CN116284253A - Antibacterial peptide targeting staphylococcus aureus and having dual functions of inhibiting quorum sensing signals and antibacterial actions - Google Patents
Antibacterial peptide targeting staphylococcus aureus and having dual functions of inhibiting quorum sensing signals and antibacterial actions Download PDFInfo
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- CN116284253A CN116284253A CN202310480820.4A CN202310480820A CN116284253A CN 116284253 A CN116284253 A CN 116284253A CN 202310480820 A CN202310480820 A CN 202310480820A CN 116284253 A CN116284253 A CN 116284253A
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- 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
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- 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
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
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- 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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Abstract
The invention belongs to the technical field of biological medicines, and particularly relates to an antibacterial peptide which targets staphylococcus aureus and has double functions of inhibiting quorum sensing signals and resisting bacteria. The invention firstly synthesizes a sultone-free AIP-III variant CP7 with targeting and QS signal path inhibiting in staphylococcus aureus cells, synthesizes FP13-2 antibacterial peptide with broad-spectrum antibacterial activity, and then successfully synthesizes a compound peptide containing a CP7 structural domain and a FP13-2 structural domain. The compound peptide disclosed by the invention can be used for targeted killing of staphylococcus aureus, is also effective on methicillin-resistant staphylococcus aureus, and has higher antibacterial activity and moderate toxicity as shown in vivo and in vitro. In addition, the action mechanism of the compound peptide is different from that of the traditional antibiotics, so that the compound peptide is not easy to generate drug resistance, and a certain guiding value is provided for treating staphylococcus aureus infection and drug-resistant bacteria.
Description
Technical Field
The invention belongs to the technical field of biological medicines, and particularly relates to an antibacterial peptide which targets staphylococcus aureus and has double functions of inhibiting quorum sensing signals and resisting bacteria.
Background
Gram positive bacteria staphylococcus aureus (Staphylococcus aureus, s.aureus) is a common colonization of human hosts. It is the most common pathogen in clinical infections and can cause systemic transmission in humans and animals, such as skin and soft tissue infections, urinary tract infections, pneumonia, osteomyelitis, endocarditis, and the like. In recent years, antibiotic resistance has become increasingly common. To address this impending global infection crisis, there is a strong need to explore alternative methods of treating staphylococcus aureus infections with different mechanisms.
In addition to obtaining resistance genes, staphylococcus aureus can synthesize several virulence factors and produce biofilms, closely related to bacterial resistance and pathogenicity of staphylococcus aureus. Virulence factors and biofilm formation are tightly controlled by the accessory gene regulatory factor (agr) system, which is found in almost all staphylococci. The agr system is the main regulator of the staphylococcus aureus Quorum Sensing (QS) system, controlling the expression of foreign and surface proteins. The system consists of RNAII and RNAIII transcription units under the control of the P2 promoter and the P3 promoter, respectively. Wherein the P2 operon regulates a four gene operon agrBDCA which biosynthesizes self-induced peptides (AIPs) and processes AgrC and AgrA proteins. The P3 operon drives the expression of RNAIII units, the primary effector of Staphylococcus aureus expression virulence factors. Staphylococcus aureus detects the extracellular self-inducing peptide of bacteria by the AgrC system, thereby mediating the bacterial agr system to control its virulence factors. Up to now, four specific agr groups have been identified in staphylococcus aureus with different AIP (I-IV) sequences, based on the agr operon. Binding of homologous AIP to AgrC activates the agr response and induces virulence factors through the regulatory factors of RNAIII, whereas non-homologous AIP competitively binds to AgrC to inhibit the agr response while still allowing bacterial growth. Thus, variants of AIPs that inhibit the production of AgrC receptors and related staphylococcus aureus toxins are of interest as new strategies against staphylococcus aureus and related infections.
Natural antimicrobial peptides (AMPs) are endogenous defenses in the innate immune system that protect the host against a variety of pathogenic agents. Moreover, drug resistance is not easy to occur. Therefore, the development of methods for treating staphylococcus aureus infections based on AIPs variants and natural antimicrobial peptides has important application value.
Disclosure of Invention
In order to overcome the defects in the prior art, the invention designs an AIP-III variant named CP7, which is combined with a transmembrane protein AgrC extra-loop-II domain in staphylococcus aureus type I and can weaken the expression of a virulence factor of staphylococcus aureus in a non-biocidal manner; meanwhile, FP (Fusogenic Peptides) with potent antibacterial activity was designed and identified; finally, a CP7-FP13-2 composite peptide containing a CP7 targeted agr system domain and a FP13-2 broad-spectrum antibacterial domain is successfully constructed based on the CP7 and the FP.
In order to achieve the above purpose, the present invention is realized by the following technical scheme:
the first aspect of the invention provides an antibacterial peptide, which is CP7-FP13-2 and has the amino acid sequence shown as follows: (L-L-F-A-Dab) -N-I-G-G-C-6MA-G-I-K-N-L-W-K-K-M-I-K-L-W-Y. The structural formula of CP7-FP13-2 is shown below:
the invention firstly designs an AIP-III variant named CP7, which is combined with a transmembrane protein AgrC extra-loop-II domain in staphylococcus aureus type I, and can weaken the expression of a staphylococcus aureus virulence factor in a non-biocidal mode. Meanwhile, based on the prior study of the subject group, several potent AMPs (Wu, w.; lin, d.; shen, x.et al, new influenza A Virus Entry Inhibitors Derived from the Viral Fusion peptides.plos One 2015,10 (9), e 0138326. Doi: 10.1371/journ.fine.0138326) were generated by replacing the negatively charged or neutral FP residues with positively charged lysines. Here, based on these peptides, an antibacterial peptide FP13-2 consisting of 13 amino acids, which has fewer amino acid residues but still has potent antibacterial activity, including anti-MRSA activity, was designed. Finally, the two N ends of the CP7 and the FP13-2 are connected through G-GGC 6MA (6-maleimide caproic acid), and the CP7-FP13-2 composite peptide containing the CP7 targeted agr system domain and the FP13-2 broad-spectrum antibacterial domain is successfully constructed.
In a second aspect, the invention provides the use of an antimicrobial peptide according to the first aspect for the manufacture of a medicament for inhibiting staphylococcus aureus.
As a preferred embodiment of the present invention, the staphylococcus aureus comprises methicillin-resistant staphylococcus aureus (MRSA).
In a third aspect, the invention provides the use of an antimicrobial peptide according to the first aspect for the manufacture of a medicament for the treatment of an infectious disease of staphylococcus aureus.
Through researches, the CP7-FP13-2 can not only retain the function of a CP7 domain but also retain the antibacterial function of an FP13-2 domain through specifically inhibiting a staphylococcus aureus QS signal path. In vivo studies have shown that CP7-FP13-2 enhances survival in Staphylococcus aureus infected Kunming mice. In conclusion, successful synthesis of CP7-FP13-2 provides a new therapeutic strategy for the treatment of Staphylococcus aureus infections, including MRSA.
Preferably, the antimicrobial peptide acts to inhibit staphylococcus aureus by inhibiting the formation of a staphylococcus aureus biofilm.
The antibacterial peptide is combined with an extra-loop-II domain of AgrC-I transmembrane protein, so as to play a role in inhibiting staphylococcus aureus.
Preferably, the antimicrobial peptide acts to treat staphylococcus aureus infectious diseases by down regulating the expression of staphylococcus aureus RNAIII.
Preferably, the antimicrobial peptide acts to treat staphylococcus aureus infectious diseases by reducing the expression levels of staphylococcus aureus hla and psm-alpha.
Preferably, the antimicrobial peptide acts to treat staphylococcus aureus infectious diseases by inhibiting hemolysis of staphylococcus aureus.
Preferably, the medicament further comprises a pharmaceutically acceptable carrier. The carrier is a functional pharmaceutical adjuvant available in the pharmaceutical field and comprises a surfactant, a suspending agent, an emulsifying agent and a plurality of novel pharmaceutical polymer materials, such as cyclodextrin, chitosan, polylactic acid (PLA), polyglycolic acid-polylactic acid copolymer (PLGA), hyaluronic acid and the like.
Preferably, the dosage forms of the medicine comprise injection, tablet, granule, capsule, dripping pill, sustained release agent, oral liquid, ointment, patch and other preparations.
Preferably, the administration mode of the medicine comprises injection, oral administration, external application and the like. Pharmaceutical formulations may be administered orally or parenterally (e.g., intravenously, subcutaneously, intraperitoneally, or topically), as well as by external application, and if some drugs are unstable under gastric conditions, they may be formulated as enteric coated tablets.
Compared with the prior art, the invention has the beneficial effects that:
the invention discloses a novel composite peptide CP7-FP13-2, which consists of a targeting peptide (CP 7) of a targeted staphylococcus aureus agr system domain and a broad-spectrum antibacterial peptide (FP 13-2). The invention firstly synthesizes a sultone-free AIP-III variant CP7 with targeting and QS signal path inhibiting in staphylococcus aureus cells, and synthesizes FP13-2 antibacterial peptide with broad-spectrum antibacterial activity. Then, a complex peptide containing the CP7 domain and the FP13-2 domain was successfully synthesized. The compound peptide disclosed by the invention can be used for targeted killing of staphylococcus aureus, is also effective on methicillin-resistant staphylococcus aureus, and has higher antibacterial activity and moderate toxicity as shown in vivo and in vitro. In addition, the action mechanism of the compound peptide is different from that of the traditional antibiotics, the drug resistance is not easy to generate, and the compound peptide is effective to the strain which has generated drug resistance to the traditional antibiotics, so that the compound peptide can effectively treat staphylococcus aureus infection, and provides a certain guiding value for treating staphylococcus aureus infection and drug-resistant bacteria.
Drawings
FIG. 1 is a synthetic process, chemical structure and HPLC chromatogram of a complex peptide. Wherein a is the synthesis process of CP7-FP 13-2: 1) 25% piperidine/DMF, 5+25min; TBTU, HOBt, DIEA Fmoc protected amino acids, 4h; 2) TFA, tis, EDT, H 2 O,3h; 3) DIEA, DMF, 24h; 4) TBTU, HOBt, DIEA, DMF, 24h; 5) 25% piperidine/DMF, 5+25min; TFA, tis, EDT, H2O,3H. bChemical structures of CP7, FP13-2 and CP7-FP13-2. c is the HPLC chromatograms of CP7, FP13-2 and CP7-FP13-2.
FIG. 2 shows the antibacterial activity and cytotoxicity of antibacterial peptides.
FIG. 3 shows that CP7 and CP7-FP13-2 show specific anti-Staphylococcus aureus virulence factors. After 24 hours of treatment with 10nM, 100 μM or 1 μM CP7, RNAIII expression in (a) Staphylococcus aureus, (b) Staphylococcus epidermidis and (c) Staphylococcus saprophyticus was detected by qRT-PCR; CP7 significantly inhibited RNAIII expression in staphylococcus aureus in a concentration-dependent manner, but did not inhibit RNAIII expression in staphylococcus epidermidis or staphylococcus saprophyticus; CP7 and CP7-FP13-2 specifically inhibited RNAIII expression in Staphylococcus aureus, but did not inhibit RNAIII expression in Staphylococcus epidermidis or Staphylococcus saprophyticus at a concentration of 0.78. Mu.M; other peptides do not inhibit the expression of RNAIII in Staphylococcus aureus, staphylococcus epidermidis or Staphylococcus saprophyticus. In addition, CP7 and CP7-FP13-2 inhibit the virulence factors (g) hla and (h) psm- α of Staphylococcus aureus; CP7 and CP7-FP13-2 inhibit hemolytic toxicity (i) caused by staphylococcus aureus. * P <0.05, < P <0.01, < P <0.001.
FIG. 4 shows the killing effect of peptides at a concentration of 6.25. Mu.M on (a) Staphylococcus aureus, (b) Staphylococcus epidermidis, (c) Cryptogenic, (d) Streptococcus pneumoniae, and (e) Escherichia coli for 120 minutes; time killing effect (f) of compound at 6.25. Mu.M on Staphylococcus aureus after 10min pretreatment with 31.25. Mu.M CP7.
FIG. 5 is an illustration of the inhibitory effect of peptides on Staphylococcus aureus biofilm formation.
FIG. 6 shows the structure of Staphylococcus aureus biofilm observed under a 400X fluorescence microscope.
FIG. 7 shows tryptophan fluorescence emission spectra of FP13-2 (a) and CP7-FP13-2 (b) detected by fluorescence spectrometry after incubation with 4mg/mL of Staphylococcus aureus membrane lipid, respectively.
Figure 8 shows (a) the cumulative survival of mice and (b) the change in weight of mice within 7 days after s.aureus infection. In figure a, normal control mice were not infected with staphylococcus aureus virus, and after 1 hour of infection of each of the remaining mice with staphylococcus aureus virus, the corresponding drugs were intraperitoneally injected 1 time, respectively, and survival rates of the mice were calculated within 7 days after infection. In panel b, all mice in the normal control group survived and body weight was continuously increased; the body weight of the infected mice continued to drop 3 days after staphylococcus aureus infection, and the surviving mice began to gain weight on day 5.
Detailed Description
The following describes the invention in more detail. The description of these embodiments is provided to assist understanding of the present invention, but is not intended to limit the present invention. In addition, the technical features of the embodiments of the present invention described below may be combined with each other as long as they do not collide with each other.
The experimental methods in the following examples, unless otherwise specified, are conventional, and the experimental materials used in the following examples, unless otherwise specified, are commercially available.
EXAMPLE 1 Synthesis of antimicrobial peptides and Studies of their anti-Staphylococcus aureus action
1. Materials and methods
1.1 materials
Wang resin (100-200 mesh, substitution degree: 0.77 mmol.g) -1 ) Purchased from western security sunlight resin new materials limited in china; amide type MHBA resin (100-200 mesh, substitution degree: 0.65 mmol.g) -1 ) Purchased from NankaiHecheng Sci.&Tech Co., ltd (Tianjin, china); (Fmoc) protected amino acids were purchased from tin-free Asian peptide biotechnology Co., ltd (China tin-free); catalysts for 2- (1H-benzotriazol-1-yl) -1, 3-tetramethyluronium hexafluoro-phosphate (HBTU) and 1-hydroxy-benzotriazol (HOBt) were purchased from GL Biochem ltd (Shanghai, china); n, N-Diisopropylethylamine (DIEA) was purchased from Macklin Biochemical Co., ltd. (Shanghai, china); 3- (4, 5-dimethylthiazol-2-yl) -2, 5-diphenyltetrazolium bromide (MTT), dulbecco's modified Eagle's Medium (DEME), and trypsin-TPCK were all purchased from Gibco (U.S.). Fetal Bovine Serum (FBS) was purchased from Gibco, usa; dimethyl sulfoxide (DMSO) and N-Dimethylformamide (DMF) were purchased from the dijin jingdong day positive precision chemical reagent factory, china; ampicillin and vancomycinAre all available from Mym biotechnology limited (united states); viability and toxic bacterial staining kits were purchased from friendship landi biotechnology limited (su state, china); propidium Iodide (PI) was purchased from Yeasen (Shanghai, china); column chromatography used sephadex LH-20 (Amersham Pharmacia Biotech) and reverse phase silica gel C18 (40-63 μm, merck).
1.2, strains and cell lines
Staphylococcus aureus ATCC6538p, staphylococcus aureus ATCC43300, staphylococcus aureus ATCC12600, staphylococcus epidermidis ATCC12228, staphylococcus saprophyticus ATCC BAA-750, streptococcus pneumoniae ATCC49619, streptococcus mutans UA159, streptococcus mutans ATCC25175, escherichia coli (e.coli) ATCC49619, all bacterial strains were stored at-80 ℃. Streptococcus mutans was cultured with brain-heart perfusion (BHI) broth at 37deg.C under anaerobic conditions, and the other strains were cultured with Mueller-hint (MH) broth at 37deg.C under aerobic conditions. Mouse monocyte macrophages (RAW 264.7) were obtained from the American Type Culture Collection (ATCC), grown in DMEM, supplemented with 10% Fetal Bovine Serum (FBS).
1.3 design and Synthesis of peptides
The peptide sequences are shown in Table 1.
1.3.1 Synthesis of FPs
The linear polypeptides were synthesized using solid phase synthesis, wherein the antibacterial peptide FPs were synthesized using Rink Amide MHBA resin and the targeting peptide was synthesized using Wang resin. The specific method for solid phase synthesis is as follows:
(1) Activating resin:
first, 0.1mmol of the resin was weighed, 1.5mL of DCM was added to the reactor to swell and activate the resin, and the mixture was shaken for 5 minutes 2 times. The DCM was removed and 2mL of DMF was added and washed 2 times with 10 minutes of shaking.
(2) Deprotection:
1) Rink Amide MHBA resin: add 25% piperidine/DMF 2mL first, shake for 5min and remove: then adding the same 25% piperidine/DMF 2mL, and activating for 25 minutes; then 2mL DMF was added and washed 6 times to remove residual piperidine.
2) Wang resin: no deprotection is required.
(3) Linking nitrogen-based acid:
generally, 0.1mmol of resin is taken as a solid phase carrier for polypeptide synthesis:
1) Rink Amide MHBA resin: fmoc amino acid: TBTU: HOBt: DIEA feed ratio (mass ratio of substances) was 1:3:3:3:6, appropriate amount of DMF (1 mL) was added as solvent and the reaction was shaken for 4 hours.
2) Wang resin: fmoc amino acid to HOBt to DIEA feed ratio (mass ratio) of 1:4:4:4; appropriate amount of DMF (1 mL) was added as solvent and the reaction was allowed to react with shaking for 6 hours.
(4) The reaction was checked:
1) Rink Amide MHBA resin: firstly, weighing 0.3g of ninhydrin, and dissolving with 15mL of absolute ethyl alcohol to prepare a 2% ninhydrin solution; sucking more than ten resin particles into a glass tube by a pipette, washing resin for 2 times by DMF and DCM in sequence, adding 200uL of 2% ninhydrin indicator, shaking and mixing uniformly, heating for 3-5 minutes, and observing the color change of the resin.
2) Wang resin: after 6 hours a white solid was seen (urea formation) indicating that the reaction had been performed, with 50% DCM/MeOH wash 4-6 times, DMF wash 4 times (wash off residual MeOH), the reaction was squeezed off, and the resin in the reactor was washed 2 times with DMF and DCM in sequence (limited to the first amino acid ligation, followed by amino acid ligation with reference to Rink Amide MHBA resin).
(5) And (3) completing connection:
the resin is completely reacted without changing blue, the reaction liquid is squeezed out, the resin in the reactor is washed 2 times by DMF and DCM respectively, and the steps (2) - (4) are repeated to connect the next amino acid in sequence.
(6) Cleavage of the polypeptide:
after completion of the amino acid synthesis, the Fmoc group on the last amino acid was optionally removed, the resin was washed 2 times with DMF, DCMM: meOH (1:1, V/V), DCM, and air dried. With 5% EDT, 2.5% tis, 87.5% TFA and 5% ddH 2 Preparing a lysate (prepared on ice and used at present), adding the lysate (2 mL) into a reactor, and reacting for 4 hours; during the preparation, a precipitating agent is prepared, namely petroleum ether, methyl tertiary butyl ether and methyl tertiary butyl ether are respectively added into a 10mL precipitating agent by taking another test tube, the lysate is extruded into the precipitating agent, fully and uniformly mixed, and centrifuged at 5000rpm/min for 10 minutes, and the supernatant is removedAdding 5mL of precipitant, mixing by vortex, centrifuging at 8000-9000rpm/min, removing supernatant, and air drying overnight.
(7) Drying and checking
The next day, the polypeptide was dried by rotary evaporation and dried for 24 hours in a vacuum pump. The compound was prepared at 1mM concentration with methanol solution and checked for purity by high performance liquid chromatography, the mobile phase consisting of solvent A (TFA: ddHO=0.075:1), solvent B (TFA: chromatomethanol: chromatoacetonitrile=0.075:0.5:0.5). After a sample is loaded by using a 15% B liquid balance chromatographic column, separating by using the chromatographic column, wherein the flow rate is 0.8mL/min, the gradient duration is 42 minutes, and the elution gradient is as follows according to the proportion of the mobile phase solvent B: 0-2 minutes, 15% -20%;2-12 minutes, 20% -60%;12-18 minutes, 60% -80%;18-24 minutes, 80% -90%;24-30 minutes, 90%;30-30.5 min, 90% -70%;30.5-32 min, 70% -40%;32-34 minutes, 40% -15%;34-40 minutes, 15%;40-42 min, 15%. The molecular weight of the compounds was confirmed by mass spectrometer analysis.
1.3.2 cyclization of targeting peptides
The natural AIP-II consists of 7 amino acid residues (P7), a thiolactone macrocycle containing 5 residues. The following takes natural AIP-III as a template to synthesize CP7, and the concrete steps are as follows:
(1) Synthesizing P7 on Wang resin according to a solid phase synthesis method;
(2) 25% piperidine removed Fmoc of isoleucine, washed 6 times with DMF to remove piperidine;
(3) Adding 10 times volume of excess Ac 2 O,10 times volume excess DIEA, solvent DMF (1 mL), stirring to react for 20 minutes to acetylate the amino group on isoleucine, and detecting whether the reaction is complete by ninhydrin test;
(4) If the reaction is complete, extruding out the reaction liquid, washing the resin in the reactor with DMF and DCM for 2 times in sequence, and airing;
(5) Cracking the resin by using a cracking solution, and airing;
(6) The next day, the polypeptide is pumped out by a rotary evaporator and dried for 24 hours by a vacuum pump to obtain linear AC2O-P7;
(7)AC 2 O-P7:Hobt:TBTU:DIEA feed ratio (mass ratio) of 1:1.5:1.5:3, DMF as solvent (1 mL) was thoroughly dissolved in the round bottom flask;
(8) The reaction is carried out for 12 hours under the protection of nitrogen, the carboxyl on leucine and the amino on Dab (2, 3-diaminobutyric acid) are condensed into a ring in the solution to obtain CP7, and finally the gel column is used for purification.
1.3.3 Synthesis of Complex peptides
The synthetic composite peptide CP7-FP13-2 (synthetic route is shown in FIG. 1 a) is specifically as follows:
(1) P7 was synthesized on Wang resin by solid phase synthesis, glycine (G), and cysteine (C) were added in this order to synthesize CGG-P7, and Fmoc on cysteine was retained. 1mL of the lysate was added and reacted for 3 hours, and the mixture was cut from the resin for use.
(2) FP13-2 was synthesized on Rink Amide MHBA resin by solid phase synthesis and G and 6-maleimidocaproic acid (6 MA) were added sequentially.
(3) 2-fold (2-fold of the amount of resin material) of excess CGG-P7 (dissolved in DMF), 10-fold excess DIEA and nitrogen were added and reacted at room temperature for 24 hours. The carbon-carbon double bond on 6MA and the sulfhydryl group on CGG-P7 are subjected to addition reaction on the resin.
(4) After the completion of the addition reaction, the reaction mixture was squeezed out, washed 6 times with DMF, and the excess DIEA and CGG-P7 were removed.
(5) TBTU, HOBt and DIEA were added in a 3-fold excess (3-fold amount of resin material) and a 6-fold excess in DMF (1 mL) were added in this order, and the amino group on Dab was reacted with the carboxyl group on leucine at room temperature for 24 hours to form a ring.
(6) Adding 25% piperidine/DMF 2mL, shaking for 5min, removing, adding the same 25% piperidine/DMF 2mL, activating for 25min, removing Fmoc protecting group on cysteine, adding 1mL lysate, reacting for 3h, and cleaving from Rink Amide MHBA resin to complete the synthesis of the compound peptide CP7-FP13-2.
(7) The synthesized complex peptide was dried by rotary evaporator and dried in a vacuum pump for 24 hours.
The synthetic route for CP7-FP13-2 is shown in FIG. 1 a. Ext> Asext> aext> controlext>,ext> randomext> polypeptideext> (ext> Lext> -ext> Gext> -ext> Aext> -ext> Kext> -ext> Aext> -ext> Gext> -ext> Gext> -ext> Gext>)ext> andext> linearext> polypeptideext> Pext> 7ext> wereext> alsoext> synthesizedext>,ext> coupledext> toext> FPext> 13ext> -ext> 2ext>,ext> purifiedext> byext> gelext> columnext> chromatographyext> combinedext> withext> preparativeext> liquidext> chromatographyext>,ext> andext> purifiedext> byext> HPLCext>.ext>
TABLE 1 amino acid sequences of peptides synthesized
| Peptide name | Sequence |
| P7 | I-N-Dab-A-F-L-L |
| CP7 | Ac-I-N-(Dab-A-F-L-L) |
| FP13-2 | I-K-N-L-W-K-K-M-I-K-L-W-Y |
| CP7-FP13-2 | (L-L-F-A-Dab)-N-I-G-G-C-6MA-G-I-K-N-L-W-K-K-M-I-K-L-W-Y |
| P7-FP13-2 | L-L-F-A-Dab-N-I-G-G-G-I-K-N-L-W-K-K-M-I-K-L-W-Y |
| Random-FP13-2 | L-G-A-K-A-G-G-G-I-K-N-L-W-K-K-M-I-K-L-W-Y |
* Letters represent amino acid codes.
1.4, MIC and MBC assays
Serial dilutions (two-fold) of the complex peptides were added to 96-well plates at a concentration of 100 μl/well. The final concentration of peptide was 0.78-100. Mu.M. Bacteria in log phase were diluted to 2X 10 in MH broth or BHI broth 5 CFU/mL. 100. Mu.L of the bacterial suspension was added to wells containing peptide in 96-well plates. The plates were then incubated at 37℃for 18 to 24 hours. MIC determinations were then performed, with the lowest inhibitory concentration (MIC) being the lowest drug concentration at which no bacterial growth was observed. After MIC determination, 50. Mu.L of culture broth was taken from MIC, 2 XMIC, 4 XMIC wells, respectively, and plated on BHI or MH agar plates. The agar plates were incubated at 37℃for 24 hours, and the bacterial colony numbers on the plates were counted separately. Minimum Bactericidal Concentration (MBC) is defined as being capable of killing>99.9% initial bacterial content%<100 CFU/mL) the experiment was repeated 3 times.
1.5 in vitro cytotoxicity test
In 96-well plates (4X 10) 4 Individual cells/well) RAW 264.7 cells were cultured overnight to allow them to adhere. The supernatant was then removed, washed three times with PBS, added to fresh DMEM medium containing various concentrations (125, 62.5, 31.25, 15.6, 7.8, 3.9, 1.9, 0.9. Mu.M) of the complex peptide, and incubated at 37℃for 48 hours. A further 100. Mu.LMTT solution (0.5 mg/mL) was added, the plate was incubated at 37℃for 4 hours, and then the supernatant was removed, and 150. Mu.L of DMSO solution was added to the plate. OD at 570nm was measured on a microplate reader.
1.6, bacterial Total RNA extraction and real-time quantitative polymerase chain reaction (qRT-PCR)
The expression of RNAIII, key factors in the staphylococcal agr system and associated virulence factors hla and psm-alpha were detected using the QRT-polymerase chain reaction. After incubation of the bacteria with the peptide for 18 hours, the bacterial cells were collected by centrifugation (12,000Xg, room temperature, 5 min) and resuspended in PBS (pH 7.4). Lysozyme (100. Mu.g/mL) and lysostaphin (100. Mu.g/mL) were added and the bacterial suspension incubated at 37℃for 20 minutes. The total RNA was then isolated and purified using bacterial RNA kit (GBCBIO, guangdong, china) according to the manufacturer's instructions. The yield and purity of RNA was assessed by spectrophotometric analysis. Using only a 260/280nm ratio of 1.8Samples in the range of-2.0 for subsequent reverse transcription. cDNA was synthesized using PrimeScript RT Master Mix (Takara, shigella, japan). QRT-PCR was performed using RR420A SYBR premix Ex Taq (Takara). By using 2 -ΔΔCt The method normalizes the data to housekeeping genes (16S rRNA) to determine relative gene expression profiles. All samples were analyzed three times. The QRT-PCR primer sequence is 5'-AAACTAAAKGAATTGACGG-3' (16-s rRNA forward direction); 5'-CTACRCCGAGCTGAC-3' (16-s rRNA reverse); 5'-TTCACTGTGTCGATAATCCA-3' (RNA iii staphylococcus aureus forward); 5'-TGATTTCAATGGCACAAGAT-3' (RNA iii staphylococcus aureus reversal); 5'-TGAGTTGTTGAGCCATCCA-3' (staphylococcus epidermidis RNA iii forward); 5'-ACCTAACACTGAGTCCAAGAAACTA-3' (staphylococcus epidermidis RNAIII reverse); 5'-ACGACCTTCACTTGTATCC-3' (Staphylococcus saprophyticus RNAIII type Forward); 5'-GCTACGGCATCTTCTTCTA-3' (RNA iii Staphylococcus saprophyticus reverse); 5'-ATGGATAGAAAAGCATCCAACA-3' (HLA forward); 5'-TTTCCAATTTGTTGAAGTCCAAT-3' (HLA reverse); 5'-TATCAAAAGCTTAATCGAACAATTC-3' (PSM-alpha forward); 5'-CCCCCCCCTCAAATAAGATGTTCATATC-3' (PSM-alpha reverse).
1.7 hemolysis test
5mL of venous blood of a rat (Experimental animal center of the university of south medical science) was collected from the inferior vena cava, and centrifuged at 1200rpm/min for 10 minutes. The supernatant was then removed. The resulting erythrocytes were washed 3 times with PBS buffer, diluted to 2% (volume fraction) with PBS, and then stored at 4 ℃. Staphylococcus aureus ATCC6538p (10) was cultured at 37℃with or without treatment with the compound (peptide) 5 CFU/ml) for 20 hours, the culture was centrifuged (12,000Xg, 15 min) and the supernatant was sterilized by filtration through a microporous filter (0.22 μm). mu.L of the supernatant was added to 500. Mu.L of the 2% erythrocyte suspension prepared previously, and cultured in a constant temperature shaker at 37℃for 1 hour. The samples were then centrifuged (12,000Xg, 10 min) to pellet out intact erythrocytes and 150. Mu.L of supernatant was transferred to 96 well plates to determine OD at 570 nm. The hemolytic activity of the sterile medium was set to 0% and the hemolytic activity of the supernatant of staphylococcus aureus without any treatment was set to 100%. Percent inhibition of hemolysis of control cultures was calculated: 1- (Experimental group OD/untreated golden yellow)Staphylococcus chromogenes supernatant OD values).
1.8, kinetic test of sterilization
Diluting bacteria in logarithmic growth phase to 5×10 5 CFU/mL and treated with compound (peptide) at a concentration of 6.25. Mu.M (2 XMIC). 50. Mu.L of each bacterial solution was taken at different time intervals (5 s, 10s, 30s, 1min, 5min, 10min and 30 min) and diluted to 1:10, 1:100 and 1:1000. mu.L of each dilution was then plated onto MH or BHI agar plates. After 24 hours of incubation at 37℃with or without oxygen, the bacterial colony count was counted. In addition, 31.25. Mu.M of CP7 (100. Mu.L) was combined with 5X 10 5 After CFU/mL s.aureus pre-incubation for 30 minutes, the bactericidal kinetics against s.aureus were determined by plate colony counting. The above results were all obtained in three independent experiments.
1.9 biofilm formation inhibition test
The ability of the complex peptides to inhibit biofilm formation was assessed by crystal violet staining. Peptides at concentrations of 1/2 XMIC, 1/4 XMIC, 1/8 XMIC and 1/16 XMIC were added to MH broth and Staphylococcus aureus suspension (1X 10) 6 CFU/mL) and untreated bacterial suspension was used as a control. Each suspension was dispensed into 96-well microtiter plates (200. Mu.L/well) and incubated at 37℃for 24 hours. The supernatant was then removed and the resulting biofilm was washed three times with PBS and fixed in 4% paraformaldehyde for 30 minutes. After air drying, the biofilm was stained with 0.2% crystal violet for 30 minutes, rinsed with water, air dried, and eluted with 33% acetic acid (v/v). OD at 595nm wavelength was determined.
Meanwhile, the formation of bacterial biofilm was also observed under a fluorescence microscope by a live/dead staining test. Staphylococcus aureus was grown on sterile slides (8X 8 mm) in 24-well plates at 37℃with or without peptide treatment, to form biofilms. The slides were then rinsed twice with 0.85% sodium chloride solution and stained with a bacterial viability/toxicity assay kit (Bioscience, china) and observed by fluorescence microscopy (Axio o bserver A1, ZEISS, germany) at 400 x magnification.
1.10 isothermal titration quantitative thermal experiment
The amino acid sequences of the three AgrC-I exocyclic domains are synthesized by adopting a solid phase synthesis method, and the purity of the three AgrC-I exocyclic domains is confirmed by using a High Performance Liquid Chromatography (HPLC), wherein the amino acid sequences of the three AgrC-I are respectively: agrC-I extra-loop-I NH2-GIKYSKLDYF; agrC-I extra-loop-II NH2-ayitkidsi; agrC-I extra-loop-III: NH2-SQINSDEAKVIRQ. Then, they were dissolved in 1% dmso to a concentration of 100 μm, respectively. The peptides were also prepared in 1% DMSO solvent at a concentration of 1mM, and after degassing for 10min, the thermal change of the test peptides interacting with the three exocyclic domains of AgrC-I was determined by Isothermal Titration Calorimetry (ITC) (MicroCal PEAQ-ITC, malvern Panalytical, malvern, UK). The ITC reaction conditions were: temperature: reference power (μcal/s) at 25 ℃): 10, stirring speed (rpm): 750, initial delay(s): 60, sampling times: 19. the data were analyzed using MicroCal PEAQ-ITC software to obtain thermodynamic parameters.
1.11 tryptophan fluorescence Spectroscopy
Logarithmic staphylococcus aureus ATCC6538p cells were collected by centrifugation (4,000Xg, 15 min), washed twice with PBS and resuspended in 10mL PBS (pH 7.4). Then 20mL of chloroform-methanol (1:2, v/v) mixed solvent was added and stirred for 18 hours, and 10mL of chloroform-water (1:1, v/v) was added and further stirred for 30 minutes. Collecting chloroform phase by using a separating funnel; the solvent was removed by rotary evaporation and dried in vacuo for 24 hours to give Staphylococcus aureus cell membrane lipids.
The prepared Staphylococcus aureus cell lipids were dissolved with 5% DMSO to a concentration of 4mg/mL, then treated with 100. Mu.M CP7-FP13-2 or FP13-2 for 1 hour, and 100. Mu.M CP7-FP13-2 and FP13-2 were dissolved with PBS, respectively, as controls. Fluorescence emission spectra of tryptophan were detected using fluorescence spectroscopy (FLS 980, edinburgh, england, excitation wavelength: 280nm, scan range: 300 to 400 nm), with slit widths of excitation and emission beams of 3nm.
1.12 in vivo experiments in mice
Male Kunming mice (16-20 g, without specific pathogenic bacteria) of 4 weeks old were purchased from the university laboratory animal center of south medical science (license number: SYXK (Guangdong) 2016-0167, guangzhou, china). During the experiment, all mice were fed standard laboratory food and water was provided ad libitum. The experiment was performed according to the standard procedures of animal protection committee of university of medical science, south and animal welfare method.
Mice were randomly divided into 5 groups of 10 mice each. Mice not infected with staphylococcus aureus virus served as normal control groups, and the remaining groups were intraperitoneally injected with 0.5mL (1.0X10) 8 CFU/mL) staphylococcus aureus ATCC6538p, a staphylococcus aureus infection mouse model was established. After 1 hour, normal and staphylococcus aureus infected control mice were respectively injected intraperitoneally with 0.9% physiological saline, 15mg/kg CP7-FP13-2 was injected in the low dose group, 45mg/kg CP7-FP13-2 was injected in the high dose group, and 5mg/kg vancomycin was injected in the positive group. All mice were injected only 1 time for 7 consecutive days and mice survival time and body mass changes were recorded. Survival analysis was performed using log pad Prism 8.0 software (GraphPad Software inc., la Jolla, CA, USA), p-value was tested using the log rank (Mantel-Cox) test<0.05 was defined as a significant difference.
1.13 statistical analysis
Each experiment was independently repeated at least three times and the results are expressed as average SD. All data were tested using unpaired student's t-test or one-way anova followed by Duncan test using SPSS 25.0 software. The graph was drawn using GraphPad Prism8 software. Statistical significance is defined as * P<0.05， ** P<0.01， *** P<0.001。
2. Experimental results
2.1 design and Synthesis of peptides
All staphylococcus aureus AIP signals have two main structural features: i) Macrocyclic thioesters formed from conserved cysteines with C-terminal carboxylic acids, ii) N-terminal exocyclic tails. This example uses natural AIP-III as the lead compound for proof of concept studies, and constructs an AIP-III variant, called CP7, with stability enhanced by replacement of Asp with Ala and Cys with Dab, water solubility reduced and synthetic difficulties reduced. Meanwhile, an antibacterial peptide FP-13-2 is constructed and consists of 13 amino acids. Next, the N-terminal ends of the two peptide chains of CP7 and FPs were joined together by Gly-Gly-Cys-6MA-Gly linker using bifunctional molecule 6MA (6-maleimidocaproic acid), and then cyclized on resin to form lactam ring by amino group of Dab residue and carboxyl group of CP7 domain, to give complex peptide CP7-FP13-2. FIGS. 1a and 1b show the synthetic procedure and chemical structure of these peptides and their purity was checked by HPLC (FIG. 1 c).
2.2 the compound peptide has strong antibacterial activity
The antimicrobial activity of these peptides against a group of bacterial strains including the gram positive bacteria Staphylococcus aureus, staphylococcus epidermidis, staphylococcus saprophyticus, streptococcus pneumoniae and Streptococcus mutans, and the gram negative bacteria Escherichia coli, was tested using the MIC and MBC methods. As can be seen from FIG. 2, FP13-2 has a broad spectrum of antibacterial activity, whereas CP7 is inactive within the range of the assay. After the CP7-FPs are coupled with the CP7, the coupling agent has the same MIC and MBCs as the FPs, which shows that the coupling agent has stronger antibacterial effect in an antibacterial domain.
2.3 Complex peptides showed moderate cytotoxicity
The MTT method was used to assess the cytotoxicity of AMPs against RAW 264.7 cells. As shown in FIG. 2, CP7 and FP13-2 were non-cytotoxic and CC of CP7-FP13-2 was not cytotoxic at concentrations up to 125. Mu.M 50 71.13.+ -. 2.13. Mu.M. The above results indicate that CP7-FP13-2 should be safe in the concentration range required for effective treatment of Staphylococcus aureus.
2.4, CP7 can specifically inhibit the QS signal pathway of staphylococcus aureus
Inhibition of staphylococcus aureus QS signal pathway by CP7 was assessed by measuring RNAIII transcript levels of staphylococcus aureus ATCC6538p 24 hours after CP7 treatment. As shown in FIG. 3a, CP7 down-regulates Staphylococcus aureus RNAIII expression in a concentration-dependent manner. Thus, downregulation of staphylococcus aureus RNAIII was due to a non-biocidal pattern, as CP7 had no antibacterial activity (fig. 2). To verify whether CP7 inhibited QS signaling against other staphylococci, its effects on staphylococcus epidermidis and staphylococcus saprophyticus were determined. As shown in fig. 3b and 3c, at the same concentration, CP7 had no inhibitory effect on RNAIII expression in staphylococcus epidermidis and staphylococcus saprophyticus, indicating that CP7 has a specific inhibitory effect on the QS signal pathway of staphylococcus aureus.
2.5, CP7-FP13-2 specific Staphylococcus aureus QS Signal System at non-bactericidal concentration
Based on the MIC value of CP7-FP13-2, the inhibition of RNAIII expression by CP7-FP13-2 was evaluated using a non-bactericidal concentration of 0.78. Mu.M (1/4 XMIC). As shown in FIGS. 3d-f, CP7-FP13-2 significantly down-regulates the expression of Staphylococcus aureus RNAIII, but has no inhibitory effect on the expression of Staphylococcus epidermidis RNAIII or Staphylococcus saprophyticus RNAIII, indicating that CP7-FP13-2 retains the function of the CP7 domain in the targeted inhibition of Staphylococcus aureus QS signaling system.
2.6, CP7-FP13-2 inhibiting expression of virulence factors
The anti-virulence effect of the peptides was evaluated by qRT-PCR to determine virulence factor levels after treatment of staphylococcus aureus with the peptides. As shown in FIGS. 3d-h, CP7 and CP7-FP13-2 significantly reduced the expression level of RNAIII from Staphylococcus aureus and hla and psm-alpha. In contrast, FP13-2 and Random-FP13-2 did not down-regulate the expression of these factors, indicating that CP7-FP13-2 has an anti-virulence effect on Staphylococcus aureus viruses due to the presence of the CP7 domain.
2.7, CP7 and CP7-FP13-2 inhibit the hemolysis of Staphylococcus aureus
The hemolysin is encoded by hla gene and is one of the most important virulence factors in staphylococcus aureus areas. Haemolysin produced by staphylococcus aureus can be assessed using an erythrocyte bacterial haemolysis assay. As shown in FIG. 3i, CP7-FP13-2 had a significant inhibitory effect on suspended erythrocyte hemolysis caused by Staphylococcus aureus, whereas FP13-2 and Random-FP13-2 had no inhibitory effect.
2.8, CP7-FP13-2 accelerates the killing kinetics of Staphylococcus aureus
The killing kinetics of the peptides against the different bacterial strains were determined within 30 minutes. As shown in FIGS. 4a-e, the CP7-FP13-2 compound showed a faster killing kinetics against Staphylococcus aureus than FP13-2 at a concentration of 6.25. Mu.M for 20 minutes, while there was no killing tendency against Staphylococcus epidermidis, staphylococcus saprophyticus, streptococcus pneumoniae or Escherichia coli. The kinetics of killing of Staphylococcus aureus by CP7-FP13-2 was also determined within 120 minutes after pretreatment of bacteria with CP7 at a concentration of 31.25. Mu.M for 10 minutes. As shown in FIG. 4f, CP7 had no bactericidal effect during the experiment, while CP7-FP13-2 showed similar killing kinetics to FP13-2, indicating that CP7 reduced the bactericidal killing rate of CP7-FP13-2 against Staphylococcus aureus.
2.9 peptides inhibit Staphylococcus aureus biofilm formation
Biofilm formation is an important factor in the development of drug resistance by staphylococcus aureus. As shown in fig. 5, at concentrations of 1.56, 0.78 μm, all peptides inhibited the formation of staphylococcus aureus biofilm; at a concentration of 0.39. Mu.M, CP7 and CP7-FP13-2 each inhibited the formation of Staphylococcus aureus biofilm. At the same time, the formation of Staphylococcus aureus biofilm was also observed under a fluorescence microscope. As shown in fig. 6, the control staphylococcus aureus formed a uniform and dense biofilm with a small amount of dead bacteria. In the peptide-treated group, the dense structure of the biofilm was destroyed, and scattered biofilm defects were seen. In addition, the number of red fluorescence representing dead bacterial cells in the biofilm increases significantly.
2.10, CP7 and CP7-FP13-2 may bind to the AgrC-I protein Extra-loop-II in Staphylococcus aureus
Next, to explore the possible binding domains of AgrC-I protein in staphylococcus aureus, thermodynamic parameters of interactions between CP7, CP7-FP13-2 and the three exocyclic peptides were measured using an ITC instrument. ITC isotherms clearly show that CP7 and CP7-FP13-2 can bind more efficiently to extra-loop-II, extra-loop-I or extra-loop-III (table 2), indicating that P7 and FP13-2 have some interaction with both extra-loop-I, extra-loop-II, and extra-loop-III. As shown in Table 2, the binding affinity Kd between CP7 and extra-loop-II was calculated to be 2.32X10 - The binding affinity Kd between 3M, CP7-FP13-2 and extra-loop-II was 3.78X10 -3 M, it is shown that CP7 and CP7-FP13-2 may bind to the extra-loop-II domain of AgrC-I transmembrane protein and may target the extra-loop-II domain of AgrC-I transmembrane protein of Staphylococcus aureus.
TABLE 2 thermodynamic parameters of peptide interactions with AgrC-extra-loop-II, and extra-loop-III
2.11 interaction of CP7-FP13-2 with Staphylococcus aureus, blue shift of tryptophan spectrum
Tryptophan fluorescence spectroscopy was used to study the possible antimicrobial modes of action and to verify the interaction between peptides and bacterial cell membranes. The FP13-2 and CP7-FP13-2 contain tryptophan residues and have maximum absorption peaks at emission wavelengths of 300-400nm at an excitation wavelength of 280 nm. As shown in FIGS. 7a-b, the emission wavelength of tryptophan residues in FP13-2 changed from 365.83.+ -. 2.25nm to 355.17.+ -. 2.75nm, blue shifted by 10.50.+ -. 1.00nm; the emission wavelength of CP7-FP13-2 was changed from 368.83 + -1.04 nm to 358.50 + -0.50 nm, blue-shifted by 10.33+ -1.57 nm.
2.12, CP7-FP13-2 shows antibacterial Activity in vivo
The in vivo effects of CP7-FP13-2 were evaluated using male Kunming mice (16-20 g) of 4 to 6 weeks of age. As shown in fig. 8a, all mice in the normal control group survived, body weight continued to increase, while those in the staphylococcus aureus infected group died in less than 3 days. About 20% and 50% of mice survived in the low dose group and the high dose group, respectively. About 90% of the mice in the vancomycin group survived. All staphylococcus aureus infected mice continued to lose weight within 3 days post infection, and surviving mice began to gain weight on day 5 (fig. 8 b). The survival time of the mice is prolonged, and the survival rate is improved.
In summary, the present invention uses staphylococcus aureus AIP-III as a lead compound, wherein Asp is substituted with Ala, followed by Dab to replace Cys in the macrocycle and acylating it to the ring, resulting in a new staphylococcus aureus AIP-III variant, designated CP7.QRT-PCR experiments demonstrated that CP7 inhibited the expression of staphylococcus aureus RNAIII in the range of 10nM to 1 μm, but not staphylococcus epidermidis RNAIII or saprophyticus RNAIII. The biofilm inhibition experiment shows that the CP7 also has an inhibition effect on the formation of staphylococcus aureus biofilm. Thus, CP7 inhibits the lethal virulence of staphylococcus aureus, providing a new therapeutic strategy for the treatment of staphylococcus aureus infections. Meanwhile, FP containing 13 amino acid residues, which has potent antibacterial activity, was designed and identified. MIC/MBC experiments show that FP13-2 has strong broad-spectrum antibacterial activity and higher antibacterial effect on staphylococcus aureus (including MRSA). FP13-2 was therefore selected for subsequent study. Finally, CP7 is coupled with FP13-2 to construct a dual-function composite peptide with antitoxic factors and anti-staphylococcus aureus. Firstly, respectively synthesizing-GGC-P7 and-6 MAG FPs, then, connecting the N-terminal of two peptides through an addition reaction of a carbon-carbon double bond on 6MA and a sulfhydryl group on Cys, acylating the amino group of Dab with the carboxyl group on Leu, and forming a ring in the P7 domain, so that the 6MA is successfully connected with the N terminal of CP7 and FPs. After that, the inhibition of QS signal by CP7-FP13-2 was examined by qRT-PCR. The result shows that the CP7-FP13-2 has an inhibitory effect on the expression of staphylococcus aureus RNAIII at the concentration of 1/4 xMIC, and is suitable for the CP7. Furthermore, CP7 and CP7-FP13-2 significantly down-regulated RNAIII, hla and psm-alpha expression, whereas FP13-2 did not. The hemolysin is an important staphylococcus aureus virulence factor and is encoded by hla genes, and CP7-FP13-2 are found to inhibit staphylococcus aureus hemolysis through researches. These results indicate that the CP7 domain in CP7-FP13-2 retains the function of resisting Staphylococcus aureus virulence factors. In addition, CP7-FP13-2 also has strong binding capacity to AgrC-I protein extra-loop-II, but weak binding capacity to extra-loop-I and extra-loop-III.
MIC experiments showed that FP13-2 and CP7-FP13-2 had similar anti-Staphylococcus aureus activity. At a concentration of 6.25. Mu.M, CP7-FP13-2 kills Staphylococcus aureus more rapidly than FP13-2. While the same trend was not observed in Staphylococcus epidermidis, staphylococcus saprophyticus or Escherichia coli. In addition, CP7 reduces the rate of sterilization of CP7-FP13-2 against Staphylococcus aureus. All these results indicate that the FP13-2 antibacterial domain in CP7-FP13-2 still retains antibacterial effect.
The primary antimicrobial mechanism of AMPs is to disrupt the stability of the bacterial membrane, resulting in bacterial membrane disruption, which is accomplished through annular holes, barrel walls, and carpet models. To further evaluate the therapeutic effect of CP7-FP13-2, an in vivo study was performed to determine its in vivo anti-Staphylococcus aureus activity. The results show that CP7-FP13-2 can effectively improve survival rate of Kunming mice after staphylococcus aureus infection. The results show that the composite peptide synthesized by the invention is an antibacterial peptide targeting staphylococcus aureus and having double functions of inhibiting quorum sensing signals and antibacterial actions, and provides a certain guiding value for treating staphylococcus aureus and drug-resistant bacteria.
The embodiments of the present invention have been described in detail above, but the present invention is not limited to the described embodiments. It will be apparent to those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, and yet fall within the scope of the invention.
Claims (10)
1. An antibacterial peptide, which is characterized in that the antibacterial peptide is CP7-FP13-2, and the amino acid sequence of the antibacterial peptide is shown as follows: (L-L-F-A-Dab) -N-I-G-G-C-6MA-G-I-K-N-L-W-K-K-M-I-K-L-W-Y.
2. The use of the antibacterial peptide of claim 1 for the preparation of a medicament for inhibiting staphylococcus aureus.
3. The use of the antibacterial peptide according to claim 1 for the preparation of a medicament for the treatment of staphylococcus aureus infectious diseases.
4. The use according to claim 2, wherein the antimicrobial peptide acts to inhibit staphylococcus aureus by inhibiting the formation of a biofilm of staphylococcus aureus.
5. The use according to claim 2, wherein said antimicrobial peptide binds to the extra-loop-II domain of the AgrC-I transmembrane protein, thereby exerting an inhibitory effect on staphylococcus aureus.
6. The use according to claim 3, wherein the antimicrobial peptide acts to treat staphylococcus aureus infectious diseases by down regulating the expression of staphylococcus aureus RNAIII.
7. The use according to claim 3, wherein the antimicrobial peptide acts to treat staphylococcus aureus infectious diseases by reducing the expression levels of staphylococcus aureus hla and psm- α.
8. The use according to claim 3, wherein the antimicrobial peptide acts to treat staphylococcus aureus infectious diseases by inhibiting the hemolysis of staphylococcus aureus.
9. The use according to claim 2 or 3, wherein the medicament further comprises a pharmaceutically acceptable carrier.
10. The use according to claim 2 or 3, wherein the pharmaceutical dosage form comprises injection, tablet, granule, capsule, dripping pill, sustained release preparation, oral liquid, ointment, patch.
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