CN116063390B - Antibacterial peptide VCK-17 and application of antibacterial peptide VCK-17 and citronellal combined as antibacterial drug - Google Patents
Antibacterial peptide VCK-17 and application of antibacterial peptide VCK-17 and citronellal combined as antibacterial drug Download PDFInfo
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- NEHNMFOYXAPHSD-UHFFFAOYSA-N citronellal Chemical compound O=CCC(C)CCC=C(C)C NEHNMFOYXAPHSD-UHFFFAOYSA-N 0.000 title claims abstract description 184
- 229930003633 citronellal Natural products 0.000 title claims abstract description 92
- 235000000983 citronellal Nutrition 0.000 title claims abstract description 92
- 239000003910 polypeptide antibiotic agent Substances 0.000 title claims abstract description 83
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- 238000004519 manufacturing process Methods 0.000 claims abstract 2
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- 239000001289 litsea cubeba fruit oil Substances 0.000 description 1
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/11—Aldehydes
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
- A61K38/10—Peptides having 12 to 20 amino acids
-
- 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
-
- 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)
- Life Sciences & Earth Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- Animal Behavior & Ethology (AREA)
- Organic Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Epidemiology (AREA)
- Genetics & Genomics (AREA)
- Biochemistry (AREA)
- Immunology (AREA)
- Engineering & Computer Science (AREA)
- Gastroenterology & Hepatology (AREA)
- Molecular Biology (AREA)
- Biophysics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Communicable Diseases (AREA)
- Oncology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Agricultural Chemicals And Associated Chemicals (AREA)
Abstract
The invention discloses an antibacterial peptide VCK-17 and application of the antibacterial peptide VCK-17 and citronellal as antibacterial drugs, and particularly relates to antibacterial drugs. The sequence of the antibacterial peptide VCK-17 is shown as SEQ ID NO. 1. The invention reasonably distributes the effective molar component proportion of the two medicines of VCK-17 and citronellal, so that the combined use can exert a more rapid broad-spectrum synergistic antibacterial effect, obviously reduce the dosage and toxic and side effects of the antibacterial medicines of each component, and effectively reduce the generation of bacterial drug resistance. The invention is helpful for providing a new application view for the actual production and application of plant extracts such as antibacterial peptide and Chinese herbal medicine, is helpful for further promoting the application and development of the plant extracts such as antibacterial peptide and Chinese herbal medicine on the strategy of 'tikang', and provides materials and new ideas for the design of novel antibacterial medicines.
Description
Technical Field
The invention belongs to the technical field of medicines, and particularly relates to an antibacterial peptide VCK-17 and application of the antibacterial peptide VCK-17 and citronellal as antibacterial medicines.
Background
Antibiotic combination therapy has been an important means of clinical treatment in the face of bacterial infections in the body caused by pathogenic bacteria. Many researches show that by selecting different types of antibacterial drugs for combined use, the overall antibacterial performance can be effectively improved, the dosage and toxic and side effects of the drugs are reduced, and the development of bacterial drug resistance can be obviously reduced. In addition, certain pharmaceutical combinations are also capable of producing synergistic antimicrobial effects, thereby maximizing antimicrobial efficacy. Therefore, the combination of antibacterial drugs has great significance in clinical application. However, in recent years, as the problem of antibiotic resistance becomes more and more serious and the development of new antibiotics becomes more delayed, especially with respect to rapid horizontal transfer of antibiotic-induced drug resistance genes, this results in that antibiotic combination therapies also face serious risks for clinical application. Therefore, the selection of an antibacterial drug which is rich in variety and is not prone to developing drug resistance to construct a novel drug combination therapy may be an effective strategy for avoiding the current risk of resistance to antibiotics by heavy taurus. Meanwhile, the combination therapy of the novel antibacterial drug also lays a foundation for developing the novel drug.
As an important component of the innate immune system of multicellular organisms, antibacterial peptides have a rich diversity within and among species, and more than 12,000 antibacterial peptides have been found in nature or artificially and organically synthesized. It has been found that most antimicrobial peptides have a typical "membrane-disrupting" antimicrobial mechanism, which is of great importance in limiting the evolution of bacterial resistance, and these advantages make them one of the most potential "tie-agents" at present. As the research goes deep, many studies now find that multicellular organisms kill pathogenic bacteria together at the site of infection by secreting multiple antibacterial peptides. Meanwhile, in vitro experiments also find that a strong synergistic antibacterial effect exists among part of antibacterial peptides. These cues not only indicate that the synergistic mechanism of action of the antimicrobial peptides may be an important mode of action for thousands of years in the natural environment to effectively resist pathogenic microorganisms, but also demonstrate the potential of the antimicrobial peptides as antimicrobial potency potentiators of other antimicrobial agents.
On the other hand, the plant essential oil is taken as an effective secondary metabolite of the plant for resisting the invasion of pathogenic microorganisms in the nature, and has extremely rich species diversity and good sterilization effect. It has been found that plant essential oils are capable of killing pathogenic microorganisms through a variety of antimicrobial modes. In addition, the plant essential oil can inhibit the virulence factor of bacteria to limit the formation of biological film and quorum sensing effect, so as to reduce the mutation of the drug resistance gene of the bacteria and effectively inhibit the drug resistance evolution process of the bacteria. As the main active ingredients of citronella oil, litsea cubeba oil and eucalyptus oil, citronellal has good insecticidal and antibacterial activities, and is mainly used for preparing essence, food additives, preservatives and the like at present.
While the novel antibacterial agents described above exhibit potential "tie-down" advantages, the expensive synthetic cost of antibacterial peptides, the weak antibacterial activity of plant extracts and the large drug dose greatly limit their further development in clinical therapies. Here, we consider that the combination therapy between the two can effectively solve the above-mentioned disadvantages, however, the combination therapy between the two has not been reported.
Disclosure of Invention
1. Based on the defects of the prior art, the invention aims to provide an antibacterial peptide VCK-17 which has high antibacterial activity and low toxicity, wherein the amino acid sequence is shown as SEQ NO.1, the C end of the antibacterial peptide VCK-17 is amidated by adopting amino groups, and the molecular formula of the antibacterial peptide VCK-17 is shown as formula (I):
another object of the present invention is to provide an application of the antibacterial peptide VCK-17 in combination with citronellal in the preparation of a medicament for treating infectious diseases caused by gram-negative bacteria or gram-positive bacteria, which can be used as a combination medicament for treating microbial infections of various pathogenic bacteria in vitro, and can significantly reduce the dosage of the antibacterial peptide.
Further, by the application, the effective components can effectively kill escherichia coli, salmonella, staphylococcus epidermidis, enterococcus faecalis, methicillin-resistant staphylococcus aureus, carbapenem-resistant escherichia coli 15A9 and beta-lactamase escherichia coli T18 according to the medicine combination of 1 part of the antibacterial peptide VCK-17 and 1000-4000 parts of citronellal, wherein the medicine concentration of 1 part of the antibacterial peptide VCK-17 is 0.5-4 mu M.
Further, by the application, the effective components can effectively kill pseudomonas aeruginosa, acinetobacter baumannii and beta-lactamase enterobacter T17 according to the medicine combination of 1 part of antibacterial peptide VCK-17 and 100-500 parts of citronellal, wherein 1 part of antibacterial peptide VCK-17 refers to the medicine concentration of 8-16 mu M.
Further, by the application, the medicine combination of the effective components according to the proportion of 1 part of the antibacterial peptide VCK-17 and 8000-60000 parts of citronellal can effectively kill Klebsiella pneumoniae, staphylococcus aureus and carbapenem-resistant enterobacteria 14E3, wherein 1 part of the antibacterial peptide VCK-17 refers to the medicine concentration of 0.25-4 mu M.
Further, for the above-described application, the gram-negative bacteria are E.coli, salmonella, carbapenem-resistant E.coli, beta-lactamase E.coli, pseudomonas aeruginosa, acinetobacter baumannii or Klebsiella pneumoniae.
Further, for the application described above, the gram positive bacteria are staphylococcus epidermidis, enterococcus faecalis or methicillin-resistant staphylococcus aureus.
The beneficial effects and advantages of the invention are as follows: the antibacterial peptide VCK-17 and citronellal are cooperated to be used as combined medicines for treating various pathogenic bacteria microbial infections in vitro, and the dosage of the antibacterial peptide can be obviously reduced, so that the toxic and side effects and the synthesis cost of the medicines are reduced, the bacterial killing rate is greatly improved, and the generation of bacterial drug resistance is further reduced. And the antibacterial pharmaceutical composition can effectively reduce cytotoxicity to human kidney cells HEK293T and human erythrocytes under the concentration of exerting antibacterial activity. The antibacterial pharmaceutical composition is capable of killing bacteria through multiple modes of action, including destroying the inner and outer membranes of bacteria and inhibiting energy metabolism of bacteria. The synergistic effect can exert a rapid broad-spectrum antibacterial effect and remarkably reduce the generation of bacterial drug resistance, thereby maximizing the antibacterial effect. The results show that the combined application of the antibacterial peptide VCK-17 and citronellal has high scientific research value and good application prospect.
Drawings
FIG. 1 is a mass spectrum of antibacterial peptide VCK-17;
FIG. 2 is a graph showing the detection of the sterilization rate of the model E.coli ATCC25922 by the antibacterial peptide VCK-17 and citronellal, alone and in combination, wherein the values in brackets are the concentrations of the added antibacterial agent;
FIG. 3 is a graph showing the detection of resistance to the continuous induction mode fungus E.coli ATCC25922 by the antibacterial peptide VCK-17 and citronellal alone and in combination over 30 days, wherein polymyxin B and ciprofloxacin served as positive controls for resistance induction;
FIG. 4 is a graph showing cytotoxicity assays of various cell lines with the antimicrobial peptides VCK-17 and citronellal alone and in combination, wherein a represents the assay of cell line RAW264.7, HEK293T and IPEC-J2 cell viability with the antimicrobial peptide VCK-17 at various concentrations, b represents the assay of cell line RAW264.7, HEK293T and IPEC-J2 cell viability with the citronellal at various concentrations, c represents the assay of cell line RAW264.7, HEK293T and IPEC-J2 cell viability with the antimicrobial peptide VCK-17 and citronellal in combination;
FIG. 5 is a diagram for a comparison of the results of a study of the antibacterial mechanism of the combination of the antibacterial peptide VCK-17 and citronellal with respect to the model bacteria E.coli ATCC25922, wherein a represents the effect of the combination of the antibacterial peptide VCK-17 and citronellal on the permeability of the outer membrane of the bacteria, b represents the effect of the combination of the antibacterial peptide VCK-17 and citronellal on the permeability of the inner membrane of the bacteria, and c represents the effect of the combination of the antibacterial peptide VCK-17 and citronellal on the energy metabolism of the bacteria;
Detailed Description
The present invention will be described in further detail with reference to examples and drawings, but embodiments of the present invention are not limited thereto.
Example 1:
the amino acid sequence of the antimicrobial peptide is shown in table 1:
TABLE 1
The molecular formula is shown as formula (I):
the chemical synthesis of the antibacterial peptide VCK-17 comprises the following specific steps of:
1. the preparation of the antibacterial peptide is carried out from the C end to the N end one by one and is completed by a polypeptide synthesizer. Fmoc-X (X is the first amino acid at the C-terminal of each antibacterial peptide) is firstly accessed into Wang resin, and then Fmoc groups are removed to obtain X-Wang resin; fmoc-Y-Trt-OH (9-fluorenylmethoxycarbonyl-trimethyl-Y, Y being the second amino acid at the C-terminus of each antimicrobial peptide); sequentially synthesizing from the C end to the N end according to the procedure until the synthesis is completed, and obtaining the side chain protected resin from which Fmoc groups are removed;
2. adding a cutting reagent into the obtained peptide resin, reacting for 2 hours at 20 ℃ in a dark place, and filtering; washing precipitated TFA (trifluoroacetic acid), mixing the washing solution with the filtrate, concentrating by a rotary evaporator, adding precooled anhydrous diethyl ether with volume about 10 times, precipitating at-20 ℃ for 3 hours, separating out white powder, centrifuging for 10 minutes at 2500g, collecting precipitate, washing the precipitate with anhydrous diethyl ether, and vacuum drying to obtain polypeptide, wherein the cutting reagent is formed by mixing TFA, water and TIS (triisopropylchlorosilane) according to a mass ratio of 95:2.5:2.5;
3. performing column balancing with 0.2mol/L sodium sulfate (pH=7.5 is adjusted to phosphoric acid) for 30min, dissolving polypeptide with 90% acetonitrile water solution, filtering, performing C18 reverse phase normal pressure column, performing gradient elution (eluent is methanol and sodium sulfate water solution are mixed according to volume ratio of 30:70-70:30), performing flow rate of 1mL/min, detecting wave of 220nm, collecting main peak, and freeze-drying; further purification using a reverse phase C18 column, eluent a was 0.1% tfa/water; eluting with 0.1% TFA/acetonitrile solution, eluting with 25-40% B for 12min at flow rate of 1mL/min, collecting main peak, and lyophilizing;
4. identification of antibacterial peptides: the obtained antibacterial peptide is analyzed by an electrospray mass spectrometry, and the molecular weight (shown in the attached drawing) in the mass spectrum is basically consistent with the theoretical molecular weight in the table 2, and the purity of the antibacterial peptide is more than 95%.
Example 2:
determination of antibacterial Activity of antibacterial peptide VCK-17 and citronellal alone or in combination
1. Antibacterial activity assay: the antibacterial peptide VCK-17 and citronellal are respectively prepared into a storage solution with a certain concentration for use. The minimum inhibitory concentration (MIC value) of several antimicrobial peptides was determined using a micro broth dilution method. Serial gradients of antimicrobial solutions were sequentially prepared using a double dilution method with 0.01% acetic acid (0.2% bsa) as the diluent. 100. Mu.L of the above solution was placed in 96-well cell culture plates, and then an equal volume of the bacterial liquid to be tested (5X 10) was added to each 5 and/mL) in each well. Positive controls (containing bacterial solution without antibacterial agent) and negative controls (containing neither bacterial solution nor antibacterial agent) were set separately. Culturing at 37deg.C for 20-24 hr, and determining minimum inhibitory concentration by detecting light absorption value at 492nm with enzyme-labeled instrument or without turbidity at bottom of hole.
From the results in Table 2, we found that the MIC of the antibacterial peptide VCK-17 against all other test bacteria was in the range of 4-128. Mu.M, except that the antibacterial activity against Pseudomonas aeruginosa, acinetobacter baumannii and Klebsiella pneumoniae was greater than 128. Mu.M. Whereas citronellal has a weaker antibacterial activity than the antibacterial peptide VCK-17, its MIC for all other bacteria tested was in the range 32768-26261440. Mu.M, except that the MIC for Pseudomonas aeruginosa was greater than 2621440. Mu.M.
Table 2 shows the antibacterial activity of the antibacterial peptide VCK-17 and citronellal alone and in combination against 20 common pathogenic microorganisms
In-table MIC VCK-17 [MIC Combination of two or more kinds of materials ]Middle MIC VCK-17 Refers to the minimum inhibitory concentration, [ MIC ] of the antimicrobial peptide when used alone Combination of two or more kinds of materials ]Refers to the minimum inhibitory concentration, MIC, of the antibacterial peptide VCK-17 when the antibacterial peptide is combined with citronellal Citronellal [MIC Combination of two or more kinds of materials ]MIC in Citronellal Refers to the minimum inhibitory concentration, [ MIC ] of citronellal when used alone Combination of two or more kinds of materials ]Refers to the minimum inhibitory concentration of citronellal when the antimicrobial peptide VCK-17 is used in combination with citronellal. The upper corner mark a refers to the minimum inhibitory concentration of the medicine, the upper corner mark b refers to the grading inhibitory index of the medicine combination, the upper corner mark c refers to the multiple of the reduction of the minimum inhibitory concentration of one medicine when the minimum inhibitory concentration of the other medicine is reduced by 8 times in the medicine combination, the upper corner mark d refers to the carbapenem-resistant enterobacteria, the upper corner mark e refers to the beta-lactamase-resistant enterobacteria, and the upper corner mark f refers to the methicillin-resistant staphylococcus aureus.
2. Determination of synergistic antibacterial Activity of antibacterial peptide VCK-17 and citronellal: based on MIC values of the antibacterial peptide VCK-17 and citronellal obtained by the antibacterial activity measurement, the antibacterial peptide VCK-17 and citronellal were prepared as a stock solution at a concentration of 2 XMIC for use. Serial gradient antibacterial drug solutions were sequentially prepared in 96-well plates using a double dilution method with 0.01% acetic acid (containing 0.2% bsa) as a diluent, using a micro broth dilution method in which the antibacterial peptide VCK-17 to be used was sequentially diluted from right to left, and citronellal to be used was sequentially diluted from bottom to top, and the remaining steps were identical to the antibacterial activity measurement method. Wherein the FIC index is calculated as follows: FIC index = FIC A +FIC B ＝MIC AB /MIC A +MIC BA /MIC B . Wherein MIC A MIC alone for Compound A, MICAB is MIC for Compound A in combination with Compound B, MIC B Is a compound B singleSingle bond MIC, MIC BA MIC, FIC for binding compound B to compound a A And FIC B FIC of Compound A and Compound B are shown, respectively. The synergy is defined as FIC.ltoreq.0.5, additive (0.5)<FICI<1) Irrelevant actions (FICI 1)<4) And antagonism (FICI)>4). The specific detection results are shown in Table 2.
From the test results, it was found that the antibacterial peptide VCK-17 and citronellal were able to produce a potent synergistic effect (FIC.ltoreq.0.5) on all bacteria tested, wherein the antibacterial activity of the antibacterial peptide VCK-17 on all bacteria tested was increased 4-128 times under conditions where the MIC of citronellal was reduced 8 times. On the contrary, the antibacterial activity of citronellal is improved by 2-512 times.
3. Determination of the synergistic antibacterial rate of the antibacterial peptide VCK-17 and citronellal: model bacteria E.coli ATCC25922 were used as the subject at 5X 10 5 To E.coli in each mL, VCK-17 (final concentrations 1/32 and 1-fold MIC), citronellal (final concentrations 1/64 and 1-fold MIC) and VCK-17 (1/32-fold MIC) and citronellal (1/64-fold MIC) combinations were added, respectively, and timing was started, with the mixtures collected at various time intervals (1, 5, 10, 20, 30, 60 and 120 minutes) and diluted to appropriate multiples, and plated onto broth agar solid dishes. Finally, the antibacterial rate of the antibacterial agent was assessed by colony counting.
The results in FIG. 2 show that at MIC concentration, 32. Mu.M of the antibacterial peptide VCK-17 was able to kill bacteria within 30min, whereas 131072. Mu.M citronellal alone was able to inhibit bacterial growth. Under the condition of FIC concentration, 1 mu M of antibacterial peptide VCK-17 and 2048 mu M of citronellal alone failed to inhibit the growth of bacteria, and the colony count of the control group was leveled. In the case of the combination of 1. Mu.M of the antibacterial peptide VCK-17 and 2048. Mu.M of citronellal, all bacteria were completely killed within 10 min. These results indicate that the combination of the antibacterial peptide VCK-17 and citronellal not only kills pathogenic bacteria at low concentrations, but also increases the rate of bacterial killing.
4. Determination of bacterial resistance to the antibacterial peptides VCK-17 and citronellal, alone or in combination: detection of mode bacteria E.coli ATCC25922 on polymyxin B, ciprofloxacin, VCK-17, citronellal and VCK-17 and incense by continuous cultureMao Quan resistance to the combination. First, the MIC method for measuring the antibacterial activity of the above-mentioned antibacterial agent was used to measure the antibacterial activity of the above-mentioned antibacterial agent against escherichia coli 25922. Bacterial suspensions were then removed from the turbid sub-MIC wells and diluted to appropriate concentrations (5X 10) with fresh MHB medium 5 and/mL), the next generation MIC determination was performed. In this way, 30 days are measured consecutively and MIC values for each antibacterial agent are recorded.
The results in FIG. 3 show that E.coli has 8-fold and 4-fold increased antimicrobial activity under conditions of 30 days of continuous stimulation of the antimicrobial peptides VCK-17 and citronellal, respectively, compared to ciprofloxacin and polymyxin B, which indicates that the antimicrobial peptides VCK-17 and citronellal alone have lower ability to induce bacterial resistance than antibiotics. The antibacterial activity is unchanged under the condition that the antibacterial peptide VCK-17 and citronellal are used in combination, which shows that the combination of the antibacterial peptide VCK-17 and citronellal is helpful for further reducing the inducibility of bacterial drug resistance.
Example 3:
cytotoxicity assays of the antibacterial peptide VCK-17 and citronellal on different cell lines, alone or in combination: cytotoxicity of VCK-17, citronellal and combinations thereof on RAW264.7, HEK293T and IPEC-J2 was assessed using the MTT dye reduction method. First, cultured 2X 10 5 Inoculating cells at a ratio of one mL in 96-well plate for 12 hr, adding equal volumes of VCK-17, citronellal and their combinations with different gradient concentrations into 96-well plate containing cultured cell line at 37deg.C and 5% CO 2 And incubated for 4h. 50. Mu.L MTT (0.5 mg/mL) was then added to each well of the 96-well plate and incubated for another 4h. Finally, 1000g was centrifuged for 5min, the supernatant was discarded, 150. Mu. LDMSO was added to each well and the mixture was subjected to an enzyme-labeled instrument (TECAN GENios F129004; TECAN, australia). Wherein cells which are not treated by the antibacterial agent are used as positive control, and whole culture medium holes are used as negative control.
The results in FIG. 4 show that at the highest concentration (2 MIC), the cell viability of the three cell lines after incubation with the antibacterial peptide VCK-17 was greater than 50%, indicating good biosafety. Although RAW264.7 and HEK293T cells survived more than 50% under co-incubation with citronellal, the cell viability of IPEC-J2 decreased significantly, indicating that there was some cytotoxicity of high concentrations of citronellal to IPEC-J2. Furthermore, although HEK293T cell viability was reduced at 2MIC concentrations of VCK-17 in combination with 1 MIC concentration of citronellal. However, under the combined use condition (1/16+1/32 MIC) of 2 times of FIC concentration, the survival rate of all cell lines is significantly higher than 50%, which indicates that the combined use of the antibacterial peptide VCK-17 and citronellal can maintain good cell states of three cell lines under the concentration of exerting antibacterial activity, which is helpful for reducing the dosage of the antibacterial peptide VCK-17 and the citronellal drug, thereby indirectly improving the survival rate of the cells of the combined use of the antibacterial peptide VCK-17 and citronellal.
Example 4:
determination of antibacterial mechanism of model bacteria E.coli ATCC25922 using antibacterial peptide VCK-17 in combination with citronellal
1. Effects of the combination of antibacterial peptide VCK-17 and citronellal on bacterial outer membrane permeability:
the effect of different concentrations of VCK-17, citronellal and combinations thereof on bacterial outer membrane permeability was detected using NPN probe method. First, the E.coli ATCC25922 cells of the model bacteria in the logarithmic growth phase were diluted to OD 600 =0.4 and incubated for 30min with NPN at a final concentration of 10 μm in the dark. VCK-17, citronellal and combinations thereof were then serially diluted in HEPES buffer (ph=7.4) containing 5mM glucose according to the double dilution method, the above antibacterial drug and the prepared bacterial suspension were mixed and added to 96-well plates and incubated for 30 minutes. Immediately detected under a fluorescence spectrometer emitting λ=420 nm (Tecan Infinite 200pro, china). Wherein a bacterial suspension treated with a 2-fold MIC of melittin was used as a positive control and an untreated bacterial suspension was used as a negative control.
From the results of FIG. 5a, it was found that the fluorescence intensity of VCK-17 treated bacteria increased with increasing concentration, while the fluorescence intensity of citronellal treated bacteria was hardly changed, as compared with the control. Furthermore, the combination of VCK-17 and citronellal has a stronger fluorescence intensity than the bacteria treated with VCK-17 alone, which suggests that VCK-17 may play a major role in disrupting bacterial outer membrane permeability during sterilization, while citronellal may have an enhanced ability to disrupt bacterial outer membrane by VCK-17.
2. Effects of the combination of antibacterial peptide VCK-17 and citronellal on bacterial endomembrane permeability: the effect of different concentrations of VCK-17, citronellal, and combinations thereof on bacterial endomembrane permeability was detected using the PI Probe method (Thermo Fisher Scientific, catalyst No. P1304MP). First, the E.coli ATCC25922 cells of the model bacteria in the logarithmic growth phase were diluted to OD 600 =0.4 and incubated for 30min with NPN at a final concentration of 200nM under light-protected conditions. VCK-17, citronellal, and combinations thereof were then serially diluted in PBS (ph=7.4) buffer according to the multiple dilution method, the above antibacterial agents were mixed with the prepared bacterial suspension, and added to 96-well plates for incubation for 30 minutes. Finally, the fluorescence intensities of all bacterial samples were measured at an excitation wavelength of 535nm and an emission wavelength of 615 nm. Wherein a 2-fold MIC of the melittin and rifampicin-treated bacterial suspension was used as positive control and an untreated bacterial suspension was used as negative control.
From the results in FIG. 5b, it was found that the fluorescence intensity of both VCK-17 and citronellal alone treated bacteria increased with increasing concentration compared to the control. Furthermore, the combination of VCK-17 and citronellal resulted in a stronger increase in bacterial fluorescence intensity, which suggests that both agents could result in an increase in bacterial endomembrane permeability, while the combination of both could result in a stronger disruption of the bacterial endomembrane.
3. Effects of the combination of antibacterial peptide VCK-17 and citronellal on bacterial energy metabolism: different concentrations of VCK-17, citronellal, and combinations thereof treated model E.coli ATCC25922 intracellular and extracellular ATP levels were measured using a synergistic ATP assay kit (Beyotime, catalyst No. S0027). First, bacterial cells in the log growth phase were diluted to OD with PBS solution (pH 7.4) 600 =0.4, bacterial suspensions were transferred into 10mL EP tubes and incubated for 90min with different concentrations of VCK-17, citronellal, and combinations thereof. The bacterial suspension was then centrifuged at 12000 rpm for 5 minutes at 4 ℃, the supernatant was discarded, and bacterial pellet was lysed using lysozyme (20 mg/mL), and centrifuged at 12000 rpm at 4 ℃, the supernatant was collected and the intracellular ATP levels of the bacteria were determined according to the ATP kit protocol. Self-luminescence of the sample was measured with a fluorescence spectrometer (Tecan Infinite 200pro, china). In addition, bee venom and rifampicin-treated bacterial suspensions were also used as positive controls at 2-fold MIC, while untreated bacterial suspensions were used as negative controls.
As shown in the results of FIG. 5c, we found that VCK-17 alone resulted in a decrease in intracellular ATP content of bacteria with increasing drug concentration compared to the control, whereas citronellal alone in combination with VCK-17 or citronellal resulted in a significant dip in bacterial ATP content. The decrease in the gradient of intracellular ATP content of the bacteria due to VCK-17 alone may be due to the leakage of intracellular ATP caused by the disruption of bacterial inner and outer membrane permeability by VCK-17. However, the sudden drop in bacterial ATP content caused by citronellal alone, in addition to the ATP reduction caused by the leakage of the inner and outer membranes, also suggests that citronellal can directly inhibit bacterial ATP synthesis. Meanwhile, the ATP content in bacterial cells treated by VCK-17 or citronellal is also obviously reduced, and the citronellal has a dominant effect on inhibiting bacterial energy metabolism.
The results of the action mechanism show that the antibacterial peptide VCK-17 can help citronellal pass through the outer membrane of the escherichia coli, and can act on the inner membrane of the escherichia coli together with the citronellal to cause greater membrane damage. Further, a large amount of citronellal enters the E.coli cells, thereby inhibiting ATP synthesis, leading to breakdown of E.coli energy metabolism and ultimately bacterial death. The results also provide experimental basis for researching the synergistic action mechanism of other antibacterial peptides and plant essential oil.
In summary, the present example explores the antibacterial mechanism of the combined application of two drugs by fluorescent dye labeling. The result proves that a unique synergistic antibacterial mechanism of 'reciprocity' exists between the antibacterial peptide VCK-17 and citronellal, namely the antibacterial peptide VCK-17 can help the citronellal to pass through the outer membrane of the escherichia coli, and can act on the inner membrane of the escherichia coli together with the citronellal to cause greater membrane damage. Further, a large amount of citronellal enters the E.coli cells, thereby inhibiting ATP synthesis, leading to breakdown of E.coli energy metabolism and ultimately bacterial death. The results also provide experimental basis for researching the synergistic action mechanism of other antibacterial peptides and other plant extracts.
Claims (4)
1. Use of the antibacterial peptide VCK-17 in combination with citronellal for the manufacture of a medicament for the treatment of an infectious disease caused by a gram negative or gram positive bacterium, said gram negative bacterium being escherichia coli, salmonella, carbapenem-resistant enterobacteria, beta-lactamase enterobacteria, pseudomonas aeruginosa, acinetobacter baumannii or klebsiella pneumoniae; the gram positive bacteria are staphylococcus epidermidis, enterococcus faecalis or methicillin-resistant staphylococcus aureus; the amino acid sequence of the antibacterial peptide VCK-17 is shown as SEQ ID NO.1, the C end adopts amino amidation, and the molecular formula is shown as formula (I):
2. the use according to claim 1, characterized in that: the effective components are calculated according to the mole fraction, and the medicine combination of 1 part of antibacterial peptide VCK-17 and 1000-4000 parts of citronellal can effectively kill escherichia coli, salmonella, staphylococcus epidermidis, enterococcus faecalis, methicillin-resistant staphylococcus aureus, carbapenem-resistant escherichia coli 15A9 and beta-lactamase escherichia coli T18, wherein 1 part of antibacterial peptide VCK-17 refers to the medicine concentration of 0.5-4 mu M.
3. The use according to claim 1, characterized in that: the effective components can effectively kill pseudomonas aeruginosa, acinetobacter baumannii and beta-lactamase enterobacter jejuni T17 according to the medicine combination of 1 part of antibacterial peptide VCK-17 and 100-500 parts of citronellal, wherein the 1 part of antibacterial peptide VCK-17 refers to the medicine concentration of 8-16 mu M.
4. The use according to claim 1, characterized in that: the active components can effectively kill Klebsiella pneumoniae, staphylococcus aureus and carbapenem-resistant enterobacteria 14E3 according to the medicine combination of 1 part of antibacterial peptide VCK-17 and 8000-60000 parts of citronellal, wherein 1 part of antibacterial peptide VCK-17 refers to the medicine concentration of 0.25-4 mu M.
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| CN104974228A (en) * | 2015-06-15 | 2015-10-14 | 四川合泰新光生物科技有限公司 | Small molecule polypeptide ZY4 and application thereof |
| CN114057835A (en) * | 2021-11-24 | 2022-02-18 | 辽宁师范大学 | Antibacterial peptide analogue and preparation method and application thereof |
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| CN102871996A (en) * | 2012-09-10 | 2013-01-16 | 中国医学科学院医药生物技术研究所 | Antibiotic composition and application thereof |
| CN104974228A (en) * | 2015-06-15 | 2015-10-14 | 四川合泰新光生物科技有限公司 | Small molecule polypeptide ZY4 and application thereof |
| JP2018521041A (en) * | 2015-06-15 | 2018-08-02 | 四川合泰新光生物科技有限公司Ｓｉｃｈｕａｎ Ｈｅｔａｉ Ｓｙｎｌｉｇｈｔ Ｂｉｏｔｅｃｈ Ｌｔｄ | Low molecular polypeptide ZY4 and use thereof |
| US10208088B1 (en) * | 2015-06-15 | 2019-02-19 | Sichuan Synlight Biotech Ltd. | Low molecular polypeptide ZY4 and applications thereof |
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