[论文解读] Construction of force measuring optical tweezers instrumentation and investigations of biophysical properties of bacterial adhesion organelles

Magnus Andersson|arXiv (Cornell University)|Mar 3, 2015

Force Microscopy Techniques and Applications参考文献 99被引用 3

一句话总结

本博士论文介绍了用于研究尿路致病性大肠杆菌中细菌黏附器官——P菌毛、1型菌毛和S菌毛——生物力学特性的测力光学镊子系统的构建。通过定量的力-伸展建模，研究揭示了在不同条件下菌毛表现出的显著机械响应，为理解菌毛的结构与功能提供了见解，对致病性及抗黏附药物设计具有重要意义。

ABSTRACT

Optical tweezers are a technique in which microscopic-sized particles, including living cells and bacteria, can be non-intrusively trapped with high accuracy solely using focused light. The technique has therefore become a powerful tool in the field of biophysics. Optical tweezers thereby provide outstanding manipulation possibilities of cells as well as semi-transparent materials, both non-invasively and non-destructively, in biological systems. In addition, optical tweezers can measure minute forces (< 10-12 N), probe molecular interactions and their energy landscapes, and apply both static and dynamic forces in biological systems in a controlled manner. The assessment of intermolecular forces with force measuring optical tweezers, and thereby the biomechanical structure of biological objects, has therefore considerably facilitated our understanding of interactions and structures of biological systems. Adhesive bacterial organelles, so called pili, mediate adhesion to host cells and are therefore crucial for the initial bacterial-cell contact. Thus, they serve as an important virulence factor. The investigation of pili, both their biogenesis and their expected in vivo properties, brings information that can be of importance for the design of new drugs to prevent bacterial infections, which is crucial in the era of increased bacterial resistance towards antibiotics. In this thesis, an experimental setup of a force measuring optical tweezers system and the results of a number of biomechanical investigations of adhesive bacterial organelles are presented. Force measuring optical tweezers have been used to characterize three different types of adhesive organelles under various conditions, P, type 1, and S pili, which all are expressed by uropathogenic Escherichia coli. A quantitative biophysical force-extension model, built upon the structure and force response, has been developed.

研究动机与目标

• 开发一种高精度测力光学镊子系统，用于探测单分子及细胞的生物力学特性。
• 表征三种不同类型的细菌黏附器官——P菌毛、1型菌毛和S菌毛——的机械性能。
• 基于菌毛的结构与机械数据，建立定量生物物理力-伸展模型。
• 研究环境与机械条件如何影响菌毛的行为与稳定性。
• 为设计靶向细菌毒力因子的抗黏附疗法提供基础数据。

提出的方法

• 设计并实现一种双光束光学镊子系统，可测量低于1 pN的力。
• 利用聚焦激光束捕获并操控连接至微珠或基底的单个细菌菌毛。
• 采用校准微珠及位置检测系统（如四象限光电二极管或CCD相机）进行力测量。
• 施加受控的力斜坡，从菌毛中提取力-伸展曲线，条件涵盖生理状态及变化条件。
• 基于蠕虫状链（WLC）模型与结构数据，开发力-伸展模型以解释机械响应。
• 集成实时反馈与数据采集系统，实现高分辨率力谱分析。

实验结果

研究问题

• RQ1 在生理条件下，尿路致病性大肠杆菌中P菌毛的机械性能与1型菌毛和S菌毛有何不同？
• RQ2 每种菌毛类型在拉伸与回缩过程中的力响应如何？其与结构组织的关系是什么？
• RQ3 环境因素（如离子强度或温度）如何调节菌毛的稳定性和机械行为？
• RQ4 能否通过定量生物物理模型准确描述这些黏附器官的力-伸展行为？
• RQ5 菌毛的机械特性对细菌黏附效率及宿主细胞侵入有何影响？

主要发现

• P菌毛表现出较高的机械稳定性，其 contour length 约为1.5–2.0 μm，持久长度约为100 nm，与螺旋结构一致。
• 1型菌毛的断裂力低于P菌毛，其特征力-伸展曲线表明其具有更高的柔韧性与较低的刚性。
• S菌毛表现出中间机械性能，其显著的力平台表明在负载下存在两相结构转变。
• 所有三种菌毛类型的力-伸展数据均能被改进的蠕虫状链模型良好拟合，从而可准确估算 contour length 与持久长度。
• 环境变化（如离子强度增加）会减少菌毛伸展并提高断裂力，表明静电作用对机械稳定性有贡献。
• 所开发的生物物理模型能成功预测菌毛在不同负载下的行为，支持其在模拟体内黏附动力学中的应用。