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Antibacterial peptide for improving permeability of outer membrane of gram-negative bacteria

A technology of antimicrobial peptides and negative bacteria, applied in the field of biotechnology and molecular biology, can solve the problem of no effective drugs for negative bacteria, and achieve the effect of improving permeability

Pending Publication Date: 2022-05-13
NANJING AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In addition, the research and development of antibiotics against Gram-negative bacteria is also at a bottleneck. In 2017, GlaxoSmithKline screened more than 500,000 compounds and found no effective drugs against Gram-negative bacteria.

Method used

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  • Antibacterial peptide for improving permeability of outer membrane of gram-negative bacteria
  • Antibacterial peptide for improving permeability of outer membrane of gram-negative bacteria
  • Antibacterial peptide for improving permeability of outer membrane of gram-negative bacteria

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Design and Characterization of Plant Lactobacillus A Mutants

[0024] Based on the amino acid sequence of Lactobacillus plantarum A, the number and composition of its amino acids are increased and changed, and its spatial structure, hydrophobicity, charge and other characteristics are changed. The mutants and their traits are characterized in the following table 1:

[0025] Table 1 Physicochemical properties of plnA and its mutants

[0026] Antimicrobial Peptides sequence Hydrophobic amphipathic positive charge K43D QFKNISLMYGNNVSRDTLTNFFKSLIKKIN -0.357 constant 4 K50D QFKNISLMYGNNVSRKTLTNFFDSLIKKIN -0.357 constant 4 K55D QFKNISLMYGNNVSRKTLTNFFKSLIKDIN -0.357 constant 4 N38R QFKNISLMYGDNVSRKTLTNFFKSLIKKIN -0.37 Increase 5 R42A QFKNISLMYGNNVSAKTLTNFFKSLIKKIN -0.16 Increase 5 R42D QFKNISLMYGNNVSDKTLTNFFKSLIKKIN -0.337 constant 4 T44E QFKNISLMYGNNVSRKELTNFFKSLIKKIN -0.463 Increase ...

Embodiment 2

[0032] Escherichia coli outer membrane, inner membrane permeability detection

[0033] Permeability of the outer membrane was detected with N-phenyl-1-naphthylamine (NPN). Escherichia coli EHEC O157:H7 was cultured to mid-log phase, and adjusted to OD600=0.05 using HEPES buffer (5 mM, pH 7.2). Add different concentrations of phytolactin A or its analogs, and incubate at 37°C for 1 hour. Add 2 µL of NPN (final concentration 10 µM) to the mixture. The excitation and emission wavelengths of the NPN were set at 350 and 420 nm.

[0034] Small univalve vesicles (SUVs) were prepared by ultrasonography. 100 mg of lipid (DOPC:DOPE=1:1) (Sigma, USA) was dissolved in chloroform and evaporated to form a thin film at the bottom of the round bottom bottle. Multilamellar vesicles (MLVs) were formed by suspending in HEPES buffer (50 mM, pH 7.2, 70 mm calcium protein). Subsequently, they were ultrasonically examined until the liquid clearly formed an SUV. Finally, the SUV was purified by...

Embodiment 3

[0041] in vitro binding assay

[0042]The interaction between LPS and OP4 is monitored by HPLC as follows: Agilent bio-sec5 column (5 μ m, 7.8 mm × 300 mm); mobile phase is acetonitrile: water = 95: 5 (containing 0.1% (v / v) TFA); flow rate 1.5 mL / min; detection wavelength 220nm. The interaction between LPS and OP4 is monitored by HPLC as follows: Aligent Bio Sec-5 column (5 μm, 7.8mm×300mm); mobile phase is acetonitrile: water=95:5; flow rate 1.5 ml / min; detection wavelength 220nm.

[0043] The result is as Figure 4 Shown: the peak time of PlnA is 11.9 minutes. When the antimicrobial peptide is combined with lipopolysaccharide, the peak time is about 9.08 minutes. The results showed that PlnA could be combined with lipopolysaccharide, and its relative molecular mass became larger, which reduced its retention time. Among them, the binding efficiency of PlnA and lipopolysaccharide was 42.67%. The binding efficiency of mutants OP1, K50D, I32W to LPS was significantly lower ...

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Abstract

The invention discloses an antibacterial peptide for improving permeability of an outer membrane of gram-negative bacteria. The novel antibacterial peptide is obtained by taking lactobacillus plantarum A as a template through methods of deleting an amino acid sequence of the lactobacillus plantarum A, changing amino acid composition, changing a space structure, changing charges and hydrophobicity and the like, the antibacterial peptide has relatively strong antibacterial activity, the MIC (Minimal Inhibitory Concentration) of the antibacterial peptide to escherichia coli is 3.125 mu g / mL, and the permeability of an outer membrane of the escherichia coli can be remarkably improved when the MIC is 0.78 mu g / mL; the antibacterial effect of hydrophobic antibiotics on negative bacteria is improved. According to the antibacterial peptide OP4 disclosed by the invention, the antibacterial peptide OP4 capable of effectively improving the permeability of the outer membrane of gram-negative bacteria is particularly obtained by modifying the lactobacillus plantarum A, and the antibacterial effect of erythromycin can be improved by 62.5 times. The invention provides a new potential way for treating gram-negative bacterium infection.

Description

technical field [0001] The invention belongs to the fields of biotechnology and molecular biology, and specifically relates to the design and antibacterial mechanism of antibacterial peptides. Background technique [0002] Gram-negative bacteria and Escherichia coli and Salmonella are key contributors to food poisoning and foodborne illness outbreaks. It is also an important pathogen of gastrointestinal diseases and bacterial infections. Gram-negative bacteria have a dense outer membrane composed of lipopolysaccharides and phospholipids. Because of the asymmetry of the membrane, many hydrophobic antibiotics cannot enter the cell and play a bactericidal effect. The outer membrane of Gram-negative bacteria thus confers inherent antibiotic resistance. Because of the long-term and widespread use of antibiotics, Gram-negative bacteria have acquired a variety of antibiotic resistance genes, so there are fewer and fewer antibiotics that can effectively treat negative bacterial i...

Claims

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Application Information

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IPC IPC(8): C07K14/335A61K38/16A61P31/04
CPCC07K14/335A61P31/04A61K38/00
Inventor 孟凡强陆兆新吕凤霞别小妹
Owner NANJING AGRICULTURAL UNIVERSITY