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

摘要/Abstract

摘要： 旨在考察鼠源抗菌肽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。采用兔血红细胞悬液的溶血性和小鼠腹腔巨噬细胞(RAW264.7)的细胞毒性考察CRAMP的安全性；采用不同温度、蛋白酶、金属离子和酸碱梯度对CRAMP抗菌活性的影响，考察其稳定性；通过在6孔细胞培养板中构建铜绿假单胞菌(PAO1株)成熟生物被膜，以人源抗菌肽LL-37和3种抗菌药为对照，采用结晶紫染色法检测生物被膜量，平板菌落计数法对生物被膜活菌数进行计数，苯酚-硫酸法结合Folin-酚试剂法检测生物被膜胞外基质含量，激光扫描共聚焦显微镜(CLSM)观察CRAMP干预PAO1生物被膜形态学的变化。结果显示，CRAMP在所测试浓度下对兔血红细胞溶血率均<2%；在62.5~125 mg·L^-1时对 RAW264.7没有细胞毒性。温度(25~100 ℃)、两种盐离子(Na⁺、K⁺)以及pH为5~10时对CRAMP的抗菌活性没有影响；胃蛋白酶、木瓜蛋白酶、胰蛋白酶均不同程度影响了CRAMP的抗菌活性。6孔细胞培养板中，4倍MIC 的CRAMP极显著降低了PAO1生物被膜量(减少率为76.74%，P<0.01)，极显著减少了生物被膜中的活菌数量(减少了1.2个CFU·mL^-1，P<0.01)，效果优于LL-37和3种抗菌药。CRAMP组的胞外基质显著低于空白对照组和LL-37组(P<0.05)。CLSM结果显示：与对照组相比较，4MIC浓度下，CRAMP组的细菌总荧光强度(PI+SYTO)显著低于空白对照组和LL-37组(P<0.05)；与空白对照组相比，CRAMP组和LL-37组的死菌荧光强度/活菌荧光强度比值 (PI/SYTO)均极显著增大(P<0.01)。综上表明，CRAMP具有低溶血性、低细胞毒性，除蛋白酶作用不稳定外，具有较好的稳定性，对PAO1成熟生物被膜具有明显的清除作用，且效果优于LL-37，具有良好的药物开发前景。

关键词: 生物被膜, 鼠源抗菌肽CRAMP, 细胞毒性, 稳定性, 激光扫描共聚焦显微镜

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

中图分类号:

李会, 熊静, 梅翠, 王士源, 周洋, 李晓芬, 付贵花, 张阳, 程鹏, 何玉张, 陈红伟. 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[J]. 畜牧兽医学报, 2022, 53(5): 1576-1586.

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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