| 1. |
- Andersson, Magnus, et al.
(author)
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Differentiating pili expressed by enterotoxigenic and uropathogenic escherichia coli with optical tweezers
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Other publication (other academic/artistic)abstract
- 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.
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| 2. |
- Axner, Ove, 1957-, et al.
(author)
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Assessing bacterial adhesion on an individual adhesin and single pili level using optical tweezers
- 2011
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In: Bacterial adhesion. - Berlin : Springer Berlin/Heidelberg. ; , s. 301-313
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Book chapter (peer-reviewed)abstract
- Optical tweezers (OT) are a technique that, by focused laser light, can both manipulate micrometer sized objects and measure minute forces (in the pN range) in biological systems. The technique is therefore suitable for assessment of bacterial adhesion on an individual adhesin-receptor and single attachment organelle (pili) level. This chapter summarizes the use of OT for assessment of adhesion mechanisms of both non-piliated and piliated bacteria. The latter include the important helix-like pili expressed by uropathogenic Escherichia coli (UPEC), which have shown to have unique and intricate biomechanical properties. It is conjectured that the large flexibility of this type of pili allows for a redistribution of an external shear force among several pili, thereby extending the adhesion lifetime of bacteria. Systems with helix-like adhesion organelles may therefore act as dynamic biomechanical machineries, enhancing the ability of bacteria to withstand high shear forces originating from rinsing flows such as in the urinary tract. This implies that pili constitute an important virulence factor and a possible target for future anti-microbial drugs.
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| 3. |
- Axner, Ove, et al.
(author)
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Unraveling the secrets of bacterial adhesion organelles using single molecule force spectroscopy
- 2010. - 96
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In: Springer series in chemical physics. - : Springer Verlag. - 9783642025969 ; , s. 337-362
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Book chapter (other academic/artistic)abstract
- 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.
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| 4. |
- Björnham, Oscar, 1976-
(author)
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A study of bacterial adhesion on a single-cell level by means of force measuring optical tweezers and simulations
- 2009
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Doctoral thesis (other academic/artistic)abstract
- 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.
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| 5. |
- Björnham, Oscar, 1976-, et al.
(author)
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Catch-Bond behavior of bacteria binding by slip bonds
- 2010
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In: Biophysical Journal. - : Elsevier BV. - 0006-3495 .- 1542-0086. ; 99:5, s. 1331-1341
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Journal article (peer-reviewed)abstract
- It is shown that multipili-adhering bacteria expressing helix-like pili binding by slip bonds can show catch-bond behavior. When exposed to an external force, such bacteria can mediate adhesion to their hosts by either of two limiting means: sequential or simultaneous pili force exposure (referring to when the pili mediate force in a sequential or simultaneous manner, respectively). As the force is increased, the pili can transition from sequential to simultaneous pili force exposure. Since the latter mode of adhesion gives rise to a significantly longer bacterial adhesion lifetime than the former, this results in a prolongation of the lifetime, which shows up as a catch-bond behavior. The properties and conditions of this effect were theoretically investigated and assessed in some detail for dual-pili-adhering bacteria, by both analytical means and simulations. The results indicate that the adhesion lifetime of such bacteria can be prolonged by more than an order of magnitude. This implies that the adhesion properties of multibinding systems cannot be directly conveyed to the individual adhesion-receptor bonds.
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| 6. |
- Björnham, Oscar, 1976-, et al.
(author)
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Dynamic force spectroscopy of the Helicobacter pylori BabA-Lewis b binding
- 2009
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In: Biophysical Chemistry. - Amsterdam : Elsevier. - 0301-4622 .- 1873-4200. ; 143:1-2, s. 102-105
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Journal article (peer-reviewed)abstract
- 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.
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| 7. |
- Björnham, Oscar, 1976-, et al.
(author)
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Measurements of the binding force between the Helicobacter pylori adhesin BabA and the Lewis b blood group antigen using optical tweezers
- 2005
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In: Journal of Biomedical Optics. - Bellingham, WA : SPIE-Intl Soc Optical Eng. - 1083-3668 .- 1560-2281. ; 10:4, s. 044024-044032
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Journal article (peer-reviewed)abstract
- 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 ~100 pN/s, 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.
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| 8. |
- Björnham, Oscar, 1976-, et al.
(author)
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Methods and error estimations of uncertainties in single-molecule dynamic force spectroscopy
- 2009
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In: European Biophysics Journal. - : Springer Science and Business Media LLC. - 0175-7571 .- 1432-1017. ; 38:7, s. 911-922
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Journal article (peer-reviewed)abstract
- 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.
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| 9. |
- Björnham, Oscar, 1976-, et al.
(author)
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Modeling of the elongation and retraction of Escherichia coli P pili under strain by Monte Carlo simulations
- 2008
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In: European Biophysics Journal. - : Springer Science and Business Media LLC. - 0175-7571 .- 1432-1017. ; 37:4, s. 381-391
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Journal article (peer-reviewed)abstract
- 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.
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| 10. |
- Björnham, Oscar, 1976-, et al.
(author)
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Multipili Attachment of Bacteria with Helix–like Pili Exposed to Stress
- 2009
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In: Journal of Chemical Physics. - : AIP Publishing. - 0021-9606 .- 1089-7690. ; 130, s. 235102-
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Journal article (peer-reviewed)abstract
- 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.
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