An antimicrobial peptide specifically targeting Gram-negative bacteria and its application

Abstract

The present invention provides an antibacterial peptide specifically targeting Gram-negative bacteria and its application. The antibacterial peptide specifically targeting Gram-negative bacteria has an amino acid sequence of: Lys-Trp-Lys-Leu-Phe-Lys-Lys-Ile-(X-Pro) n -Arg-Lys-Val-Arg-Gly-Pro-Pro-NH2, where (X-Pro) n is a linker. In the present invention, the truncated peptide Ce(1-8) of Cecropin A is used as the antibacterial domain, and its sequence is Lys-Trp-Lys-Leu-Phe-Lys-Lys-Ile. The 28-34th residues Lf(28-34) of human lactoferrin are used as the targeting domain, and its sequence is Arg-Lys-Val-Arg-Gly-Pro-Pro. Through a rigid linker, the two functional domains are covalently linked to obtain an antibacterial peptide specifically targeting Gram-negative bacteria, which is expected to become a new antibacterial drug for treating infections caused by Gram-negative bacteria and has good application prospects in clinical practice.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to an antimicrobial peptide specifically targeting Gram-negative bacteria and an application thereof. Background Art

[0002] Antibiotics play an important role in controlling and treating bacterial infections. However, the problem of antibiotic resistance caused by the irrational use of antibiotics not only affects the life and health of humans and animals, but also seriously threatens agricultural livelihoods and global food security, and has become a global public health and economic issue. Among all the problems of bacterial resistance, Gram-negative pathogens are particularly worrying. It has been reported that Gram-negative bacteria account for about 70% of clinically isolated resistant bacteria. The increasing rate of infections caused by multidrug-resistant Gram-negative bacteria has made treatment more and more complicated.

[0003] Antimicrobial peptides are a class of small molecule peptides widely found in nature and are an important component of the body's innate immune system. Antimicrobial peptides are safe, highly effective, have a unique mechanism of action, and are not prone to drug resistance, making them ideal candidates for the development of new antimicrobial agents. However, the indiscriminate inhibitory effect of broad-spectrum antimicrobial peptides on all microorganisms can easily cause an imbalance in the distribution of microbial flora. For example, melittin, as a conventional broad-spectrum antimicrobial peptide, is a polypeptide composed of 26 amino acid residues, and its amino acid sequence is Gly-Ile-Gly-Ala-Val-Leu-Lys-Val-Leu-Thr-Thr-Gly-Leu-Pro-Ala-Leu-Ile-Ser-Trp-Ile-Lys-Arg-Lys-Arg-Gln-Gln-NH 2 Melittin is the main active ingredient of bee venom, has a broad-spectrum antibacterial activity, and has a strong inhibitory effect on both Gram-positive and Gram-negative bacteria. However, low concentrations of melittin can cause high cytotoxicity, limiting its application. Therefore, the development of antimicrobial peptides that can specifically inhibit Gram-negative bacteria is of great clinical significance. Summary of the invention

[0004] In order to solve the problem that the broad-spectrum antimicrobial peptides in the prior art have an indiscriminate inhibitory effect on all microorganisms and are prone to cause an unbalanced distribution of microbial flora, the present invention provides an antimicrobial peptide specifically targeting Gram-negative bacteria and its application. The present invention uses the truncated peptide Ce (1-8) of CecropinA as the antimicrobial domain, whose sequence is Lys-Trp-Lys-Leu-Phe-Lys-Lys-Ile, and the 28-34 residues Lf (28-34) of human lactoferrin as the targeting domain, whose sequence is Arg-Lys-Val-Arg-Gly-Pro-Pro. The two functional domains are covalently linked by a rigid linker to obtain an antimicrobial peptide specifically targeting Gram-negative bacteria, which is expected to become a new type of antimicrobial drug for treating infections caused by Gram-negative bacteria and has good application prospects in clinical practice.

[0005] The technical solution adopted by the present invention is:

[0006] An antimicrobial peptide specifically targeting Gram-negative bacteria, whose amino acid sequence is: Lys-Trp-Lys-Leu-Phe-Lys-Lys-Ile-(X-Pro) n -Arg-Lys-Val-Arg-Gly-Pro-Pro-NH 2 , where the sequence Lys-Trp-Lys-Leu-Phe-Lys-Lys-Ile is the antibacterial domain, which is the truncated peptide Ce(1-8) of Cecropin A; the sequence Arg-Lys-Val-Arg-Gly-Pro-Pro is the targeting domain, which is the 28th to 34th residues Lf(28-34) of human lactoferrin; (X-Pro) n For linker.

[0007] Preferably, X is leucine Leu, and n=1-3.

[0008] The application of antimicrobial peptides that specifically target Gram-negative bacteria in the preparation of Gram-negative antimicrobial drugs.

[0009] Preferably, the Gram-negative bacteria include Escherichia coli, Pseudomonas aeruginosa, Pseudomonas fluorescens, Salmonella typhimurium, Salmonella enteritidis, Shigella sonnei and Shewanella putrefaciens.

[0010] Preferably, the minimum inhibitory concentration (MICs) of the antimicrobial peptide is in the range of 4-32 μM.

[0011] The preparation method of the above-mentioned antimicrobial peptide specifically targeting Gram-negative bacteria comprises the following steps: using the truncated peptide Ce (1-8) of CecropinA as the antimicrobial domain, the sequence of which is Lys-Trp-Lys-Leu-Phe-Lys-Lys-Ile, using the 28th to 34th residues Lf (28-34) of human lactoferrin as the targeting domain, the sequence of which is Arg-Lys-Val-Arg-Gly-Pro-Pro, and covalently linking the two functional domains through a rigid linker to obtain an antimicrobial peptide specifically targeting Gram-negative bacteria.

[0012] Beneficial Effects

[0013] (1) The antimicrobial peptide specifically targeting Gram-negative bacteria in the present invention can specifically inhibit Gram-negative bacteria, has no obvious activity against Gram-positive bacteria, and has low cytotoxicity. It is expected to become a new type of antimicrobial drug for treating infections caused by Gram-negative bacteria and has good application prospects in clinical practice.

[0014] (2) The antimicrobial peptide specifically targeting Gram-negative bacteria in the present invention has good thermal stability and still retains activity against Gram-negative bacteria after being treated at 80-100°C for 1 hour.

[0015] (3) The antimicrobial peptides specifically targeting Gram-negative bacteria in the present invention cause cell death of Gram-negative bacteria by destroying the integrity of bacterial cell membranes. This mechanism of action is not likely to induce drug resistance in bacteria.

[0016] (4) The antibacterial domain Ce (1-8) used in the present invention (sequence: Lys-Trp-Lys-Leu-Phe-Lys-Lys-Ile) has a moderate inhibitory effect on Gram-positive and Gram-negative bacteria. n The linker covalently connects it to the targeting domain Lf (28-34) (sequence: Arg-Lys-Val-Arg-Gly-Pro-Pro), and the anti-Gram-negative bacteria activity of the obtained antimicrobial peptides CL5-CL7 is significantly improved compared with the antimicrobial domain Ce (1-8), especially the antimicrobial peptide CL5, which is 2-16 times higher than the antimicrobial domain Ce (1-8). Importantly, the antimicrobial peptides CL5-CL7 in the present invention can selectively inhibit Gram-negative bacteria, but have no effect on Gram-positive bacteria, indicating that the antimicrobial peptides CL5-CL7 have both targeting and antibacterial functions, can achieve targeted inhibition of pathogens, and reduce drug side effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is the ESI-MS image of the antimicrobial peptide specifically targeting Gram-negative bacteria in Example 1;

[0018] Figure 2 This is the RP-HPLC chart of the antimicrobial peptide specifically targeting Gram-negative bacteria in Example 1;

[0019] Figure 3 This is a cytotoxicity graph of the antimicrobial peptide specifically targeting Gram-negative bacteria in Example 3;

[0020] Figure 4 This is the killing kinetics diagram of the antimicrobial peptide CL5 specifically targeting Gram-negative bacteria in Example 5;

[0021] Figure 5 These are scanning electron microscope images of E. coli ATCC25922 before and after treatment with the antimicrobial peptide CL5 specifically targeting Gram-negative bacteria in Example 6. DETAILED DESCRIPTION

[0022] The present invention is further described in detail below in conjunction with embodiments.

[0023] Example 1 Design and synthesis of antimicrobial peptides specifically targeting Gram-negative bacteria

[0024] The sequence KWKLFKKI was used as the antibacterial domain and the sequence RKVRGPP was used as the targeting domain. The two functional domains were covalently linked through a rigid linker, i.e., (LP)n, to obtain a group of hybrid antimicrobial peptides, as shown in Table 1.

[0025] Table 1 Amino acid sequences of antimicrobial peptides

[0026] Antimicrobial peptides sequence Molecular weight (Da) CL5 <![CDATA[KWKLFKKILPRKVRGPP-NH 2 ]]> 2090.64 CL6 <![CDATA[KWKLFKKILPLPRKVRGPP-NH 2 ]]> 2300.91 CL7 <![CDATA[KWKLFKKILPLPLPRKVRGPP-NH 2 ]]> 2511.19

[0027] The above antimicrobial peptides were commissioned to Shanghai Yaxian Chemical Co., Ltd. to be synthesized by solid phase synthesis. The C-terminus was amidated and the purity was greater than 95%. Afterwards, we verified the peptides CL5-CL7 by ESI-MS and RP-HPLC (see Figure 1 , Figure 2 ).

[0028] Example 2 Determination of Minimum Inhibitory Concentration (MIC) of Antimicrobial Peptides Specific for Gram-negative Bacteria

[0029] The detection steps are as follows:

[0030] 1) Dissolving the peptide: dissolving the lyophilized powder of the antimicrobial peptide specifically targeting Gram-negative bacteria in sterile deionized water to prepare a stock solution of the antimicrobial peptide specifically targeting Gram-negative bacteria with a concentration of 512 μM;

[0031] 2) Preparation of bacterial suspension: Culture the bacteria at 37°C and 180 rpm with shaking until the logarithmic growth phase, and dilute the bacterial suspension with Luria-Bertani (LB) liquid medium until the final concentration is (1×10 5)-(5×10 5 )CFU / mL;

[0032] 3) Add the peptide stock solution to a 96-well plate, dilute it by two-fold dilution method, and then inoculate the bacterial solution, with the group without antimicrobial peptide as the positive control and the group without bacteria as the negative control. Incubate the culture plate in a 37°C incubator for 20-24 hours, and measure the optical density (OD) value at 630nm by a microplate reader. The minimum inhibitory concentration is defined as the lowest concentration of the peptide that completely inhibits bacterial growth.

[0033] Table 2 Minimum inhibitory concentration of antimicrobial peptides

[0034]

[0035] As can be seen from Table 2, the antimicrobial peptides specifically targeting Gram-negative bacteria of the present invention can specifically inhibit Gram-negative bacteria, such as Escherichia coli (E. coli ATCC 25922, E. coli CMCC 44102), Pseudomonas aeruginosa (P. aeruginosa ATCC 27853, P. aeruginosa ATCC 9027, P. aeruginosa CMCC10104), Pseudomonas fluorescens (P. fluorescens BNCC 336632), Salmonella typhimurium (S. typhimurium ATCC 14028), Salmonella enteritidis (S. enteritidis CVCC 3375), Shigella sonnei (S. sonnei ATCC 25931), Shewanella putrefaciens (S. putrefaciens BNCC 336632), and Salmonella typhimurium (S. typhimurium ATCC 14028). 337021), MICs were 4-32μM; while it had no significant inhibitory effect on Gram-positive bacteria, such as Staphylococcus aureus (S.aureus ATCC 43300, S.aureus ATCC 25923) and Bacillus subtilis (B.subtilis ATCC 6633) (MICs>128μM).

[0036] Example 3 Cytotoxicity detection of antimicrobial peptides specifically targeting Gram-negative bacteria

[0037] The MTT method was used to determine the cytotoxicity of peptides to human embryonic kidney cells HEK-293. HEK-293 cells were plated at 10 4The cells were inoculated at a density of 100 μM in a 96-well plate and incubated at 37°C for 24 h. Then, antimicrobial peptides with a final concentration of 2-512 μM were added to the 96-well plate and incubated for another 24 h. After that, 20 μL MTT (5 mg / mL) was added to each well and cultured in the dark for 4 h. The formazan crystals formed were dissolved with 150 μL DMSO, and the optical density (OD) value at 490 nm was measured using a microplate reader. The group without antimicrobial peptides was used as the control group, and the group without inoculated cells was used as the blank group. The cell survival rate was calculated by the following formula: Cell survival rate (%) = [(treatment OD 490 -Blank OD 490 )]÷[(control OD 490 -Blank OD 490 )]×100.

[0038] from Figure 3 It can be seen that the antimicrobial peptides CL5-CL7 have low toxicity to HEK-293 cells, and at the highest tested concentration of 512 μM, the cell survival rate is above 60%.

[0039] Example 4 Thermal stability test of antimicrobial peptides specifically targeting Gram-negative bacteria

[0040] In order to evaluate the effect of heat on the antibacterial activity of the peptides, peptides CL5-CL7 were treated at different temperatures (60, 80, 100°C) for 60 min. After cooling to room temperature, the minimum inhibitory concentration of the peptides against E. coli ATCC 25922 was determined according to the method of Example 2.

[0041] The experimental results showed that the minimum inhibitory concentration of CL5-CL7 against E.coli ATCC25922 after treatment at different temperatures (60, 80, 100°C) was consistent with that of the untreated group, indicating that CL5-CL7 has good thermal stability.

[0042] Example 5 Bactericidal kinetics of antimicrobial peptides specifically targeting Gram-negative bacteria

[0043] Taking the antimicrobial peptide CL5 as an example, its killing kinetics against E. coli ATCC 25922 and P. aeruginosa ATCC 27853 was investigated. After the bacteria were cultured to the logarithmic phase, they were diluted to (1×10 5 )-(5×10 5 ) CFU / mL, and then peptide CL5 with a final concentration of 1× and 4× MIC was added to the bacterial dilution. The mixture was incubated at 37°C, and at each determined time point, 10 μL of the bacterial solution was diluted and 20 μL of the dilution was evenly spread on Luria-Bertani agar (LBA) medium, and the colonies were counted after incubation at 37°C for 20-24 hours.

[0044] The experimental results are as follows Figure 4 As shown in the figure, compared with the control group, CL5 at 4×MIC killed approximately 2-log and 7-log E. coli in 30min and 1h, respectively, and completely eliminated E. coli in 2h. At lower concentrations (1× or 2×MIC), approximately 3-log E. coli were killed after 1h of incubation, while significant growth was observed after 2h, indicating that CL5 was mainly inhibitory at lower concentrations. For Pseudomonas aeruginosa, CL5 showed bactericidal effects at 2× and 4×MIC, while it was mainly inhibitory at 1×MIC.

[0045] Example 6 Scanning electron microscope photos of E. coli ATCC 25922 before and after treatment with antimicrobial peptides specifically targeting Gram-negative bacteria

[0046] Taking the antimicrobial peptide CL5 as an example, its effect on the morphology of Escherichia coli was studied. The process is as follows:

[0047] 1) Preparation of bacteria: Escherichia coli in the logarithmic growth phase were centrifuged at 3,000 rpm for 5 min, the bacteria were collected, washed three times with 10 mM PBS (pH 7.4), and resuspended to OD 600 =0.2.

[0048] 2) Antimicrobial peptide treatment: Antimicrobial peptide CL5 was added to the above bacterial solution. The final concentration of CL5 was 1× and 4× MIC. The mixture was incubated at 37°C for 1 h. The untreated bacterial solution was used as a control.

[0049] 3) Sample fixation: The incubated bacterial solution was centrifuged at 3,000 rpm for 5 min, washed three times with PBS, fixed with 2.5% glutaraldehyde at 4°C overnight, centrifuged, and the supernatant was removed.

[0050] 4) Rinsing: After fixation, centrifuge the cells, wash three times with PBS buffer, each time for 15 minutes, then wash twice with pure water, centrifuge, and remove the supernatant.

[0051] 5) Dehydration: Dehydrate with 30%, 50%, 70%, and 90% ethanol in a gradient manner for 10-15 min each, and dehydrate with 100% ethanol twice, each for 15 min.

[0052] 6) Replacement: Replace with ethanol: tert-butanol (2:1, 1:1, v / v) or 100% tert-butanol once, each time for 10 min. Centrifuge and discard the supernatant after each dehydration step. Prepare tert-butanol bacterial suspension in the last step.

[0053] 7) Freeze drying: Add appropriate concentration of tert-butyl alcohol suspension onto aluminum foil, pre-cool at 80°C and then put into freeze dryer for drying.

[0054] 8) Machine testing: The samples were freeze-dried and then gold-plated, and observed using a Quanta 250FEG SEM (FEI, American) at 3.0 kV.

[0055] like Figure 5 As shown, the untreated E. coli cells (control group) are uniform, complete and plump. After treating E. coli with 1×MIC CL5 for 1 hour, the cell membranes of some bacteria became rough and shrunken. As the CL5 concentration increased to 4×MIC, the number of E. coli with abnormal morphology increased significantly, the degree of cell membrane damage became more obvious, and the E. coli showed obvious ruptures, collapses, and fragments.

[0056] Comparative Example 1

[0057] According to the literature Kim et al. (Kim, H., Jang, JH, Kim, SC, & Cho, JH (2016). Enhancement of the antimicrobial activity and selectivity of GNU7 against Gram-negativebacteria by fusion with LPS-targeting Peptides, 82, 60-66), using a flexible linker (Gly-Gly-Gly) to covalently link the targeting domain (Gly-Leu-Arg-Arg-Leu-Leu-Arg-Lys-Ile-Arg-Gly-Arg-Trp-Lys) and the antibacterial domain (Arg-Leu-Leu-Arg-Pro-Leu-Leu-Gln-Leu-Leu-Lys-Gln-Lys-Leu-Arg), and a hybrid peptide was also obtained. Although this hybrid peptide can effectively inhibit Gram-negative bacteria, it can also inhibit Gram-positive bacteria at the same time, and cannot specifically inhibit Gram-negative bacteria.

[0058] Comparative Example 2

[0059] The linker (X-Pro) in the antimicrobial peptide specifically targeting Gram-negative bacteria in this application n The X in is replaced by Lys, that is, the sequence is

[0060] Lys-Trp-Lys-Leu-Phe-Lys-Lys-Ile-(X-Pro) n -Arg-Lys-Val-Arg-Gly-Pro-Pro-NH 2 , among which (X-Pro) nis a linker, and X is Lys. When X is Lys, Lys provides a positive charge to the entire peptide sequence, destroying the balance of the antimicrobial peptide structural parameters, thereby reducing the activity of inhibiting Gram-negative bacteria.

[0061] Comparative Example 3

[0062] The linker (X-Pro) in the antimicrobial peptide specifically targeting Gram-negative bacteria in this application n The X in is replaced by Ala, that is, the sequence is

[0063] Lys-Trp-Lys-Leu-Phe-Lys-Lys-Ile-(X-Pro) n -Arg-Lys-Val-Arg-Gly-Pro-Pro-NH 2 , among which (X-Pro) n is a linker, and X is Ala. When X is Ala, due to the flexibility of Ala, the rigid conformation of the rigid linker is weakened, resulting in a decrease in the ability of the linker to separate the domains, and ultimately resulting in the impairment of the activity of inhibiting Gram-negative bacteria.

[0064] Comparative Example 4

[0065] The linker (X-Pro) in the antimicrobial peptide specifically targeting Gram-negative bacteria in this application n Remove, that is, the sequence is Lys-Trp-Lys-Leu-Phe-Lys-Lys-Ile-Arg-Lys-Val-Arg-Gly-Pro-Pro-NH 2 The direct hybridization of the antibacterial domain and the targeting domain leads to misfolding of the polypeptide, thus affecting the respective functions of the functional domains, and ultimately resulting in impaired activity in inhibiting Gram-negative bacteria.

[0066] Comparative Example 5

[0067] The linker (X-Pro)n in the antimicrobial peptide specifically targeting Gram-negative bacteria in the present application is replaced with the linker Gly-Gly-Gly-Gly-Gly-Ser, that is, the sequence is Lys-Trp-Lys-Leu-Phe-Lys-Lys-Ile-Gly-Gly-Gly-Gly-Ser-Arg-Lys-Val-Arg-Gly-Pro-Pro-NH2, wherein Gly-Gly-Gly-Gly-Gly-Ser is a flexible linker. Since the flexible linker allows certain interactions between functional domains, the functional domains cannot be efficiently separated, resulting in mutual interference between the functional domains, which weakens the activity of inhibiting Gram-negative bacteria.

[0068] Although the present invention has been described in detail in the embodiments through general explanations, specific implementation methods and experiments, modifications or improvements can still be made without departing from the core of the present invention, and all belong to the scope of protection required by the present invention.

Claims

1. An antimicrobial peptide that specifically targets Gram-negative bacteria, Features: Its amino acid sequence is: Lys-Trp-Lys-Leu-Phe-Lys-Lys-Ile-(X-Pro) n -Arg-Lys-Val-Arg-Gly-Pro-Pro-NH 2 , where the sequence Lys-Trp-Lys-Leu-Phe-Lys-Lys-Ile is the antibacterial domain, which is the truncated peptide Ce(1-8) of Cecropin A; the sequence Arg-Lys-Val-Arg-Gly-Pro-Pro is the targeting domain, which is the 28th to 34th residues Lf(28-34) of human lactoferrin; (X-Pro) n For linker; The X is leucine Leu, and n=1-3.

2. The use of the antimicrobial peptide specifically targeting Gram-negative bacteria according to claim 1, Features: The antimicrobial peptide is used in the preparation of antimicrobial drugs for Gram-negative bacteria.

3. The use of the antimicrobial peptide specifically targeting Gram-negative bacteria according to claim 2, Features: The Gram-negative bacteria include Escherichia coli, Pseudomonas aeruginosa, Pseudomonas fluorescens, Salmonella typhimurium, Salmonella enteritidis, Shigella sonnei and Shewanella putrefaciens.

4. Use of the antimicrobial peptide specifically targeting Gram-negative bacteria according to claim 2, Features: The minimum inhibitory concentration (MICs) of the antimicrobial peptides ranged from 4 to 32 μM.

5. The method for preparing an antimicrobial peptide specifically targeting Gram-negative bacteria according to claim 1, It is characterized in that The method comprises the following steps: using the truncated peptide Ce (1-8) of Cecropin A as the antibacterial domain with the sequence of Lys-Trp-Lys-Leu-Phe-Lys-Lys-Ile, using the 28th to 34th residues of human lactoferrin Lf (28-34) as the targeting domain with the sequence of Arg-Lys-Val-Arg-Gly-Pro-Pro, and covalently connecting the two functional domains through a rigid linker to obtain an antibacterial peptide specifically targeting Gram-negative bacteria.

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