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Construction of force measuring optical tweezers instrumentation and investigations of biophysical properties of bacterial adhesion organelles
Umeå University, Faculty of Science and Technology, Physics.
2007 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

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. It is found, that this model describes the biomechanical properties for all three pili in an excellent way. Various parameters in their energy landscape, e.g., bond lengths and transition barrier heights, are assessed and the difference in behavior is compared. The work has resulted in a method that in a swift way allows us to probe different types of pili with high force and high spatial resolution, which has provided an enhanced understanding of the biomechanical function of these pili.

Abstract [sv]

Optisk pincett är en teknik i vilken mikrometerstora objekt, inkluderande levande celler och bakterier, beröringsfritt kan fångas och förflyttas med hög noggrannhet enbart med hjälp av ljus. Den optiska pincetten har därmed blivit ett kraftfullt verktyg inom biofysiken, som möjliggör enastående precisions-manipulering av celler och semi-transparenta objekt. Dessutom kan denna manipulation göras intracellulärt, dvs. utan att fysiskt öppna eller penetrera cellernas membran. Den optiska pincetten kan även mäta mycket små krafter och interaktioner (< 10-12 N) samt applicera både statiska och dynamiska krafter i biologiska system med utmärkt precision. Optisk pincett är därför en utmärkt teknik för mätning av intermolekylära krafter och för bestämning av biomekaniska strukturer och dess funktioner.

Vissa typer av bakterier har specifika vidhäftningsorganeller som kallas för pili. Dessa förmedlar vidhäftningen till värdceller och är därför viktiga vid bakteriens första kontakt. En djupare förståelse av pilis uppbyggnad och biomekanik kan därmed ge information, som kan vara vital i framtagandet av nya mediciner som förhindrar bakteriella infektioner. Detta är av stor vikt i skenet av den ökande antibiotikaresistensen i vårt samhälle.

I denna avhandling presenteras konstruktionen av en experimentell uppställning av kraftmätande optiskt pincett tillsammans med resultat från biomekaniska undersökningar av vidhäftande bakteriella organeller. Kraftmätande optisk pincett har använts för att karakterisera tre olika typer av pili, P, typ 1, och S pili, vilka kan uttryckas av uropatogena Escherichia coli. En kvantitativ biofysikalisk modell som beskriver deras förlängningsegenskaper under pålagd kraft har konstruerats. Modellen bygger på pilis strukturella uppbyggnad samt på dess respons som uppmäts med den kraftmätande optiska pincetten. Modellen beskriver de biomekaniska egenskaperna väl för alla tre pili. Dessutom kan ett antal specifika bindnings- och subenhetsparametrar bestämmas, t.ex. interaktionsenergier och bindningslängder. Skillnaden mellan dessa parametrar hos de tre pilis samt deras olika kraftrespons har jämförts. Detta arbete har dels resulterat i en förbättrad förståelse av pilis biomekaniska funktion och dels i en metod som, med hög noggrannhet, tillåter oss att bestämma ett antal biomekaniska egenskaper hos olika organeller på ett effektivt sätt.

Place, publisher, year, edition, pages
Umeå: Fysik , 2007. , 79 p.
Keyword [en]
optical tweezers, biological physics, unfolding, Escherichia coli, force measurements, energy landscape, dynamic force spectroscopy, manipulation, polymers, pili
National Category
Physical Sciences
Identifiers
URN: urn:nbn:se:umu:diva-1425ISBN: 978-91-7264-435-9 (print)OAI: oai:DiVA.org:umu-1425DiVA: diva2:141012
Public defence
2007-11-30, N450, Naturvetarhuset, Umeå Universitet Campus, Umeå, 13:00 (English)
Opponent
Supervisors
Available from: 2007-11-08 Created: 2007-11-08 Last updated: 2009-08-19Bibliographically approved
List of papers
1. A sticky chain model of the elongation and unfolding of escherichia coli P pili under stress
Open this publication in new window or tab >>A sticky chain model of the elongation and unfolding of escherichia coli P pili under stress
2006 (English)In: Biophysical Journal, ISSN 0006-3495, E-ISSN 1542-0086, Vol. 90, no 5, 1521-1534 p.Article in journal (Refereed) Published
Abstract [en]

A model of the elongation of P pili expressed by uropathogenic Escherichia coli exposed to stress is presented. The model is based upon the sticky chain concept, which is based upon Hooke’s law for elongation of the layer-to-layer and head-to-tail bonds between neighboring units in the PapA rod and a kinetic description of the opening and closing of bonds, described by rate equations and an energy landscape model. It provides an accurate description of the elongation behavior of P pili under stress and supports a hypothesis that the PapA rod shows all three basic stereotypes of elongation/unfolding: elongation of bonds in parallel, the zipper mode of unfolding, and elongation and unfolding of bonds in series. The two first elongation regions are dominated by a cooperative bond opening, in which each bond is influenced by its neighbor, whereas the third region can be described by individual bond opening, in which the bonds open and close randomly. A methodology for a swift extraction of model parameters from force-versus-elongation measurements performed under equilibrium conditions is derived. Entities such as the free energy, the stiffness, the elastic elongation, the opening length of the various bonds, and the number of PapA units in the rod are determined.

Identifiers
urn:nbn:se:umu:diva-2744 (URN)10.1529/biophysj.105.074674 (DOI)
Available from: 2007-11-08 Created: 2007-11-08 Last updated: 2017-12-14Bibliographically approved
2. Dynamic Force Spectroscopy of E. coli P Pili
Open this publication in new window or tab >>Dynamic Force Spectroscopy of E. coli P Pili
2006 (English)In: Biophysical Journal, ISSN 0006-3495, E-ISSN 1542-0086, Vol. 91, no 7, 2717-2725 p.Article in journal (Refereed) Published
Abstract [en]

Surface organelles (so-called pili) expressed on the bacterial membrane mediate the adhesion of Escherichia coli causing urinary tract infection. These pili possess some extraordinary elongation properties that are assumed to allow a close bacterium-to-host contact even in the presence of shear forces caused by urine flow. The elongation properties of P pili have therefore been assessed for low elongation speeds (steady-state conditions). This work reports on the behavior of P pili probed by dynamic force spectroscopy. A kinetic model for the unfolding of a helixlike chain structure is derived and verified. It is shown that the unfolding of the quaternary structure of the PapA rod takes place at a constant force that is almost independent of elongation speed for slow elongations (up to ~0.4 μm/s), whereas it shows a dynamic response with a logarithmic dependence for fast elongations. The results provide information about the energy landscape and reaction rates. The bond length and thermal bond opening and closure rates for the layer-to-layer bond have been assessed to ~0.76 nm, ~0.8 Hz, and ~8 GHz, respectively. The results also support a previously constructed sticky-chain model for elongation of the PapA rod that until now had been experimentally verified only under steady-state conditions.

Identifiers
urn:nbn:se:umu:diva-2745 (URN)10.1529/biophysj.106.087429 (DOI)
Available from: 2007-11-08 Created: 2007-11-08 Last updated: 2017-12-14Bibliographically approved
3. Force measuring optical tweezers system for long time measurements of P pili stability
Open this publication in new window or tab >>Force measuring optical tweezers system for long time measurements of P pili stability
2006 (English)In: Imaging, Manipulation, and Analysis of Biomolecules, Cells, and Tissues IV / [ed] Farkas, DL, Nicolau, DV, Leif, RC, 2006, Vol. 6088, 608810- p.Conference paper, Published paper (Refereed)
Abstract [en]

A force-measuring optical tweezers instrumentation and long time measurements of the elongation and retraction of bacterial fimbriae from Uropathogenic E. coli (UPEC) under strain are presented. The instrumentation is presented in some detail. Special emphasis is given to measures taken to reduce the influence of noise and drifts in the system and from the surrounding, which makes long term force measurements possible. Individual P pili from UPEC bacteria were used as a biological model system for repetitive unfolding and refolding cycles of bacterial fimbriae under equilibrium conditions. P pili have evolved into a three-dimensional helix-like structure, the PapA rod, that can be successively and significantly elongated and/or unfolded when exposed to external forces. The instrumentation is used for characterization of the force-vs.-elongation response of the PapA rod of individual P pili, with emphasis on the long time stability of the forced unfolding and refolding of the helical structure of the PapA rod. The results show that the PapA rod is capable of withstanding extensive strain, leading to a complete unfolding of the helical structure, repetitive times during the life cycle of a bacterium without any noticeable alteration of the mechanical properties of the P pili. This function is believed to be importance for UPEC bacteria in vivo since it provides a close contact to a host cell (which is an initial step of invasion) despite urine cleaning attempts.

Series
Proceedings of the society of photo-optical instrumentation engineers (SPIE), ISSN 0277-786X ; 6088
Keyword
force measuring optical tweezers, bacterial fimbriae, E. coli, P pili, PapA, unfolding and refolding, macromolecules
National Category
Other Physics Topics
Identifiers
urn:nbn:se:umu:diva-2746 (URN)10.1117/12.642266 (DOI)000237107800032 ()2-s2.0-33646201092 (Scopus ID)0-8194-6130-X (ISBN)978-081946130-8 (ISBN)
Conference
Conference on Imaging, Manipulation, and Analysis of Biomolecules, Cells, and Tissues IV. San Jose, CA. JAN 23-25, 2006. SPIE.
Available from: 2007-11-08 Created: 2007-11-08 Last updated: 2014-11-18Bibliographically approved
4. Optical tweezers based force measurement system for quantitating binding interactions: system design and application for the study of bacterial adhesion
Open this publication in new window or tab >>Optical tweezers based force measurement system for quantitating binding interactions: system design and application for the study of bacterial adhesion
Show others...
2004 (English)In: Biosensors & bioelectronics, ISSN 0956-5663, E-ISSN 1873-4235, Vol. 19, no 11, 1429-1437 p.Article in journal (Refereed) Published
Abstract [en]

An optical force measurement system for quantitating forces in the pN range between micrometer-sized objects has been developed. The system was based upon optical tweezers in combination with a sensitive position detection system and constructed around an inverted microscope. A trapped particle in the focus of the high numerical aperture microscope-objective behaves like an omnidirectional mechanical spring in response to an external force. The particle’s displacement from the equilibrium position is therefore a direct measure of the exerted force. A weak probe laser beam, focused directly below the trapping focus, was used for position detection of the trapped particle (a polystyrene bead). The bead and the condenser focus the light to a distinct spot in the far field, monitored by a position sensitive detector. Various calibration procedures were implemented in order to provide absolute force measurements. The system has been used to measure the binding forces between Escherichia coli bacterial adhesins and galabiose-functionalized beads

Keyword
Force measurements, Optical tweezers, Bacterial adhesion
Identifiers
urn:nbn:se:umu:diva-2747 (URN)10.1016/j.bios.2003.12.029 (DOI)
Available from: 2007-11-08 Created: 2007-11-08 Last updated: 2017-12-14Bibliographically approved
5. Dual-trap technique for reduction of low-frequency noise in force measuring optical tweezers
Open this publication in new window or tab >>Dual-trap technique for reduction of low-frequency noise in force measuring optical tweezers
2007 (English)In: Applied Optics, ISSN 1559-128X, E-ISSN 2155-3165, Vol. 46, no 3, 405-412 p.Article in journal (Refereed) Published
Abstract [en]

High-resolution long-time force measurements by optical tweezers are often limited by low- frequency (1/f) noise. A dual-trap technique is presented that can reduce such noise in the force signal. It incorporates a second trap (a reference trap) that probes the noise in the system and it is based upon the assumption that the low-frequency parts of the noise from the two traps are correlated. A subtraction of the low-frequency signal from the reference trap from the signal from the force measuring trap will therefore yield a net signal that is significantly less influenced by noise. It is shown that this dual-trap technique can reduce the noise in the force signal up to 60% depending on detection bandwidth.

Identifiers
urn:nbn:se:umu:diva-2748 (URN)10.1364/AO.46.000405 (DOI)
Available from: 2007-11-08 Created: 2007-11-08 Last updated: 2017-12-14Bibliographically approved
6. Modeling of the elongation and retraction of Escherichia coli P pili under strain by Monte Carlo simulations
Open this publication in new window or tab >>Modeling of the elongation and retraction of Escherichia coli P pili under strain by Monte Carlo simulations
2008 (English)In: European Biophysics Journal, ISSN 0175-7571, E-ISSN 1432-1017, Vol. 37, no 4, 381-391 p.Article in journal (Refereed) Published
Abstract [en]

P pili are fimbrial adhesion organelles expressed by uropathogenic Escherichia coli in the upper urinary tract. They constitute a stiff helix-like polymer consisting of a number of subunits joined by head-to-tail bonds. The elongation and retraction properties of individual P pili exposed to strain have been modeled by Monte Carlo (MC) simulations. The simulation model is based upon a three-state energy landscape that deforms under an applied force. Bond opening and closure are modeled by Bells theory while the elongation of the linearized part of the pilus is described by a worm-like chain model. The simulations are compared with measurements made by force measuring optical tweezers. It was found that the simulations can reproduce pili elongation as well as retraction, under both equilibrium and dynamic conditions, including entropic effects. It is shown that the simulations allow for an assessment of various model parameters, e.g. the unfolding force, energy barrier heights, and various distances in the energy landscape, including their stochastic spread that analytical models are unable to do. The results demonstrate that MC simulations are useful to model elongation and retraction properties of P pili, and therefore presumably also other types of pili, exposed to strain and/or stress. MC simulations are particularly suited for description of helix-like pili since these have an intricate self-regulating mechanical elongation behavior that makes analytical descriptions non-trivial when dynamic processes are studied, or if additional interactions in the rod or the behavior of the adhesion tip needs to be modeled.

Keyword
P pili, Escherichia coli, Optical tweezers, Monte Carlo simulations, Unfolding, Force spectroscopy
Identifiers
urn:nbn:se:umu:diva-2749 (URN)10.1007/s00249-007-0223-6 (DOI)
Available from: 2007-11-08 Created: 2007-11-08 Last updated: 2017-12-14Bibliographically approved
7. The biomechanical properties of E. coli pili for urinary tract attachment reflect the host environment
Open this publication in new window or tab >>The biomechanical properties of E. coli pili for urinary tract attachment reflect the host environment
2007 (English)In: Biophysical Journal, ISSN 0006-3495, E-ISSN 1542-0086, Vol. 93, no 9, 3008-3014 p.Article in journal (Refereed) Published
Abstract [en]

Uropathogenic Escherichia coli express pili that mediate binding to host tissue cells. We demonstrate with in situ force measuring optical tweezers that the ability of P and type 1 pili to elongate by unfolding under exposure to stress is a shared property with some differences. The unfolding force of the quaternary structures under equilibrium conditions is similar, 28 ± 2 and 30 ± 2 pN for P pili and type 1 pili, respectively. However, type 1 pili are found to be more rigid than P pili through their stronger layer-to-layer bonds. It was found that type 1 pili enter a dynamic regime at elongation speeds of 6 nm/s, compared to 400 nm/s for P pili; i.e., it responds faster to an external force. This possibly helps type 1 to withstand the irregular urine flow in the urethra as compared to the more constant urine flow in the upper urinary tract. Also, it was found that type 1 pili refold during retraction at two different levels that possibly could be related to several possible configurations. Our findings highlight functions that are believed to be of importance for the bacterial ability to sustain a basic antimicrobial mechanism of the host and for bacterial colonization.

Keyword
Bacterial Adhesion/*physiology, Biomechanics, Escherichia coli/*physiology, Fimbriae; Bacterial/*chemistry/*physiology, Humans, Microscopy; Atomic Force, Optical Tweezers, Urinary Tract Infections/microbiology, Urinary Tract Physiology
Identifiers
urn:nbn:se:umu:diva-2750 (URN)10.1529/biophysj.107.110643 (DOI)
Available from: 2007-11-08 Created: 2007-11-08 Last updated: 2017-12-14Bibliographically approved
8. Physical Properties of Biopolymers Assessed by Optical Tweezers: Analysis of folding and refolding of bacterial pili
Open this publication in new window or tab >>Physical Properties of Biopolymers Assessed by Optical Tweezers: Analysis of folding and refolding of bacterial pili
Show others...
2008 (English)In: ChemPhysChem, ISSN 1439-4235, E-ISSN 1439-7641, Vol. 9, no 2, 221-235 p.Article in journal (Refereed) Published
Abstract [en]

Bacterial adhesion to surfaces mediated by specific adhesion organelles that promote infections, as exemplified by the pili of uropathogenic E. coli, is studied mostly at the level of cell-cell interactions and thereby reflects the averaged behavior of multiple pili. The role of pilus rod structure has therefore only been estimated from the outcome of experiments involving large numbers of organelles at the same time. It has, however, lately become clear that the biomechanical behavior of the pilus shafts play an important, albeit hitherto rather unrecognized, role in the adhesion process. For example, it has been observed that shafts from two different strains, even though they are similar in structure, result in large differences in the ability of the bacteria to adhere to their host tissue. However, in order to identify all properties of pilus structures that are of importance in the adhesion process, the biomechanical properties of pili must be assessed at the single-molecule level. Due to the low range of forces of these structures, until recently it was not possible to obtain such information. However, with the development of force-measuring optical tweezers (FMOT) with force resolution in the low piconewton range, it has lately become possible to assess forces mediated by individual pili on single living bacteria in real time. FMOT allows for a more or less detailed mapping of the biomechanical properties of individual pilus shafts, in particular those that are associated with their elongation and contraction under stress. This Mi- nireview presents the FMOT technique, the biological model system, and results from assessment of the biomechanical properties of bacterial pili. The information retrieved is also compared with that obtained by atomic force microscopy.

Keyword
bacterial adhesion, force spectroscopy, mechanical properties, optical tweezers´, single-molecule studies
Identifiers
urn:nbn:se:umu:diva-17336 (URN)doi:10.1002/cphc.200700389 (DOI)
Available from: 2008-01-25 Created: 2008-01-25 Last updated: 2017-12-14Bibliographically approved
9. Characterization of S pili — investigation of their mechanical properties
Open this publication in new window or tab >>Characterization of S pili — investigation of their mechanical properties
2007 (English)Manuscript (preprint) (Other (popular science, discussion, etc.))
Identifiers
urn:nbn:se:umu:diva-2752 (URN)
Available from: 2007-11-08 Created: 2007-11-08 Last updated: 2012-04-25Bibliographically approved

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