A cd14 protein-derived peptide 10w sensitive to staphylococcus aureus and use thereof

Abstract

The application discloses a CD14 protein derivative peptide 10W sensitive to staphylococcus aureus and application thereof, and obtains a new alpha helix derivative peptide 10W by replacing amino acids based on a CD14 protein fragment, the minimum inhibitory concentration (MIC) of the peptide 10W to staphylococcus aureus is 20 μM, and the hemolysis rate is extremely low, the peptide 10W can be combined with the negatively charged phospholipid on the bacterial membrane, increase the membrane permeability, disturb the ion distribution inside and outside the membrane, affect the electron transfer of the respiratory chain on the membrane, and especially has a special antibacterial mode, the derivative peptide 10W forms an ion transporter structure on the liposome simulating the bacterial membrane, specifically mediates the transportation of chloride ions and other anions, and has ion selectivity on the basis of the traditional hole model of the antibacterial peptide.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to a CD14 protein-derived peptide 10W sensitive to Staphylococcus aureus and its applications. Background Technology

[0002] Staphylococcus aureus is commonly found in skin wound infections and can cause serious diseases such as endocarditis, pneumonia, and sepsis, once posing a significant challenge in clinical medicine. Against the backdrop of resistance to traditional antibiotics and adverse reactions from combined antibiotic use, antimicrobial peptides have attracted widespread attention as novel antimicrobial agents. In recent years, obtaining protein binding site sequence fragments or their modified peptides through molecular cloning or chemical synthesis has become a research hotspot. Summary of the Invention

[0003] In view of the prior art, the object of the present invention is to provide a derived peptide 10W with ion transporter activity and to exert an antibacterial effect against Staphylococcus aureus.

[0004] To achieve the aforementioned objectives, the present invention provides a technical solution: a CD14 protein-derived peptide 10W sensitive to Staphylococcus aureus, wherein the amino acid sequence of the derived peptide 10W is SEQ ID NO.1: AlaAlaArg IleProSerArgIleLeuTrpGlyAlaLeuArgValLeu-NH2. The derived peptide 10W is obtained by replacing amino acids of the original peptide obtained from the CD14 protein, wherein the amino acid sequence of the original peptide obtained from the CD14 protein is SEQ ID NO.2: AlaAlaArg Ile ProSerArg IleLeuPheGlyAlaLeuArgValLeu-NH2. Increasing the α-helix degree and hydrophobicity of the peptide without changing its positive charge number is beneficial for its binding and insertion into the bacterial cell membrane, thus enhancing its antibacterial activity. The relatively long phenylalanine side chain at position 10 of the original amino acid sequence hinders the formation of the α-helix. Studies have shown that W plays an important role in the antibacterial selectivity of antimicrobial peptides. Therefore, without altering its hydrophobic surface, we replaced the 10-position phenylalanine (Phe, F) with tryptophan (Trp, W), resulting in the 10W sequence AARIPSRILWGALRVL. To improve the net charge and stability of the peptides, all designed antimicrobial peptides underwent C-terminal amidation modification.

[0005] The derivative peptide 10W of the present invention is characterized by:

[0006] (a) The amino acid sequence extracted from the CD14 protein is AlaAlaArgIle Pro SerArgIle Leu Phe GlyAlaLeuArgValLeu-NH2;

[0007] (b) By substituting one amino acid in the amino acid sequence of (a), a polypeptide derived from (a) that is sensitive to Staphylococcus aureus and has low hemolytic activity was obtained. The amino acid sequence is AlaAlaArg IlePro SerArg IleLeuTrp GlyAlaLeuArgValLeu-NH2. The derivative peptide 10W carries 3 positive charges and has an α-helix secondary structure with a helicity of 85.12%.

[0008] This invention modifies the amino acid sequence of the CD14 protein, effectively improving its physicochemical properties such as hydrophobicity and helicity. The resulting modified peptide exhibits excellent antibacterial activity and low hemolytic activity, making it a novel antibacterial agent with a very high safety range and narrow spectrum against Staphylococcus aureus, showing promising application prospects.

[0009] This invention investigates the therapeutic effect of the derived peptide 10W on Staphylococcus aureus infection at the mouse animal level.

[0010] The results showed that after treatment with Derivative Peptide 10W, the number of bacteria at the epidermal infection site in mice decreased significantly, and the wound area decreased and recovered, indicating that Derivative Peptide 10W can effectively treat skin infections caused by Staphylococcus aureus.

[0011] This invention provides that the derived peptide 10W increases the permeability of Staphylococcus aureus membranes without causing membrane rupture, indicating that the derived peptide 10W may exert its antibacterial effect using a pore model.

[0012] This invention demonstrates that the derived peptide 10W can disrupt the proton motive force (PMF) on the bacterial membrane by detecting changes in ΔΨ and ΔpH on the bacterial membrane using a method of loading fluorescent probes.

[0013] Furthermore, the derived peptide 10W has an inhibitory effect on components related to the membrane electron respiratory chain and induces the accumulation of intracellular ROS in bacteria.

[0014] The present invention provides a 10W helical structure and membrane insertion properties of the derived peptide, which facilitates its application as an anion transporter on liposomes.

[0015] This invention identified the specific anion transport mediated by the derived peptide 10W on liposomes by constructing a liposome model simulating the cell membrane of Gram-positive bacteria, with a transport efficiency of Br. - >I - >Cl - SO4 2- .

[0016] This invention demonstrates that the derived peptide 10W cannot induce differential cation transport on liposomes.

[0017] This invention provides a concentration-dependent method for the transport of chloride ions on liposomes by a 10W derived peptide, and the direction thereof is Cl. - and OH - Reverse transport.

[0018] Based on the above-described solution and explanation, the present invention has at least the following advantages:

[0019] (1) This invention obtains a derived peptide 10W by replacing amino acids in a fragment of CD14 protein, which effectively improves the physicochemical properties such as hydrophobicity and helicity. This derived peptide 10W carries 3 positive charges, its secondary structure is an α-helix, and its helicity is 85.12%. Experimental verification has shown that it has good antibacterial activity and low hemolytic activity, and has a therapeutic effect on Staphylococcus aureus infection. It can effectively treat skin infections caused by Staphylococcus aureus.

[0020] (2) The 10W helical structure and membrane insertion properties of the derived peptide facilitate its application as an anion transporter on liposomes without causing differential cation transport on liposomes.

[0021] The above description is only an overview of the technical solution of the present invention. In order to better understand the technical means of the present invention, it can be implemented according to the contents of the specification to verify it. Attached Figure Description

[0022] To make the content of this invention easier to understand, the invention will be further described in detail below with reference to specific embodiments and accompanying drawings.

[0023] Figure 1 The spiral wheel diagram and CD spectrum of the derived peptide 10W of this invention are shown.

[0024] Figure 2 This demonstrates the in vivo antibacterial activity of the derived peptide 10W of the present invention.

[0025] Figure 3 This invention demonstrates that the derived peptide 10W affects bacterial membrane permeability.

[0026] Figure 4 This invention demonstrates how the derived peptide 10W disrupts the proton dynamic potential on the membrane.

[0027] Figure 5 This invention demonstrates the effect of its derived peptide 10W on the bacterial electronic respiratory chain.

[0028] Figure 6 Ion transport in a liposome model. Detailed Implementation

[0029] The present invention will be further described below with reference to the accompanying drawings and specific embodiments. It should be noted that, where there is no conflict, the embodiments and features in the embodiments of this application can be combined with each other.

[0030] The method for obtaining 10W of the derived peptides of this invention:

[0031] (a) The amino acid sequence extracted from the CD14 protein is AARIPSRILFGALRVL-NH2;

[0032] (b) Substitution of one amino acid in the amino acid sequence in (a) yields a polypeptide derived from (a) that is sensitive to Staphylococcus aureus and has low hemolytic activity, namely the derivative peptide 10W with the amino acid sequence AARIPSRILWGALRVL-NH2. The derivative peptide 10W carries three positive charges and has an α-helix secondary structure with a helicity of 85.12%.

[0033]

Example 1

[0034] The derived peptide 10W was subjected to antibacterial activity tests against Staphylococcus aureus (NCTC10788), methicillin-resistant Staphylococcus aureus (NCTC12493), methicillin-resistant Staphylococcus aureus (ATCC43300), Escherichia coli (NCTC10418), and Pseudomonas aeruginosa (ATCC27853). Antibacterial activity (MIC / MBC) and hemolytic activity (HC) were also tested. 50 The results are shown in Table 1.

[0035] Table 1. Antimicrobial activity (MIC / MBC) and hemolytic activity (HC) of the derived peptide 10W against standard strains 50 )

[0036]

[0037]

[0038] like Figure 1 The secondary structure of the derived peptide 10W is a typical α-helix. In vitro activity screening showed that the derived peptide 10W was more effective against Gram-positive bacteria than against Gram-negative bacteria. It inhibited the growth of Staphylococcus aureus and methicillin-resistant Staphylococcus aureus at a concentration of 20 μM, and exhibited bactericidal activity at a concentration of 25 μM. The derived peptide 10W showed extremely low cytotoxicity, with a hemolysis rate of less than 50% at 250 μM, i.e., HC... 50 >250μM.

[0039] [Example 2] Animal Experiment

[0040] Healthy male ICR mice aged 8 weeks were used as animals. Before infection, the mice were anesthetized with 4% chloral hydrate, and their backs were shaved. Silicone bands were sutured on both sides, and the epidermis of the inner band area was excised. A 1*10⁻⁶ concentration was used during the logarithmic growth phase. 8 After centrifugation, the bacterial solution (CFU / mL) was rinsed with normal saline, and 20 μL of the solution was applied to each wound for infection. Infection was continued for 3 days until the bacterial count in the wound stabilized. After successful establishment of the infection model, the control group was treated with 20 μL of normal saline daily, while the drug-treated group was treated with 10W of derived peptide dissolved in 20 μL of normal saline (administration concentrations of 20 μg / day and 40 μg / day). Healing was observed and wound bacterial counts were monitored.

[0041] Figure 2 The results showed that the number of bacteria in the wound was significantly reduced on the third day after medication, with the order of magnitude of the bacteria decreasing from 10 to 10. 8 -10 9 Dropped to 10 5 -10 4 (A) The wound is healing significantly, and the area has decreased to approximately 20 mm. 2 (B,C). In the group receiving 40μg daily, no bacteria were detected at the wound site on day 5, and the wound area decreased to 15mm. 2 The scabs fell off (D), and the mice's mental state improved. This indicates that the derived peptide 10W has a good therapeutic effect on Staphylococcus aureus infection of the mouse epidermis.

[0042] [Example 3] Staining of live and dead bacteria

[0043] Bacteria cultured to the logarithmic growth phase were diluted with physiological saline to a concentration of 1 x 10⁻⁶. 7 CFU / mL. 1 mL of bacterial culture was inoculated into each well of a sterile 12-well plate containing a prepared smear and treated with a derivatized peptide at 37°C for 1 h at 10 W. The bacteria were then collected, resuspended in physiological saline, and processed using Live / DeadBacLight. TM The kit was used for staining, with SYTO9 and PI added at a 1:1 (v / v) ratio. The mixture was incubated in the dark for 30 min, washed, and then slides were prepared. Fluorescence was detected using an inverted fluorescence microscope: SYTO9 dye (green fluorescence) was detected at 510–540 nm, and PI dye (red fluorescence) was detected at 620–650 nm.

[0044] Scanning electron microscopy (SEM) was used to observe the effects of the derived peptide 10W on bacteria.

[0045] Take the logarithmic phase bacterial culture, centrifuge at 8000 rpm for 10 min at 4℃, and dilute with PBS to a concentration of 1*10. 7CFU / mL, 1 mL of bacterial culture was inoculated into each well of a sterile 12-well plate, and peptide was added to achieve a final concentration of MIC. The plates were incubated on a shaker at 37°C and 200 rpm for 1 h, then centrifuged and washed twice with sterile PBS. The bacterial plaques were fixed with 2.5% glutaraldehyde solution, dehydrated with a gradient of 20%, 50%, 70%, 80%, 90%, and 100% ethanol, dried at room temperature, sputter-coated with gold, and micrographs were recorded using a scanning electron microscope.

[0046] like Figure 3 As shown in Figure A, the effect of derived peptide 10W on cell membrane integrity is observed under an inverted fluorescence microscope; green fluorescence represents the total number of bacteria, and red fluorescence represents bacteria with damaged membranes. Figure B shows the effect of derived peptide 10W on cell membrane morphology as observed by SEM. Figure C shows the effect of derived peptide 10W on the antibacterial activity when interacting with different membrane components PC, PG, PE, and LPS. It can be seen that at the MIC concentration, derived peptide 10W damages a large number of bacterial membranes, leading to increased bacterial uptake of PI, with a significantly larger red area compared to the control group. Scanning electron microscopy shows slight indentation on the bacterial membrane surface, largely preserving its original shape, and the membrane does not undergo carpet-like rupture. Therefore, it is speculated that derived peptide 10W may exert its antibacterial effect through a pore model. In addition, when different types of phospholipids PG, PC, PE and lipopolysaccharide LPS were added to the system in which the derived peptide 10W was co-incubated with Staphylococcus aureus, it was observed that as the PG concentration increased to 32 mg / L and 126 mg / L, the MIC value of the derived peptide 10W increased to 2 times and 4 times the original value, respectively. This means that the derived peptide 10W specifically interacts with the bacterial inner membrane component PG, leading to increased membrane permeability.

[0047] [Example 4] Detection of Staphylococcus aureus proton dynamics (PMF)

[0048] The membrane potential was detected using the fluorescent probe disc3(5). Staphylococcus aureus was cultured to the logarithmic phase, and the bacterial culture was centrifuged and reconstituted in 10 mL of 5 mM HEPES (containing 20 mM glucose and 100 mM KCl, pH = 7.2). 100 μL of the bacterial culture was inoculated into each well of a sterile 96-well plate, and 1 μL of disc3(5) was added to each well to a final concentration of 5 μM. The plate was quenched in the dark for 30 min, and the basic fluorescence was detected at 622 nm / 670 nm. Immediately after peptide addition, the fluorescence change in each well was detected at 622 nm / 670 nm for 10 min.

[0049] The fluorescent probe BCECFAM was used to detect ΔpH. The bacterial culture was centrifuged and resuspended in HEPES buffer containing 10 mM glucose. 1 mol / L BCECFAM fluorescent probe was added, and the mixture was incubated in a shaker at 37°C in the dark for 1 h. The culture was washed twice with HEPES buffer to remove unloaded fluorescent probe. 10 W of the derived peptide was added to bring the final concentrations to MIC and 2 MIC. The fluorescence value was immediately measured using a fluorometer at an excitation wavelength of 502 nm and an emission wavelength of 525 nm.

[0050] The results of the proton dynamic potential of Staphylococcus aureus are as follows: Figure 4 As shown in Figure A, the bacterial membrane potential was detected by the disc3(5) fluorescent probe, and Figure B shows the change in membrane pH detected by the BCECFAM probe. It can be seen that the derived peptide 10W can release disc3(5) from the membrane, increase fluorescence, and dissipate membrane potential; it can also reduce the concentration dependence of membrane pH and disrupt the proton kinetic potential.

[0051] [Example 5] Effect of 10W Derived Peptide on Bacterial Electron Respiratory Chain (ETC)

[0052] Determination of NADH content in bacteria

[0053] Add equal volumes of peptide solutions of different concentrations to a 96-well plate, adding 1*10 to each well. 8 200 μL of CFU / mL logarithmic growth phase bacterial culture was incubated with peptides at MIC and 2MIC concentrations on a shaker at 37°C and 200 rpm for 1 h. Then, under dark conditions, 2 mg / mL of resazurin was added to each well to bring the final concentration to 0.1 mg / mL. The fluorescent dye and bacterial culture were thoroughly mixed, and fluorescence changes were detected at 550 / 590 nm wavelengths.

[0054] RNA extraction and real-time quantitative polymerase chain reaction (RT-PCR)

[0055] The derived peptide 10W was incubated with Staphylococcus aureus under specified conditions, centrifuged at 4°C, and the precipitate was collected, repeatedly frozen and thawed in liquid nitrogen, and then resuspended in 1 mL TRIzol. Total RNA was extracted using chloroform extraction and isopropanol precipitation, and the obtained RNA was dissolved in enzyme-free water for later use. cDNA was synthesized using random primers and reverse transcriptase (Invitgen). RT-PCR was performed using SYBR II Green SuperMix (BioRed) to detect the expression of Staphylococcus aureus respiratory chain-related genes.

[0056] Detecting ROS accumulation

[0057] Incubate *Staphylococcus aureus* overnight for 10-12 hours until the culture becomes turbid. Take 500 μL of this culture and inoculate it into sterile MHB medium. Incubate at 37°C with a shaker until the exponential growth phase (OD = 0.5-0.55). Wash twice with sterile PBS solution and reconstitute in 5 mL of PBS. Add 10 μg / mL of the DCFH-DA probe. The blank control group does not contain DCFH-DA; the difference in fluorescence between the two groups indicates successful probe loading. After shaking at 37°C for 1 hour, wash twice with MHB and reconstitute in 5 mL of MHB. Then add 10W of the derived peptide (final concentration MIC / MBC), incubate at 37°C for 3 hours, and measure the fluorescence value using a microplate reader.

[0058] The results are as follows Figure 5 As shown, compared with the control group, the fluorescence intensity and NADH content in the drug-treated group were significantly reduced (A). NADH dehydrogenase II (encoded by ndh2) and cytochrome aa3 oxidase (encoded by qoxBCD) are important components of the Staphylococcus aureus respiratory chain, and ATPase (encoded by atpA) also promotes proton motive force (PMF) generation by pumping out protons during ATP hydrolysis. RT-PCR results showed that the expression of ndh2, qoxBCD, and atpA genes was significantly reduced in the drug-treated group compared with the control group, indicating that the derived peptide 10W can affect bacterial metabolic processes by inhibiting the gene expression of important respiratory chain enzyme components (B).

[0059] Influence on electron transport in the bacterial respiratory chain and inhibition of oxidative phosphorylation lead to the accumulation of intracellular ROS. Oxidative stress, in addition to causing bacterial death, also triggers a series of indirect reactions, such as lipid peroxidation and oxidative damage to DNA and RNA. Treatment with 10W of the derived peptide increased intracellular ROS levels in Staphylococcus aureus, and the accumulation of ROS increased with increasing peptide concentration (C).

[0060] [Example 6] Detection of ion transport in a liposome model

[0061] Liposomes of phospholipid POPC:POPG (4:1) were prepared by repeated freeze-thaw cycles.

[0062] cationic selectivity

[0063] The liposome contains approximately 0.1 mM HPTS buffer (HEPES 10 mM, NaCl 100 mM, pH = 7.2) internally and externally contains HEPES 10 mM, MCl (M n+ =Li + Na + ,K +100mM, pH=6.8. Mix 10μL of peptide solutions of different concentrations with 50μL of liposome solution, and bring the total volume to 2mL with HEPES buffer. Detect the fluorescence intensity (I1, E1) using a fluorescence spectrometer. X =454nm, Em=510nm, excitation slit width and emission slit width are both 10.0nm), and fluorescence changes were monitored. Then, the surfactant TritonX-100 (20μL, 20%, v / v) was added, causing liposome lysis and complete release of the internally encapsulated HPTS, resulting in the maximum fluorescence intensity (I2). Normalization was performed using the following formula. Only the type of cations on the exterior of the liposomes was changed, and the directional transport of cations was determined by the fluorescence changes of the system.

[0064] R(%)=(I1-I0) / (I2-I0)×100%

[0065] Anion selectivity

[0066] The liposomes contain approximately 0.1 mM HPTS buffer (HEPES 10 mM, NaCl 100 mM, pH = 7.2), while the external system consists of HEPES 10 mM and NaX (X = Cl... - ,Br - I - NO3 - SO4 2- 100 mM, pH = 6.8. Using the same system and method as described above, only the type of anion outside the system was changed, and fluorescence changes were detected.

[0067] like Figure 6 When the derived peptide 10W interacts with liposomes mimicking bacterial membranes, it maintains the α-helix structure, a condition necessary for the formation of ion channels within the bacterial membrane by the antimicrobial peptide (A). When the derived peptide 10W acts on single-compartment liposomes, the fluorescence changes are largely consistent with the addition of different cations to the system (B). Conversely, the derived peptide 10W exhibits selectivity for anion transport, with the highest transport efficiency for Br... - Secondly, I - and Cl - SO4 2- The efficiency was lowest (C). Chloride ions are the most abundant anion in cells, and the derived peptide 10W can induce chloride ion transport in liposomes in a concentration-dependent manner (D). Furthermore, FCCP was used to further clarify the transport direction of chloride ions. The results showed that the fluorescence intensity increased by 20% when FCCP, a hydrogen ion transporter, was added alone, indicating the presence of H+. + The fluorescence intensity remained essentially unchanged after the addition of FCCP and 10W of the derived peptide, suggesting that Cl- occurs in the system when the derived peptide 10W is present. - and OH- The opposing transport (E). The action model is shown in Figure (F).

[0068] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

[0069]

Claims

1. A CD14 protein-derived peptide 10W sensitive to Staphylococcus aureus, characterized in that: The amino acid sequence of the derived peptide 10W is shown in SEQ ID NO.

1. The derived peptide 10W is sensitive to Staphylococcus aureus and has low hemolytic activity. The derived peptide 10W is obtained by replacing the amino acids of the original peptide obtained from CD14 protein. The replacement method is to replace the phenylalanine (Phe, F) at position 10 of the amino acid sequence of the original peptide with tryptophan (Trp, W).

2. The application of the derived peptide 10W according to claim 1 in the preparation of a low-hemolytic antibacterial agent, characterized in that: The antibacterial agent is effective against Staphylococcus aureus NCTC10788, methicillin-resistant Staphylococcus aureus NCTC12493, methicillin-resistant Staphylococcus aureus ATCC43300, Escherichia coli NCTC10418, and Pseudomonas aeruginosa ATCC27853.

3. The application of the derived peptide 10W according to claim 1 in forming anion transporters on liposomes.

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