CN114702554A - Application of antibacterial peptide NZX in preparation of streptococcus agalactiae antibacterial drugs - Google Patents
Application of antibacterial peptide NZX in preparation of streptococcus agalactiae antibacterial drugs Download PDFInfo
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- CN114702554A CN114702554A CN202210483672.7A CN202210483672A CN114702554A CN 114702554 A CN114702554 A CN 114702554A CN 202210483672 A CN202210483672 A CN 202210483672A CN 114702554 A CN114702554 A CN 114702554A
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- Prior art keywords
- nzx
- streptococcus agalactiae
- antibacterial
- streptococcus
- agalactiae
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/04—Antibacterial agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Medicinal Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- General Chemical & Material Sciences (AREA)
- Biophysics (AREA)
- Molecular Biology (AREA)
- Biochemistry (AREA)
- Gastroenterology & Hepatology (AREA)
- Communicable Diseases (AREA)
- Oncology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Genetics & Genomics (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Pharmacology & Pharmacy (AREA)
- Animal Behavior & Ethology (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention provides application of antibacterial peptide NZX in preparation of a streptococcus agalactiae antibacterial drug. The invention discovers that the antibacterial peptide NZX has an inhibiting effect on streptococcus agalactiae for the first time. Experiments show that NZX has high-efficiency, rapid sterilization and low drug resistance effects on streptococcus agalactiae. The antibacterial activity and the sterilization rate of the antibacterial peptide NZX on the streptococcus agalactiae are obviously better than those of the florfenicol, the hemolysis rate is lower, and the stability of blood plasma and blood serum is higher. NZX acts by killing bacteria by inhibiting bacterial cell wall synthesis; the inhibition rate of the initial biofilm at the concentration of 64 multiplied by MIC is 49.0 percent; the haemolysis rate at 256 × MIC is only 0.71%; NZX retained 97.8% and 98.9% activity, respectively, after 60min of incubation with 25% serum and plasma. NZX is expected to be developed as a novel agent for the treatment of streptococcal diseases caused by Streptococcus agalactiae.
Description
Technical Field
The invention relates to the field of biological medicines, in particular to application of antibacterial peptide NZX in preparation of a streptococcus agalactiae antibacterial drug.
Background
Streptococcus agalactiae (streptococcus. agalactiae) is a pathogen for zoonosis, can induce not only vaginal infection of pregnant women, neonatal septicemia and meningitis, and cow mastitis, but also fish streptococcosis, and is one of the main pathogenic bacteria of tilapia streptococcosis. Tilapia streptococcicosis is characterized by septicemia, meningoencephalitis and eyeball protrusion, the morbidity and mortality are very high, the economic impact on the tilapia industry exceeds 10 hundred million dollars, and the tilapia streptococcicosis is a great obstacle in global tilapia culture. The drug resistance brought by the abuse of antibiotics by conventional treatment means is more and more common, in the latest research, streptococcus agalactiae generates high-level drug resistance to macrolide drugs, tetracycline) and penicillin G, and the time cost and the application cost of the research and development of the vaccine serving as a new prevention and control means limit the large-scale application of the vaccine, so that the development of a novel antibacterial drug with high bactericidal activity and low drug resistance to streptococcus agalactiae is imperative.
The antibacterial peptides (AMPs) are natural small molecules with antibacterial activity, and become one of hot spots in candidate drugs for replacing antibiotics due to the characteristics of broad-spectrum antibacterial, low toxicity and low drug resistance, Plectasin is a first defensin cationic antibacterial peptide separated from ascosphaera humicola pseudopygmata nigrella, has strong activity on gram-positive bacteria (such as staphylococcus aureus and streptococcus), a derivative peptide NZX with better antibacterial activity is obtained by mutating three sites of D9, M13 and K32 of the Plectasin, and a high-expression-quantity system is established in Pichia pastoris X-33. The peptide has better in vitro and in vivo (mouse infection model) antibacterial activity than rifampicin on acute mycobacterium tuberculosis. NZX reports on the antibacterial aspect of the streptococcus agalactiae are not found.
Disclosure of Invention
The invention aims to provide a new application of antibacterial peptide NZX in inhibiting streptococcus agalactiae.
In order to achieve the object, in a first aspect, the invention provides an application of the antibacterial peptide NZX in preparation of a streptococcus agalactiae (streptococcus agalactiae) antibacterial drug or composition.
In the present invention, the antimicrobial peptide NZX comprises or consists of the amino acid sequence as follows:
i) 1, SEQ ID NO; or
ii) an amino acid sequence obtained by connecting a tag to the N-terminal and/or C-terminal of i).
In a second aspect, the present invention provides the use of antimicrobial peptide NZX in the manufacture of a biological product for the treatment or prevention of streptococcus agalactiae infection and diseases associated with infection with streptococcus agalactiae.
In a third aspect, the present invention provides the use of antimicrobial peptide NZX in the treatment or prevention of infection by Streptococcus agalactiae, and in the treatment or prevention of diseases associated with infection by Streptococcus agalactiae.
The disease refers to related diseases caused by streptococcus agalactiae and a biological membrane generated by the streptococcus agalactiae, such as streptococcosis (tilapia streptococcosis).
In a fourth aspect, the present invention provides the use of antimicrobial peptide NZX for inhibiting biofilm caused by Streptococcus agalactiae, including inhibition of primary biofilm,
in the invention, the streptococcus agalactiae can be a clinical strain obtained by separating in vivo the streptococcicosis-suffering tilapia, and can also be a standard strain.
The streptococcus agalactiae includes but is not limited to the following strains: ACCC61733, ATCC 13813, CAU-FRI 1, CAU-FRI 2, CAU-FRI 3, CAU-FRI 4, PBSA 0903.
In a fifth aspect, the present invention provides an application of the antibacterial peptide NZX in preparing an antibacterial drug or composition of Streptococcus dysgalactiae (Streptococcus dysgalactiae).
In a sixth aspect, the present invention provides the use of antimicrobial peptide NZX in the manufacture of a biological product for the treatment or prevention of Streptococcus dysgalactiae infection and related diseases caused by infection with Streptococcus dysgalactiae.
In a seventh aspect, the present invention provides the use of antimicrobial peptide NZX in the treatment or prevention of Streptococcus dysgalactiae infection, and in the treatment or prevention of diseases associated with infection therewith.
The disease refers to related diseases caused by streptococcus dysgalactiae and biomembranes produced by the streptococcus dysgalactiae, such as cow mastitis.
The streptococcus dysgalactiae includes but is not limited to the strain CVCC 3938.
By means of the technical scheme, the invention at least has the following advantages and beneficial effects:
the invention discovers that the antibacterial peptide NZX has an inhibiting effect on streptococcus agalactiae for the first time. Experiments show that NZX has high-efficiency, rapid sterilization and low drug resistance effects on streptococcus agalactiae. Antimicrobial activity and bactericidal rate of antimicrobial peptide NZX against streptococcus agalactiae were significantly better than florfenicol (Ff), NZX showed better antimicrobial activity against s.agalactiae ACCC61733, with MIC values of 0.11 μ M, much lower than 5.59 μ M for Ff, mpc (mutant preventi On conjugation) of NZX of 1.82 μ M, selection index SI of 16, both lower than Ff, indicating NZX with lower preventive resistance than the latter, and furthermore with hemolysis rate of 256 × MIC of only 0.71%, NZX retained 97.8% and 98.9% activity, respectively, after 60min of incubation with 25% serum and plasma. NZX shows potential as a novel formulation for the treatment of tilapia streptococcal disease caused by streptococcus agalactiae.
Drawings
FIG. 1 is a graph showing the bactericidal kinetics of NZX against Streptococcus agalactiae ACCC61733 in the preferred embodiment of the present invention.
FIG. 2 is the result of measuring the hemolytic activity of antibacterial peptide NZX on red blood cells of tilapia mossambica in the preferred embodiment of the present invention.
FIG. 3 is a graph showing the stability of antibacterial peptide NZX in tilapia serum in accordance with a preferred embodiment of the present invention.
FIG. 4 is a graph showing the stability of antimicrobial peptide NZX in tilapia mossambica plasma in a preferred embodiment of the present invention.
FIG. 5 is a graph showing the effect of NZX on early stage biofilm formation in Streptococcus agalactiae ACCC61733 in a preferred embodiment of the invention.
FIG. 6 is a graph showing the effect of NZX on the cell membrane integrity of Streptococcus agalactiae ACCC61733 in a preferred embodiment of the present invention.
FIG. 7 is a graph showing the effect of scanning electron microscopy on NZX on the cell morphology of Streptococcus agalactiae ACCC61733 in a preferred embodiment of the present invention.
FIG. 8 is a graph showing the effect of incubation on the bactericidal capacity of NZX.
FIG. 9 is a super-resolution microscope showing the localization of antimicrobial peptide NZX in Streptococcus agalactiae ACCC61733 in accordance with a preferred embodiment of the present invention.
Detailed Description
The invention provides application of a derivative peptide-NZX of fungal defensin Plectasin in killing streptococcus agalactiae (such as a clinical isolate strain streptococcus agalactiae ACCC 61733). Antibacterial peptide NZX was used as a research object, and Streptococcus agalactiae was used as an indicator bacterium, and its antibacterial activity, serum/plasma stability, hemolytic property and primary biofilm inhibiting ability were examined. And analyzing cell membrane integrity by using a flow cytometer; observing the morphological change of the bacteria under the action of NZX by a scanning electron microscope; the location of the resolution microscopy NZX in the cell, etc. elucidate its mechanism of action and target. Experiments show that NZX has high-efficiency, rapid sterilization and low drug resistance effects on streptococcus agalactiae. NZX the mechanism of action is to kill bacteria by inhibiting bacterial cell wall synthesis; the inhibition rate of the initial biofilm at the concentration of 64 multiplied by MIC is 49.0 percent; the haemolysis rate at 256 × MIC is only 0.71%; NZX retained 97.8% and 98.9% activity, respectively, after 60min of incubation with 25% serum and plasma.
The antibacterial peptide NZX has better antibacterial activity and sterilization rate on streptococcus agalactiae than florfenicol, has lower hemolytic rate and higher plasma and serum stability, has a unique sterilization mechanism targeting cell walls, has stronger in-vitro sterilization effect on streptococcus agalactiae, and has the sterilization characteristics of quick sterilization, no rebound and strong drug resistance. NZX is expected to be developed into a novel preparation for treating tilapia streptococcicosis caused by streptococcus agalactiae.
The invention adopts the following technical scheme:
the invention provides application of antibacterial peptide NZX in preparation of a streptococcus agalactiae antibacterial drug.
The invention also provides application of the antibacterial peptide NZX in preparing biological products for treating or preventing streptococcus agalactiae infection and related diseases caused by the infection.
Further, the disease refers to related diseases caused by streptococcus agalactiae.
The disease comprises tilapia streptococcicosis.
The present invention also provides the principle that antibacterial peptide NZX inhibits streptococcus agalactiae.
The invention also provides the application of the antibacterial peptide NZX in inhibiting the biofilm caused by streptococcus agalactiae, including the inhibition effect on the initial biofilm.
In the present invention, Streptococcus agalactiae can be a clinical strain isolated from streptococcicosis-affected tilapia, or a standard strain, such as ATCC 13813.
The strains, media and main instruments used in the following examples are as follows:
streptococcus agalactiae (S.agalactiae) ACCC61733 was obtained from the fishery institute in south China sea, the institute for aquatic sciences, and was deposited at the center for agricultural culture Collection (ACCC) in China. Agalactiae ATCC 13813 was purchased from American Type Culture Collection (ATCC). S.agalactiae CAU-FRI 1, CAU-FRI 2, CAU-FRI 3 and CAU-FRI 4 isolated from bovine mastitis tissue were from university of agriculture in china. Agalactiae PBSA0903 isolated from tilapia, from university of hainan. Streptococcus dysgalactiae CVCC 3938 was purchased from the Chinese Veterinary Culture Collection (CVCC).
The antibacterial peptide NZX is prepared by recombinant expression of pichia pastoris in the gene engineering laboratory of the feed research institute of Chinese academy of agricultural sciences, and the purity is more than 95 percent. All other chemicals used were of analytical grade.
Tryptone Soy Agar (TSA) medium, Tryptone Soy Broth (TSB) medium were purchased from tokyo obozin biotechnology, ltd; florfenicol (Ff) was purchased from the chinese veterinary medicine institute; other reagents all belong to domestic analytical purity grade. High speed refrigerated bench top centrifuge (Sigma 3K15) was purchased from Sigma company; a whole temperature shaking incubator (ZHHWY-211D) was purchased from Shanghai sperm macro laboratory Equipment Ltd; super clean bench (SWCJ-2FD) purchased from Sujing group facilities, Inc.; FACS alibur flow cytometry was purchased from BD corporation; scanning Electron microscopy (QUANTA 2100PRO) was purchased from Philips, Netherlands; super-resolution microscopy (N-SIMS) was purchased from Nikon, Japan.
The amino acid sequence of the antibacterial peptide LF5 is as follows: FKAFRWAWRWKKLAAPS are provided.
The amino acid sequence of the antibacterial peptide N2 is as follows: AFCWNVCVYRNAVRVCHRRCN are provided.
Example 1 determination of minimum inhibitory concentration of s.agalactiae by antimicrobial peptides NZX, LF5, N2
A trace broth dilution method is adopted to determine NZX bacteriostatic activity on pathogenic bacteria. The culture medium for preparing S.agalactiae liquid in exponential phase is diluted to 1 × 105CFU/mL bacterial suspension, antibacterial peptides NZX, LF5, N2 and antibiotic control florfenicol (Ff) powder after dissolving with 0.01mol/L PBS (pH7.4) are prepared into peptide solution of 0.0625-128 μ g/mL according to 2-fold gradient concentration; the peptide solution and the bacterial suspension are respectively added into a 96-well plate according to the volume ratio of 1:9, and then the plate is placed in an incubator at 37 ℃ for aseptic culture for 18-24h, wherein a blank control, a positive control and a negative control are respectively sterile TSB broth, Ff and PBS, each concentration is set to be 3 times, and the minimum concentration of the drug which can not be seen turbid by naked eyes is taken as a MIC value. The results are shown in table 1:
TABLE 1 MIC values for 1 NZX against bacteria
Example 2 determination of minimum prophylactic drug resistance inhibitory concentration (MPC) of antimicrobial peptide NZX on s.agalactiae ACCC61733
On the basis of MIC, NZX peptide solution and the sterilized solid culture medium TSA are mixed and prepared into a drug plate by 2-fold gradient, the final concentration of the drug is from 1 × MIC to 64 × MIC, and the concentration of S.agalactiae ACCC61733 bacterial liquid in the logarithmic growth phase is concentrated to 3.0 × 1010CFU/mL. 100. mu.L of the suspension was pipetted and applied to NZX drug plates at the above-mentioned concentration ratios. And incubated at a constant temperature of 37 ℃. MPC was used as the lowest drug concentration at which no colony growth occurred for 72 h. The results are shown in Table 2. The ratio of MPC to MIC (Selection index) SI reflects the ability of an antibacterial drug to limit Selection of drug resistant mutants, the lower the SI value the stronger the ability to prevent drug resistance, the NZX has a Selection index of 16, Ff>64, prevention of instruction NZXThe resistance mutation ability is larger than Ff.
Table 2 NZX MPC and SI values for s.agalactiae ACCC61733
Example 3 bactericidal kinetics curves of antimicrobial peptide NZX versus s.agalactiae ACCC61733
Dilution of s.agalactiae ACCC61733 in logarithmic growth phase to 1 × 105CFU/ml bacterial suspension was then mixed with NZX solutions at final concentrations of 1, 2, 4 × MIC at 37 deg.C (250rpm), PBS as a blank control, 2 × MIC Ff as a positive control, 100 μ l bacterial samples were taken at different time points (0, 0.5, 1, 1.5, 2, 4, 6, 8, 10, 12, 22, 24h), counted with solid medium TSA, and a NZX time sterilization curve was plotted. As shown in fig. 1, the sterilization rate of NZX is concentration-dependent, the sterilization rate of NZX to s.agalactiae ACCC61733 is higher than that of Ff, the sterilization rate of NZX with 1 × MIC-4 × MIC to pathogenic bacteria is 99% within 1-2h, and no rebound occurs, while the Ff with 1 × MIC only can inhibit s.agalactiae ACCC61733, and the killing rate can reach 99% within 6h under 2 × MIC, and NZX has significant sterilization rate and sterilization effect advantage over Ff.
Example 4 hemolytic assay of antimicrobial peptide NZX on red blood cells of tilapia
Collecting blood from tail vein of healthy tilapia, collecting whole blood by an anticoagulation tube, gently shaking, centrifuging for 5min at 1500rpm and 4 ℃, gently washing red blood cells for three times by using 0.9% physiological saline until supernatant is colorless and transparent, diluting the supernatant into suspension with the concentration of red blood cells being 8%, dissolving antibacterial peptide NZX in 0.01M PBS (pH7.4) to prepare 2-512 mu g/ml peptide solution, mixing 100 mu l of each of the red blood cell suspension and the peptide solution with different concentrations, adding the mixture into a 96-well plate, taking physiological saline and 0.1% Triton X-100 as blank control and 100% hemolytic positive control respectively, incubating for 1h at 37 ℃, centrifuging for 5min at 4 ℃ and 1500rpm, taking supernatant into the 96-well plate, and detecting ultraviolet absorbance value at 540nm by using a microplate reader. The hemolysis rate is calculated as:
hemolysis rate (%) - (Abs540 nm)NZX－Abs540nmPhysiological saline)/(Abs540nm0.1％Triton X-100－Abs540nmPhysiological saline)]×100％
As shown in FIG. 2, NZX having a concentration in the range of 1 to 256. mu.g/ml had almost no hemolytic effect on red blood cells; when the peptide concentration reaches 256 mug/ml, the hemolysis rate is only 0.72 percent, which indicates that the antibacterial peptide NZX has almost no hemolysis and is relatively safe to be used as a medicament for intraperitoneal and intravenous injection.
Example 5 stability of antimicrobial peptide NZX in Tilapia serum and plasma
Collecting blood from tail vein of tilapia, collecting whole blood by anticoagulation tube/procoagulant tube, gently shaking, centrifuging at 1500rpm and 4 deg.C for 5min to obtain tilapia serum and plasma, preparing NZX into peptide solution with concentration of 200 μ g/mL by 0.01M PBS, diluting serum and plasma to 50%, mixing 50% serum/plasma with peptide solution in equal volume, preparing into mixed solution with final concentration of 25% serum and plasma and final concentration of NZX of 100 μ g/mL, and incubating at 37 deg.C. Logarithmic phase S.agalactiae ACCC61733 was prepared to a final concentration of 1X 10 by adding sterilized MHA solid medium6And (3) dripping 30 mu L of mixed solution on the prepared MHA plate at different time points by using a CFU/ml plate, culturing for 16-24h at 37 ℃, and determining the diameter of the inhibition zone. NZX (100. mu.g/mL) prepared in PBS was used as a positive control, and 25% serum/plasma was used as a negative control. The results are shown in fig. 3 and 4, and after NZX was incubated with 25% serum and plasma for 60min, NZX retained 97.8% and 98.9% activity on s.agalactiae ACCC61733, respectively, indicating that NZX had higher stability in tilapia serum and plasma.
Example 6 determination of the Effect of antimicrobial peptide NZX on the Primary biofilm of S.agalactiae ACCC61733
Diluting S.agalactiae in logarithmic growth phase to a concentration of 1X 108CFU/ml, 180. mu.l were added to a 96-well plate for incubation, followed by 2-fold gradient dilutions of NZX and Ff to final concentrations of 0.25, 0.5, 1, 2, 4 and 8 × MIC, respectively. PBS was used as negative control and blank medium as blank control. 37After incubation at C for 24h, the supernatant was carefully aspirated from the wells, washed gently with PBS 3 times, and air-dried. The results of determination of biofilm content per well by crystal violet staining after air drying are shown in fig. 5, and after treatment with 8 × MIC NZX for 24h, the primary biofilm of s.agalactiae decreased to 0.23%, and after 8 × MIC Ff treatment for 24h, the primary biofilm of s.agalactiae decreased to 32.5%, indicating that NZX had a higher ability to inhibit early membrane formation than Ff, indicating that NZX was able to significantly inhibit the formation of a primary biofilm of streptococcus agalactiae.
Example 7 analysis of cell Membrane integrity of S.agalactiae ACCC61733 by antimicrobial peptide P2
S.agalactiae of logarithmic growth phase was diluted to OD with TSB broth6001 (about 1 × 10)8CFU/mL), adding NZX to the final concentration of 1 × MIC, 2 × MIC and 4 × MIC, respectively incubating for 30min and 2h at 37 ℃, taking Ff as a positive control and PBS as a negative control, centrifuging the incubated sample at 4000rpm for 5min, and discarding the supernatant; adding PBS for repeated soft washing for 3 times, adding 0.22 μ M filtered PBS for resuspension for the last time, adding Propidium Iodide (PI) dye solution with final concentration of 50 μ g/mL before flowing on a flow cytometer, standing and incubating for 15min at room temperature, and finally detecting on a machine. As shown in fig. 6, PI penetration into cell membrane was 97.3% after Nisin treatment of 2 × MIC, whereas PI signals in s.agalactiae ACCC61733 cells treated with PBS, 4 × MIC Ff, and 4 × MIC NZX were all around 1%, indicating that the bacterial plasma membrane was intact and that PI hardly entered the interior of the bacteria. NZX shows that the sterilization mechanism of the non-penetration effect of the streptococcus agalactiae cell membrane is a non-membrane-breaking mechanism.
Example 8 Effect of antimicrobial peptide NZX on cell morphology of S.agalactiae ACCC61733
Diluting S.agalactiae of logarithmic growth phase to 1 × 10 with TSB liquid medium8Adding NZX with the final concentration of 4 xMIC into CFU/mL, taking PBS with the same volume as blank control and Ff as positive control, incubating for 2h at 37 ℃, centrifuging the incubated sample at 4000rpm for 5min, and discarding the supernatant; the thalli is lightly washed by 0.01mol/L PBS for three times, 2.5 percent glutaraldehyde buffer solution prepared by 0.01mol/L PBS is slowly added after the supernatant is discarded, the thalli is resuspended, and is placed and fixed at 4 ℃ for more than 2 hours. After fixation, the mixture is sequentially used by 50-70-85-95% (2 times) -100%And (3) performing gradient dehydration rinsing by using ethanol for 15min each time, drying the dehydrated sample by using a critical point dryer, coating the surface of the sample by using an ion sputtering instrument, and observing by using an S-4800 type scanning electron microscope.
As shown in fig. 7, untreated s.agalactiae ACCC61733 cells were morphologically plump and smooth, with no content leakage and morphological changes. Under the action of Ff, the cell surface of the streptococcus agalactiae is swollen and deformed, some filamentous adhesions appear on the cell surface, and the cells cannot normally divide, but the number of bacteria is reduced after NZX treatment, the cell morphology has no obvious change, and the sterilization target point NZX is not intracellular.
Example 9 Effect of incubation Environment on the Bactericidal Capacity of antimicrobial peptide NZX
S.agalactiae ACCC61733 in logarithmic growth phase was diluted to (1X 10) with PBS and TSB medium, respectively6CFU/mL), NZX, florfenicol, penicillin, gentamicin, streptomycin and nisin were added to a final concentration of 4 XMIC, incubated at 37 ℃ for 2h, and 100. mu.L of samples were taken for colony counting on TSA plates after the incubation was completed.
As shown in FIG. 8, gentamicin, streptomycin, nisin, etc. have bactericidal activity of non-cell wall targeting mechanism, and they showed similar bactericidal ability in PBS and TSB. Agalactiae ACCC61733 treated with NZX showed little change in bacterial numbers when suspended in PBS. NZX the bactericidal power of the streptococcus agalactiae cells treated in the TSB environment is obviously improved, and the colony number is obviously reduced by 5.6log10 compared with the CK group. The results were similar to the cell wall-targeting penicillin treated group (3.93 log reduction 10 in the TSB environment). It is suggested that NZX may act on cell walls.
Example 10 localization of antimicrobial peptide NZX in Streptococcus agalactiae ACCC61733
Agalactiae ACCC61733 dilution to 1 × 10 in log phase s8CFU/mL, incubated with 4 × MIC FITC-NZX for 30min at 37 deg.C, then washed twice in PBS (0.01M, pH7.4), stained with 10 μ g/mL DAPI and PI at 4 deg.C for 15min, washed 2 additional times, and resuspended in PBS. Add 5. mu.L of sample and 2. mu.L of antifluorescent quencher to poly TM microscope slide and cover glassThe slides were sealed and finally observed by N-SIMS (super resolution microscope), cells not treated with FITC-peptide served as a blank. As shown in fig. 9, only blue fluorescence (DAPI) derived from cell nuclei was detected in the control group. After treatment with FITC-NZX, green fluorescence was localized to the cell wall of the mitotic cells. This suggests that NZX can bind to the cell wall of dividing cells preventing them from continuing to divide, the cell wall being the primary bactericidal target of NZX.
Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
Reference:
[1]Barony,G.M.；Tavares,G.C.；Pereira,F.L.；Carvalho,A.F.；Dorella,F.A.；Leal,C.A.G.；Figueiredo,H.C.P.Large-scale genomic analyses reveal the population structure and evolutionary trends of Streptococcus agalactiae strains in Brazilian fish farms.Sci Rep 2017,7,13538.
[2]Hernandez,L.；Bottini,E.；Cadona,J.；Cacciato,C.；Monteavaro,C.；Bustamante,A.；Sanso,A.M.Multidrug resistance and molecular characterization of Streptococcus agalactiae isolates from dairy cattle with mastitis.Front Cell Infect Microbiol 2021,11:647324.
[3]Gao,J.；Yu,F.Q.；Luo,L.P.；He,J.Z.；Hou,R.G.；Zhang,H.Q.；Li,S.M.；Su,J.L.；Han,B.Antibiotic resistance of Streptococcus agalactiae from cows with mastitis.Vet J 2012,194,423-424.
[4]Chideroli,R.T.；Amoroso,N.；Mainardi,R.M.；Suphoronski,S.A.；de Padua,S.B.；Alfieri,A.F.；Alfieri,A.A.；Mosela,M.； Moralez,A.T.P.；de Oliveira,A.G.；Zanolo,R.；Di Santis,G.B.；Pereira,U.P.Emergence of a new multidrug-resistant and highly virulent serotype of Streptococcus agalactiae in fish farms from Brazil.Aquaculture 2017,479,45-51.
[5]Joshi,R.；Skaaurd,A.；Tola Alvarez,A.Experimental validation of genetic selection for resistance against Streptococcus agalactiae via different routes of infection in the commercial Nile tilapia breeding programme.J Anim Breed Genet 2021, 138,338-348.
[6]Kaminska D,Ratajczak M,
A,Dlugaszewska J,Nowak-Malczewska DM,Gajecka M.Increasing Resistance and Changes in Distribution of Serotypes of Streptococcus agalactiae in Poland.Pathogens 2020；9(7):526.
[7]Brogden,Antimicrobial peptides:pore formers or metabolic inhibitors in bacteriaNat.Rev.Microbiol 2005,3:238–250. [8]Mygind P.H.,Fischer R.L.,Schnorr K.M.,et al.Plectasin is a peptide antibiotic with therapeutic potential from a saprophytic fungus.Nature 2005,437:975-980.
[9]Tenland E,Krishnan N,
A,Kalsum S,Puthia M,
M,Davoudi M,Otrocka M,Alaridah N, Glegola-Madejska I,A novel derivative of the fungal antimicrobial peptide plectasin is active against Mycobacterium tuberculosis.Tuberculosis 2018,113:231–238
[10]Liu H,Yang N,Mao R,Teng D,Hao Y,Wang X,Wang J.A new high-yielding antimicrobial peptide NZX and its antibacterial activity against Staphylococcus hyicus in vitro/vivo.Appl Microbiol Biotechnol 2020,104(4):1555-1568。
sequence listing
<110> institute of feed of Chinese academy of agricultural sciences
Application of <120> antibacterial peptide NZX in preparation of streptococcus agalactiae antibacterial drugs
<130> KHP221114857.1
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 40
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 1
Gly Phe Gly Cys Ser Gly Pro Trp Asn Glu Asp Asp Ile Arg Cys His
1 5 10 15
Asn His Cys Lys Ser Ile Lys Gly Tyr Lys Gly Gly Tyr Cys Ala Lys
20 25 30
Gly Gly Phe Val Cys Lys Cys Tyr
35 40
Claims (10)
1. Use of antimicrobial peptide NZX in the preparation of an antibacterial medicament or composition against streptococcus agalactiae (streptococcus agalactiae);
antimicrobial peptide NZX comprises or consists of the amino acid sequence:
i) 1, SEQ ID NO; or
ii) an amino acid sequence obtained by connecting a tag to the N-terminal and/or C-terminal of i).
2. The use of antimicrobial peptide NZX in the manufacture of a biological product for the treatment or prevention of Streptococcus agalactiae infection and related diseases caused by infection; wherein the antimicrobial peptide NZX is as described in claim 1.
3. The use according to claim 2, wherein the disease is a related disease caused by Streptococcus agalactiae and the biofilm produced thereby.
4. The use according to claim 2, wherein the disease comprises streptococcal disease.
5. The use according to any one of claims 1 to 4, wherein the Streptococcus agalactiae comprises the following strains: ACCC61733, ATCC 13813, CAU-FRI 1, CAU-FRI 2, CAU-FRI 3, CAU-FRI 4, PBSA 0903.
6. The application of the antibacterial peptide NZX in preparing antibacterial medicines or compositions of Streptococcus dysgalactiae (Streptococcus dysgalaciae); wherein the antimicrobial peptide NZX is as described in claim 1.
7. The use of antimicrobial peptide NZX in the manufacture of a biological product for the treatment or prevention of Streptococcus dysgalactiae infection and related diseases caused by infection; wherein the antimicrobial peptide NZX is as described in claim 1.
8. The use according to claim 7, wherein the disease is a related disease caused by Streptococcus dysgalactiae and the biofilms produced thereby.
9. The use according to claim 7, wherein the disease comprises cow mastitis.
10. The use according to any one of claims 6 to 9, wherein streptococcus dysgalactiae comprises strain CVCC 3938.
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| WO2024025209A1 (en) * | 2022-07-26 | 2024-02-01 | 주식회사 인트론바이오테크놀로지 | Antibacterial protein composition for treatment of bovine mastitis |
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| US20190359673A1 (en) * | 2018-05-23 | 2019-11-28 | Huazhong Agricultural University | Polypeptide derivatives from grass carp interferon and application thereof |
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| US20190359673A1 (en) * | 2018-05-23 | 2019-11-28 | Huazhong Agricultural University | Polypeptide derivatives from grass carp interferon and application thereof |
| CN111298100A (en) * | 2020-03-26 | 2020-06-19 | 中国农业科学院饲料研究所 | Application of antibacterial peptide NZ2114 in preparation of streptococcus agalactiae antibacterial drugs |
| CN111518187A (en) * | 2020-04-16 | 2020-08-11 | 中国农业科学院饲料研究所 | Antibacterial peptide DN6NH2And uses thereof |
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| WO2024025209A1 (en) * | 2022-07-26 | 2024-02-01 | 주식회사 인트론바이오테크놀로지 | Antibacterial protein composition for treatment of bovine mastitis |
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