Johan Zakrisson

The Shaft of the Type 1 Fimbriae Regulates an External Force to Match the FimH Catch Bond

Type 1 fimbriae mediate adhesion of uropathogenic Escherichia coli (UPEC) to host cells. It has been hypothesized that fimbriae can, by their ability to uncoil under exposure to force, reduce fluid shear stress on the adhesin-receptor interaction by which the bacterium adheres to the surface. In this work we develop a model that describes how the force on the adhesin-receptor interaction of a type 1 fimbriae varies as a bacterium is affected by a time dependent fluid flow mimicking in vivo conditions. The model combines in vivo hydrodynamic conditions with previously assessed biomechanical properties of the fimbriae. Numerical methods are used to solve for the motion and adhesion force under the presence of time dependent fluid profiles. It is found that a bacterium tethered with a type 1 pilus will experience significantly reduced shear stress for moderate to high flow velocities and that the maximum stress the adhesin will experience is limited to ~120 pN, which is sufficient to activate the conformational change of the FimH adhesin into its stronger state but also lower than the force required for breaking it under rapid loading. Our model thus supports the assumption that the type 1 fimbriae shaft and the FimH adhesin-receptor interaction are optimized to each other, and that they give piliated bacteria significant advantages in rapidly changing fluidic environments.

Cell shape identification using digital holographic microscopy.

We present a cost-effective, simple, and fast digital holographic microscopy method based upon Rayleigh-Sommerfeld backpropagation for identification of the geometrical shape of a cell. The method was tested using synthetic hologram images generated by ray-tracing software and from experimental images of semitransparent spherical beads and living red blood cells. Our results show that, by only using the real part of the back-reconstructed amplitude, the proposed method can provide information of the geometrical shape of the object and at the same time accurately determine the axial position of the object under study. The proposed method can be used in flow chamber assays for pathophysiological studies where fast morphological changes of cells are studied in high numbers and at different heights.

Rigid multibody simulation of a helix-like structure: the dynamics of bacterial adhesion pili

We present a coarse-grained rigid multibody model of a subunit assembled helix-like polymer, e.g., adhesion pili expressed by bacteria, that is capable of describing the polymer’s force-extension response. With building blocks representing individual subunits, the model appropriately describes the complex behavior of pili expressed by the gram-negative uropathogenic Escherichia coli bacteria under the action of an external force. Numerical simulations show that the dynamics of the model, which include the effects of both unwinding and rewinding, are in good quantitative agreement with the characteristic force-extension response as observed experimentally for type 1 and P pili. By tuning the model, it is also possible to reproduce the force-extension response in the presence of anti-shaft antibodies, which dramatically changes the mechanical properties. Thus, the model and results in this work give enhanced understanding of how a pilus unwinds under the action of external forces and provide a new perspective of the complex bacterial adhesion processes.

Detecting Bacterial Surface Organelles on Single Cells Using Optical Tweezers

Bacterial cells display a diverse array of surface organelles that are important for a range of processes such as intercellular communication, motility and adhesion leading to biofilm formation, infections, and bacterial spread. More specifically, attachment to host cells by Gram-negative bacteria are mediated by adhesion pili, which are nanometers wide and micrometers long fibrous organelles. Since these pili are significantly thinner than the wavelength of visible light, they cannot be detected using standard light microscopy techniques. At present, there is no fast and simple method available to investigate if a single cell expresses pili while keeping the cell alive for further studies. In this study, we present a method to determine the presence of pili on a single bacterium. The protocol involves imaging the bacterium to measure its size, followed by predicting the fluid drag based on its size using an analytical model, and thereafter oscillating the sample while a single bacterium is trapped by an optical tweezer to measure its effective fluid drag. Comparison between the predicted and the measured fluid drag thereby indicate the presence of pili. Herein, we verify the method using polymer coated silica microspheres and Escherichia coli bacteria expressing adhesion pili. Our protocol can in real time and within seconds assist single cell studies by distinguishing between piliated and nonpiliated bacteria.

Realizing Large-Area Arrays of Semiconducting Fullerene Nanostructures with Direct Laser Interference Patterning

We present a laser interference patterning method for the facile fabrication of large-area and high-contrast arrays of semiconducting fullerene nanostructures, which does not rely on a tedious application of sacrificial photoresists or photomasks. A solution-deposited phenyl-C61-butyric acid methyl ester (PCBM) fullerene thin film is exposed to a spatially modulated illumination intensity, as realized by a two-beam laser interference. The PCBM molecules exposed to strong intensity are photochemically transformed into a low-solubility dimeric state, so that the nontransformed PCBM molecules can be selectively removed in a subsequent solution-based development step. Following brief exposure to green laser light (λ = 532 nm, t = 5 s, p = 0.17 W cm-2) in the designed two-beam interference setup, and a 1 min development in a tuned acetone-chloroform solution, we realize well-defined and ordered PCBM nanostripe patterns with a fwhm line width of ∼200 nm and a repetition rate of ∼2.900 lines mm-1 over a large area of 1 cm2. We demonstrate that a desired high contrast is effectuated because the initial PCBM-dimer transformation rate is dependent on the square of the illumination intensity. The semiconducting functionality of the patterned fullerene is verified in a field-effect transistor experiment, where a typical PCBM nanostripe featured an electron mobility of 5.3 × 10-3 cm2 V-1 s-1 and an on/off ratio of 3 × 103.