The mechanical behavior of individual P pili of uropathogenic Escherichia coli has been investigated using optical tweezers. P pili, whose main part constitutes the PapA rod, composed of approximately 10(3) PapA subunits in a helical arrangement, are distributed over the bacterial surface and mediate adhesion to host cells. They are particularly important in the pathogenesis of E. coli colonizing the upper urinary tract and kidneys. A biological model system has been established for in situ measurements of the forces that occur during mechanical stretching of pili. A mathematical model of the force-versus-elongation behavior of an individual pilus has been developed. Three elongation regions of pili were identified. In region I, P pili stretch elastically, up to a relative elongation of 16 +/- 3%. The product of elasticity modulus and area of a P pilus, EA, was assessed to 154 +/- 20 pN (n=6). In region II, the quaternary structure of the PapA rod unfolds under a constant force of 27 +/- 2 pN (n approximately 100) by a sequential breaking of the interactions between adjacent layers of PapA subunits. This unfolding can elongate the pilus up to 7 +/- 2 times. In region III, pili elongate in a nonlinear manner as a result of stretching until the bond ruptures.

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.

A design for complete beam steering (in three dimensions) of one or two optical tweezers traps is presented. The two most important requirements for efficient and stable movement of an optical trap are identified. A detailed recipe for the construction of a movable optical tweezers trap that fulfills these requirements is given (exemplified with an inverted microscope). The system has been found to allow for precise and free movements of both traps in all three dimensions in a dual-trap optical tweezers configuration and to be robust and reliable, as well as forgiving of small misalignments in the optical system.

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.

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 approximately 0.4 mum/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 approximately 0.76 nm, approximately 0.8 Hz, and approximately 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.