Pleurocidin及其优化多肽抑制常见致龋菌的研究
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摘要
尽管人们已经做了许多努力来控制和预防龋病，但是其依然是影响人类生活、学习、工作的三大流行传染病之一。龋病的主要病原体目前被确定为变异链球菌和远缘链球菌，并且粘性放线菌和链球菌戈登是牙齿表面的早期定殖菌[1]。但是杀灭这些细菌是非常困难的，因为口腔微生物能够形成牙菌斑生物膜。这种生物膜能够为细菌提供一定的保护能力，可以增加细菌对抗菌药物的抵抗能力[2]。机械去除（刷牙、龈上下洁治术）菌斑依旧是首先选择的治疗方法，但是其也并不能完全去除菌斑，因此目前的研究主要集中于如何在机械去除菌斑的基础上使用抗菌剂抑制、杀灭细菌。为此本实验对天然多肽pleurocidin的抗菌能力进行了研究，并在此基础上改造、修饰优化多肽，使其能够更好的发挥抗菌的能力。
1、Pleurocidin对口腔常见致龋菌抑菌作用研究
Pleurocidin是从Pleuronectes americanus的表皮中分离出来的天然多肽。其具有较为广谱的抗菌活性，能够抵抗多种细菌和真菌。此外，与其他天然多肽相比，在体外毒性研究中pleurocidin表现出较低的溶血性，具有潜在的治疗价值。
在第一部分实验，我们对pleurocidin能否作为一种有效的抗菌多肽运用于龋病的治疗进行了检测。多肽对常见致龋微生物的抗菌机理也进行了研究。我们检测了pleurocidin的最小抑菌浓度（MIC），最小杀菌浓度（MBC）和杀菌动力学的特性，并进行了唾液影响pleurocidin抑菌效果的抑菌环实验。BioFlux系统用于生成可控剪切力下细菌生物膜的形成及pleurocidin对生物膜的抑制作用研究。荧光显微镜和共聚焦激光扫描显微镜（CLSM）用来分析和观察生物膜，扫描电子显微镜用于观察细菌细胞膜。
MIC和MBC结果表明，pleurocidin对不同的口腔细菌有不同的抗菌活性。虽然唾液中的成分可能会影响抗菌活性，溶解于唾液中的pleurocidin仍然具有对口腔微生物的抗菌作用。此外体外模拟实验显示，pleurocidin对于变异链球菌生物膜具有一定的抗生物膜能力。我们的研究结果表明，pleurocidin具有杀死口腔生物膜，防止龋齿的运用潜力。
2、基于Pleurocidin的新型抗菌多肽的优化设计
鱼类具有强有力的天然免疫系统，从而能够应对大量的病原微生物。我们在之前已经证明α螺旋线性阳离子多肽Pleurocidin具有抗致龋菌、真菌的能力，并对变异链球菌生物膜有一定的抑制作用。但是pleurocidin的长度为25个氨基酸，过长的氨基酸提高了成本和降低了其对细菌生物膜的渗透能力，因此开发出新的具有高抗菌活性的短肽就更为必要。
本研究在确定pleurocidin活性中心的基础上（GW18），新设计十几种多肽。经过我们的检测发现，GW18来源的pc1、pc8、pc9、pc11、pc12这几种对于口腔的几种常见致病菌有较强的抗菌能力。其中pc9是抗菌效果最好的多肽，pc12是具有抗菌能力里面最短的多肽。我们通过对多肽物理性质的分析，确定疏水性和多肽长度对抗菌能力最为重要。最后通过检测多肽的杀菌动力学特性，发现该类型多肽属于快速杀菌多肽的类型。
3、新型多肽Pc9、Pc12的抗菌原理及细胞毒性研究
很多多肽都有一定细胞毒性，甚至有很强的溶血作用。理想中的多肽是在具有抗菌能力的前提下，对细胞、宿主没有任何毒副作用，不会引起机体的过敏反应。实验的第二部分确定了pc9、pc12的抗菌能力，为此我们通过电镜、细胞实验更进一步探讨研究这一新型多肽的抗菌机理和细胞毒性。
扫描电镜显示用多肽Pc9、Pc12处理过后，变异链球菌和血链球菌的细胞膜受到多肽的影响。抗菌多肽使细菌表面失去原有性状，变得粗糙甚至有出现孔隙。该孔隙直径大约为10nm左右，说明该多肽是通过在细菌表面形成离子通道，引起细菌内容物的流出，从而导致细菌的死亡。这种机理与原来设计多肽的时候设计是相一致的。细胞毒性显示当使用8×MIC的浓度的pc9和pc12处理HGFs，共培养2小时后，均显示对细胞有一定的影响。但是短时间处理时（5分钟、1小时），没有统计学上的差异。说明多肽的细胞毒性还是时间依赖型的。并且由于pc12在处理变异链球菌1小时以后，细菌均已经死亡。说明该多肽在杀菌时间内对细胞的影响轻微，有一定的临床运用价值。
1、Antimicrobial and antibiofilm activity of pleurocidin againstcariogenic microorganisms
Pleurocidin, produced by Pleuronectes americanus, is an antimicrobialpeptide that exerts broad-spectrum activity against pathogenic bacteria and fungi.Moreover, pleurocidin shows less hemolysis and is less toxic than other naturalpeptides. In the present study, we investigated whether pleurocidin is aneffective antibiotic peptide against common cariogenic microorganisms andperformed a preliminary study of the antimicrobial mechanism. We assayedminimal inhibitory concentration (MIC), minimal bactericide concentration(MBC) and bactericidal kinetics and performed a spot-on-lawn assay. TheBioFlux system was used to generate bacterial biofilms under controllable flow.Fluorescence microscopy and confocal laser scanning microscopy (CLSM) wereused to analyze and observe biofilms. Scanning electron microscopy was used toobserve the bacterial membrane. MIC and MBC results showed that pleurocidin had different antimicrobial activities against the tested oral strains. Althoughcomponents of saliva could affect antimicrobial activity, pleurocidin dissolved insaliva still showed antimicrobial effects against oral microorganisms.Furthermore, pleurocidin showed a favorable killing effect against BioFlux flowbiofilms in vitro. Our findings suggest that pleurocidin has the potential to killdental biofilms and prevent dental caries
2Design and antimicrobial activity of new peptides againstcariogenic microorganisms
Fish possess a strong innate immune system that acts as the first line ofdefense against a broad spectrum of pathogens. We have demonstrated thatα-helix linear cationic peptide pleurocidin is sensitive to cariogenic bacteria,fungi, and have antibiofilm of Streptococcus mutans biofilms. However,pleurocidin is composed by25amino acid residue, and synthetic cost is highand has the poor ability to penetrate the entire bacterial biofilm, therefore thedevelopment of new peptide is even more necessary.
In this study, GW18, activity centers of pleurocidin, was confirmed and aseries of new peptides were designed. Pc1, pc8, pc9, pc11and pc12, congenersof pleurocidin, had antibacterial ability against several oral microorganisms. Pc9had the best antibacterial activity, while pc12was the shortest peptide..Hydrophobicity and the length of the peptide are related to antibacterial ability.Duo to the short time killing effect of, it was suggested that pc12was a ‘rapidkill’peptide.
3The antibacterial mechanism and the cytotoxic of the new designed peptides Pc9, Pc12
Many peptides are cytotoxic and even have a strong hemolytic effect. Theideal peptide does not have any side effects or allergic reactions of the immunesystem. In this part of the experiment, electron microscopy was used to observethe membrane damage of pc9, pc12and cell experiments was taken to study thecytotoxic of new designed peptides.
Electron microscopy showed that after exposed under Pc9and Pc12,membrane of Streptococcus mutans and Streptococcus sanguis is damaged bythe peptide. The bacteria lose their original shape, and transmembrane poreappeared. The pore diameter is about10nm, the formation of ion channels in thebacterial surface caused the outflow of the bacterial contents, resulting in thedeath of the bacteria. HGFs were treated with8xMIC of pc9and pc12. Aftercultured for2hours, there was some influence to the cell. However, shortexpose (5minutes,1hour) showed no statistically significant difference to thecontrol group. However, pc12had the ability to kill Streptococcus mutans in onehour. Therefore, our findings suggest that pc9has the potential to prevent dentalcaries
1、Pleurocidin对口腔常见致龋菌抑菌作用研究
Pleurocidin是从Pleuronectes americanus的表皮中分离出来的天然多肽。其具有较为广谱的抗菌活性，能够抵抗多种细菌和真菌。此外，与其他天然多肽相比，在体外毒性研究中pleurocidin表现出较低的溶血性，具有潜在的治疗价值。
在第一部分实验，我们对pleurocidin能否作为一种有效的抗菌多肽运用于龋病的治疗进行了检测。多肽对常见致龋微生物的抗菌机理也进行了研究。我们检测了pleurocidin的最小抑菌浓度（MIC），最小杀菌浓度（MBC）和杀菌动力学的特性，并进行了唾液影响pleurocidin抑菌效果的抑菌环实验。BioFlux系统用于生成可控剪切力下细菌生物膜的形成及pleurocidin对生物膜的抑制作用研究。荧光显微镜和共聚焦激光扫描显微镜（CLSM）用来分析和观察生物膜，扫描电子显微镜用于观察细菌细胞膜。
MIC和MBC结果表明，pleurocidin对不同的口腔细菌有不同的抗菌活性。虽然唾液中的成分可能会影响抗菌活性，溶解于唾液中的pleurocidin仍然具有对口腔微生物的抗菌作用。此外体外模拟实验显示，pleurocidin对于变异链球菌生物膜具有一定的抗生物膜能力。我们的研究结果表明，pleurocidin具有杀死口腔生物膜，防止龋齿的运用潜力。
2、基于Pleurocidin的新型抗菌多肽的优化设计
鱼类具有强有力的天然免疫系统，从而能够应对大量的病原微生物。我们在之前已经证明α螺旋线性阳离子多肽Pleurocidin具有抗致龋菌、真菌的能力，并对变异链球菌生物膜有一定的抑制作用。但是pleurocidin的长度为25个氨基酸，过长的氨基酸提高了成本和降低了其对细菌生物膜的渗透能力，因此开发出新的具有高抗菌活性的短肽就更为必要。
本研究在确定pleurocidin活性中心的基础上（GW18），新设计十几种多肽。经过我们的检测发现，GW18来源的pc1、pc8、pc9、pc11、pc12这几种对于口腔的几种常见致病菌有较强的抗菌能力。其中pc9是抗菌效果最好的多肽，pc12是具有抗菌能力里面最短的多肽。我们通过对多肽物理性质的分析，确定疏水性和多肽长度对抗菌能力最为重要。最后通过检测多肽的杀菌动力学特性，发现该类型多肽属于快速杀菌多肽的类型。
3、新型多肽Pc9、Pc12的抗菌原理及细胞毒性研究
很多多肽都有一定细胞毒性，甚至有很强的溶血作用。理想中的多肽是在具有抗菌能力的前提下，对细胞、宿主没有任何毒副作用，不会引起机体的过敏反应。实验的第二部分确定了pc9、pc12的抗菌能力，为此我们通过电镜、细胞实验更进一步探讨研究这一新型多肽的抗菌机理和细胞毒性。
扫描电镜显示用多肽Pc9、Pc12处理过后，变异链球菌和血链球菌的细胞膜受到多肽的影响。抗菌多肽使细菌表面失去原有性状，变得粗糙甚至有出现孔隙。该孔隙直径大约为10nm左右，说明该多肽是通过在细菌表面形成离子通道，引起细菌内容物的流出，从而导致细菌的死亡。这种机理与原来设计多肽的时候设计是相一致的。细胞毒性显示当使用8×MIC的浓度的pc9和pc12处理HGFs，共培养2小时后，均显示对细胞有一定的影响。但是短时间处理时（5分钟、1小时），没有统计学上的差异。说明多肽的细胞毒性还是时间依赖型的。并且由于pc12在处理变异链球菌1小时以后，细菌均已经死亡。说明该多肽在杀菌时间内对细胞的影响轻微，有一定的临床运用价值。
1、Antimicrobial and antibiofilm activity of pleurocidin againstcariogenic microorganisms
Pleurocidin, produced by Pleuronectes americanus, is an antimicrobialpeptide that exerts broad-spectrum activity against pathogenic bacteria and fungi.Moreover, pleurocidin shows less hemolysis and is less toxic than other naturalpeptides. In the present study, we investigated whether pleurocidin is aneffective antibiotic peptide against common cariogenic microorganisms andperformed a preliminary study of the antimicrobial mechanism. We assayedminimal inhibitory concentration (MIC), minimal bactericide concentration(MBC) and bactericidal kinetics and performed a spot-on-lawn assay. TheBioFlux system was used to generate bacterial biofilms under controllable flow.Fluorescence microscopy and confocal laser scanning microscopy (CLSM) wereused to analyze and observe biofilms. Scanning electron microscopy was used toobserve the bacterial membrane. MIC and MBC results showed that pleurocidin had different antimicrobial activities against the tested oral strains. Althoughcomponents of saliva could affect antimicrobial activity, pleurocidin dissolved insaliva still showed antimicrobial effects against oral microorganisms.Furthermore, pleurocidin showed a favorable killing effect against BioFlux flowbiofilms in vitro. Our findings suggest that pleurocidin has the potential to killdental biofilms and prevent dental caries
2Design and antimicrobial activity of new peptides againstcariogenic microorganisms
Fish possess a strong innate immune system that acts as the first line ofdefense against a broad spectrum of pathogens. We have demonstrated thatα-helix linear cationic peptide pleurocidin is sensitive to cariogenic bacteria,fungi, and have antibiofilm of Streptococcus mutans biofilms. However,pleurocidin is composed by25amino acid residue, and synthetic cost is highand has the poor ability to penetrate the entire bacterial biofilm, therefore thedevelopment of new peptide is even more necessary.
In this study, GW18, activity centers of pleurocidin, was confirmed and aseries of new peptides were designed. Pc1, pc8, pc9, pc11and pc12, congenersof pleurocidin, had antibacterial ability against several oral microorganisms. Pc9had the best antibacterial activity, while pc12was the shortest peptide..Hydrophobicity and the length of the peptide are related to antibacterial ability.Duo to the short time killing effect of, it was suggested that pc12was a ‘rapidkill’peptide.
3The antibacterial mechanism and the cytotoxic of the new designed peptides Pc9, Pc12
Many peptides are cytotoxic and even have a strong hemolytic effect. Theideal peptide does not have any side effects or allergic reactions of the immunesystem. In this part of the experiment, electron microscopy was used to observethe membrane damage of pc9, pc12and cell experiments was taken to study thecytotoxic of new designed peptides.
Electron microscopy showed that after exposed under Pc9and Pc12,membrane of Streptococcus mutans and Streptococcus sanguis is damaged bythe peptide. The bacteria lose their original shape, and transmembrane poreappeared. The pore diameter is about10nm, the formation of ion channels in thebacterial surface caused the outflow of the bacterial contents, resulting in thedeath of the bacteria. HGFs were treated with8xMIC of pc9and pc12. Aftercultured for2hours, there was some influence to the cell. However, shortexpose (5minutes,1hour) showed no statistically significant difference to thecontrol group. However, pc12had the ability to kill Streptococcus mutans in onehour. Therefore, our findings suggest that pc9has the potential to prevent dentalcaries
引文
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