抗菌肽CRAMP的安全性、稳定性及其在清除铜绿假单胞菌生物被膜中的作用

doi:10.11843/j.issn.0366-6964.2022.05.025

Abstract

Abstract: This study aimed to evaluate the safety and stability of the mouse-derived antimicrobial peptide CRAMP and its role in eradicating Pseudomonas aeruginosa biofilms. The hemolysis of CRAMP against rabbit red blood cells and the cytotoxicity against murine macrophage cells (RAW264.7.) were determined. The effects of temperature, proteases, metal ions, and pH on the antimicrobial activity of the CRAMP, and the stability of the CRAMP were investigated. The mature biofilm of P. aeruginosa (PAO1 strain) was constructed in the 6-well cell culture plate, and the human antimicrobial peptide LL-37 and three kinds of antibacterial drugs were used as controls. The biofilm biomass was detected by the crystal violet staining method. The number of viable bacteria in biofilms was counted, and the extracellular polysaccharide content of the biofilm was detected by the Phenol-sulfuric acid and Foline-phenol reagent method, and the morphological changes of the PAO1 biofilm were observed by the laser scanning confocal microscope (CLSM). The results showed that the hemolysis rate of CRAMP in rabbits was below 2% at the tested concentrations; The CRAMP had no cytotoxicity to RAW264.7 at 62.5-125 mg·L^-1. Temperature (25-100℃), two salt ions (Na⁺, K⁺), and pH value 5-10 do not affect the antibacterial activity of CRAMP; Pepsin, papain, and trypsin affected the antibacterial activity of CRAMP to varying degrees. In the 6-well cell culture plate, 4-fold MIC of the CRAMP extremely significantly reduced the PAO1 biofilm (decreased rate is 76.74%), and reduced the number of viable bacteria in the biofilm (reduced by 1.2 lg CFU·mL^-1), and the effect is better than that of the LL-37 and three kinds of antibacterial drugs. The extracellular polysaccharide of the CRAMP group was significantly lower than that of the blank control group and LL-37 group. CLSM results showed that:Compared with the control group, the total fluorescence intensity (PI+SYTO) of the bacteria in the CRAMP group was significantly lower than the blank control group and the LL-37 group at a 4-fold MIC concentration. Compared with the blank control group, the ratio of dead to living fluorescence intensity (PI/SYTO) in the CRAMP group and LL-37 group was significantly increased. The above results suggest that the CRAMP has low hemolysis and cytotoxicity, and it has greatstability in addition to the unstable action of protease. The CRAMP has an obvious eradication effect on the mature biofilm of PAO1, and the effect is better than LL-37, which implies that the CRAMP has a good prospect for drug development.

Key words: biofilm, the mouse-derived antimicrobial peptide CRAMP, cytotoxicity, stability, CLSM

CLC Number:

LI Hui, XIONG Jing, MEI Cui, WANG Shiyuan, ZHOU Yang, LI Xiaofen, FU Guihua, ZHANG Yang, CHENG Peng, HE Yuzhang, CHEN Hongwei. The Safety and Stability of the Antimicrobial Peptide CRAMP and Its Role in Eradicating Pseudomonas aeruginosa Biofilms[J]. Acta Veterinaria et Zootechnica Sinica, 2022, 53(5): 1576-1586.

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The Safety and Stability of the Antimicrobial Peptide CRAMP and Its Role in Eradicating Pseudomonas aeruginosa Biofilms

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