Instrumentation and methodologies for single molecule force spectroscopy on bacterial adhesion organelles by the
use of force measuring optical tweezers have been developed. A thorough study of the biomechanical properties of
fimbrial adhesion organelles expressed by uropathogenic <i>E. coli</i>, so-called pili, is presented. Steady-state as well as
dynamic force measurements on P pili, expressed by <i>E. coli</i> causing pyelonephritis, have revealed, among other things,
various unfolding and refolding properties of the helical structure of P pili, the PapA rod. Based on these properties an
energy landscape model has been constructed by which specific biophysical properties of the PapA rod have been
extracted, e.g. the number of subunits, the length of a single pilus, bond lengths and activation energies for bond
opening and closure. Moreover, long time repetitive measurements have shown that the rod can be unfolded and
refolded repetitive times without losing its intrinsic properties. These properties are believed to be of importance for
the bacteria's ability to maintain close contact with host cells during initial infections. The results presented are
considered to be of importance for the field of biopolymers in general and the development of new pharmaceuticals
towards urinary tract infections in particular. The results show furthermore that the methodology can be used to gain
knowledge of the intrinsic biomechanical function of adhesion organelles. The instrumentation is currently used for
characterization of type 1 pili, expressed by <i>E. coli</i> causing cystitis, i.e. infections in the bladder. The first force
spectrometry investigations of these pili will be presented.
The rapid development of the optical tweezers technique has broadened the applicability of the technique from physics to biology. Although the construction of an optical tweezers system only requires basic skills in optics, the realization of an optical tweezers set up is not always as easy as it seems. A number of designs for moveable traps have been presented in the literature. It is not clear to the readers, however, how the movability of the trap affects its quality. We have therefore scrutinized and compared the most commonly used techniques for steering of an optical trap in terms of the aberrations they introduce. The study shows that a moveable trap based on the movement of a lens introduces significantly more aberration than the systems based on fiber optics or the tilting of a mirror.
A force-measuring optical tweezers instrumentation and long time measurements of the elongation and retraction of bacterial fimbriae from Uropathogenic <i>E. coli</i> (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.
Optical tweezers have previously been used to characterize the force-vs.-elongation dependence of the PapA rod of uropathogenic <i>E. coli</i> P pili. It was found that the PapA rod elongates in several elongation regions. In the two first, the elongation originates from an elastic stretching and a sequential unfolding of the layer-to-layer bonds (and thereby of the helical structure). Region III is characterized by an elongation that originates from an elastic stretching and an opening of the head-to-tail bonds in the linearized PapA rod. The opening of these bonds takes place in a random order, wherefore the response in this region is affected by entropy. Since the entropic softening of a macromolecule depends on the number of units, the shape of this region can be used to assess the number of PapA units. We provide in this work a recipe for how this can be done solely from the form of region III. An advantage with this technique is that it does not require a continuous monitoring of the elongation of a single PapA rod from unstretched conditions, which often is difficult because of simultaneous multi-pili binding; it suffices to detect it in the third region at which binding often is mediated by only one pilus. Another advantage is that it does not require any prior knowledge about (or assessment of) any physical entity of the PapA rod; the number of PapA units can be assessed solely from the shape of the curve in the third elongation region.
The ability of uropathogenic Escherichia coli (UPEC) to cause urinary tract infections is dependent on their ability to colonize the uroepithelium. Infecting bacteria ascend the urethra to the bladder and then kidneys by attaching to the uroepithelial cells via the differential expression of adhesins. P pili are associated with pyelonephritis, the more severe infection of the kidneys. In order to find means to treat pyelonephritis, it is therefore of interest to investigate the properties P pili. The mechanical behavior of individual P pili of uropathogenic Escherichia coli has recently been investigated using optical tweezers. P pili, whose main part constitutes the PapA rod, composed of ~1000 PapA subunits in a helical arrangement, are distributed over the bacterial surface and mediate adhesion to host cells. We have earlier studied P pili regarding its stretching/elongation properties where we have found and characterized three different elongation regions, of which one constitute an unfolding of the quaternary (helical) structure of the PapA rod. It was shown that this unfolding takes place at an elongation independent force of 27 ± 2 pN. We have also recently performed studies on its folding properties and shown that the unfolding/folding of the PapA rod is completely reversible. Here we present a study of the dynamical properties of the PapA rod. We show, among other things, that the unfolding force increases and that the folding force decreases with the speed of unfolding and folding respectively. Moreover, the PapA rod can be folded-unfolded a significant number of times without loosing its characteristics, a phenomenon that is believed to be important for the bacterium to keep close contact to the host tissue and consequently helps the bacterium to colonize the host tissue.
Optical tweezers together with a position sensitive detection system allows measurements of forces in the pN range between micro-sized biological objects. A prototype force measurement system has been constructed around in inverted microscope with an argon-ion pumped Ti:sapphire laser as light source for optical trapping. A trapped particle in the focus of the high numerical aperture microscope-objective behaves like an omni-directional mechanical spring if an external force displaces it. The displacement from the equilibrium position is a measure of the exerted force. For position detection of the trapped particle (polystyrene beads), a He-Ne laser beam is focused a small distance below the trapping focus. An image of the bead appears as a distinct spot in the far field, monitored by a photosensitive detector. The position data is converted to a force measurement by a calibration procedure. The system has been used for measuring the binding forces between <i>E-coli</i> bacterial adhesin and their receptor sugars.