Combination of Host Defense Peptide Mimicking Peptide Polymer and Proteinase K against Biofilms
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摘要: 采用双（三甲基硅基）胺基锂（LiHMDS）引发的α-氨基酸N-羧酸酐（NCA）快速开环聚合方法合成宿主防御肽模拟聚合物，该聚合物呈现窄分子量分布。抗菌实验结果表明, 该聚合物对耐甲氧西林金黄色葡萄球菌（MRSA）浮游菌和持留菌有高杀菌活性，且遵循破膜杀菌机制。实验证明，1.25 U/mL的蛋白酶K在人工尿液环境中可分散MRSA成熟生物被膜。蛋白酶K与聚合物协同作用实现了人工尿液中成熟生物被膜的清除。Abstract: Staphylococcus aureus (S. aureus) is one of the main pathogens causing urinary catheter-related infections, and its biofilm has strong resistance to immune clearance and antibiotics. Effective measures should be taken to solve catheter biofilm-related infections fundamentally. In this study, the host defense peptide mimicking peptide polymer was synthesized by rapid ring-opening polymerization of α-amino acid N-carboxyanhydride (NCA), which was initiated by lithium bis(trimethylsilyl)amide (LiHMDS). The resulting polymer showed a narrow molecular weight distribution. Antimicrobial experiment showed that this peptide polymer had a high bactericidal activity against the planktonic and persister cells of methicillin-resistant Staphylococcus aureus (MRSA). Bactericidal mechanism showed that this peptide polymer killed MRSA by destroying the integrity of bacterial cell membrane. Protein is one of the basic components of extracellular polymeric matrix (EPS), which plays an important role in bacterial colonization and biofilm development. The dispersing effect of proteinase K on EPS is beneficial to the penetration of antimicrobial agents into biofilms. 1.25 U/mL proteinase K dispersed mature MRSA biofilms in artificial urine, and the remaining cell viability was about 55% of the control group. The combination of proteinase K and polymer further eradicated mature MRSA biofilms in artificial urine, which confirmed the potential of synergism in eradicating the biofilms inside urinary catheter. The optimal combination reduced the cell viability within biofilms to about 10% of the control group.
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Key words:
- peptide polymer /
- proteinase K /
- biofilm /
- persister cell /
- drug synergy
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图 1 蛋白酶K和P（DLL-co-BLG）联合作用清除MRSA生物被膜和持留菌的示意图
Figure 1. Schematic diagram of combined action of protease K and P（DLL-co-BLG） to eradicate MRSA biofilms and persister cells
图 3 P（DLL-co-BLG）的（a）GPC曲线和（b）1H-NMR图谱
Figure 3. （a）GPC curve and （b）1H-NMR spectrum of P（DLL-co-BLG）
图 4 P（DLL-co-BLG）（a）对金黄色葡萄球菌的MIC值和（b）对S. aureus USA300的MBC值
Figure 4. （a）MIC against S. aureus and （b）MBC against S. aureus USA300 of P（DLL-co-BLG）
图 5 P（DLL-co-BLG）与S. aureus USA300作用后DiSC3（5）荧光强度随时间的变化
Figure 5. Fluorescence of DiSC3（5） in the presence of S. aureus USA300 and P（DLL-co-BLG） as a function of the incubation time
图 6 浓度为4×MIC的P（DLL-co-BLG）对S. aureus USA300持留菌的杀菌动力学曲线
Figure 6. Time-kill curves for P（DLL-co-BLG） with final concentrations of 4×MIC against S. aureus USA300 persister cells
图 7 （a）不同浓度的蛋白酶K对人工尿中S. aureus USA300成熟生物膜细菌活力的影响；（b）MTT法获得人工尿中P（DLL-co-BLG）对S. aureus USA300成熟生物被膜的清除活性（*P<0.05, **P<0.01, ***P<0.001，显著性差异是通过与未经聚合物处理的对照组对比得到的）；（c）人工尿液介质中，蛋白酶K和P（DLL-co-BLG）联合作用后，S. aureus USA300成熟生物被膜内部的细菌活力（图中小写字母表示各实验组间有显著性差异（P<0.05）柱状图上方为药物处理和未处理的经过MTT染色后的生物被膜的图像）
Figure 7. （a） Effects of proteinase K on mature S. aureus USA300 biofilms in artificial urine; （b） Eradication activity of P（DLL-co-BLG） in artificial urine against mature S. aureus USA300 biofilms obtained by MTT assay （*P<0.05, **P<0.01, ***P<0.001, significant difference analysis results were obtained when compared with the control groups that had not been treated with polymers）;（c） Remaining cell viability within mature S. aureus USA300 biofilm after the combined action of proteinase K and P（DLL-co-BLG） in artificial urine （Different lowercase letters indicate significant difference （P< 0.05） between different experimental groups, above the bar diagram are the corresponding images of treated and untreated biofilms after MTT staining and precipitation dissolution）
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