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A study of bacterial adhesion on a single-cell level by means of force measuring optical tweezers and simulations
Umeå University, Faculty of Science and Technology, Applied Physics and Electronics. (Optical Tweezers Center)
2009 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The intriguing world of microbiology is nowadays accessible for detailed exploration at a single–molecular level. Optical tweezers are a novel instrument that allows for non–invasive manipulation of single cells by the sole use of laser light and operates on the nano– and micrometer scale which corresponds to the same length scale as living cells. Moreover, forces within the field of microbiology are typically in the picoNewton range which is in accordance with the capability of force measuring optical tweezers systems. Both these conformabilities imply that force measuring optical tweezers is suitable for studies of single living cells. This thesis focuses on the mechanisms of bacterial attachments to host cells which constitute the first step in bacterial infection processes. Bacteria bind specifically to host receptors by means of adhesins that are expressed either directly on the bacterial membrane or on micrometer–long adhesion organelles that are called pili. The properties of single adhesin–receptor bonds that mediate adherence of the bacterium Helicobacter pylori are first examined at various acidities. Further on, biomechanical properties of P pili expressed by Escherichia coli are presented to which computer simulations, that capture the complex kinetics of the pili structure and precisely replicate measured data, are applied. Simulations are found to be a powerful tool for investigations of adhesive attributes of binding systems and are utilized in the analyses of the specific binding properties of P pili on a single–pilus level. However, bacterial binding systems generally involve a multitude of adhesin–receptor bonds. To explore bacterial attachments, the knowledge from single–pilus studies is brought into a full multipili attachment scenario which is analyzed by means of theoretical treatments and simulations. The results are remarkable in several aspects. Not only is it found that the intrinsic properties of P pili are composed in an optimal combination to promote strong multipili bindings. The properties of the pili structure itself are also found to be optimized with respect to its in vivo environment. Indeed, the true meaning of the attributes derived at a single–pilus level cannot be unraveled until a multipili–binding system is considered. Whereas detailed studies are presented for the helix–like P pili expressed by Gram–negative Escherichia coli, conceptual studies are presented for the open coil–like T4 pili expressed by Gram–positive Streptococcus pneumoniae. The structural and adhesive properties of these two types of pili differ considerably. These dissimilarities have far–reaching consequences on the adhesion possibilities at both single–pilus and multipili levels which are discussed qualitatively. Moreover, error analyses of conventional data processing methods in dynamic force spectroscopy as well as development of novel analysis methods are presented. These findings provide better understanding of how to perform refined force measurements on single adhesion organelles as well as how to improve the analyses of measurement data to obtain accurate parameter values of biomechanical entities. In conclusion, this thesis comprises a study of bacterial adhesion organelles and the way they cooperate to establish efficient attachment systems that can successfully withstand strong external forces that acts upon bacteria. Such systems can resist, for instance, rinsing effects and thereby allow bacteria to colonize their host. By understanding the complexity, and thereby possible weaknesses, of bacterial attachments, new targets for combating bacterial infections can be identified.

Place, publisher, year, edition, pages
Umeå: Print & Media , 2009. , 108 p.
National Category
Physical Sciences
Research subject
Physics; cellforskning; Molecular Cellbiology
Identifiers
URN: urn:nbn:se:umu:diva-21493ISBN: 978-91-7264-765-7 (print)OAI: oai:DiVA.org:umu-21493DiVA: diva2:211352
Public defence
2009-05-08, N420, Naturvetarhuset, Umeå Universitet, Umeå, 10:00 (English)
Opponent
Supervisors
Available from: 2009-04-17 Created: 2009-04-14 Last updated: 2009-04-17Bibliographically approved
List of papers
1. Methods and error estimations of uncertainties in single-molecule dynamic force spectroscopy
Open this publication in new window or tab >>Methods and error estimations of uncertainties in single-molecule dynamic force spectroscopy
2009 (English)In: European Biophysics Journal, ISSN 0175-7571, E-ISSN 1432-1017, Vol. 38, no 7, 911-922 p.Article in journal (Refereed) Published
Abstract [en]

In dynamic force spectroscopy, access to the characteristic parameters of single molecular bonds requires non-trivial measurements and data processing as the rupture forces are found not only to be distributed over a wide range, but are also dependent on the loading rate. The choice of measurement procedure and data processing methods has a considerable impact on the accuracy and precision of the final results. We analyse, by means of numerical simulations, methods to minimize and assess the magnitude of the expected errors for different combinations of experimental and evaluation methods. It was found that the choice of fitting function is crucial to extract correct parameter values. Applying a Gaussian function, which is a common practice, is equivalent to introducing a systematic error, and leads to a consequent overestimation of the thermal off-rate by more than 30%. We found that the precision of the bond length and the thermal off-rate, in presence of unbiased noise, were improved by reducing the number of loading rates for a given number of measurements. Finally, the results suggest that the minimum number of measurements needed to obtain the bond strength, with acceptable precision, exceeds the common number of ~100 reported in literature.

 

Keyword
Specific Binding, DFS, Receptor-ligand, Error Propagation, Bond Length, Thermal off-rate, Monte Carlo Simulation
National Category
Other Physics Topics
Research subject
Physics
Identifiers
urn:nbn:se:umu:diva-21485 (URN)10.1007/s00249-009-0471-8 (DOI)000269055500009 ()
Available from: 2009-04-14 Created: 2009-04-14 Last updated: 2016-05-27Bibliographically approved
2. Physical properties of the specific PapG–galabiose binding in E. coli P pili-mediated adhesion
Open this publication in new window or tab >>Physical properties of the specific PapG–galabiose binding in E. coli P pili-mediated adhesion
2009 (English)In: European Biophysics Journal, ISSN 0175-7571, E-ISSN 1432-1017, Vol. 38, no 2, 245-254 p.Article in journal (Refereed) Published
Abstract [en]

Detailed analyses of the mechanisms thatmediate binding of the uropathogenic Escherichia coli tohost cells are essential, as attachment is a prerequisite forthe subsequent infection process. We explore, by means offorce measuring optical tweezers, the interaction betweenthe galabiose receptor and the adhesin PapG expressed byP pili on single bacterial cells. Two variants of dynamicforce spectroscopy were applied based on constant andnon-linear loading force. The specific PapG–galabiosebinding showed typical slip-bond behaviour in the forceinterval (30–100 pN) set by the pilus intrinsic biomechanicalproperties. Moreover, it was found that the bondhas a thermodynamic off-rate and a bond length of2.6×10-3 s-1 and 5.0 Å , respectively. Consequently, thePapG–galabiose complex is significantly stronger thanthe internal bonds in the P pilus structure that stabilizes thehelical chain-like macromolecule. This finding suggeststhat the specific binding is strong enough to enable the Ppili rod to unfold when subjected to strong shear forces inthe urinary tract. The unfolding process of the P pili rodpromotes the formation of strong multipili interaction,which is important for the bacterium to maintain attachmentto the host cells.

Place, publisher, year, edition, pages
New York: Springer, 2009
Keyword
Escherichia coli, Non-covalent single bond, Slip-bond, Dynamic force spectroscopy, Receptor–ligand interaction
National Category
Cell Biology Biochemistry and Molecular Biology
Research subject
Physics
Identifiers
urn:nbn:se:umu:diva-19734 (URN)10.1007/s00249-008-0376-y (DOI)000262671600011 ()
Available from: 2009-03-10 Created: 2009-03-10 Last updated: 2016-05-27Bibliographically approved
3. Measurements of the binding force between the Helicobacter pylori adhesin BabA and the Lewis b blood group antigen using optical tweezers
Open this publication in new window or tab >>Measurements of the binding force between the Helicobacter pylori adhesin BabA and the Lewis b blood group antigen using optical tweezers
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2005 (English)In: Journal of Biomedical Optics, ISSN 1560-2281 (Online), Vol. 10, no 4, 044024- p.Article in journal (Refereed) Published
Abstract [en]

Helicobacter pylori is a world-wide spread bacterium that causes persistent infections and chronic inflammations that can develop into gastritis and peptic ulcer disease. It expresses several adhesin proteins on its surface that bind to specific receptors in the gastric epithelium. The most well-known adhesin is BabA, which has previously been shown to bind specifically to the fucosylated blood group antigen Lewis b (Leb). The adhesion forces between BabA and the Leb antigen are investigated in this work and assessed by means of optical tweezers. A model system for in situ measurements of the interaction forces between individual bacteria and beads coated with Leb is developed. It is found that the de-adhesion force in this model system, measured with a loading rate of approximately 100 pNs, ranges from 20 to 200 pN. The de-adhesion force appears predominantly as multiples of an elementary force, which is determined to 25+/-1.5 pN and identified as the unbinding force of an individual BabA-Leb binding. It is concluded that adhesion in general is mediated by a small number of bindings (most often 1 to 4) despite that the contact surface between the bacterium and the bead encompassed significantly more binding sites.

Place, publisher, year, edition, pages
Bellingham, WA: , 2005
Identifiers
urn:nbn:se:umu:diva-12120 (URN)16178657 (PubMedID)
Available from: 2007-12-18 Created: 2007-12-18 Last updated: 2016-05-27Bibliographically approved
4. Dynamic force spectroscopy of the Helicobacter pylori BabA-Lewis b binding
Open this publication in new window or tab >>Dynamic force spectroscopy of the Helicobacter pylori BabA-Lewis b binding
2009 (English)In: Biophysical Chemistry, ISSN 0301-4622, E-ISSN 1873-4200, Vol. 143, no 1-2, 102-105 p.Article in journal (Refereed) Published
Abstract [en]

The binding strength of the Helicobacter pylori adhesin–receptor complex BabA-ABO/Lewis b has been analyzed by means of dynamic force pectroscopy. High-resolution measurements of rupture forces were performed in situ on single bacterial cells, expressing the high-affinity binding BabA adhesin, by the use of force measuring optical tweezers. The resulting force spectra revealed the mechanical properties of a single BabA–Leb bond. It was found that the bond is dominated by one single energy barrier and that it is a slipbond. The bond length and thermal off-rate were assessed to be 0.86±0.07 nm and 0.015±0.006 s−1, respectively.

Place, publisher, year, edition, pages
Amsterdam: Elsevier, 2009
Keyword
Optical tweezers, Single bond dissociation, Receptor–ligand interaction, Bond strength, Lewis b receptor
National Category
Physical Sciences Biochemistry and Molecular Biology
Research subject
Physics
Identifiers
urn:nbn:se:umu:diva-21486 (URN)10.1016/j.bpc.2009.03.007 (DOI)000267006000012 ()
External cooperation:
Available from: 2009-04-14 Created: 2009-04-14 Last updated: 2016-08-31Bibliographically approved
5. pH regulated H. pylori adherence: implications for persistent infection and disease
Open this publication in new window or tab >>pH regulated H. pylori adherence: implications for persistent infection and disease
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

Helicobacter pylori’s BabA adhesin binds strongly to gastric mucosal ABH/Leb glycans on the stomach epithelium and overlying mucus, materials continuously shed into the acidic gastric lumen. Here we report that this binding is acid labile, acid inactivation is fully reversible; and acid lability profiles vary with BabA sequence and correlate with disease patterns. Isogenic H. pylori strains from the gastric antrum and more acidic corpus were identified that differed in acid lability of receptor binding and in sequence near BabA’s carbohydrate binding domain. We propose that reversible acid inactivation of receptor binding helps H. pylori avoid clearance by mucosal shedding, and that strain differences in acid lability affect tissue tropism and the spectrum of associated gastric diseases.

National Category
Microbiology in the medical area
Identifiers
urn:nbn:se:umu:diva-21487 (URN)
Available from: 2009-04-14 Created: 2009-04-14 Last updated: 2016-05-27Bibliographically approved
6. Unraveling the secrets of bacterial adhesion organelles using single molecule force spectroscopy
Open this publication in new window or tab >>Unraveling the secrets of bacterial adhesion organelles using single molecule force spectroscopy
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2010 (English)In: Springer series in chemical physics: single molecule spectroscopy in chemistry, physics and biology, Springer Verlag , 2010, 96, 337-362 p.Chapter in book (Other academic)
Abstract [en]

Many types of bacterium express micrometer-long attachment organelles (so called pili) whose role is to mediate adhesion to host tissue. Until recently, little was known about their function in the adhesion process. Forcemeasuring  ptical tweezers (FMOT) have since then been used to unravel the  iomechanical properties of various types of pili, primarily those from uropathogenic E. coli, in particular their force-vs.-elongation response, but lately also some properties of the adhesin situated and the distal end of the pilus. This knowledge provides an understanding of how piliated bacteria can sustain external shear forces caused by rinsing processes, e.g. urine flow. It has been found that anytypes of pilus exhibit unique and complex force-vs.-elongation responses. It has been conjectured that their dissimilar properties impose significant differences in their ability to sustain external forces and that different types of pilus therefore have dissimilar predisposition to withstand different types of rinsing conditions. An understanding of these properties is of high importance since it can serve as a basis for finding new means to combat bacterial adhesion, including that caused by antibiotic-resistance bacteria. This work presents a review of the current status of the assessment of biophysical properties of individual pili on single bacteria exposed to strain/stress, primarily by the FMOT technique. It also addresses, for the first time, how the elongation and retraction properties of the rod couple to the adhesive properties of the tip adhesin.

Place, publisher, year, edition, pages
Springer Verlag, 2010 Edition: 96
Series
Nobel Symposium 138: Single Molecule Spectroscopy in Chemistry, Physics and Biosciences, ISSN 0172-6218 ; 138
National Category
Condensed Matter Physics
Identifiers
urn:nbn:se:umu:diva-21488 (URN)978-3-642-02596-9 (ISBN)
Available from: 2009-04-14 Created: 2009-04-14 Last updated: 2016-05-27Bibliographically approved
7. 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, 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: 2009-08-19Bibliographically approved
8. Characterization of the Biomechanical Properties of T4 Pili Expressed by Streptococcus pneumoniae – A Comparison between Helix-like and Open Coil-like Pili
Open this publication in new window or tab >>Characterization of the Biomechanical Properties of T4 Pili Expressed by Streptococcus pneumoniae – A Comparison between Helix-like and Open Coil-like Pili
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2009 (English)In: ChemPhysChem, ISSN 1439-4235, E-ISSN 1439-7641, Vol. 10, no 9-10, 1533-1540 p.Article in journal (Refereed) Published
Abstract [en]

Bacterial adhesion organelles, known as fimbria or pili, are expressed by Gram–positive as well as Gram–negative bacteria families. These appendages play a key role in the first steps of the invasion and infection processes, and they therefore provide bacteria with pathogenic abilities. To improve the knowledge of pili-mediated bacterial adhesion to host cells and how these pili behave under the presence of an external force, we first characterize, using force measuring optical tweezers, open coil-like T4 pili expressed by Gram–positive Streptococcus pneumoniae with respect to their biomechanicalproperties. It is shown that their elongation behavior can be well described by the worm-like chain model and that they possess a large degree of flexibility. Their properties are then compared with those of helix-like pili expressed by Gram–negative uropathogenic Escherichia coli (UPEC), which have different pili architecture. The differences suggest that these two types of pili have distinctly dissimilar mechanisms to adhere and sustain external forces. Helix-like pili expressed by UPEC bacteria adhere to host cells by single adhesins located at the distal end of the pili while their helix-like structures act as shock absorbers to dampen the irregularly shear forces induced by urine flow and to increase the cooperativity of the pili ensemble. Open coil-like pili expressed by S. pneumoniae adhere to cells by a multitude of adhesins distributed along the pili. It is hypothesized that these two types of pili represent different strategies of adhering to host cells in the presence of external forces. When exposed to significant forces, bacteria expressing helix-like pili remain attached bydistributing the external force among a multitude of pili, whereas bacteria expressing open coil-like pili sustain large forces primarily by their multitude of binding adhesins.

National Category
Other Physics Topics
Identifiers
urn:nbn:se:umu:diva-21489 (URN)10.1002/cphc.200900195 (DOI)000267928100027 ()19565578 (PubMedID)
Available from: 2009-04-14 Created: 2009-04-14 Last updated: 2016-05-27Bibliographically approved
9. Multipili Attachment of Bacteria with Helix–like Pili Exposed to Stress
Open this publication in new window or tab >>Multipili Attachment of Bacteria with Helix–like Pili Exposed to Stress
2009 (English)In: Journal of Chemical Physics, ISSN 0021-9606, Vol. 130, 235102- p.Article in journal (Refereed) Published
Abstract [en]

A number of biomechanical properties of various types of pili expressed by Escherichia coli, predominantly their force–vs.–elongation behavior, have previously been assessed in detail on a single pilus level. In vivo, however, bacteria bind in general to host cells by a multitude of pili, which presumably provides them with adhesion properties that differs from those of single pili. Based upon the previously assessed biomechanical properties of individual pili, this work presents a theoretical analysis of the adhesion properties of multipili–attaching bacteria expressing helix–like pili exposed to an external force. Expressions for the adhesion lifetime of dual– and multipili–attaching bacteria are derived and their validity is verified by Monte Carlo simulations. It is shown that the adhesion lifetime of a multipili–binding bacterium depends to a large degree on the cooperativity of the attaching pili, which, in turn, depends strongly on their internal biomechanical properties, in particular their helix–like structure and its ability to elongate, which, in turn, depend on the intrinsic properties of the bonds, e.g. their lengths and activation energies. It is shown, for example, that a decrease in the length of a layer–to–layer bond in the rod of P pili, expressed by E. coli, by 50 % leads to a decrease in the adhesion lifetime of a bacterium attaching by 10 pili and exposed to a force of 500 pN by three orders of magnitude. The results indicate moreover that the intrinsic properties of the rod for this particular type of pili are optimized for multipili attachment under a broad range of external forces and presumably also to its in vivo environment. Even though the results presented in this work apply quantitatively to one type of pilus, they are assumed to apply qualitatively to all helix–like pili systems expressing slip bonds.

Identifiers
urn:nbn:se:umu:diva-21490 (URN)10.1063/1.3148027 (DOI)
Available from: 2009-04-14 Created: 2009-04-14 Last updated: 2010-03-03
10. Differentiating pili expressed by enterotoxigenic and uropathogenic escherichia coli with optical tweezers
Open this publication in new window or tab >>Differentiating pili expressed by enterotoxigenic and uropathogenic escherichia coli with optical tweezers
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

Enterotoxigenic Escherichia coli (ETEC) attach to the host epithelium in the intestinal tract via specific adhesion organelles expressed on the cell membrane. We investigate, by force measuring optical tweezers, the intrinsic biomechanical properties and kinetics of the colonization factor I (CFA/I) at a single pilus level. The measurements indicate that CFA/I pili are helix-like structures that can both be unraveled to a linearized polymer by applying a small external force, 7.5 ± 1.5 pN but also regain its helix-like structure when the applied force is reduced. The data confirm that layer-to-layer interactions, that stabilize the helix-like structure, are much weaker than the interactions found in pili expressed by Uropathogenic Escherichia coli (UPEC). It is also found, contrary to previous results assessed from UPEC pili, that the CFA/I undergo in some cases a sudden structural change, a force drop of ~2 pN, when unraveled from the helix-like configuration to an open helical linearized fiber. These data suggest a rotation of the filament about its helical axis, followed by a region in which the force required to extend the pili further increases rapidly. During this final elongation to a super-extended fiber, CFA/I pili do not show any structural transition as seen for UPEC pili. In addition, the CFA/I pili show faster kinetics than UPEC pili that allows for a larger dynamic regime of in vivo shear forces. The unfolding and refolding possibility points toward an organelle that has evolved to allow for dynamic damping of external forces and handling of harsh motion without breaking.

National Category
Physical Sciences
Identifiers
urn:nbn:se:umu:diva-21492 (URN)
Available from: 2009-04-14 Created: 2009-04-14 Last updated: 2012-05-14Bibliographically approved

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