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 “bacterial probes”. 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 Adhäsion an abiotischen Oberflächen: 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 Verständnis der Adhäsion von Bakterien an abiotischen Substratoberflächen ist von größter Bedeutung für medizinische Belange. Kraftmessungen mit Rasterkraftmikroskopen und Bakteriensonden stellen den modernsten Stand quantitativer Erforschung bakterieller Adhäsion dar. In dieser Arbeit wurde eine einfache und reproduzierbare Methode entwickelt, um Bakteriensonden mit einzelnen Zellen herzustellen. Mit diesen Sonden wurden daraufhin Adhäsionsmechanismen von Bakterien an abiotischen Substratoberflächen untersucht. Um die molekularen Mechanismen der Bakterienadhäsion besser zu verstehen, wurden zudem Monte Carlo Simulationen durchgeführt. Dadurch konnten grundlegende Haftungsmechanismen bestimmt werden: Die Adhäsion von Bakterien beruht auf der Bindung von Zellwandpolymeren, wobei die Stärke der Haftung eines Bakteriums durch die Eigenschaften und die Anzahl der an eine Oberfläche bindenden Polymere bestimmt wird. Zum Beispiel wird die Adhäsion von Bakterien auf hydrophoben Substraten durch thermisch fluktuierende Zellwandproteine hervorgerufen, die in großer Zahl an die Oberfläche binden. Im Gegensatz dazu ist die Adhäsion von Bakterien auf hydrophilen Substraten wesentlich schwächer, aufgrund einer geringeren Anzahl bindender Polymere. Die stark unterschiedliche Adhäsionskraft von Bakterien auf hydrophoben und hydrophilen Oberflächen war die Grundlage der Entwicklung einer neuen Technik zur Messung der Kontaktfläche zwischen Bakterien und Oberflächen

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 “bacterial probes”. 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 “neutrophil extracellular traps” (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