标题: 应用奈米技术探讨克雷白氏肺炎杆菌第三型线毛的黏附与延展特性
Using Nanotechniques to Explore the Adhesion and Extension Properties of Klebsiella pneumoniae Type 3 Fimbriae
作者: 詹佳翰
Chan, Chia-Han
徐琅
Hsu, Long
电子物理系所
关键字: 克雷白氏肺炎杆菌;第三型线毛;奈米技术;雷射镊夹;电子显微镜;机械特性;Klebsiella pneumoniae;type 3 fimbria;nanotechniques;optical tweezers;transmission electron microscopy;mechanical propertie
公开日期: 2011
摘要: 细菌藉由其表面线毛黏附宿主细胞,因线毛具螺旋弹簧结构的伸缩特性,能降低环境冲刷力的影响,使细菌与宿主保持紧密黏附。克雷白氏肺炎杆菌(Klebsiella pneumoniae)是一株伺机性感染细菌,近年来临床上发现多种抗药性菌株,使得治疗方式趋于困难,而其表面第三型线毛引起细胞制造生物膜,进而产生抗药性。本论文将以克雷白氏肺炎杆菌主要致病的因子第三型线毛作为材料,利用雷射镊夹(OT)、电子显微镜(TEM)、原子力显微镜(AFM)、基因重组技术、基因标定技术、胺基酸定点突变技术等奈米技术,从生物物理的角度,探讨单一根第三型线毛的特性,并进一步分析影响线毛主要蛋白MrkA组装的重要残基。我们发现第三型线毛的长度约为1微米,线毛的螺旋截面并不对称,截面最大宽度7.4奈米和最小宽度4.2奈米。第三型线毛顶端黏附蛋白有四个黏附因子,其中MrkDv3与胶原蛋白的黏附力最大,约为4皮牛顿,是黏附蛋白的重要因子。第三型线毛受力伸展曲线具有三个阶段:(1)第一阶段表现线性的受力伸长特性,弹性系数为60.9皮牛顿/微米,杨氏模数为100皮牛顿/微米2;(2)第二阶段表现固定力伸展特性,野生株的线毛以固定力66 ± 4皮牛顿拆开并伸展数微米长;(3)第三阶段表现非线性的受力伸长特性,其受力伸展曲线转折点为特征力102 ± 9皮牛顿。另外,针对第三型线毛骨干主要蛋白MrkA单体,我们筛选十二个MrkA关键的胺基酸进行定点突变,以分析其受力伸展物理特性的变化。其中G189A突变株具特殊力学性能,其第二阶段的拆解力呈现持续增加的拆解力(57~68皮牛顿),不同于其他突变株或一般株以固定66皮牛顿力拆解。这样的结果显示,G189胺基酸对MrkA的结构拆解力稳定度扮演重要的角色。本论文提供一个研究各种线毛的生物力学性能的奈米技术平台,藉由线毛生物物理特性结果与发现,能提供细菌感染致病的资讯,有助于传统细菌感染与抗药性的研究。
Fimbriae exhibiting flexible and stretchable properties similar to springs may aid bacteria in remaining attached to host cells by reducing the impact of the flushes in environment. Klebsiella pneumoniae is a common pathogen causing both pneumonia and urinary tract infections. Most pathogenic Klebsiella pneumoniae strains produce type 3 fimbriae, which are critical for bacterial biofilm formation. In this thesis, we investigated the structural and mechanical properties of Klebsiella pneumoniae type 3 fimbriae, which constitute a known virulence factor for the bacterium, and to identify residues in the major pilin MrkA important for pilus assembly. Type 3 fimbria was 1 micron in length, and the largest diameter was 7.4 nm and the smallest was 4.2 nm, have an elliptically cylindrical structure with the largest diameter and smallest diameter of each ellipse-like cross section in fimbrial structures by using transmission electron microscopy. We present type 3 fimbriae MrkD adhesin variants, mrkDV3-,was the best adhesin, and the adhesive force between mrkDV3-expressed fimbriae and collagen IV is about 4 pN.The force-extension curve of type 3 fimbria exhibited a three-phase:(1) the first spring-like phase stretched with increasing force, the spring constant kp of 60.9 N/μm, the estimated Young’s moduli (E) were 100 Mpa, (2) the second uncoiling phase, it started to uncoil and extended several micrometers at a fixed force of 66 ± 4 pN, and (3) the final nonlinear phase with a characteristic force of 102 ± 9.8 pN at the inflection point. In addition, we screened twelve critical residues on major pilins MrkA of type 3 fimbriae. By contrast, the G189A mutant remained capable of producing normal amounts of fimbriae. Further investigating the mechanical properties of the G189A fimbriae revealed that the uncoiling force for MrkA-MrkA interaction varied from 58 pN to 67 pN rather than be constant like the uncoiling force of 66 pN measured from the other mutants or wild type. The work has provided a method that allows us to understand of the biomechanical function of different types of pili. The investigation of pili brings information for the design of new drugs to prevent bacterial infections, which is increased bacterial resistance towards antibiotics.
URI: http://140.113.39.130/cdrfb3/record/nctu/#GT079221804
http://hdl.handle.net/11536/40409
显示于类别:Thesis


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