Using Knock-Out Mutants to Investigate the Adhesion of Staphylococcus aureus to Abiotic Surfaces

The adhesion of Staphylococcus aureus to abiotic surfaces is crucial for establishing device related infections. With a high number of single-cell force spectroscopy measurements with geneti cally modified S. aureus cells, this study provides insights into the adhesion process of the pathogen to abiotic surfaces of different wettability. Our results show that S. aureus utilizes different cell wall molecules and interaction mechanisms when binding to hydrophobic and hydrophilic surfaces. We found that covalently bound cell wall proteins strongly interact with hydrophobic substrates, while their contribution to the overall adhesion force is smaller on hydrophilic substrates. Teichoic acids promote adhesion to hydrophobic surfaces as well as to hydrophilic surfaces. This, however, is to a lesser extent. An interplay of electrostatic effects of charges and protein composition on bacterial surfaces is predominant on hydrophilic surfaces, while it is overshadowed on hydrophobic surfaces by the influence of the high number of binding proteins. Our results can help to design new models of bacterial adhesion and may be used to interpret the adhesion of other microorganisms with similar surface properties

Impact of geometry on chemical analysis exemplified for photoelectron spectroscopy of black silicon

For a smooth surface, the chemical composition can be readily evaluated by a variety of spectroscopy techniques; a prominent example is X-ray photoelectron spectroscopy (XPS), where the relative proportions of the elements are mainly determined by the intensity ratio of the element-specific photoelectrons. This deduction, however, is more intricate for a nanorough surface, such as black silicon, since the steep slopes of the geometry mimic local variations of the local emission angle. Here, we explicitly quantify this effect via an integral geometric analysis, by using so-called Minkowski tensors. Thus, we match the chemical information from XPS with topographical information from atomic force microscopy (AFM). Our method provides reliable estimates of layer thicknesses for nanorough surfaces. For our black silicon samples, we found that the oxide layer thickness is on average comparable to that of a native oxide layer. Our study highlights the impact of complex geometries at the nanoscale on the analysis of chemical properties with implications for a broad class of spectroscopy techniques

Different binding mechanisms of Staphylococcus aureus to hydrophobic and hydrophilic surfaces

Bacterial adhesion to surfaces is a crucial step in initial biofilm formation. In a combined experimental and computational approach, we studied the adhesion of the pathogenic bacterium Staphylococcus aureus to hydrophilic and hydrophobic surfaces. We used atomic force microscopy-based single-cell force spectroscopy and Monte Carlo simulations to investigate the similarities and differences of adhesion to hydrophilic and hydrophobic surfaces. Our results reveal that binding to both types of surfaces is mediated by thermally fluctuating cell wall macromolecules that behave differently on each type of substrate: on hydrophobic surfaces, many macromolecules are involved in adhesion, yet only weakly tethered, leading to high variance between individual bacteria, but low variance between repetitions with the same bacterium. On hydrophilic surfaces, however, only few macromolecules tether strongly to the surface. Since during every repetition with the same bacterium different macromolecules bind, we observe a comparable variance between repetitions and different bacteria. We expect these findings to be of importance for the understanding of the adhesion behaviour of many bacterial species as well as other microorganisms and even nanoparticles with soft, macromolecular coatings, used e.g. for biological diagnostics

Macromolecules at interfaces : from pure protein bilayers to the adhesion of cells

Macromolecules in cell membranes and cell walls are essential for many cell functions. The use of macromolecules as possible replacements for lipids in membranes and the role of cell wall macromolecules in adhesion have not been fully explored. In this work, HFBI bilayers were shown to have extremely low water permeability while maintaining high stability to osmotic pressure. Disruption of protein order increases water permeability. The methodology of HFBI vesicle preparation was further developed, especially in terms of reproducibility and control. Also, the incorporation of HFBI into lipid membranes has been shown to stabilize the formation of pores. The role of macromolecules in Staphylococcus aureus adhesion was investigated by varying the surfaces used and using knock-out mutants. For adhesion on nano-rough surfaces, the contact area between the macromolecule and the surface is crucial. While many macromolecules, especially cell wall proteins, adhere weakly to hydrophobic surfaces, only a few cell wall macromolecules adhere strongly to hydrophilic surfaces. Electrostatic interactions play a crucial role on hydrophilic surfaces. Furthermore, it has been shown that in human retinal pigment epithelial cells, adhesion weakens as the size of focal adhesions decreases.Makromoleküle in Zellmembranen und Zellwänden sind für viele Zellfunktionen von entscheidender Bedeutung. Die Verwendung von Makromolekülen als möglicher Ersatz für Lipide in Membranen, aber auch die Rolle von Zellwandmakromolekülen bei der Adhäsion sind noch nicht vollständig erforscht. In dieser Arbeit wurde gezeigt, dass HFBI-Doppelschichten eine sehr geringe Wasserpermeabilität bei gleichzeitig hoher Stabilität gegenüber osmotischem Druck aufweisen. Durch Störung der Proteinordnung steigt die Wasserpermeabilität. Die Methodik der HFBI-Vesikelherstellung wurde insbesondere hinsichtlich Reproduzierbarkeit und Kontrolle weiterentwickelt. Ebenso wurde durch den Einbau von HFBI in Lipidmembranen eine Stabilisierung der gebildeten Poren festgestellt. Die Rolle der Makromoleküle bei der Adhäsion von Staphylococcus aureus wurde durch Variation der verwendeten Oberflächen und die Nutzung von Knock-out-Mutanten untersucht. Für die Adhäsion auf nanorauen Oberflächen ist die Kontaktfläche Makromolekül/Oberfläche entscheidend. Während auf hydrophoben Oberflächen viele Makromoleküle, insbesondere Zellwandproteine, schwach haften, tun dies nur wenige Zellwandmakromoleküle stark auf hydrophilen Oberflächen. Elektrostatische Wechselwirkungen spielen dabei auf hydrophilen Oberflächen eine entscheidende Rolle. Weiterhin konnte gezeigt werden, dass sich bei humanen retinalen Pigmentepithelzellen die Adhäsion mit abnehmender Größe der Fokalkontakte abschwächt

Hydrophobin Bilayer as Water Impermeable Protein Membrane

One of the most important properties of membranes is their permeability to water and other small molecules. A targeted change in permeability allows the passage of molecules to be controlled. Vesicles made of membranes with low water permeability are preferable for drug delivery, for example, because they are more stable and maintain the drug concentration inside. This study reports on the very low water permeability of pure protein membranes composed of a bilayer of the amphiphilic protein hydrophobin HFBI. Using a droplet interface bilayer setup, we demonstrate that HFBI bilayers are essentially impermeable to water. HFBI bilayers withstand far larger osmotic pressures than lipid membranes. Only by disturbing the packing of the proteins in the HFBI bilayer is a measurable water permeability induced. To investigate possible molecular mechanisms causing the near-zero permeability, we used all-atom molecular dynamics simulations of various HFBI bilayer models. The simulations suggest that the experimental HFBI bilayer permeability is compatible neither with a lateral honeycomb structure, as found for HFBI monolayers, nor with a residual oil layer within the bilayer or with a disordered lateral packing similar to the packing in lipid bilayers. These results suggest that the low permeabilities of HFBI and lipid bilayers rely on different mechanisms. With their extremely low but adaptable permeability and high stability, HFBI membranes could be used as an osmotic pressure-insensitive barrier in situations where lipid membranes fail such as desalination membranes

Hydrophobin Bilayer as Water Impermeable Protein Membrane

One of the most important properties of membranes is their permeability to water and other small molecules. A targeted change in permeability allows the passage of molecules to be controlled. Vesicles made of membranes with low water permeability are preferable for drug delivery, for example, because they are more stable and maintain the drug concentration inside. This study reports on the very low water permeability of pure protein membranes composed of a bilayer of the amphiphilic protein hydrophobin HFBI. Using a droplet interface bilayer setup, we demonstrate that HFBI bilayers are essentially impermeable to water. HFBI bilayers withstand far larger osmotic pressures than lipid membranes. Only by disturbing the packing of the proteins in the HFBI bilayer is a measurable water permeability induced. To investigate possible molecular mechanisms causing the near-zero permeability, we used all-atom molecular dynamics simulations of various HFBI bilayer models. The simulations suggest that the experimental HFBI bilayer permeability is compatible neither with a lateral honeycomb structure, as found for HFBI monolayers, nor with a residual oil layer within the bilayer or with a disordered lateral packing similar to the packing in lipid bilayers. These results suggest that the low permeabilities of HFBI and lipid bilayers rely on different mechanisms. With their extremely low but adaptable permeability and high stability, HFBI membranes could be used as an osmotic pressure-insensitive barrier in situations where lipid membranes fail such as desalination membranes