A LPS targeting peptide based on specific anti-Gram-negative bacteria and its application

Abstract

The present invention discloses a 16-amino acid polypeptide AA16 extracted from the LPS-binding protein CD14. 13 derivative polypeptides are obtained by amino acid replacement to increase α-helicity, improve amphiphilicity, and change the number of positive charges and hydrophobicity, thereby obtaining a targeting peptide with specific resistance to Gram-negative bacteria. Among them, the derivative polypeptide AA16-10R is specifically resistant to Gram-negative bacteria, with minimum inhibitory concentrations of 32 μg / mL and 16 μg / mL for Pseudomonas aeruginosa and Escherichia coli, respectively. It can bind to LPS on the bacterial membrane in the form of an α-helix, increase membrane permeability, and ultimately cause bacterial death, thereby having a good antibacterial effect.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to an LPS targeting peptide based on specific resistance to Gram-negative bacteria and an application thereof. Background Art

[0002] Gram-negative bacteria infection can cause nausea, vomiting, diarrhea, skin diseases, and even be fatal in severe cases. While there are many drugs that treat Gram-positive bacteria alone, such as theoxazolidinone family and daptomycin, there are very few drugs that treat Gram-negative bacteria. Currently, there are only three AMPs (one FDA-approved, Colistin or polymyxin E (natural source), two under development, Pexiganan and MSI-78 (engineered), and LL-37 (natural source), and three immunomodulatory peptides (AB103 (D-ala-p2TA) (engineered), Dusquetide (engineered), and EA-230 (engineered)) that can be used to treat Gram-negative infections. Therefore, exploring new methods to treat Gram-negative bacteria is of great practical significance. Summary of the Invention

[0003] In view of the prior art, the object of the present invention is to provide an LPS targeting peptide that is specific against Gram-negative bacteria and can bind to LPS of Gram-negative bacteria to exert an antibacterial effect.

[0004] The characteristics of the polypeptide AA16 and its 13 derived polypeptides of the present invention are:

[0005] The polypeptide AA16 is a 16-amino acid stretch with three positive charges and good α-helicity, derived from A79-L94 of the Gram-negative bacterial LPS-binding protein CD14. The amino acid sequence of the polypeptide AA16 is SEQ ID NO. 1: acetylation - Ala Ala Arg Ile Pro Ser Arg Ile Leu PheGly Ala Leu Arg Val Leu- amidation. The present invention also includes 13 derivative polypeptides of the polypeptide AA16, each of which is obtained by replacing amino acids on the polypeptide AA16, including AA16-5L, AA16-5F, and AA16-11F obtained by changing the hydrophobicity.

[0006] The amino acid sequence of the derivative polypeptide AA16-5L, SEQ ID NO. 2, is: Ala Ala Arg Ile LeuSerArg Ile Leu Phe Gly Ala Leu ArgVal Leu;

[0007] The amino acid sequence of the derived polypeptide AA16-5F, SEQ ID NO. 3, is: Ala Ala Arg Ile PheSer ArgIle Leu Phe Gly Ala Leu ArgVal Leu;

[0008] The amino acid sequence of the derived polypeptide AA16-11F is SEQ ID NO.4: Ala Ala Arg Ile Pro Ser ArgIle LeuPhe Phe Ala Leu Arg Val Leu; the positive charge is changed to obtain AA16-11R, AA16-10K, and AA16-10R;

[0009] The amino acid sequence of the derived polypeptide AA16-11R, SEQ ID NO. 5, is: Ala Ala Arg Ile Pro Ser ArgIle LeuPhe Arg Ala Leu Arg Val Leu;

[0010] The amino acid sequence of the derived polypeptide AA16-10K, SEQ ID NO. 6, is: Ala Ala Arg Ile Pro Ser ArgIle LeuLys Gly Ala Leu Arg Val Leu;

[0011] The amino acid sequence of the derived polypeptide AA16-10R, SEQ ID NO. 7, is: Ala Ala Arg Ile Pro Ser ArgIle LeuArg Gly Ala Leu Arg Val Leu;

[0012] Increasing the number of positive charges while increasing the α-helicity resulted in AA16-5L10R, AA16-5F10R, AA16-5L10S, and AA16-5L10R11R;

[0013] The amino acid sequence of the derived polypeptide AA16-5L10R, SEQ ID NO. 8, is: Ala Ala Arg Ile LeuSerArg Ile Leu Arg Gly Ala Leu ArgVal Leu;

[0014] The amino acid sequence of the derived polypeptide AA16-5F10R, SEQ ID NO. 9, is: Ala Ala Arg Ile PheSerArg Ile Leu Arg Gly Ala Leu ArgVal Leu;

[0015] The amino acid sequence of the derived polypeptide AA16-5L10S, SEQ ID NO. 10, is: Ala Ala Arg Ile LeuSerArg Ile Leu Ser Gly Ala Leu ArgVal Leu;

[0016] The amino acid sequence of the derived polypeptide AA16-5L10R11R, SEQ ID NO. 11, is: Ala Ala Arg Ile LeuSer Arg Ile Leu Arg Arg AlaLeu Arg Val Leu;

[0017] By increasing the number of positive charges and improving α-helicity while enhancing hydrophobicity, AA16-3K5L7K10K14K, AA16-5L10R11F, and AA16-5L10R11F12F were obtained;

[0018] The amino acid sequence of the derived polypeptide AA16-3K5L7K10K14K, SEQ ID NO. 12, is: Ala Ala Lys IleLeu Ser Lys Ile Leu Lys Gly Ala Leu Lys Val Leu;

[0019] The amino acid sequence of the derived polypeptide AA16-5L10R11F, SEQ ID NO. 13, is: Ala Ala Arg Ile LeuSer Arg Ile Leu Arg Phe AlaLeu Arg Val Leu;

[0020] The amino acid sequence of the derivative polypeptide AA16-5L10R11F12F, SEQ ID NO. 14, is: Ala Ala Arg IleLeu Ser Arg Ile Leu Arg Phe PheLeu Arg Val Leu.

[0021] The polypeptide AA16 according to the present invention exhibits antibacterial activity against both Gram-positive and Gram-negative bacteria, but the activity is not high, the hemolytic toxicity is negligible, it has three positive charges, an α-helix secondary structure, and a helicity of 34.8%.

[0022] The 13 derived peptides carried 3-5 positive charges and all had α-helical secondary structures.

[0023] Among them, AA16-10R can exert specific anti-Gram-negative bacteria effects, with minimum inhibitory concentrations of 32 μg / mL and 16 μg / mL against P. aeruginosa and E. coli, respectively, but the minimum inhibitory concentration against Gram-positive bacteria is greater than 128 μg / mL.

[0024] Among them, the derivative peptide AA16-10R can further exert its specific anti-Gram-negative bacteria effect by binding to LPS. Its good antibacterial activity against Gram-negative bacteria is due to its stronger penetration ability into the outer membrane of the bacterial cell wall.

[0025] The present invention modifies the amino acid sequence of a 16-amino acid polypeptide intercepted from CD14 protein A79-L94, effectively improving the hydrophobicity, positive charge and helicity, so that the resulting modified peptide has good LPS neutralization activity and low hemolysis. It can be used as a new antibacterial agent with an extremely high safety range and has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In order to make the contents of the present invention more clearly understood, the present invention is further described in detail below based on specific embodiments of the present invention in conjunction with the accompanying drawings.

[0027] Figure 1 Shown are the physicochemical properties and helical wheel diagrams of AA16 and its 13 derivative polypeptides of the present invention.

[0028] Figure 2 Shown are the CD spectra of AA16 and its 13 derivative polypeptides of the present invention.

[0029] Figure 3 The results show that AA16-10R of the present invention binds to LPS.

[0030] Figure 4 The results show the live-dead staining and inner and outer membrane binding of AA16-10R of the present invention.

[0031] Figure 5 The present invention is evaluated for the effect of AA16-10R on promoting wound healing in E. coli infected mouse epidermis. DETAILED DESCRIPTION

[0032] The present invention will be further described below with reference to the accompanying drawings and specific embodiments. It should be noted that the embodiments of the present application and the features therein may be combined with each other without conflict.

[0033] LPS is a major pathogenic factor in Gram-negative bacteria and can also trigger a series of inflammatory responses in the body. This invention primarily fragments CD14, a protein involved in inflammatory responses and also a receptor for LPS, to directly obtain polypeptide fragments that retain the ability to bind to LPS while also exhibiting antimicrobial activity.

[0034] Methods for obtaining AA16 and its 13 derivative polypeptides of the present invention:

[0035] (a) A 16-amino acid peptide AA16 was extracted from A79-L94 of the CD14 protein. The amino acid sequence is AARIPSRILFGALRVL, and its helical structure has an amphipathic distribution of natural cationic and hydrophobic amino acids.

[0036] (b) The peptide AA16 was further designed and modified to obtain 13 derivative peptides.

[0037] Example 1: Design and modification of polypeptide AA16 and its 13 derivative polypeptides

[0038] The transformation includes

[0039] By changing the hydrophobicity, we obtained AA16-5L, AA16-5F, and AA16-11F;

[0040] By changing the positive charge, we get AA16-11R, AA16-10K, and AA16-10R;

[0041] Increasing the number of positive charges while increasing the α-helicity resulted in AA16-5L10R, AA16-5F10R, AA16-5L10S, and AA16-5L10R11R;

[0042] While increasing the number of positive charges and improving the α-helicity, the hydrophobicity was enhanced to obtain AA16-3K5L7K10K14K, AA16-5L10R11F, and AA16-5L10R11F12F.

[0043] like Figure 1 Shown are the physicochemical properties and helical wheel diagrams of 13 derived polypeptides after modification of polypeptide AA16 according to the present invention.

[0044] Example 2: Antibacterial and hemolytic activity experiments of polypeptide AA16 and its 13 derivative polypeptides

[0045] The antibacterial activity of AA16 and its 13 derived peptides was tested against Staphylococcus aureus (NCTC10788), methicillin-resistant Staphylococcus aureus (ATCC43300), Escherichia coli (NCTC10418), and Pseudomonas aeruginosa (ATCC27853) strains.

[0046] The results of antibacterial activity (MIC) and hemolytic activity (HC50) are shown in Table 1, respectively.

[0047] Table 1: Antibacterial activity (MIC) and hemolytic activity (HC50) of peptide AA16 and its 13 derivative peptides against standard strains

[0048]

[0049] Example 3: Binding of AA16-10R to LPS

[0050] like Figure 3 The derivative polypeptide AA16-10R of the present invention binds to LPS.

[0051] (A) Effect of LPS at MIC concentration on the antibacterial activity of AA16-10R against Staphylococcus aureus. The control group was a group without peptide.

[0052] (B) CD spectra of AA16-10R100 in aqueous and LPS50 environments;

[0053] (C) Neutralization of LPS by AA16-10R was determined by LAL.

[0054] (D) AA16-10R depolarizes the plasma membrane of E. coli and E. coli spheroids.

[0055] Example 4: Live-dead staining and inner and outer membrane binding

[0056] like Figure 4 The antibacterial mechanism of the derivative peptide AA16-10R is shown. (A) Fluorescence microscopy shows the effect of AA16-10R on the cell membrane integrity of Pseudomonas aeruginosa. Green fluorescence indicates the number of viable bacteria, while red fluorescence indicates bacteria with membrane damage. (B) AA16-10R depolarizes the cytoplasmic membrane of Escherichia coli and Staphylococcus aureus. (C) Permeability of AA16 and AA16-10R to the outer membrane of Pseudomonas aeruginosa.

[0057] Example 5: AA16-10R promotes wound healing in E. coli-infected mouse epidermis

[0058] like Figure 5 Evaluation of the indicated derivative peptide, AA16-10R, on wound healing in mouse epidermis infected with E. coli. (A) Evaluation of the effects of AA16-10R (20 μg and 40 μg doses) on E. coli-infected mouse skin; (B) Evaluation of wound healing with AA16-10R; (C) Evaluation of the therapeutic efficacy of AA16-10R by viable bacterial counts.

[0059] Obviously, those skilled in the art may make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if such changes and modifications fall within the scope of the claims and their equivalents, the present invention is intended to include such changes and modifications.

Claims

1. An LPS targeting peptide AA16-10R based on specific anti-Gram-negative bacteria, characterized by: The amino acid sequence of AA16-10R, SEQ ID NO.7, is: Acetylation - Ala Ala Arg Ile Pro Ser Arg Ile LeuArg Gly Ala Leu Arg Val Leu - Amidation.

2. Use of the LPS targeting peptide AA16-10R based on specific anti-Gram-negative bacteria according to claim 1 in the preparation of an antibacterial agent for use against P. aeruginosa and E. coli.

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