Drug-resistant salmonella antibacterial peptide capable of penetrating intestinal barrier and application of drug-resistant salmonella antibacterial peptide

Abstract

The invention belongs to the field of biotechnology and veterinary medicine, and discloses a drug-resistant salmonella antibacterial peptide capable of penetrating an intestinal barrier and an application of the drug-resistant salmonella antibacterial peptide. The antibacterial peptide can selectively penetrate infected host cells (such as macrophages and intestinal epithelial cells) and remove intracellular parasitic bacteria, and has no obvious toxicity to the host cells. The composition has good effect stability in the local part of the intestinal tract. When the antibacterial peptide is added into poultry feed, the survival rate of poultry infected by salmonella can be remarkably increased, the bacterium carrying amount of visceral organs can be remarkably reduced, and the effect of the antibacterial peptide is remarkably superior to that of a traditional antibiotic additive. The invention provides a new scheme of a feed additive which is clear in action mechanism, efficient and safe, is not easy to induce drug resistance, and meets the requirements of green and antibiotic-free breeding.

Description

Technical Field

[0001] This invention belongs to the fields of biotechnology and veterinary medicine, specifically relating to an antimicrobial peptide against drug-resistant Salmonella that can penetrate the intestinal barrier and its applications. The antimicrobial peptide of this invention has an α-helical amphiphilic structure and a ditryptophan (WW) motif, enabling it to efficiently kill Salmonella and selectively penetrate infected host cells to eliminate intracellular parasites. Background Technology

[0002] Salmonella is an important zoonotic pathogen that causes diseases such as pullorum disease and fowl typhoid in poultry, resulting in serious economic losses. This bacterium has intracellular parasitic capabilities, allowing it to invade host cells such as macrophages and intestinal epithelial cells, evading conventional antibiotics and making treatment difficult. Currently, the livestock industry relies heavily on antibiotics such as ampicillin and ceftiofur for control, but this easily leads to increased drug resistance and drug residues, threatening food safety and public health.

[0003] Antimicrobial peptides, as natural immune components, possess advantages such as broad-spectrum antibacterial activity, rapid action, and low likelihood of inducing drug resistance, making them a hot topic in antibiotic alternative research. Antimicrobial peptides rich in tryptophan have attracted considerable attention due to their membrane affinity and penetration properties; however, existing studies often lack oral stability evaluations and host cell safety verification, and their mechanisms of action are frequently described without sufficient rigor.

[0004] Therefore, there is a need to provide an antimicrobial peptide formulation with a clear mechanism of action, good stability, high safety, and suitability for feed addition. Summary of the Invention

[0005] The purpose of this invention is to provide an antimicrobial peptide against drug-resistant Salmonella that can penetrate the intestinal barrier, wherein the amino acid sequence of the antimicrobial peptide is shown in SEQ ID NO.1.

[0006] Another object of the present invention is to provide the application of the above-mentioned antimicrobial peptide.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] This invention addresses the current problems with antimicrobial peptides by providing a stable, safe, and highly effective antimicrobial peptide that can pass through the gastrointestinal tract and eliminate intracellular Salmonella. This antimicrobial peptide exhibits stability in simulated gastrointestinal fluid, supporting its oral efficacy. While efficiently eliminating intracellular bacteria, its safety to host cells is demonstrated, elucidating its selective mechanism of action. Its action is clearly limited to the intestinal tract, avoiding systemic circulation and reducing the risk of residue. It completely kills bacteria within 2 hours at 2×MIC, maintains a low MIC against multidrug-resistant bacteria, and shows no significant increase in MIC after 20 consecutive passages, indicating it is unlikely to induce drug resistance. The amino acid sequence of this antimicrobial peptide is shown in SEQ ID NO.1.

[0009] The scope of protection of this invention also includes:

[0010] The fusion protein obtained by fusing the antimicrobial peptide shown in SEQ ID NO.1 with a protein tag.

[0011] The gene encoding the antimicrobial peptide shown in SEQ ID NO.1 or the above-mentioned fusion protein.

[0012] Expression cassettes, recombinant vectors, recombinant microorganisms, or in vitro recombinant cells containing the above-mentioned coding genes.

[0013] The application of the above-mentioned antimicrobial peptides, fusion proteins, encoding genes, or expression cassettes containing the above-mentioned encoding genes, recombinant vectors, recombinant microorganisms, or ex vivo recombinant cells in the preparation of Salmonella antimicrobial peptides;

[0014] The use of the above-mentioned antimicrobial peptides, fusion proteins, encoding genes, or expression cassettes containing the above-mentioned encoding genes, recombinant vectors, recombinant microorganisms, or ex vivo recombinant cells in the preparation of drugs for the treatment or prevention of Salmonella infection;

[0015] In the above-described applications, preferably, the Salmonella is a drug-resistant Salmonella.

[0016] The application of the above-mentioned antimicrobial peptides, fusion proteins, encoding genes, or expression cassettes containing the above-mentioned encoding genes, recombinant vectors, recombinant microorganisms, or isolated recombinant cells in the preparation of feed additives;

[0017] In the above-described applications, preferably, the feed additive is a poultry feed additive;

[0018] In the above-described applications, preferably, the dosage of the feed additive is 100–400 mg / kg;

[0019] In the above-described applications, preferably, the dosage of the feed additive is 300 mg / kg.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] 1. The antimicrobial peptide provided by this invention has a structure that combines tryptophan membrane affinity with α-helical amphiphilicity, which can penetrate infected cells to eliminate intracellular bacteria, thus solving the problem that conventional additives are ineffective against intracellular bacteria;

[0022] 2. High activity and rapid onset of action: Complete sterilization within 2 hours at 2×MIC, while maintaining low MIC against multidrug-resistant bacteria;

[0023] 3. Low risk of drug resistance: No significant increase in MIC was observed after 20 consecutive passages, making it difficult to induce drug resistance;

[0024] 4. High safety: In vitro cytotoxicity tests showed no significant toxicity to host cells at effective antibacterial concentrations; no tissue residue was found 24 hours after feeding; subchronic toxicity tests showed no adverse effects on poultry.

[0025] 5. Clear application effects: In animal experiments, an addition of 300 mg / kg resulted in a survival rate of 96% in chickens, ducks, and geese, which is significantly better than traditional antibiotics;

[0026] 6. Industrialization is feasible: It can be produced on a large scale through solid-phase synthesis, and has good stability in feed processing and storage. Attached Figure Description

[0027] Figure 1 The image shows the bactericidal kinetics curve of the antimicrobial peptide against Salmonella standard strain ATCC 14028.

[0028] Figure 2 Survival curves of chicks fed different doses of antimicrobial peptides after Salmonella challenge.

[0029] Figure 3 The curve shows the clearance rate of Salmonella in macrophages by the antimicrobial peptide.

[0030] Figure 4 The bar chart shows the activity retention rate of antimicrobial peptides at different granulation temperatures.

[0031] Figure 5 To simulate the retention rate curve of antimicrobial peptide activity after treatment with gastric / intestinal fluid.

[0032] Figure 6 The effect of antimicrobial peptide treatment on the survival rate of primary chicken macrophages (CCK-8 assay). Detailed Implementation

[0033] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments, experimental data, and accompanying drawings. The following embodiments are for illustrative purposes only and should not be considered as limitations on the invention. Unless otherwise specified, the reagents and methods used in this invention are conventional techniques in the art.

[0034] Example 1:

[0035] Chemical synthesis and quality control of antimicrobial peptides

[0036] The target polypeptide SEQ ID NO.1 was synthesized using the Fmoc-SPPS method. After cleavage and precipitation, it was purified by preparative HPLC. Analytical HPLC confirmed a purity >98%; MALDI-TOF MS identified the molecular weight with an error <0.1%; circular dichroism (CD) analysis showed that the sequence exhibited an α-helical structure in a membrane simulation environment, demonstrating its amphiphilic nature; tryptophan residue content detection (HPLC) confirmed the presence of two tryptophan residues.

[0037] Example 2:

[0038] In vitro antibacterial activity (MIC) assay

[0039] The tested strains included the standard strains *Salmonella typhimurium* ATCC 14028, *Salmonella pullorum* CVCC 583, and *Salmonella fowleri* CVCC 798, as well as three clinical isolates of multidrug-resistant (MDR) Salmonella (MDR-S1, S2, and S3). The method followed the CLSI M07-A10 broth microdilution method. The results are shown in Table 1. The antimicrobial peptides of this invention exhibited potent inhibitory activity against all strains, with MIC values ​​of 2–4 μg / mL, superior to the widely used antibiotics ampicillin (MIC = 4–64 μg / mL) and ceftiofur (MIC = 2–32 μg / mL).

[0040] Table 1. MIC values ​​of the antimicrobial peptides of the present invention and control drugs against Salmonella.

[0041] .

[0042] Example 3:

[0043] bactericidal kinetics study

[0044] Using Salmonella Typhimurium ATCC 14028 as the test strain, the following groups were set up: an experimental group (the antimicrobial peptide of this invention, final concentration 4 μg / mL, 2×MIC), a positive control group (ampicillin final concentration 8 μg / mL, ceftiofur final concentration 4 μg / mL), and a negative control group (MH broth without antimicrobial drugs). The bacterial suspension was diluted to approximately 1×10⁻⁶ using the time-kill curve method. 6 CFU / mL was mixed with drug-containing broth and incubated at 37°C with shaking. Samples were taken at 0, 0.5, 1, 2, 4, 6, 8, 12, and 24 hours to count CFU / mL. The results showed that the antimicrobial peptide of this invention could completely kill the initial bacterial load to the detection limit (1 Log) within 2 hours. 10 The concentration of CFU / mL is below 1000, while ampicillin and ceftiofur require 12 hours and 8 hours respectively (e.g., CFU / mL). Figure 1 As shown in the figure, the antimicrobial peptides of the present invention have a significantly better bactericidal rate than traditional antibiotics.

[0045] Example 4:

[0046] Animal feeding trials of antimicrobial peptides as feed additives

[0047] Experiment 1 (Validation of different dosages and infection types in chickens): A two-factor factorial design of 7 (feed treatments) × 3 (challenge strains) was used.

[0048] One hundred and fifty-five one-day-old healthy AA broiler chickens were randomly divided into 21 groups of 50 each. Feed treatments included: G1 (basal diet), G2 (ampicillin 100 mg / kg), G3 (ceftiofur 80 mg / kg), G4 (antimicrobial peptide 100 mg / kg), G5 (antimicrobial peptide 200 mg / kg), G6 (antimicrobial peptide 300 mg / kg, preferred), and G7 (antimicrobial peptide 400 mg / kg). At seven days of age, the chickens were orally challenged with Salmonella pullorum CVCC 583, Salmonella fowleri CVCC 798, and Salmonella typhimurium ATCC 14028, at a dose of 1×10⁻⁶. 8 CFU / animal. Survival rate was recorded after 14 days of post-challenge observation, and total bacterial load in the cecum and intracellular bacterial load in the liver were measured on day 7.

[0049] The results are shown in Table 2. For all three pathogenic Salmonella strains, the addition of antimicrobial peptides (G4-G7) significantly improved the survival rate of infected chicks (P<0.01) and dose-dependently reduced tissue bacterial load. The optimal antimicrobial peptide dosage was 300 mg / kg (G6), achieving a survival rate of 98.0%, significantly superior to the traditional antibiotic groups (G2, G3). The effect curves of different doses of antimicrobial peptides on chick survival rate are shown in Table 2. Figure 2 As shown.

[0050] Table 2. Protective effect of antimicrobial peptide feed additives on Salmonella-infected chicks.

[0051] .

[0052] Note: *P<0.05, **P<0.01 (compared with group G1)

[0053] Experiment 2 (Applicability Verification in Ducks and Geese): 150 healthy 1-day-old Beijing ducks and Yangzhou geese were selected and divided into 3 groups (50 birds per group): negative control (basal diet), ampicillin control (100 mg / kg), and the group receiving 300 mg / kg of the antimicrobial peptide of this invention. At 7 days of age, the birds were orally challenged with Salmonella Typhimurium ATCC 14028 (1×10⁻⁶). 8 CFU / animal), observe for 14 days and record survival rate.

[0054] The results showed that the survival rate of the antimicrobial peptide group in ducks was 96% (compared to 62% in the control group and 80% in ampicillin group), and the survival rate of the antimicrobial peptide group in geese was also 96% (compared to 60% in the control group and 80% in ampicillin group), proving that it is equally applicable to both ducks and geese.

[0055] Example 5:

[0056] Intracellular bacterial killing test

[0057] A chicken primary macrophage infection model was used, with strains ATCC 14028, CVCC 583, and CVCC 798. The experimental groups were treated with the antimicrobial peptide of this invention (concentration 2×MIC for each strain), the positive control group used the antibiotic with the best in vitro activity against each strain (2×MIC of ceftiofur for ATCC 14028, CVCC 583, and CVCC 798), and the negative control group was drug-free culture medium. Two hours after infection, extracellular bacteria were removed, and the antibiotic was added for further culture. Samples were taken at 0, 0.5, 1, 2, and 4 hours to calculate the clearance rate.

[0058] The results are shown in Table 3. The clearance rate of the antimicrobial peptides of the present invention was higher than 95% after 4 hours, while the clearance rate of traditional antibiotics was only about 28-36% (e.g., ...). Figure 3 As shown in the figure, it has a significant intracellular bactericidal advantage.

[0059] Table 3. Clearance rates of different Salmonella strains in macrophages by antimicrobial peptides and traditional antibiotics (4 hours, 2×MIC)

[0060] .

[0061] Intracellular bacterial load detection in animals: Seven days after challenge with the virus in group G6 (300 mg / kg) of Example 4, the intracellular bacterial load was detected by isolating macrophages from the liver and cecum.

[0062] The results showed that the liver size was 1.82 ± 0.25 Log. 10 CFU / g, cecum: 2.15±0.30 Log 10 CFU / g was significantly lower than that of the negative control group (liver 5.23±0.42 Log). 10 CFU / g, cecum 6.85±0.50 Log 10 (CFU / g, P<0.01), confirming that it also has excellent intracellular bactericidal effect in vivo.

[0063] Example 6:

[0064] Drug resistance induction test

[0065] Salmonella typhimurium ATCC 14028 was used as the test strain. Antimicrobial peptide group (sub-MIC=1 μg / mL) and ampicillin group (sub-MIC=2 μg / mL) were set up and passaged for 20 generations. The MIC value was measured every 5 generations.

[0066] Results: The MIC value of the antimicrobial peptide group remained at 2 μg / mL without any increase; the MIC of the ampicillin group increased from 4 μg / mL to 32 μg / mL, an 8-fold increase, indicating that the antimicrobial peptide of the present invention is not likely to induce drug resistance.

[0067] Example 7:

[0068] Safety and residue testing

[0069] Subchronic toxicity test: 200 one-day-old AA broiler chickens were randomly divided into 4 groups and fed with 0, 200, 300 and 500 mg / kg of the antimicrobial peptide of this invention, respectively, for 42 consecutive days.

[0070] Growth performance, blood biochemical indicators, and visceral pathological sections were tested. The results showed no significant differences among the groups, indicating good safety. Residue detection: Chickens fed 300 mg / kg of antimicrobial peptides had chicken meat and liver samples collected at 12, 24, and 48 hours. HPLC-MS / MS analysis showed that the residue was ≤0.05 μg / g at 12 hours and undetectable after 24 hours, meeting the requirement of no residue.

[0071] Example 8:

[0072] Processing and storage stability tests

[0073] Processing stability: Feed containing 300 mg / kg antimicrobial peptides was pelleted at 80℃, 100℃, and 120℃, and the activity was tested. The retention rates were 98.5%, 95.2%, and 90.3%, respectively (e.g., ...). Figure 4 As shown in the figure, it meets the processing requirements.

[0074] Storage stability: After being stored at 25℃ and 60% humidity for 6 months, the feed retained 88.7% of its activity, demonstrating good stability.

[0075] Example 9:

[0076] Simulated gastrointestinal fluid stability test

[0077] Simulated gastric juice (pH 2.0, pepsin 3.2 g / L) and intestinal juice (pH 6.8, trypsin 10 g / L) were prepared according to the Chinese Pharmacopoeia. The activity of the antimicrobial peptide (100 μg / mL) was detected after incubation at 37°C.

[0078] Results: Gastric juice retention was 82.5% ± 3.1% at 30 min, and intestinal juice retention was 71.2% ± 2.8% at 120 min (e.g., Figure 5 As shown in the figure, this confirms that its local window of action in the intestine is sufficient.

[0079] Example 10:

[0080] Evaluation of the toxicity of antimicrobial peptides to host cells

[0081] To comprehensively evaluate the biosafety of the antimicrobial peptides of this invention as potential feed additives, in addition to completing in vitro antimicrobial activity and in vivo animal experiments, their direct toxic effects on host cells were further systematically investigated.

[0082] Experimental Methods: Peritoneal macrophages of 1-day-old AA broiler chickens were aseptically isolated and purified by adherence to the culture medium to obtain primary macrophages. Cells were seeded into 96-well plates. After cell adhesion, the medium was replaced with fresh medium containing different concentrations of the antimicrobial peptide (SEQ ID NO. 1) of this invention. The antimicrobial peptide concentrations were set as 0 (negative control), 1×MIC (2 µg / mL), 2×MIC (4 µg / mL), 4×MIC (8 µg / mL), and 8×MIC (16 µg / mL), where the MIC value was determined based on the results of the assay for Salmonella Typhimurium ATCC 14028 (2 µg / mL). Each concentration was used in triplicate. CCK-8 reagent was added to each well 4 hours and 24 hours after drug treatment, and incubation was continued for an appropriate time. The absorbance (OD) at 450 nm was measured using a microplate reader. 450 The relative cell viability of each drug-treated group was calculated by normalizing the OD value of the cell pores without the drug to 100% viability.

[0083] Test results: such as Figure 6 As shown, after 4 hours of treatment with the antimicrobial peptide, cells maintained a viability of 77.1% ± 1.4% even at concentrations as high as 8×MIC (16 µg / mL). At a concentration of 2×MIC (4 µg / mL), the cell viability was 93.3% ± 1.2%, demonstrating no significant toxicity to host cells at this effective antimicrobial concentration.

[0084] To assess safety under longer exposure times, cell viability was measured after 24 hours of treatment, and the specific data are shown in Table 4 below. After 24 hours of treatment, cell viability remained at a high level in all concentration groups, with a viability of over 85% at a 2×MIC concentration. This result further confirms that the antimicrobial peptides of this invention maintain high safety for avian host cells over a longer period of action.

[0085] Table 4. Virulence test of different concentrations of antimicrobial peptides on primary macrophages

[0086] .

[0087] Note: Three independent parallel experiments were conducted for each concentration point (n=3), and data are expressed as mean ± standard deviation.

[0088] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. An antimicrobial peptide against drug-resistant Salmonella that can penetrate the intestinal barrier, wherein the amino acid sequence of the antimicrobial peptide is shown in SEQ ID NO.

1.

2. The fusion protein obtained by fusing the antimicrobial peptide shown in SEQ ID NO.1 with a protein tag.

3. The gene encoding the antimicrobial peptide shown in SEQ ID NO.1 or the fusion protein of claim 2.

4. An expression cassette, recombinant vector, recombinant microorganism, or in vitro recombinant cell having the gene encoding as described in claim 3.

5. The use of the antimicrobial peptide of claim 1, the fusion protein of claim 2, the encoding gene of claim 3, or an expression cassette having the encoding gene of claim 3, a recombinant vector, a recombinant microorganism, or an ex vivo recombinant cell in the preparation of Salmonella antimicrobial peptides.

6. The use of the antimicrobial peptide of claim 1, the fusion protein of claim 2, the encoding gene of claim 3, or an expression cassette having the encoding gene of claim 3, a recombinant vector, a recombinant microorganism, or an ex vivo recombinant cell in the preparation of a medicament for the treatment or prevention of Salmonella infection.

7. The application according to claim 5 or 6, wherein the Salmonella is a drug-resistant Salmonella.

8. The use of the antimicrobial peptide of claim 1, the fusion protein of claim 2, the encoding gene of claim 3, or an expression cassette having the encoding gene of claim 3, a recombinant vector, a recombinant microorganism, or an isolated recombinant cell in the preparation of feed additives.

9. The application according to claim 8, wherein the feed additive is a poultry feed additive.

10. In the application according to claim 9, the dosage of the feed additive is 100–400 mg / kg.

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