Polymorphisms in fibronectin binding protein A of Staphylococcus aureus are associated with infection of cardiovascular devices.

Medical implants, like cardiovascular devices, improve the quality of life for countless individuals but may become infected with bacteria like Staphylococcus aureus. Such infections take the form of a biofilm, a structured community of bacterial cells adherent to the surface of a solid substrate. Every biofilm begins with an attractive force or bond between bacterium and substratum. We used atomic force microscopy to probe experimentally forces between a fibronectin-coated surface (i.e., proxy for an implanted cardiac device) and fibronectin-binding receptors on the surface of individual living bacteria from each of 80 clinical isolates of S. aureus. These isolates originated from humans with infected cardiac devices (CDI; n = 26), uninfected cardiac devices (n = 20), and the anterior nares of asymptomatic subjects (n = 34). CDI isolates exhibited a distinct binding-force signature and had specific single amino acid polymorphisms in fibronectin-binding protein A corresponding to E652D, H782Q, and K786N. In silico molecular dynamics simulations demonstrate that residues D652, Q782, and N786 in fibronectin-binding protein A form extra hydrogen bonds with fibronectin, complementing the higher binding force and energy measured by atomic force microscopy for the CDI isolates. This study is significant, because it links pathogenic bacteria biofilms from the length scale of bonds acting across a nanometer-scale space to the clinical presentation of disease at the human dimension.

A template-free, more environmentally friendly approach for glass micro-texturing.

Micron and nanometer size textured silicate glass surfaces are of interest in consumer electronics, photovoltaics, and biosensing applications. Typically, texturing glass surfaces requires applying a patterned mask or a pre-etching treatment (e.g. sandblasting) on the glass substrate, followed by a mask transferring or etching process using a fluoride-containing compound. The major challenges of such a process are the complexity and cost of masking, and the safety and environmental concerns around the usage and disposal of hydrofluoric acid. Here, we describe a template-free method to construct micron-sized and submicron-sized texture on isotropic glass surfaces in one step. The new texturing mechanisms are well supported by experimental data and peridynamic simulations. With this novel strategy, the etchant uses fluoride-free chemicals such as citric acid to texture silicate glass. Etchant concentration, etch temperature, time, and additives are the primary parameters that dictate the texturing process. Surface feature size and depth can be independently controlled by tuning the leaching and chemical polishing process. We hope this study can trigger more research on novel and more environmentally friendly texturing of isotropic materials.

A tactile response in Staphylococcus aureus.

It is well established that bacteria are able to respond to temporal gradients (e.g., by chemotaxis). However, it is widely held that prokaryotes are too small to sense spatial gradients. This contradicts the common observation that the vast majority of bacteria live on the surface of a solid substrate (e.g., as a biofilm). Herein we report direct experimental evidence that the nonmotile bacterium Staphylococcus aureus possesses a tactile response, or primitive sense of touch, that allows it to respond to spatial gradients. Attached cells recognize their substrate interface and localize adhesins toward that region. Braille-like avidity maps reflect a cell's biochemical sensory response and reveal ultrastructural regions defined by the actual binding activity of specific proteins.

Bonds between fibronectin and fibronectin-binding proteins on Staphylococcus aureus and Lactococcus lactis.

Bacterial cell-wall-associated fibronectin binding proteins A and B (FnBPA and FnBPB) form bonds with host fibronectin. This binding reaction is often the initial step in prosthetic device infections. Atomic force microscopy was used to evaluate binding interactions between a fibronectin-coated probe and laboratory-derived Staphylococcus aureus that are (i) defective in both FnBPA and FnBPB (fnbA fnbB double mutant, DU5883), (ii) capable of expressing only FnBPA (fnbA fnbB double mutant complemented with pFNBA4), or (iii) capable of expressing only FnBPB (fnbA fnbB double mutant complemented with pFNBB4). These experiments were repeated using Lactococcus lactis constructs expressing fnbA and fnbB genes from S. aureus. A distinct force signature was observed for those bacteria that expressed FnBPA or FnBPB. Analysis of this force signature with the biomechanical wormlike chain model suggests that parallel bonds form between fibronectin and FnBPs on a bacterium. The strength and covalence of bonds were evaluated via nonlinear regression of force profiles. Binding events were more frequent (p < 0.01) for S. aureus expressing FnBPA or FnBPB than for the S. aureus double mutant. The binding force, frequency, and profile were similar between the FnBPA and FnBPB expressing strains of S. aureus. The absence of both FnBPs from the surface of S. aureus removed its ability to form a detectable bond with fibronectin. By contrast, ectopic expression of FnBPA or FnBPB on the surface of L. lactis conferred fibronectin binding characteristics similar to those of S. aureus. These measurements demonstrate that fibronectin-binding adhesins FnBPA and FnBPB are necessary and sufficient for the binding of S. aureus to prosthetic devices that are coated with host fibronectin.

Antibody recognition force microscopy shows that outer membrane cytochromes OmcA and MtrC are expressed on the exterior surface of Shewanella oneidensis MR-1.

Antibody recognition force microscopy showed that OmcA and MtrC are expressed on the exterior surface of living Shewanella oneidensis MR-1 cells when Fe(III), including solid-phase hematite (Fe(2)O(3)), was the terminal electron acceptor. OmcA was localized to the interface between the cell and mineral. MtrC displayed a more uniform distribution across the cell surface. Both cytochromes were associated with an extracellular polymeric substance.

Monolithically integrated micro- and nanostructured glass surface with antiglare, antireflection, and superhydrophobic properties.

Hierarchical micro- and nanostructured surfaces have previously been made using a variety of materials and methods, including particle deposition, polymer molding, and the like. These surfaces have attracted a wide variety of interest for applications including reduced specular reflection and superhydrophobic surfaces. To the best of our knowledge, this paper reports the first monolithic, hierarchically structured glass surface that combines micro- and nanoscale surface features to simultaneously generate antiglare (AG), antireflection (AR), and superhydrophobic properties. The AG microstructure mechanically protects the AR nanostructure during wiping and smudging, while the uniform composition of the substrate and the micro- and nanostructured surface enables ion exchange through the surface, so that both the substrate and structured surface can be simultaneously chemically strengthened.

Specific bonds between an iron oxide surface and outer membrane cytochromes MtrC and OmcA from Shewanella oneidensis MR-1.

Shewanella oneidensis MR-1 is purported to express outer membrane cytochromes (e.g., MtrC and OmcA) that transfer electrons directly to Fe(III) in a mineral during anaerobic respiration. A prerequisite for this type of reaction would be the formation of a stable bond between a cytochrome and an iron oxide surface. Atomic force microscopy (AFM) was used to detect whether a specific bond forms between a hematite (Fe(2)O(3)) thin film, created with oxygen plasma-assisted molecular beam epitaxy, and recombinant MtrC or OmcA molecules coupled to gold substrates. Force spectra displayed a unique force signature indicative of a specific bond between each cytochrome and the hematite surface. The strength of the OmcA-hematite bond was approximately twice that of the MtrC-hematite bond, but direct binding to hematite was twice as favorable for MtrC. Reversible folding/unfolding reactions were observed for mechanically denatured MtrC molecules bound to hematite. The force measurements for the hematite-cytochrome pairs were compared to spectra collected for an iron oxide and S. oneidensis under anaerobic conditions. There is a strong correlation between the whole-cell and pure-protein force spectra, suggesting that the unique binding attributes of each cytochrome complement one another and allow both MtrC and OmcA to play a prominent role in the transfer of electrons to Fe(III) in minerals. Finally, by comparing the magnitudes of binding force for the whole-cell versus pure-protein data, we were able to estimate that a single bacterium of S. oneidensis (2 by 0.5 microm) expresses approximately 10(4) cytochromes on its outer surface.

Correlation between fundamental binding forces and clinical prognosis of Staphylococcus aureus infections of medical implants.

Atomic force microscopy was used to "fish" for binding reactions between a fibronectin-coated probe (i.e., substrate simulating an implant device) and each of 15 different isolates of Staphylococcus aureus obtained from either patients with an infected cardiac prosthesis (invasive group) or healthy human subjects (control group). There is a strong distinction (p = 0.01) in the binding-force signature observed for the invasive versus control populations. This observation suggests that a microorganism's "force taxonomy" may provide a fundamental and practical indicator of the pathogen-related risk that infections pose to patients with implanted medical devices.

Simultaneous force and fluorescence measurements of a protein that forms a bond between a living bacterium and a solid surface.

All microbial biofilms are initiated through direct physical contact between a bacterium and a solid surface, a step that is controlled by inter- and intramolecular forces. Atomic force microscopy and confocal laser scanning microscopy were used simultaneously to observe the formation of a bond between a fluorescent chimeric protein on the surface of a living Escherichia coli bacterium and a solid substrate in situ. The chimera was composed of a portion of outer membrane protein A (OmpA) fused to the cyan-fluorescent protein AmCyan. Sucrose gradient centrifugation and fluorescent confocal slices through bacteria demonstrated that the chimeric protein was targeted and anchored to the external cell surface. The wormlike chain theory predicted that this protein should exhibit a nonlinear force-extension "signature" consistent with the sequential unraveling of the AmCyan and OmpA domains. Experimentally measured force-extension curves revealed a unique pair of "sawtooth" features that were present when a bond formed between a silicon nitride surface (atomic force microscopy tip) and E. coli cells expressing the OmpA-AmCyan protein. The observed sawtooth pair closely matched the wormlike chain model prediction for the mechanical unfolding of the AmCyan and OmpA substructures in series. These sawteeth disappeared from the measured force-extension curves when cells were treated with proteinase K. Furthermore, these unique sawteeth were absent for a mutant stain of E. coli incapable of expressing the AmCyan protein on its outer surface. Together, these data show that specific proteins exhibit unique force signatures characteristic of the bond that is formed between a living bacterium and another surface.