17 research outputs found
Stochastic binding of Staphylococcus aureus to hydrophobic surfaces
The adhesion of pathogenic bacteria to surfaces is of immense importance for health care applications.
Via a combined experimental and computational approach, we studied the initiation of contact in the
adhesion process of the pathogenic bacterium Staphylococcus aureus. AFM force spectroscopy with
single cell bacterial probes paired with Monte Carlo simulations enabled an unprecedented molecular
investigation of the contact formation. Our results reveal that bacteria attach to a surface over distances
far beyond the range of classical surface forces via stochastic binding of thermally fluctuating cell wall
proteins. Thereby, the bacteria are pulled into close contact with the surface as consecutive proteins of
different stiffnesses attach. This mechanism greatly enhances the attachment capability of S. aureus. It,
however, can be manipulated by enzymatically/chemically modifying the cell wall proteins to block their
consecutive binding. Our study furthermore reveals that fluctuations in protein density and structure are
much more relevant than the exact form of the binding potential
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
A detailed guideline for the fabrication of single bacterial probes used for atomic force spectroscopy
The atomic force microscope (AFM) evolved as a standard device in modern microbiological research. However, its capability as a sophisticated force sensor is not used to its full capacity. The AFM turns into a unique tool for quantitative adhesion research in bacteriology by using âbacterial probesâ. Thereby, bacterial probes are AFM cantilevers that provide a single bacterium or a cluster of bacteria as the contact-forming object. We present a step-by-step protocol for preparing bacterial probes, performing force spectroscopy experiments and processing force spectroscopy data. Additionally, we provide a general insight into the field of bacterial cell force spectroscopy
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
Bakterielle AdhÀsion an abiotischen OberflÀchen: Rasterkraftspektroskopie und Monte Carlo Simulationen
A profound understanding of bacterial adhesion on abiotic substrates is of great importance for health care concerns. Force measurements with an atomic force microscope (AFM) and bacterial probes are the state-of-the art method in quantitative bacterial adhesion research. In this thesis, a simple and reproducible method to produce single bacterial probes was developed and, subsequently, used to investigate bacterial adhesion mechanisms on abiotic surfaces. To deepen the understanding of the molecular mechanisms of bacterial adhesion, Monte Carlo simulations were paired with AFM experiments. By using highly controlled substrates, fundamental mechanisms of bacterial adhesion are revealed: Bacterial adhesion relies on the binding of bacterial surface polymers, and the nature and the amount of bound polymers finally determine the strength of adhesion. On hydrophobic substrates, for instance, bacterial adhesion relies on fast binding of a large number of thermally fluctuating surface proteins. In contrast, on hydrophilic substrates, bacterial adhesion is weak due to a small amount of attaching surface polymers. Thus, the individual adhesion properties of a bacterial cell rely on the interplay of the surface polymers of a cell with a substrate in close
proximity. Furthermore, the difference of bacterial adhesion strength to hydrophilic and hydrophobic substrates was utilized to develop a new technique to determine the contact area between a single bacterial cell and surface.Ein grundlegendes VerstĂ€ndnis der AdhĂ€sion von Bakterien an abiotischen SubstratoberflĂ€chen ist von gröĂter Bedeutung fĂŒr medizinische Belange. Kraftmessungen mit Rasterkraftmikroskopen und Bakteriensonden stellen den modernsten Stand quantitativer Erforschung bakterieller AdhĂ€sion dar.
In dieser Arbeit wurde eine einfache und reproduzierbare Methode entwickelt, um Bakteriensonden mit einzelnen Zellen herzustellen. Mit diesen Sonden wurden daraufhin AdhĂ€sionsmechanismen von Bakterien an abiotischen SubstratoberflĂ€chen untersucht. Um die molekularen Mechanismen der BakterienadhĂ€sion besser zu verstehen, wurden zudem Monte Carlo Simulationen durchgefĂŒhrt. Dadurch konnten grundlegende Haftungsmechanismen bestimmt werden: Die AdhĂ€sion von Bakterien beruht auf der Bindung von Zellwandpolymeren, wobei die StĂ€rke der Haftung eines Bakteriums durch die Eigenschaften und die Anzahl der an eine OberflĂ€che bindenden Polymere bestimmt wird. Zum Beispiel wird die AdhĂ€sion von Bakterien auf hydrophoben Substraten durch thermisch fluktuierende Zellwandproteine hervorgerufen, die in groĂer Zahl an die OberflĂ€che binden. Im Gegensatz dazu ist die AdhĂ€sion von Bakterien auf hydrophilen Substraten wesentlich schwĂ€cher, aufgrund einer geringeren Anzahl bindender Polymere. Die stark unterschiedliche AdhĂ€sionskraft von Bakterien auf hydrophoben und hydrophilen OberflĂ€chen war die Grundlage der Entwicklung einer neuen Technik zur Messung der KontaktflĂ€che zwischen Bakterien und OberflĂ€chen
A detailed guideline for the fabrication of single bacterial probes used for atomic force spectroscopy
The atomic force microscope (AFM) evolved as a standard device in modern microbiological research. However, its capability as a sophisticated force sensor is not used to its full capacity. The AFM turns into a unique tool for quantitative adhesion research in bacteriology by using âbacterial probesâ. Thereby, bacterial probes are AFM cantilevers that provide a single bacterium or a cluster of bacteria as the contact-forming object. We present a step-by-step protocol for preparing bacterial probes, performing force spectroscopy experiments and processing force spectroscopy data. Additionally, we provide a general insight into the field of bacterial cell force spectroscopy
Hydrophobic interaction governs unspecific adhesion of staphylococci: a single cell force spectroscopy study
Unspecific adhesion of bacteria is usually the first step in the formation of biofilms on abiotic surfaces, yet it is unclear up to now which forces are governing this process. Alongside long-ranged van der Waals and electrostatic forces, short-ranged hydrophobic interaction plays an important role. To characterize the forces involved during approach and retraction of an individual bacterium to and from a surface, single cell force spectroscopy is applied: A single cell of the apathogenic species Staphylococcus carnosus isolate TM300 is used as bacterial probe. With the exact same bacterium, hydrophobic and hydrophilic surfaces can be probed and compared. We find that as far as 50 nm from the surface, attractive forces can already be recorded, an indication of the involvement of long-ranged forces. Yet, comparing the surfaces of different surface energy, our results corroborate the model that large, bacterial cell wall proteins are responsible for adhesion, and that their interplay with the short-ranged hydrophobic interaction of the involved surfaces is mainly responsible for adhesion. The ostensibly long range of the attraction is a result of the large size of the cell wall proteins, searching for contact via hydrophobic interaction. The model also explains the strong (weak) adhesion of S. carnosus to hydrophobic (hydrophilic) surfaces
The Staphylococcus aureus Extracellular Adherence Protein Eap Is a DNA Binding Protein Capable of Blocking Neutrophil Extracellular Trap Formation
The extracellular adherence protein (Eap) of Staphylococcus aureus is a secreted protein known to exert a number of adhesive and immunomodulatory properties. Here we describe the intrinsic DNA binding activity of this multifunctional secretory factor. By using atomic force microscopy, we provide evidence that Eap can bind and aggregate DNA. While the origin of the DNA substrate (e.g., eukaryotic, bacterial, phage, and artificial DNA) seems to not be of major importance, the DNA structure (e.g., linear or circular) plays a critical role with respect to the ability of Eap to bind and condense DNA. Further functional assays corroborated the nature of Eap as a DNA binding protein, since Eap suppressed the formation of âneutrophil extracellular trapsâ (NETs), composed of DNA-histone scaffolds, which are thought to function as a neutrophil-mediated extracellular trapping mechanism. The DNA binding and aggregation activity of Eap may thereby protect S. aureus against a specific anti-microbial defense reaction from the host