Nicolas Thewes

Influence of the Subsurface Composition of a Material on the Adhesion of Staphylococci

Controlling the interface between bacteria and solid materials has become an important task in biomedical science. For a fundamental and comprehensive understanding of adhesion it is necessary to seek quantitative information about the involved interactions. Most studies concentrate on the modification of the surface (chemical composition, hydrophobicity, or topography) neglecting, however, the influence of the bulk material, which always contributes to the overall interaction via van der Waals forces. In this study, we applied AFM force spectroscopy and flow chamber experiments to probe the adhesion of Staphylococcus carnosus to a set of tailored Si wafers, allowing for a separation of short- and long-range forces. We provide experimental evidence that the subsurface composition of a substrate influences bacterial adhesion. A coarse estimation of the strength of the van der Waals forces via the involved Hamaker constants substantiates the experimental results. The results demonstrate that the uppermost layer is not solely responsible for the strength of adhesion. Rather, for all kinds of adhesion studies, it is equally important to consider the contribution of the subsurface.

Hydrophobic interaction governs unspecific adhesion of staphylococci: a single cell force spectroscopy study

Summary 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.

Reduced Adhesion of Oral Bacteria on Hydroxyapatite by Fluoride Treatment

The mechanisms of action of fluoride have been discussed controversially for decades. The cavity-preventive effect for teeth is often traced back to effects on demineralization. However, an effect on bacterial adhesion was indicated by indirect macroscopic studies. To characterize adhesion on fluoridated samples on a single bacterial level, we used force spectroscopy with bacterial probes to measure adhesion forces directly. We tested the adhesion of Streptococcus mutans , Streptococcus oralis , and Staphylococcus carnosus on smooth, high-density hydroxyapatite surfaces, pristine and after treatment with fluoride solution. All bacteria species exhibit lower adhesion forces after fluoride treatment of the surfaces. These findings suggest that the decrease of adhesion properties is a further key factor for the cariostatic effect of fluoride besides the decrease of demineralization.

Activation of Rac1 GTPase by nanoparticulate structures in human macrophages.

UNLABELLEDnInflammatory activation of alveolar macrophages by ambient particles can be facilitated via Toll-like receptors (TLR). The action of TLR agonists and antagonists has been reported to depend on the formation of nanoparticulate structures. Aim of the present study was to identify the signaling pathways induced by nanoparticulate structures in human macrophages, which might be critical for inflammatory cell activation.nnnMETHODSnStudies were performed in primary human alveolar macrophages or in differentiated THP-1 macrophages. Silica nanoparticles were prepared by Stöber synthesis and characterized by dynamic light scattering and scanning electron microscopy. Mycobacterial DNA was isolated from Mycobacterium bovis BCG, and nanoparticle formation was assessed by atomic force microscopy and dynamic light scattering. Actin polymerization was measured by phalloidin-TRITC staining, and cell activation was determined by reverse transcription quantitative PCR analysis, L929 cytotoxicity assay (cytokine induction), and pull-down assays (Rho GTPases).nnnRESULTSnIn contrast to immune stimulatory sequence ISS 1018, BCG DNA spontaneously formed nanoparticulate structures and induced actin polymerization as did synthetic silica nanoparticles. Co-incubation with silica nanoparticles amplified the responsiveness of macrophages toward the TLR9 ligand ISS 1018. The activation of Rac1 was induced by silica nanoparticles as well as BCG DNA and is suggested as the critical signaling event inducing both cytoskeleton changes as well as inflammatory cell activation.nnnCONCLUSIONnNanoparticles can induce signaling pathways, which amplify an inflammatory response in macrophages.

The extracellular adherence protein (Eap) of Staphylococcus aureus acts as a proliferation and migration repressing factor that alters the cell morphology of keratinocytes

Staphyloccocus aureus is a major human pathogen and a common cause for superficial and deep seated wound infections. The pathogen is equipped with a large arsenal of virulence factors, which facilitate attachment to various eukaryotic cell structures and modulate the host immune response. One of these factors is the extracellular adherence protein Eap, a member of the secretable expanded repertoire adhesive molecules (SERAM) protein family that possesses adhesive and immune modulatory properties. The secreted protein was previously shown to impair wound healing by interfering with host defense and neovascularization. However, its impact on keratinocyte proliferation and migration, two major steps in the re-epithelialization process of wounds, is not known. Here, we report that Eap affects the proliferation and migration capacities of keratinocytes by altering their morphology and adhesive properties. In particular, treatment of non-confluent HaCaT cell cultures with Eap resulted in cell morphology changes as well as a significant reduction in cell proliferation and migration. Eap-treated HaCaT cells changed their appearance from an oblong via a trapezoid to an astral-like shape, accompanied by decreases in cell volume and cell stiffness, and exhibited significantly increased cell adhesion. Eap had a similar influence on endothelial and cancer cells, indicative for a general effect of Eap on eukaryotic cell morphology and functions. Specifically, Eap was found to interfere with growth factor-stimulated activation of the mitogen-activated protein kinase (MAPK) pathway that is known to be responsible for cell shape modulation, induction of proliferation and migration of epithelial cells. Western blot analyses revealed that Eap blocked the phosphorylation of extracellular signal-regulated kinase 1 and 2 (Erk1/2) in keratinocyte growth factor (KGF)-stimulated HaCaT cells. Together, these data add another antagonistic mechanism of Eap in wound healing, whereby the bacterial protein interferes with keratinocyte migration and proliferation.

A detailed guideline for the fabrication of single bacterial probes used for atomic force spectroscopy

Abstract.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.Graphical abstract

Enhanced adhesion of Streptococcus mutans to hydroxyapatite after exposure to saliva: Enhanced adhesion of Streptococcus mutans to hydroxyapatite after exposure to saliva

Streptococcus mutans cells form robust biofilms on human teeth and are strongly related to caries incidents. Hence, understanding the adhesion of S.u2009mutans in the human oral cavity is of major interest for preventive dentistry. In this study, we report on atomic force microscopy–based single‐cell force spectroscopy measurements of S.u2009mutans cells to hydroxyapatite surfaces. We observe for almost all measurements a significant difference in adhesion strength for S.u2009mutans as well as for Staphylococcus carnosus cells. However, the increase in adhesion strength after saliva exposure is much higher for S.u2009mutans cells compared to S. carnosus cells. Our results demonstrate that S.u2009mutans cells are well adapted to their natural environment, the oral cavity. This ability promotes the biofilm‐forming capability of that species and hence the production of caries‐provoking acids. In consequence, understanding the fundamentals of this mechanism may pave a way towards more effective caries‐reducing techniques.

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.