南方医科大学学报

南方医科大学学报 ›› 2021, Vol. 41 ›› Issue (6): 923-930.doi: 10.12122/j.issn.1673-4254.2021.06.16

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新型抗菌肽KR-1的设计、筛选及抗菌活性评价

何佳宁，梁东生，梁悦娥，左诗雅，赵望泓   

  • 出版日期:2021-06-20 发布日期:2021-07-05

Design, screening and antibacterial activity evaluation of the novel antibacterial peptide KR-1

  • Online:2021-06-20 Published:2021-07-05

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摘要： 目的 构建高活性、低毒性的新型抗菌肽，对其抗变异链球菌及其他口腔致病菌的活性进行评价，探讨其在防治龋病中的潜在应用价值。方法 以天然抗菌肽Histatins5的类似物Dhvar4为模版合成新型抗菌肽KR-1、KR-2。通过所设计抗菌肽对变异链球菌的最小抑菌浓度（MIC）实验，兔血红细胞溶血试验以及CCK-8法，筛选出高活性、低毒性的目标抗菌肽；使用96孔板培养变异链球菌生物膜，分为实验组（用浓度梯度为0.6×、0.8×、1×、2×MICs的KR-1进行处理）与阳性对照组（用浓度梯度为0.6×、0.8×、1×、2×MICs的洗必泰进行处理），通过结晶紫染色定量法观察生物膜量的改变；使用10×MIC浓度KR-1作用于变异链球菌生物膜后通过激光共聚焦显微镜观察结构的改变；通过KR-1作用口腔链球菌后杀菌所需的时间探讨目标抗菌肽对口腔链球菌的抗菌效率。结果 实验测得KR-1及KR-2的MIC分别为3.2 μmol/L和12.8 μmol/L，有效浓度下，兔血红细胞溶血率分别为0.35%和48.8%，24 h牙龈成纤维细胞存活率分别为72.96 %和21.94 %，将活性较高、生物相容性较好的KR-1作为目标抗菌肽；经结晶紫染色后测定KR-1的50%生物膜最小抑制浓度（MBIC50）低于1.92 μmol/L，且浓度为2.56 μmol/L时效果优于阳性对照组洗必泰（P=0.001），共聚焦显微镜下观察可见经KR-1处理后生物膜结构疏松；KR-1在5 min内可杀灭约90%细菌。结论 KR-1抗菌肽具有较强的抗菌活性和较低的毒性，且有良好的抗变异链球菌生物膜作用。

关键词: 抗菌肽；变异链球菌；生物膜

Abstract: Objective To design novel antimicrobial peptides with high activity and low toxicity and evaluate their effect against Streptococcus mutans and other oral bacteria for prevention and treatment of dental caries. Methods We synthesized two antimicrobial peptides (KR-1 and KR-2) using Dhvar4 (a histatins5 mimic) as the template. The antimicrobial peptides with high activity and low toxicity were screened using minimal inhibitory concentration (MIC) test, hemolysis test, and CCK-8 assay. Streptococcus mutans biofilms cultured in 96-well plates were divided into experimental group (KR-1) and positive control group (CHX) and treated with concentration gradients (0.6×, 0.8×, 1× and 2×MICs) of KR-1 and CHX, respectively. Crystal violet staining was used for quantitative analysis of the changes of the biofilms after the treatments. The structural changes of the biofilms were observed with laser confocal microscopy after KR-1 treatment at 10 × MIC. The antimicrobial activity of KR-1 against oral Streptococcus was analyzed based on the time required for sterilization after KR-1 treatment. Results The MIC of KR-1 and KR-2 for S. mutans was 3.2 μmol/L and 12.8 μmol/L, respectively. Under the effective concentration, KR-1 and KR-2 resulted in hemolysis rates of 0.35% and 48.8% in rabbit red blood cells and lowered the survival rates of gingival fibroblasts to 88.7% and 21.94%, respectively. KR-1 treatment significantly reduced biofilm formation with a minimum biofilm inhibition concentration (MBIC50) lower than 1.92 μmol/L, and showed an even stronger antimicrobial than CHX at the concentration of 2.56 μmol/L (P=0.001). Confocal laser scanning microscopy revealed that the biofilm structure became loosened after KR-1 treatment, which was capable of killing about 90% of the bacteria within 5 min. Conclusion The antimicrobial peptide KR-1 has a stronger antibacterial activity and a low toxicity with a good inhibitory effect against S. mutans biofilm.

Key words: antibacterial peptides; Streptococcus mutans; biofilm