The accumulation and growth of Pseudomonas aeruginosa on surfaces is modulated by surface mechanics via cyclic-di-GMP signaling.

Attachment of bacteria onto a surface, consequent signaling, and accumulation and growth of the surface-bound bacterial population are key initial steps in the formation of pathogenic biofilms. While recent reports have hinted that surface mechanics may affect the accumulation of bacteria on that surface, the processes that underlie bacterial perception of surface mechanics and modulation of accumulation in response to surface mechanics remain largely unknown. We use thin and thick hydrogels coated on glass to create composite materials with different mechanics (higher elasticity for thin composites; lower elasticity for thick composites) but with the same surface adhesivity and chemistry. The mechanical cue stemming from surface mechanics is elucidated using experiments with the opportunistic human pathogen Pseudomonas aeruginosa combined with finite-element modeling. Adhesion to thin composites results in greater changes in mechanical stress and strain in the bacterial envelope than does adhesion to thick composites with identical surface chemistry. Using quantitative microscopy, we find that adhesion to thin composites also results in higher cyclic-di-GMP levels, which in turn result in lower motility and less detachment, and thus greater accumulation of bacteria on the surface than does adhesion to thick composites. Mechanics-dependent c-di-GMP production is mediated by the cell-surface-exposed protein PilY1. The biofilm lag phase, which is longer for bacterial populations on thin composites than on thick composites, is also mediated by PilY1. This study shows clear evidence that bacteria actively regulate differential accumulation on surfaces of different stiffnesses via perceiving varied mechanical stress and strain upon surface engagement.

Acetylcholinesterase Adsorption on Modified Gold: Effect of Surface Chemistry on Enzyme Binding and Activity.

Surface chemistry plays a crucial role in the performance of biosensors and biocatalysts, where enzymes directly interact with a solid support. In this work, we investigated the effect of surface charge and hydrophobicity on the binding and activity of acetylcholinesterase (AChE) following direct adsorption to modified gold surfaces. Surface modifications included self-assembled monolayers (SAMs) terminated with -COO-, -NH3+, -OH, and -CH3 functional groups at varying mole %. We also investigated the effects of positively and negatively charged helical peptides covalently coupled to the SAM. Using spectroscopic ellipsometry, we measured the surface concentration of AChE on each modified surface after 1 h of adsorption. We found that surface concentration was directly proportional to surface hydrophobicity (r = 0.76). The highest binding was observed on the more hydrophobic surfaces. We also measured the specific activity of AChE on each surface using a colorimetric assay and found that activity was inversely proportional to surface hydrophobicity (r = -0.71). The highest activity was observed on the more hydrophilic surfaces. Plotting specific activity versus surface concentration showed a similar relationship, with the highest activity observed at low AChE densities (∼20% of a monolayer) on surfaces terminated with 50% -COO- or -NH3+ and 50% -CH3 functional groups. Interestingly, this is similar to the approximate composition of hydrophobic versus hydrophilic amino acid residues on the surface of AChE. These surfaces also exhibited the highest total activity: a ∼100% improvement over bare gold due to a combination of moderate binding and high activity retention. This work highlights the importance of developing new attachment strategies beyond direct adsorption that promote, tune, and optimize both high binding and high activity retention.

Bacterial mechanosensing of surface stiffness promotes signaling and growth leading to biofilm formation by Pseudomonas aeruginosa.

The attachment of bacteria onto a surface, consequent signaling, and the accumulation and growth of the surface-bound bacterial population are key initial steps in the formation of pathogenic biofilms. While recent reports have hinted that the stiffness of a surface may affect the accumulation of bacteria on that surface, the processes that underlie bacterial perception of and response to surface stiffness are unknown. Furthermore, whether, and how, the surface stiffness impacts biofilm development, after initial accumulation, is not known. We use thin and thick hydrogels to create stiff and soft composite materials, respectively, with the same surface chemistry. Using quantitative microscopy, we find that the accumulation, motility, and growth of the opportunistic human pathogen Pseudomonas aeruginosa respond to surface stiffness, and that these are linked through cyclic-di-GMP signaling that depends on surface stiffness. The mechanical cue stemming from surface stiffness is elucidated using finite-element modeling combined with experiments - adhesion to stiffer surfaces results in greater changes in mechanical stress and strain in the bacterial envelope than does adhesion to softer surfaces with identical surface chemistry. The cell-surface-exposed protein PilY1 acts as a mechanosensor, that upon surface engagement, results in higher cyclic-di-GMP levels, lower motility, and greater accumulation on stiffer surfaces. PilY1 impacts the biofilm lag phase, which is extended for bacteria attaching to stiffer surfaces. This study shows clear evidence that bacteria actively respond to different stiffness of surfaces where they adhere via perceiving varied mechanical stress and strain upon surface engagement.

Characterizing protein-surface and protein-nanoparticle conjugates: Activity, binding, and structure.

Many sensors and catalysts composed of proteins immobilized on inorganic materials have been reported over the past few decades. Despite some examples of functional protein-surface and protein-nanoparticle conjugates, thorough characterization of the biological-abiological interface at the heart of these materials and devices is often overlooked in lieu of demonstrating acceptable system performance. This has resulted in a focus on generating functioning protein-based devices without a concerted effort to develop reliable tools necessary to measure the fundamental properties of the bio-abio interface, such as surface concentration, biomolecular structure, and activity. In this Perspective, we discuss current methods used to characterize these critical properties of devices that operate by integrating a protein into both flat surfaces and nanoparticle materials. We highlight the advantages and drawbacks of each method as they relate to understanding the function of the protein-surface interface and explore the manner in which an informed understanding of this complex interaction leads directly to the advancement of protein-based materials and technology.

Formation and Characterization of a Stable Monolayer of Active Acetylcholinesterase on Planar Gold.

Enzyme activity is the basis for many biosensors where a catalytic event is used to detect the presence and amount of a biomolecule of interest. To create a practical point-of-care biosensor, these enzymes need to be removed from their native cellular environments and immobilized on an abiological surface to rapidly transduce a biochemical signal into an interpretable readout. This immobilization often leads to loss of activity due to unfolded, aggregated, or improperly oriented enzymes when compared to the native state. In this work, we characterize the formation and surface packing density of a stable monolayer of acetylcholinesterase (AChE) immobilized on a planar gold surface and quantify the extent of activity loss following immobilization. Using spectroscopic ellipsometry, we determined that the surface concentration of AChE on a saturated Au surface in a buffered solution was 2.77 ± 0.21 pmol cm-2. By calculating the molecular volume of hydrated AChE, corresponding to a sphere of 6.19 nm diameter, divided by the total volume at the AChE-Au interface, we obtain a surface packing density of 33.4 ± 2.5% by volume. This corresponds to 45.1 ± 3.4% of the theoretical maximum monolayer coverage, assuming hexagonal packing. The true value, however, may be larger due to unfolding of enzymes to occupy a larger volume. The enzyme activity and kinetic measurements showed a 90.6 ± 1.4% decrease in specific activity following immobilization. Finally, following storage in a buffered solution for over 100 days at both room temperature and 4 °C, approximately 80% of this enzyme activity was retained. This contrasts with the native aqueous enzyme, which loses approximately 75% of its activity within 1 day and becomes entirely inactive within 6 days.