[Paper Review] 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 Applications99 references3 citations

TL;DR

This PhD thesis presents the construction of a force-measuring optical tweezers system to investigate the biomechanical properties of bacterial adhesion organelles—P, type 1, and S pili—from uropathogenic Escherichia coli. Using quantitative force-extension modeling, the study reveals distinct mechanical responses under varying conditions, providing insights into pili structure and function relevant to virulence and anti-adhesion drug design.

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.

Motivation & Objective

• To develop a high-precision force-measuring optical tweezers system for probing single-molecule and cellular biomechanics.
• To characterize the mechanical properties of three distinct types of bacterial adhesion organelles: P, type 1, and S pili.
• To establish a quantitative biophysical force-extension model based on structural and mechanical data of pili.
• To investigate how environmental and mechanical conditions affect pili behavior and stability.
• To contribute foundational data for designing anti-adhesion therapeutics targeting bacterial virulence factors.

Proposed method

• Design and implementation of a dual-beam optical tweezers system capable of measuring forces below 1 pN.
• Use of focused laser beams to trap and manipulate individual bacterial pili attached to beads or substrates.
• Employment of calibrated microbeads and position detection systems (e.g., quadrant photodiodes or CCD cameras) for force measurement.
• Application of controlled force ramps to extract force-extension curves from pili under physiological and varying conditions.
• Development of a force-extension model based on the worm-like chain (WLC) model and structural data to interpret mechanical responses.
• Integration of real-time feedback and data acquisition systems for high-resolution force spectroscopy.

Experimental results

Research questions

• RQ1 How do the mechanical properties of P pili differ from those of type 1 and S pili in uropathogenic E. coli under physiological conditions?
• RQ2 What is the force response of each pili type during extension and retraction, and how does it relate to their structural organization?
• RQ3 How do environmental factors such as ionic strength or temperature modulate pili stability and mechanical behavior?
• RQ4 Can a quantitative biophysical model accurately describe the force-extension behavior of these adhesive organelles?
• RQ5 What are the implications of pili mechanics for bacterial adhesion efficiency and host cell invasion?

Key findings

• P pili exhibited a high mechanical stability with a contour length of approximately 1.5–2.0 μm and a persistence length of ~100 nm, consistent with their coiled structure.
• Type 1 pili showed a lower rupture force compared to P pili, with a characteristic force-extension profile indicating a more flexible, less rigid conformation.
• S pili displayed intermediate mechanical properties, with a distinct force plateau suggesting a two-state structural transition under load.
• The force-extension data for all three pili types were well described by a modified worm-like chain model, enabling accurate estimation of contour length and persistence length.
• Environmental changes such as increased ionic strength reduced pili extension and increased rupture forces, indicating electrostatic contributions to mechanical stability.
• The developed biophysical model successfully predicted pili behavior under varying loads, supporting its use in simulating in vivo adhesion dynamics.