepared by solid-phase chemical synthesis according to the amino acid sequence shown in SEQ ID NO:1. The synthesized product was purified by high-performance liquid chromatography to a purity of not less than 90%, preferably not less than 95%. Subsequently, oxidative folding was performed to promote the formation of disulfide bonds between cysteine ​​residues, and the molecular weight was confirmed by mass spectrometry.

[0038] The resulting mature peptides can be prepared as freeze-dried powder or dissolved in sterile water, physiological saline, phosphate buffer, or other buffer systems acceptable for aquaculture for subsequent antimicrobial activity testing, animal infection model evaluation, and intestinal flora regulation evaluation.

[0039] Example 4: In vitro antibacterial activity of HlHepcidin

[0040] The inhibitory activity of HlHepcidin against aquaculture-related bacteria was evaluated using a two-fold microdilution method. HlHepcidin was prepared in gradient concentrations ranging from 100 μg / mL to 3.125 μg / mL and co-incubated with logarithmic-phase bacteria. The minimum inhibitory concentration (MIC) was determined based on OD600 and microbial precipitation. Exemplary results are shown in Table 1.

[0041] Table 1. Antibacterial activity of HlHepcidin against selected bacteria

[0042]

[0043] As shown in Table 1, HlHepcidin has inhibitory activity against a variety of Gram-negative and Gram-positive bacteria, especially against Staphylococcus warwickii, Citrobacter flexneri, and Shigella flexneri, and can also inhibit the growth of Aeromonas hydrophila.

[0044] Example 5: Regulation of intestinal flora in fish after Aeromonas hydrophila infection by HlHepcidin

[0045] Individuals were randomly divided into a healthy control group, an Aeromonas hydrophila infection group, and a post-infection HlHepcidin treatment group. Both the infection and treatment groups were exposed to Aeromonas hydrophila via immersion in a bath at an exemplary bacterial concentration of 1 x 10^6 CFU / mL for 2 h. Following infection, the treatment group received an intraperitoneal injection of HlHepcidin at a dose of 1.0 μg / g body weight. After 3 days of culture, intestinal contents or intestinal samples were collected for 16S rDNA sequencing analysis.

[0046] Alpha diversity analysis showed that Aeromonas hydrophila infection significantly reduced the Shannon and Simpson indices of the gut microbiota, suggesting a decrease in the richness and evenness of the gut microbiota in fish after infection. After treatment with HlHepcidin, the Shannon index increased from approximately 2.86 in the infected group to approximately 4.29, and the Simpson index increased from approximately 0.58 in the infected group to approximately 0.88. There was no significant difference between the treated groups and the healthy control group.

[0047] β-diversity analysis showed that the gut microbiota composition in the infected group deviated significantly from that of the healthy control group, while the microbiota composition in the HlHepcidin-treated group shifted towards a healthy state. At the phylum level, the relative abundance of Proteobacteria increased to approximately 87.00% in the infected group, decreasing to approximately 69.08% after treatment, approaching the level of approximately 59.69% in the healthy control group. Genus and species level analysis also showed that infection led to an abnormal increase in certain opportunistic bacteria, and HlHepcidin treatment reshaped the microbiota composition.

[0048] The above results indicate that HlHepcidin not only has direct antibacterial activity, but can also be used to restore the diversity and community structure of the gut microbiota in fish after Aeromonas hydrophila infection.

[0049] Example 6: Aquaculture composition containing HlHepcidin

[0050] In one embodiment, lyophilized HlHepcidin powder is mixed with carriers and protectants such as starch, maltodextrin, alginate, gelatin, chitosan, glycerol, trehalose, or mannitol to prepare powders, granules, or premixes. The premixes can be mixed with basal feed at an addition rate of 1 mg / kg to 1000 mg / kg for daily feeding, as an adjunct to stress control, or for prevention during periods of high pathogen incidence.

[0051] In another embodiment, HlHepcidin is prepared as a bathing agent or water treatment agent, used at a concentration of 0.01 mg / L to 100 mg / L, preferably 0.1 mg / L to 20 mg / L, for short-term immersion, temporary holding container treatment, or bacterial load control in aquaculture water.

[0052] In another embodiment, HlHepcidin is prepared as an injection at a dose of 0.01 μg / g to 10 μg / g body weight, preferably 0.1 μg / g to 2 μg / g body weight, for individual treatment or experimental disease models in high-risk infection situations.

[0053] In another embodiment, HlHepcidin is combined with probiotics, immune enhancers, plant extracts, organic acids, vitamins, minerals, chitosan, or other antimicrobial peptides to form a complex fish health aquaculture product.

[0054] Example 7: Application of HlHepcidin in the Green Control of Bacterial Diseases in Fish

[0055] HlHepcidin or its combinations can be used in the following fish farming scenarios: First, it can be added to feed as a feed additive or premix to improve the non-specific resistance to infection in fish and maintain intestinal flora homeostasis. Second, it can be used as a bathing agent during transportation, sorting, water changes, density adjustments, or during periods of high disease prevalence to reduce the risk of pathogens on the body surface and in the environment. Third, it can be used as an adjunct control product after Aeromonas hydrophila infection to promote the recovery of intestinal flora diversity and community composition. Fourth, it can be combined with pathogen monitoring, water quality management, nutritional regulation, and probiotic application to form a comprehensive disease prevention and control program.

[0056] Through the above applications, HlHepcidin can reduce the risk of bacterial diseases in fish, reduce the use of conventional antibiotics, improve the health of farmed fish, and enhance the safety of aquatic products.

[0057] sequence list

[0058] SEQ ID NO:1:QSHLSLCRYCCNCCRNKGCGYCCKF Sequence description: SEQ ID NO:1 is the mature peptide sequence of Hepcidin derived from the lips, consisting of 25 amino acids and containing eight cysteine ​​residues.

Claims

1. An HlHepcidin antimicrobial peptide derived from Hemibarbus labeo, characterized in that, The antimicrobial peptide comprises or consists of the amino acid sequence QSHLSLCRYCCNCCRNKGCGYCCKF shown in SEQ ID NO:1, wherein the antimicrobial peptide contains eight cysteine ​​residues and is capable of forming four pairs of disulfide bonds after oxidative folding.

2. The antimicrobial peptide according to claim 1, characterized in that, The antimicrobial peptide is a chemically synthesized peptide, a recombinant expressed peptide, a mature peptide obtained by enzymatic digestion of a fusion protein, a naturally isolated peptide, a lyophilized peptide, a salt-form peptide, or a peptide obtained by purification, refolding, desalting, ultrafiltration, chromatography, or oxidative folding.

3. A functional variant of the antimicrobial peptide according to claim 1, characterized in that, The functional variant has at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity with SEQ ID NO:1 and retains antibacterial activity and / or fish gut microbiota regulation activity.

4. The functional variant according to claim 3, characterized in that, The functional variants are selected from conserved amino acid substitution variants, truncated peptides, extended peptides, cyclized peptides, disulfide bond-stabilized peptides, N-terminal modified peptides, C-terminal amidated peptides, D-amino acid substitution peptides, lipopeptides, PEG-modified peptides, salt forms, fusion peptides, or aquaculture-acceptable, veterinary-acceptable, feed-acceptable, or pharmaceutically acceptable derivatives.

5. A nucleic acid molecule, a recombinant expression vector, an engineered bacterium, or a host cell, characterized in that, The nucleic acid molecule encodes the antimicrobial peptide of claim 1 or 2 or the functional variant of claim 3 or 4; the recombinant expression vector contains the nucleic acid molecule; the engineered bacteria or host cell contains the recombinant expression vector, or the nucleic acid molecule is integrated into its genome.

6. A method for preparing the antimicrobial peptide or functional variant according to any one of claims 1 to 4, characterized in that, The method includes at least one of the following steps: solid-phase chemical synthesis according to the target amino acid sequence; oxidative folding of the synthesized peptide to form disulfide bonds; obtaining a fusion protein through recombinant expression, and obtaining the antimicrobial peptide by enzyme digestion, purification, refolding or desalting; or isolating and purifying the antimicrobial peptide from labial tissue, secretions, proteome samples or peptide samples.

7. A composition, characterized in that, It includes the antimicrobial peptide or functional variant as described in any one of claims 1 to 4, as well as a carrier, excipient, stabilizer, protectant, sustained-release material or diluent that is acceptable for aquaculture, veterinary use, feed, water treatment or pharmaceutical use.

8. The composition according to claim 7, characterized in that, The composition is a powder, lyophilized powder, solution, granules, premix, feed additive, microcapsule formulation, liposome formulation, nanocarrier formulation, sustained-release formulation, bath agent, water treatment agent, environmental spray, injection, or topical formulation; and optionally further comprises at least one selected from probiotics, immune enhancers, vitamins, minerals, enzymes, plant extracts, organic acids, chitosan, alginate, cyclodextrin, gelatin, starch, trehalose, mannitol, buffer salts, and preservatives.

9. The use of the antimicrobial peptide or functional variant according to any one of claims 1 to 4, or the composition according to claim 7 or 8, in the preparation of products for regulating the intestinal flora of fish and / or preventing bacterial diseases in fish, wherein, Regulating the gut microbiota of fish includes restoring the Shannon and / or Simpson indices that are reduced due to Aeromonas hydrophila infection, improving infection-induced gut microbiota composition shifts, reducing the risk of excessive expansion of pathogens or opportunistic pathogens, or restoring the gut microbiota to a healthy fish microbiota state after infection; and the bacterial disease in fish is caused by or related to Aeromonas hydrophila, Vibrio anguillarum, Citrobacter flexneri, Escherichia coli, Shigella flexneri, Staphylococcus warwickii, Listeria monocytogenes, or other aquaculture-associated pathogens.

10. An antibacterial or microecological regulation product or non-therapeutic method for fish farming, characterized in that, The product or method includes applying the antimicrobial peptide or functional variant of any one of claims 1 to 4, or the composition of claim 7 or 8, to aquaculture water, holding containers, aquaculture facilities, fish surface, or feed to reduce bacterial load, inhibit pathogen proliferation, improve the intestinal flora status of fish, or assist in the prevention and treatment of bacterial diseases in fish.

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