Objective: This study aimed to evaluate the biological characteristics of a human specifically targeted antimi- crobial peptide C16LL-37 against Streptococcus mutans (S. mutans).
Methods: In this study, an antimicrobial peptide LL-37, a peptide derived from CSP(C16) (S. mutans competence stimulating peptide), and recombinant peptide C16LL-37 were synthesized by Fmoc-chemistry-based strategy. The selectivity and antibacterial activity of C16LL-37 were identified by the colony counting method on microbial culture plates. After treatment of C16LL-37 at 32 µmol · L⁻¹, the morphological changes in S. mutans were observed by using scanning electron microscopy (SEM). In addition, enzyme-linked immunosorbent assay was used to evaluate the hemolytic activity and antibacterial activity of C16LL-37 under different conditions.
Results: 1) The minimum inhibitory concentration of C16LL-37 was 16 µmol · L⁻¹, and the minimum bactericidal concentration was 64 μmol ·L⁻¹. 2) The survival rate of S. mutans was 3.46% after C16LL-37 treatment at 64 µmo-L⁻¹ for 30 min, whereas it was 0% at 64 µmol · L⁻¹ for 60 min. The survival rates of four other kinds of bacteria were more than 60% at any time (P < 0.05). 3) The morphological change in S. mutans was observed after C16LL-37 treatment at 32 µmol · L⁻¹ by using SEM. S. mutans presented an irregular shape, rough surface, and evident splitting. 4) The hemolysis rate of C16LL-37 (≤ 64 µmol · L⁻¹) was less than 0.33%. 5) This study showed no significant in- fluence on the antibacterial activity of C16LL-37 under different conditions, such as temperature, pH, salinity, and trypsin at low concentration (P > 0.05).
Conclusion: C16LL-37 exhibited obvious specificity for S. mutans, strong antibacterial activity, low toxicity, and high stability. Thus, C16LL-37 has good potential in caries research and clinical application.
目的: 研究针对变异链球菌（S. mutans）的人源特异性靶向抗菌肽C16LL-37的生物学特性。
方法: 通过标准固相合成技术（Fmoc保护法）合成人源抗菌肽LL-37、S. mutans感受态刺激肽（CSP）C端16个氨基酸组成的多肽CSPC16及二者的重组多肽C16LL-37；以LL-37、CSPC16为对照组，采用平板菌落计数法检测C16LL-37对S. mutans、黏性放线菌、大肠埃希菌、嗜酸乳杆菌、金黄色葡萄球菌的抗菌活性及其对S. mutans的靶向性。通过扫描电子显微镜（SEM）观察，浓度为32 µmol·L−1的C16LL-37作用后S. mutans的细胞形态学变化；利用酶联免疫吸附法测定C16LL-37的红细胞溶血率以及不同条件对C16LL-37抗菌活性的影响。
结果: 1）C16LL-37对S. mutans的最小抗菌浓度为16 µmol·L−1，最小杀菌浓度为64 µmol·L−1。2）C16LL-37浓度为64 µmol·L−1时，作用30 min后S. mutans存活率为3.46%，作用60 min后细菌存活率降至0%，其他4种细菌在各时间的存活率均大于60%（P<0.05）。3）SEM观察：经C16LL-37（32 µmol·L−1）作用后，部分S. mutans细胞形态出现不规则改变，部分细胞的细胞膜粗糙、细胞质外溢或出现细胞裂解。4）C16LL-37浓度不超过64 µmol·L−1时，红细胞溶血率低于0.33%，与阴性对照组无明显差异（P>0.05）。5）不同温度、pH值、盐浓度及低浓度胰蛋白酶处理后，C16LL-37的抗菌活性无明显变化（P>0.05）。
结论: C16LL-37对变异链球菌具有靶向特异性，并且具有较强的抗菌活性、较高的生物安全性及良好的稳定性。