Anti-pseudomonas aeruginosa nanofiber capture peptide, and preparation method and application thereof

Abstract

The application provides anti-pseudomonas aeruginosa nanofiber capture peptides and a preparation method and application thereof, belongs to the field of bioengineering, and the sequence is shown in SEQ ID No. 1 and SEQ ID No. 2. The two capture peptides 3KF and 2KF of the application can self-assemble into a three-dimensional nanofiber network under physiological conditions, and can capture and fix pseudomonas aeruginosa through mechanisms such as electrostatic interaction, hydrophilic and hydrophobic interaction and ligand-receptor recognition; meanwhile, the capture peptides 3KF and 2KF can realize enrichment on the membrane surface through the high specific surface area of the nanofiber and kill the pseudomonas aeruginosa through membrane damage. The capture and enrichment membrane damage effect can avoid the escape of pseudomonas aeruginosa, realize rapid killing of the bacteria, and effectively alleviate the generation of drug resistance. In summary, the capture peptides 3KF and 2KF are nanofiber capture peptides with high development potential and high application value for resisting pseudomonas aeruginosa.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering technology, specifically relating to nanofiber-capturing peptides against Pseudomonas aeruginosa, their preparation methods, and applications. Background Technology

[0002] *Pseudomonas aeruginosa* is a highly drug-resistant, opportunistic pathogen, particularly prevalent in immunocompromised individuals, easily causing serious illnesses such as pneumonia, sepsis, and burn wound infections. This bacterium employs multiple resistance mechanisms, including the expression of broad-spectrum β-lactamases, overactivation of the efflux pump system, and reduced outer membrane permeability, significantly diminishing the efficacy of commonly used antibiotics (including carbapenems, cephalosporins, and aminoglycosides). Furthermore, *Pseudomonas aeruginosa* can form a dense biofilm, a structure that prevents antibiotic penetration and protects the internal bacterial cells from the immune system. In addition, its secreted toxins (such as exotoxin A, elastase, and rhamnolipids) can directly damage host tissues and facilitate immune evasion through flagella and pili. These factors collectively contribute to the difficulty of traditional antibiotic treatment, persistent and recurrent infections, necessitating the development of novel, antibiotic-free anti-infection strategies. To address this challenge, nanofiber-based peptide-trapping technology offers an innovative physical defense strategy. This technology leverages the self-assembly properties of specific capture peptide molecules, spontaneously forming a three-dimensional nanofiber network under physiological conditions. This network efficiently captures and immobilizes *Pseudomonas aeruginosa*, physically restricting bacterial migration and spread, effectively addressing its escape problem. Simultaneously, the high specific surface area of ​​the nanofibers allows for the abundant accumulation of capture peptides on the bacterial surface, significantly increasing the local concentration of antimicrobial peptides and achieving a rapid and efficient bactericidal effect. Compared to traditional antibiotic therapy, this synergistic strategy of physical capture and local accumulation is less likely to induce new drug resistance. Therefore, the novel design of capture peptides capable of recognizing *Pseudomonas aeruginosa* membrane proteins and self-assembling into a nanonetwork is of great significance for finding antibiotic alternatives and treating *Pseudomonas aeruginosa* infections. Summary of the Invention

[0003] To address the aforementioned problems, the present invention aims to provide a nanofiber-capturing peptide with highly efficient antibacterial activity against *Pseudomonas aeruginosa*. This peptide can self-assemble into nanofibers in a physiological environment, exhibiting excellent capture and killing effects against *Pseudomonas aeruginosa*. Furthermore, the capture peptide of this invention can efficiently kill bacteria by disrupting their cell membrane structure.

[0004] The technical solution adopted in this invention is as follows: two nanofiber-based peptide molecules for resisting Pseudomonas aeruginosa are designed, and their amino acid sequences are shown in SEQ ID No. 1 and SEQ ID No. 2.

[0005] Another object of the present invention is to provide a method for preparing nanofiber-based peptides for combating Pseudomonas aeruginosa, comprising the following steps: Step S1: Phenylalanine is selected to provide hydrophobic interaction, glutamine and leucine are selected to form intermolecular hydrogen bonds, and lysine is selected to provide positive charge, thus satisfying the basic conditions for the capture peptide. In the capture peptide structure, consecutive lysine and phenylalanine are placed at both ends of the repeating sequences of glutamine and leucine, and the peptide molecule self-assembly is promoted through aromatic stacking, hydrophobic interaction and hydrogen bonding. Step S2: The GSGS flexible linker was used to connect with the Pseudomonas aeruginosa membrane protein recognition sequence SQRKLAAKLTSK to construct the capture peptide with the amino acid sequence shown in SEQ ID No. 1 and SEQ ID No. 2; The capture peptides were prepared using chemical solid-phase synthesis and mass spectrometry. The nanoscale morphology, hemolytic activity, in vitro cytotoxicity, bacterial capture ability and antibacterial activity of the capture peptides were then determined. Finally, they were named capture peptides 3KF and 2KF.

[0006] Furthermore, the self-assembly method for the nanofiber capturing peptides 3KF and 2KF against Pseudomonas aeruginosa, as described above, has self-assembly conditions of >16 μM and incubation at 37°C for 24 h.

[0007] Another object of the present invention is to provide the use of the nanofiber capturing peptides 3KF and 2KF against Pseudomonas aeruginosa as described above in the preparation of medicaments for treating infectious diseases caused by Pseudomonas aeruginosa.

[0008] This invention has the following advantages and beneficial effects: the nanofiber capturing peptides 3KF and 2KF of this invention, which are effective against *Pseudomonas aeruginosa*, can form nanofiber structures under physiological conditions, exhibiting excellent ability to capture and kill *P. aeruginosa*. The hemolytic activity, eukaryotic cytotoxicity, bacterial capture ability, and antibacterial activity of the prepared capturing peptides 3KF and 2KF were determined. It was found that the capturing peptides 3KF and 2KF have strong *P. aeruginosa* capture ability and antibacterial activity, and are almost toxic to erythrocytes and eukaryotic cells. In summary, 3KF and 2KF have the potential for clinical treatment of *P. aeruginosa*. Attached Figure Description

[0009] Figure 1 High-performance liquid chromatograms for capturing peptides 3KF(a) and 2KF(b); Figure 2 Mass spectra of the captured peptides 3KF (a) and 2KF (b); Figure 3 Graphs showing the critical aggregation concentration determination of the capture peptides 3KF and 2KF; Figure 4Linear fitting plots for the critical aggregation concentrations of the capture peptides 3KF and 2KF; Figure 5 Transmission electron microscopy nanocharacterization images of the captured peptides 3KF(a) and 2KF(b); Figure 6 Nanoparticle size distribution of the trapping peptides 3KF and 2KF; Figure 7 Zeta potential measurements of the 3KF and 2KF trapping peptides; Figure 8 Diagrams showing the secondary structures of the capture peptides 3KF and 2KF; Figure 9 Graphs showing the hemolytic activity assays of the capture peptides 3KF and 2KF; Figure 10 Graphs showing the cytotoxicity assays of the capture peptides 3KF and 2KF; Figure 11 To capture peptides 3KF(a) and 2KF(b) against Pseudomonas aeruginosa at 6 h P. aeruginosa PAO1 capture effect graph, unit: μM; Figure 12 Capture effect of capture peptides 3KF(a) and 2KF(b) on Pseudomonas aeruginosa 27853 at 6 h, unit: μM; Figure 13 To capture peptides 3KF(a) and 2KF(b) against Pseudomonas aeruginosa at 6 h P. aeruginosa The capture effect of 25625, unit: μM; Figure 14 To capture Pseudomonas aeruginosa using peptides 3KF and 2KF. P. aeruginosa PAO1, P. aeruginosa 27853 and P. aeruginosa Graph showing changes in colony count in the supernatant after 25625 hours; Figure 15 To capture the effects of peptides 3KF and 2KF on the outer membrane permeability of Pseudomonas aeruginosa PAO1, different letters represent significant differences ( P < 0.05); Figure 16 To capture peptides 3KF and 2KF against Pseudomonas aeruginosa P. aeruginosa The effect of PAO1 plasma membrane potential; Figure 17 To capture peptides 3KF and 2KF against Pseudomonas aeruginosa P. aeruginosa The effect of PAO1 membrane structure integrity (scale bar: 100 µm). Figure 18 To observe the effects of captured peptides 3KF and 2KF on Pseudomonas aeruginosa using scanning electron microscopy P. aeruginosa The morphological effects of PAO1 were observed in (a) the blank control group, (b) the 3KF treatment group, and (c) the 2KF treatment group. Detailed Implementation

[0010] The present invention will be further described in detail below with reference to the embodiments and accompanying drawings, but the embodiments of the present invention are not limited thereto. Example 1

[0011] Design of nanofiber-based trapping peptides 3KF and 2KF against Pseudomonas aeruginosa: Phenylalanine was selected to provide hydrophobic interaction, glutamine and leucine were selected to form intermolecular hydrogen bonds, and lysine was selected to provide positive charge, satisfying the basic conditions for trapping peptides. In terms of structure, consecutive lysine and phenylalanine were placed at both ends of the repeating sequences of glutamine and leucine, and molecular self-assembly was promoted through aromatic stacking, hydrophobic interaction, and hydrogen bonding. The trapping peptides 3KF and 2KF were connected to the membrane recognition sequence of Pseudomonas aeruginosa membrane protein -SQRKLAAKLTSK- using a GSGS flexible linker. The parameters of the trapping peptides 3KF and 2KF are shown in Table 1.

[0012] Table 1. Parameters of nanofiber-trapping peptides 3KF and 2KF against Pseudomonas aeruginosa Example 2

[0013] Synthesis of nanofiber-scavenging peptides 3KF and 2KF against Pseudomonas aeruginosa using solid-phase synthesis: (1) Preparation of solid support and initial amino acid ligation: Weigh the resin and add it to the solid synthesis reaction column. Add N,N-dimethylformamide (DMF) and soak at room temperature for 30 minutes to allow the resin to swell fully (the volume increases to about 2-3 times the original volume). Then, dry the DMF. Dissolve the C-terminal Fmoc-protected amino acid in DMF. Add 1-hydroxybenzotriazole and N,N'-diisopropylcarbodiimide and activate at room temperature for 10 minutes. Add the activated solution to the reaction column and react with shaking at room temperature for 2 hours. Then, dry the reaction solution and wash three times with DMF to remove unreacted amino acids.

[0014] (2) Fmoc deprotection: Add 20% piperidine / DMF solution to the reaction column, shake at room temperature for 1 minute and then dry.

[0015] Add another 5 mL of 20% piperidine / DMF solution, shake at room temperature for 20 minutes to completely remove the Fmoc group, generating a dibenzofuran-piperidine adduct. Then, dry the deprotection solution, wash 5 times with DMF, and finally wash 2 times with dichloromethane (DCM), and dry for later use.

[0016] (3) Activation and coupling of amino acids: Fmoc-Ala-OH was dissolved in DMF, and HOBt and O-benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate were added. After dissolution, N,N-diisopropylethylamine was added. The above activation solution was added to the reaction column and the reaction was carried out at room temperature with shaking for 1.5 hours. A small amount of resin was taken, and ninhydrin reagent (1% ninhydrin / ethanol solution) was added. The mixture was heated at 100°C for 1 minute. If the resin was colorless, the coupling was complete. The reaction solution was dried under vacuum, washed 3 times with DMF and 2 times with DCM, and then dried under vacuum.

[0017] (4) Repeat “deprotection-activation coupling-washing”: Following step (2) (20% piperidine / DMF deprotection) and step (3) (amino acid activation coupling), add the target sequence amino acids sequentially (from C-terminus to N-terminus). Wash and ninhydrin detection are required after each round of coupling until all amino acid coupling is completed, and finally obtain “resin-fully protected polypeptide”.

[0018] (5) Peptide cleavage and side chain deprotection: The cleavage buffer was prepared using trifluoroacetic acid (TFA), triisopropylsilane (TIS), and water. The cleavage buffer was added to the reaction column, and the reaction was carried out with shaking at room temperature for 2.5 hours. The cleavage buffer was collected into a centrifuge tube containing ice-cold diethyl ether (pre-cooled at -20°C). At this time, a white precipitate of peptide was precipitated. The reaction column was washed twice with TFA, and the washes were combined into a centrifuge tube and vortexed to mix. The column was centrifuged at 5000 rpm / min for 10 minutes at 4°C, and the supernatant was discarded. The precipitate was washed three times with ice-cold diethyl ether to remove residual TFA and protecting group fragments.

[0019] (6) Peptide purification and lyophilization: Add 0.1% TFA / water solution to the precipitate in the centrifuge tube and sonicate to dissolve the peptide. Use a reverse-phase C18 column with mobile phase A of 0.1% TFA / water and mobile phase B of 0.1% TFA / acetonitrile for gradient elution. Detect at a wavelength of 220 nm and collect the eluent of the target peak. Concentrate the purified eluent using a rotary evaporator, transfer it to a lyophilization bottle, pre-freeze at 80°C for 2 hours, and then lyophilize for 24 hours to obtain high-purity peptide powder.

[0020] (7) Identification: The captured peptides obtained above were analyzed by electrospray mass spectrometry (e.g., Figure 1 As shown), the purity of the captured peptide is greater than 95% (e.g. Figure 2 (As shown). Example 3

[0021] Determination of critical aggregation concentrations of nanofiber-trapping peptides 3KF and 2KF against Pseudomonas aeruginosa: The CAC value of the captured peptide was detected using a 1-aniline-8-naphthalenesulfonate (1,8-ANS) fluorescent probe. The captured peptide was serially diluted in PBS solution (10 mM, pH = 7.4) to a gradient concentration ranging from 128 µM to 2 µM. The 1,8-ANS fluorescent probe powder was dissolved to a concentration of 1 mM using N,N-dimethylformamide (DMF) as a solvent. In 96-well plates, probe concentrations of 20 µM were added to different concentrations of captured peptide solutions. After incubation at 37°C in the dark for 30 min, fluorescence intensity was measured in the 400–700 nm wavelength range using a fluorescence spectrophotometer (Hitachi, Japan) with an excitation wavelength of 369 nm.

[0022] The results are as follows Figure 3 and Figure 4 As shown, when the concentration of the capturing peptide exceeds 16 μM, the fluorescence value increases sharply, indicating that the hydrophobic groups in the peptide molecule aggregate rapidly, representing the beginning of the self-assembly behavior of the capturing peptide molecule. Finally, the self-assembly threshold concentrations of capturing peptides 3KF and 2KF in PBS were calculated to be 15.25 μM and 15.27 μM, respectively. Example 4

[0023] Transmission electron microscopy nanoscale characterization images of nanofiber-trapping peptides 3KF and 2KF against Pseudomonas aeruginosa: Nanoscale morphology analysis: To further analyze the nanoscale morphology of the nanofiber-capturing peptides 3KF and 2KF against *Pseudomonas aeruginosa*, the peptides (2.56 mM) were diluted to 16 μM in phosphate-buffered saline (PBS) and incubated at 37°C for 24 h. The capture peptide solution was then placed on a copper grid plate for 2 min and stained with 0.1% phosphotungstic acid for 3 s. Finally, the morphology of the air-dried peptides was examined using a Hitachi H-7650 transmission electron microscope (Hitachi H-7650, Japan). The results are shown in the figure. Figure 5 .

[0024] from Figure 5 It can be seen that the 16 μM capturing peptides 3KF and 2KF both formed cross-linked short nanofibers. Example 5

[0025] Nanostructure size distribution and zeta potential determination of nanofiber-trapping peptides 3KF and 2KF for resistance to Pseudomonas aeruginosa: The captured peptide solution was adjusted to 64 µM in PBS (10 mM, pH = 7.4) and incubated overnight at 37°C in the dark. The hydrodynamic size distribution and Zeta potential of the captured peptide were determined using a Zetasizer Nano Z90 instrument (Malvin Instruments, Worcestershire, UK). The incubated captured peptide solution was pipetted into cuvettes, with viscosity and solute reflectance set to PBS and protein, respectively. Each sample was measured three times.

[0026] Capture peptide nanoparticles with a diameter of such as Figure 6 As shown, 3KF and 2KF form nanofiber structures of varying lengths, all distributed in the 10-400 nm range; furthermore, the zeta potential of the trapping peptide is as follows: Figure 7 As shown, the zeta potentials of the capturing peptides 3KF and 2KF in PBS solution were both positive, ranging from 7.19 mV to 8.78 mV. Example 6

[0027] Secondary structure determination of nanofiber-trapping peptides 3KF and 2KF against Pseudomonas aeruginosa: PBS (10 mM, pH = 7.4), 30 mM SDS solution, and 50% TFE were used as solvents for capturing peptides, and the concentration of the capturing peptide solution was adjusted to 64 µM in these three solvents. A Jasco CD spectrometer was used to scan the broad spectrum in the range of 190 nm to 260 nm at a 1 mm optical path. Each sample was analyzed three times, and the average value was taken. The results are expressed as the average elliptic residue rate, using the formula: ; Where θ M The average residue ellipticity (deg·cm) 2 ·mdol -1 ), θ obs The measured value of ellipticity is given, c represents the concentration of the trapping peptide solution (µM), n represents the number of amino acids in the trapping peptide, and l is the optical path length (mm).

[0028] The results are as follows Figure 8 As shown, 3KF and 2KF exhibit regular α-helical structure characteristics in TFE and SDS, except for the α-helical structure with signal shift in PBS. Example 7

[0029] Hemolytic activity and in vitro eukaryotic cytotoxicity assays of the capture peptide 2KF: Hemolytic activity assay: 1 mL of fresh healthy human blood was centrifuged (4℃, 1000 × g, 5 min), washed three times with PBS phosphate buffer (pH = 7.4), the supernatant was discarded, and the precipitated blood cells were collected and resuspended in PBS phosphate buffer to 10 mL. Then, an equal volume of the diluted red blood cell suspension was placed in a 96-well plate and mixed with serially diluted capture peptide solutions (128 μM–1 μM). After incubation at 37℃ for 1 h, the plate was centrifuged (1000 × g, 10 min), and 50 μl of supernatant was transferred from each well to a new 96-well plate. Wells treated with 0.1% Triton X-100 served as positive controls, and wells without capture peptide treatment served as negative controls. The absorbance at 570 nm was measured using a microplate reader. The capture peptide concentration that caused 5% hemolysis of red blood cells was defined as the minimum hemolytic concentration (MHC).

[0030] ; Where A is the absorbance value of the captured peptide sample at 570 nm, A0 is the absorbance value of the negative control, and A t The absorbance value is for the positive control.

[0031] pass Figure 9 It can be seen that even at a concentration of 128 μM, the hemolysis rate caused by the capture peptides 3KF and 2KF is less than 5%, which indicates that both capture peptides 3KF and 2KF have good biocompatibility.

[0032] Eukaryotic cytotoxicity assay: RAW 264.7 and IPEC-J2 cells were cultured at 37°C and 5% CO2 in medium containing 10% fetal bovine serum and 1% penicillin-dextrose antibiotics. After reaching 85% confluence, the cells were passaged. Following culture, the cells were digested with 0.25% trypsin, and the cell suspension concentration was adjusted to 2–5 × 10⁻⁶ cells / mL using culture medium. 5 Cells / ml: 50 μL of cell suspension was mixed with 50 μL of different concentrations of capture peptide solution and incubated at 37°C with 5% CO2 for 24 h. Then, 50 μL of MTT (5 mg / ml) was added to each well, followed by incubation under the same conditions for 4 h. After incubation, the supernatant was discarded, and 100 μL of DMSO was added to each well to dissolve the bottom precipitate. Finally, the absorbance of each well was measured at 570 nm using a microplate reader. The positive control was cell suspension without capture peptide solution, and the negative control was culture medium. The cell viability formula is as follows: ; Where A is the absorbance value of the captured peptide sample at 570 nm, A0 is the absorbance value of the negative control, and A tThe absorbance value is for the positive control.

[0033] like Figure 10 As shown, even under treatment with high concentrations (128 μM) of the capture peptides 3KF and 2KF, the survival rate of RAW 264.7 and IPEC-J2 cells remained above 80%, indicating that 3KF and 2KF have good biocompatibility and the potential to become alternative drugs to antibiotics. Example 8

[0034] Determination of the capture ability of nanofiber-based Pseudomonas aeruginosa-resistant peptides 3KF and 2KF against Pseudomonas aeruginosa: To evaluate the capture ability of the nanofiber-based anti-Pseudomonas aeruginosa peptide 2KF against Pseudomonas aeruginosa, Pseudomonas aeruginosa and the peptide were mixed in a small dish, and the phenomenon was observed. Pseudomonas aeruginosa cultured in MHB liquid medium was then used. P. aeruginosa PAO1 cells were collected by centrifugation at 3000 rpm for five minutes, and the cells were adjusted to OD using sterile phosphate-buffered saline (PBS) solution. 600 nm = 0.4, added to a small dish, and the concentration of the captured peptide was adjusted to 128 μM, 64 μM, 32 μM, 16 μM and 8 μM in the small dish respectively. The observation and photography were recorded at 0 h, 0.5 h, 1 h, 2 h, 4 h and 6 h respectively. The untreated bacterial solution was used as a positive control.

[0035] This study preliminarily explored the in vitro bacterial capture ability and sedimentation effect of the capture peptides using a bacterial agglutination assay. This assay was evaluated by measuring the turbidity of the solution and the colony-forming units (CFU) in the supernatant. Figure 11 , Figure 12 and Figure 13 As can be seen, compared with the control group, the turbidity of the supernatant in the capture peptide treatment group was significantly reduced, and this effect showed a clear dose-dependent characteristic. The colony count results of the supernatant are as follows: Figure 14 As shown, all concentrations of the capture peptides significantly reduced the bacterial colony count in the supernatant, exhibiting a time- and concentration-dependent gradient. High concentrations (> 64 μM) of the capture peptides 3KF and 2KF both reduced the bacterial colony count in the supernatant to 0. 3KF showed a slightly better capture rate than 2KF, achieving near-complete sedimentation of bacteria within 0.5 h.

[0036] Subsequently, different peptide concentrations were used to treat the effects over one hour. P. aeruginosa The overall agglutination efficiency was calculated by weighting the average agglutination percentages of PAO1, 27853, and 25625, and this was used to evaluate the capture ability of each capture peptide. As shown in Table 2, the overall bacterial agglutination efficiency of 3KF and 2KF exceeded 90%, indicating that this series of capture peptides has good Pseudomonas aeruginosa capture ability.

[0037] Table 2. Effects of captured peptides on the body at 1 h P. aeruginosa PAO1, P. aeruginosa 27853 and P. aeruginosa The overall coagulation efficiency is 25625. Example 9

[0038] This study evaluated the antibacterial activity of the capture peptides based on the minimum bactericidal concentration (MBC). The specific steps are as follows: (1) Cell preparation: Take the cryopreserved bacterial strain, streak it on MHA solid medium, and incubate it in a 37℃ incubator for 12 h. Pick a single colony from the plate and place it in MHB medium. Shake it in a 37℃, 220 r / min incubator. Plot the growth curve by absorbance value. After the culture reaches the logarithmic growth phase, adjust the bacterial concentration to OD. 600 nm = 0.4, further diluted 1000-fold with PBS to 1×10 6 CFU / mL available for use.

[0039] (2) Preparation of captured peptide samples: Add 95 µL of PBS to row A of a 96-well plate, and add 50 µL of PBS to each of the other rows. Add 5 µL of the captured peptide sample with an initial concentration of 2.56 mM to row A, adjusting the captured peptide concentration in row A to 64 µM. After thoroughly mixing the captured peptide solution in row A, transfer 50 µL to row B, and so on, serially diluting to row G. After mixing, transfer 50 µL of the solution from row G and discard it. Set up three replicates for each captured peptide sample.

[0040] (3) Inoculation of bacterial culture: Mix the diluted bacterial culture evenly, and add 50 µL to all wells in rows A to G and wells 1 to 6 in row F as sample detection wells and positive control wells. Add 50 µL of PBS to wells 7 to 12 in row F as negative control wells. After mixing, incubate in a 37°C incubator for 3 h.

[0041] (4) Sample processing: After the incubation is complete, mix the samples evenly in each well and transfer 50 µL of the sample to an EP tube containing PBS for dilution. Spread the diluted solution on MHA solid medium and incubate at 37°C for 16 h.

[0042] (5) Determination of minimum bactericidal concentration: The number of colonies on the solid culture medium after the culture is completed is counted, and the concentration of the capture peptide that kills 99.9% of the bacteria is defined as the minimum bactericidal concentration (MBC).

[0043] The results are shown in Table 3. The capturing peptides 3KF and 2KF exhibited excellent antibacterial activity against all six tested Pseudomonas aeruginosa strains. The geometric mean (GM) of the MBC values ​​for Pseudomonas aeruginosa was [data missing]. MBC They are 1.41 and 1.78 respectively.

[0044] Table 3. MBC values ​​(μM) of nanofiber-scavenging peptides 3KF and 2KF against Pseudomonas aeruginosa.

[0045] Note: a This represents the geometric mean of the MBC values ​​of each capture peptide against the six tested Pseudomonas aeruginosa species. Example 10

[0046] With Pseudomonas aeruginosa P. aeruginos Taking PAO1 as an example, the changes in MBC of the captured peptide in different physiological salt ion environments and serum at different concentrations were measured to assess the physiological salt ion stability and serum stability of the captured peptide.

[0047] (1) Preparation of bacterial cells: as described in Example 9.

[0048] (2) Capture peptide samples: 5 mM HEPES buffer (pH = 7.2, containing 20 mM glucose) was used as the solvent to prepare various physiological saline solutions, including 150 mM NaCl, 4.5 mM KCl, 1 mM MgCl2, 8 μM ZnCl2, 6 μM NH4Cl, and 4 μM FeCl3. The capture peptides were serially diluted using different physiological saline solutions, with the capture peptide concentrations adjusted sequentially from 64 μM to 1 μM. Simultaneously, the serum concentrations were adjusted to 100%, 50%, and 25% using sterile water. The capture peptides with an initial concentration of 2.56 mM were mixed thoroughly with equal volumes of serum at different concentrations and incubated at 37°C for 18–24 h. After incubation, the capture peptides were mixed thoroughly with the serum and serially diluted with PBS, with the capture peptide concentrations adjusted sequentially from 64 μM to 1 μM.

[0049] (3) Inoculation with bacterial solution: as described in Example 9.

[0050] (4) Sample preparation: as described in Example 9.

[0051] (5) Determination of minimum bactericidal concentration: as described in Example 9.

[0052] The results are shown in Table 4. The antibacterial activity of the trapped peptides was generally less affected by the physiological salt particle environment. Specifically, the trapped peptide 3KF showed the best performance in the Ca2+ environment. 2+In the environment, MBC increased to 8 times its original value, while in other physiological salt ions, MBC decreased to 2 times its original value. Meanwhile, the trapping peptide 2KF, except in Ca... 2+ and K + Apart from the MBC concentration in the environment increasing to 4 times and 2 times the original value, respectively, the antibacterial activity of the remaining salt particles was not significantly affected, demonstrating higher physiological salt ion environment stability than 3KF.

[0053] Furthermore, the 3KF trapping peptide was more significantly affected by serum conditions than the 2KF peptide. In 50% and 25% serum environments, the MBC (metabolite concentration) of 3KF increased by 8 times and 4 times, respectively, while the MBC of 2KF only increased by 2 times in both environments. In conclusion, both 3KF and 2KF exhibited good physiological salt particle stability and serum stability, indicating their potential for in vivo application.

[0054] Table 4. Effects of nanoparticles against Pseudomonas aeruginosa in physiological saline and serum environments. P. aeruginosa PAO1 MBC (μM) Example 11 With Pseudomonas aeruginosa P. aeruginos Taking PAO1 as an example, the effects of the trapping peptides 3KF and 2KF on the outer membrane permeability of this bacterium were determined: Prepare a suspension of Pseudomonas aeruginosa ATCC PAO1, and adjust the suspension to 1 × 10⁻⁶ using HEPES buffer. 5 CFU / mL. This was then mixed with an N-phenyl-1-naphthylamine (NPN, Sigma-Aldrich) fluorescent probe and incubated in the dark at 30°C for 30 min. It was then added to serially diluted capture peptide solutions of different concentrations (50 μL). Fluorescence intensity was measured using an F-4500 fluorescence spectrophotometer at an excitation wavelength of 350 nm and an emission wavelength of 420 nm.

[0055] from Figure 15 The results show that 3KF and 2KF can significantly increase fluorescence intensity, indicating that 3KF and 2KF can increase the permeability of the bacterial outer membrane. Example 12

[0056] With Pseudomonas aeruginosa P. aeruginos Taking PAO1 as an example, the effects of the trapping peptides 3KF and 2KF on the bacterial plasma membrane potential were determined: The ability of the capture peptide to depolarize the bacterial plasma membrane was detected using the membrane potential-sensitive dye 3,3'-dipropylthiadicarbocyanine iodide (DiSC3-5).

[0057] (1) Bacterial samples: samples to be frozen in an ultra-low temperature freezer P. aeruginosa PAO1 was inoculated onto MHA solid medium and incubated overnight at 37°C. The next day, single colonies from the medium were picked and transferred to MHB medium, and cultured at 37°C and 220 rpm / min on a shaker until the logarithmic growth phase. P. aeruginosa PAO1 was centrifuged at low temperature (4℃, 1000 × g), washed three times with HEPES buffer (5 mM, pH = 7.4, containing 20 mM glucose), and the final bacterial suspension concentration was adjusted to OD. 600 nm = 0.05. Next, DiSC3-5 dye was added to the bacterial suspension to make the final dye concentration 0.4 μM. The mixture was incubated at 37°C for 1 hour in the dark. Then, 0.1 M KCl solution was added and the mixture was incubated at 37°C in the dark for another 30 min.

[0058] (2) Sample preparation: After incubation, take 2 mL of the bacterial suspension containing dye and add it to a 24-well plate. Set up the bacterial suspension without the addition of the capture peptide solution as the negative control.

[0059] (3) Results determination: After mixing evenly, the fluorescence intensity was measured using an F-4500 fluorescence spectrophotometer (Hitachi, Japan) at an excitation wavelength of 622 nm and an emission wavelength of 670 nm. After the fluorescence intensity value of the negative control stabilized, capture peptide solutions with final concentrations of 1 μM, 2 μM and 4 μM were added to the mixture, and the fluorescence intensity changes were continuously recorded over 1000 seconds.

[0060] The results are as follows Figure 16 As shown, the fluorescence intensity remained stable without the addition of 3KF and 2KF. After the addition of 3KF and 2KF, the fluorescence intensity increased in a time- and concentration-dependent manner, indicating that 3KF and 2KF have the effect of increasing fluorescence intensity. P. aeruginosa The ability of the PAO1 plasma membrane to rapidly depolarize. Example 13

[0061] With Pseudomonas aeruginosa P. aeruginos Taking PAO1 as an example, the formation of nanofibers containing the trapping peptides 3KF and 2KF was investigated, and the effects of the trapping peptides 3KF and 2KF on the integrity of the bacterial membrane structure were explored: (1) Bacterial samples: The test procedures described in Example 9 were followed.

[0062] (2) In a 1 mL bacterial culture system, adjust the concentrations of the capture peptides 3KF and 2KF to 32 μM and mix thoroughly. Set up a blank control with no added capture peptides. Incubate at 37℃ for 2 h. After incubation, take the incubated samples and centrifuge at low temperature with PBS buffer (4℃, 1000 × g, 10 min), and repeat the washing three times. After the last centrifugation, resuspend the bacterial cells in 100 μL of PBS. Add ThT solution to a final concentration of 25 μM and PI to 5 μg / mL in the dark to each sample, and continue to incubate at 37℃ in the dark for 30 min. After the incubation, take 15 μL of each sample, spread it evenly on a glass slide, and observe and record the results using a fluorescence microscope.

[0063] The results are as follows Figure 17 As shown, no green or red fluorescence was detected in the blank control without the added capture peptide, indicating that the control... P. aeruginosa No amyloid fibrils or bacterial cells with damaged membrane structures were observed in the PAO1 sample. However, samples treated with the capture peptides 3KF and 2KF both showed significant green and red fluorescence, and... P. aeruginosa The aggregation of PAO1 is evident. This indicates that both 3KF and 2KF generate nanofibers through self-assembly, thereby... P. aeruginosa PAO1 aggregates and traps, thus disrupting the integrity of its membrane structure. Example 14

[0064] Effects of 3KF and 2KF on cell morphology: Prepared Pseudomonas aeruginosa cultured in MHB liquid medium P. aeruginosa Collect bacterial cells by centrifugation at 3000 rpm for five minutes using PAO1, and adjust the bacterial cells to OD200 using sterile phosphate-buffered saline (PBS) solution. 600 nm = 0.4, add capture peptides 3KF and 2KF to a final concentration of 8 μM, and incubate at 37℃ for 1 hour. After incubation, collect the bacterial cells by centrifugation at 1000× g for 5 minutes at 4℃, resuspend the cells in 600 μL of 2.5% glutaraldehyde fixative, and fix overnight at 4℃. After dehydration of the bacterial cells sequentially (50%, 70%, 90%, and 100%), replace the cells with 1000 μL of ethanol / tert-butanol (1:1) mixture and pure tert-butanol for 15 minutes each. After freeze-drying, fix the cells to the sample stage with conductive adhesive and perform metal coating using ion sputtering. Finally, observe the samples using a Hitachi S-4800 scanning electron microscope.

[0065] pass Figure 18 It can be observed that Pseudomonas aeruginosa that has not been treated with 3KF and 2KF... P. aeruginosaPAO1 cells exhibit a complete and smooth cell membrane structure, with the inner and outer membranes tightly adhered and intracellular material evenly distributed. In contrast, after 1 hour of treatment with 3KF and 2KF, the cell surface of the bacteria showed significant shrinkage, the integrity of the cell membrane was disrupted, leading to leakage of intracellular material.

Claims

1. Nanofiber-based peptides 3KF and 2KF for combating Pseudomonas aeruginosa, characterized in that: Its amino acid sequence is shown in SEQ ID No. 1 and SEQ ID No.

2. Its C-terminus is linked to the Pseudomonas aeruginosa membrane protein recognition sequence SQRKLAAKLTSK via the flexible linker GSGS, so as to achieve the killing of Pseudomonas aeruginosa.

2. The self-assembly method of nanofiber-trapping peptides 3KF and 2KF against Pseudomonas aeruginosa according to claim 1, characterized in that, The self-assembly conditions are as follows: the concentration of the captured peptide is >16 μM, and it is incubated at 37°C for 24 hours in a phosphate buffer environment.

3. The method for preparing nanofiber-trapping peptides 3KF and 2KF against Pseudomonas aeruginosa according to claim 1, characterized in that, The steps are as follows: S1: A nanofiber scaffold is formed by using a repeating sequence of glutamine and leucine, and adjacent phenylalanine and lysine to provide positive charge and hydrophobic interaction. The scaffold is then connected to the sequence SQRKLAAKLTSK via a flexible linker GSGS, and the amino acid sequence of the capture peptide is finally obtained as shown in SEQ ID No. 1 and SEQ ID No.

2. S2: The capture peptides were prepared by solid-phase chemical synthesis and mass spectrometry identification. The nanomorphic morphology, secondary structure, hemolytic activity, in vitro cytotoxicity, capture ability, bactericidal activity, outer membrane permeability, plasma membrane potential, and bacterial morphology and membrane structure were then measured and the capture peptides were finally named 3KF and 2KF.

4. The use of the nanofiber capturing peptides 3KF and 2KF against Pseudomonas aeruginosa as described in claim 1 in the preparation of medicaments for treating diseases caused by Pseudomonas aeruginosa infections.

Images