Huabin Wang

Self-organization of bacterial biofilms is facilitated by extracellular DNA

Twitching motility-mediated biofilm expansion is a complex, multicellular behavior that enables the active colonization of surfaces by many species of bacteria. In this study we have explored the emergence of intricate network patterns of interconnected trails that form in actively expanding biofilms of Pseudomonas aeruginosa. We have used high-resolution, phase-contrast time-lapse microscopy and developed sophisticated computer vision algorithms to track and analyze individual cell movements during expansion of P. aeruginosa biofilms. We have also used atomic force microscopy to examine the topography of the substrate underneath the expanding biofilm. Our analyses reveal that at the leading edge of the biofilm, highly coherent groups of bacteria migrate across the surface of the semisolid media and in doing so create furrows along which following cells preferentially migrate. This leads to the emergence of a network of trails that guide mass transit toward the leading edges of the biofilm. We have also determined that extracellular DNA (eDNA) facilitates efficient traffic flow throughout the furrow network by maintaining coherent cell alignments, thereby avoiding traffic jams and ensuring an efficient supply of cells to the migrating front. Our analyses reveal that eDNA also coordinates the movements of cells in the leading edge vanguard rafts and is required for the assembly of cells into the “bulldozer” aggregates that forge the interconnecting furrows. Our observations have revealed that large-scale self-organization of cells in actively expanding biofilms of P. aeruginosa occurs through construction of an intricate network of furrows that is facilitated by eDNA.

Atomic Force Microscopy Reveals the Mechanobiology of Lytic Peptide Action on Bacteria

Increasing rates of antimicrobial-resistant medically important bacteria require the development of new, effective therapeutics, of which antimicrobial peptides (AMPs) are among the promising candidates. Many AMPs are membrane-active, but their mode of action in killing bacteria or in inhibiting their growth remains elusive. This study used atomic force microscopy (AFM) to probe the mechanobiology of a model AMP (a derivative of melittin) on living Klebsiella pneumoniae bacterial cells. We performed in situ biophysical measurements to understand how the melittin peptide modulates various biophysical behaviors of individual bacteria, including the turgor pressure, cell wall elasticity, and bacterial capsule thickness and organization. Exposure of K. pneumoniae to the peptide had a significant effect on the turgor pressure and Youngs modulus of the cell wall. The turgor pressure increased upon peptide addition followed by a later decrease, suggesting that cell lysis occurred and pressure was lost through destruction of the cell envelope. The Youngs modulus also increased, indicating that interaction with the peptide increased the rigidity of the cell wall. The bacterial capsule did not prevent cell lysis by the peptide, and surprisingly, the capsule appeared unaffected by exposure to the peptide, as capsule thickness and inferred organization were within the control limits, determined by mechanical measurements. These data show that AFM measurements may provide valuable insights into the physical events that precede bacterial lysis by AMPs.

Role of Capsular Polysaccharides in Biofilm Formation: An AFM Nanomechanics Study.

Bacteria form biofilms to facilitate colonization of biotic and abiotic surfaces, and biofilm formation on indwelling medical devices is a common cause of hospital-acquired infection. Although it is well-recognized that the exopolysaccharide capsule is one of the key bacterial components for biofilm formation, the underlying biophysical mechanism is poorly understood. In the present study, nanomechanical measurements of wild type and specific mutants of the pathogen, Klebsiella pneumoniae, were performed in situ using atomic force microscopy (AFM). Theoretical modeling of the mechanical data and static microtiter plate biofilm assays show that the organization of the capsule can influence bacterial adhesion, and thereby biofilm formation. The capsular organization is affected by the presence of type 3 fimbriae. Understanding the biophysical mechanisms for the impact of the structural organization of the bacterial polysaccharide capsule on biofilm formation will aid the development of strategies to prevent biofilm formation.

Terahertz Biosensing Based on a Polarization-Insensitive Metamaterial

A polarization-insensitive metamaterial biosensor composed of metallic square ring resonators is proposed and is shown to be especially suitable to work together with the linear polarized femtosecond laser pumped terahertz time-domain spectroscopy. A symmetric slit is distributed on every side of the ring to render the biosensor insensitive to the polarization direction of the incident terahertz wave. The proposed structure is analyzed with the finite-element method and investigated experimentally. The results reveal that a minimal resolution of 17.7 μmol/L in the detection of bovine serum albumin is obtained when the biosensor is oriented arbitrarily in the plane containing the electric field vector.

Stigmergy: a key driver of self-organization in bacterial biofilms

Bacterial biofilms are complex multicellular communities that are often associated with the emergence of large-scale patterns across the biofilm. How bacteria self-organize to form these structured communities is an area of active research. We have recently determined that the emergence of an intricate network of trails that forms during the twitching motility mediated expansion of Pseudomonas aeruginosa biofilms is attributed to an interconnected furrow system that is forged in the solidified nutrient media by aggregates of cells as they migrate across the media surface. This network acts as a means for self-organization of collective behavior during biofilm expansion as the cells following these vanguard aggregates were preferentially confined within the furrow network resulting in the formation of an intricate network of trails of cells. Here we further explore the process by which the intricate network of trails emerges. We have determined that the formation of the intricate network of furrows is associated with significant remodeling of the sub-stratum underlying the biofilm. The concept of stigmergy has been used to describe a variety of self-organization processes observed in higher organisms and abiotic systems that involve indirect communication via persistent cues in the environment left by individuals that influence the behavior of other individuals of the group at a later point in time. We propose that the concept of stigmergy can also be applied to describe self-organization of bacterial biofilms and can be included in the repertoire of systems used by bacteria to coordinate complex multicellular behaviors.

AFM lateral force calibration for an integrated probe using a calibration grating

Atomic force microscopy (AFM) friction measurements on hard and soft materials remain a challenge due to the difficulties associated with accurately calibrating the cantilever for lateral force measurement. One of the most widely accepted lateral force calibration methods is the wedge method. This method is often used in a simplified format but in so doing sacrifices accuracy. In the present work, we have further developed the wedge method to provide a lateral force calibration method for integrated AFM probes that is easy to use without compromising accuracy and reliability. Raw friction calibration data are collected from a single scan image by continuous ramping of the set point as the facets of a standard grating are scanned. These data are analysed to yield an accurate lateral force conversion/calibration factor that is not influenced by adhesion forces or load deviation. By demonstrating this new calibration method, we illustrate its reliability, speed and ease of execution. This method makes accessible reliable boundary lubrication studies on adhesive and heterogeneous surfaces that require spatial resolution of frictional forces.

Influence of Fimbriae on Bacterial Adhesion and Viscoelasticity and Correlations of the Two Properties with Biofilm Formation

The surface polymers of bacteria determine the ability of bacteria to adhere to a substrate for colonization, which is an essential step for a variety of microbial processes, such as biofilm formation and biofouling. Capsular polysaccharides and fimbriae are two major components on a bacterial surface, which are critical for mediating cell-surface interactions. Adhesion and viscoelasticity of bacteria are two major physical properties related to bacteria-surface interactions. In this study, we employed atomic force microscopy (AFM) to interrogate how the adhesion work and the viscoelasticity of a bacterial pathogen, Klebsiella pneumoniae, influence biofilm formation. To do this, the wild-type, type 3 fimbriae-deficient, and type 3 fimbriae-overexpressed K. pneumoniae strains have been investigated in an aqueous environment. The results show that the measured adhesion work is positively correlated to biofilm formation; however, the viscoelasticity is not correlated to biofilm formation. This study indicates that AFM-based adhesion measurements of bacteria can be used to evaluate the function of bacterial surface polymers in biofilm formation and to predict the ability of bacterial biofilm formation.

Study of substrate-directed ordering of long double-stranded DNA molecules on bare highly oriented pyrolytic graphite surface based on atomic force microscopy relocation imaging

Usually, long double-stranded DNA molecules exhibit an aggregated or a random spreading behavior when deposited on a highly ordered pyrolytic graphite (HOPG) substrate. In this article, the authors report a novel phenomenon where randomly oriented DNA strands can gradually be rearranged into two-dimensional ordered nanostructures under the operation of repeatedly rotating a water droplet on the DNA sample. The process of DNA rearrangement was traced by using atomic force microscopy relocation imaging. The orientation of the ordered DNA strands shows a threefold symmetry consistent with the underlying atomic lattice of the HOPG substrate, signifying a substrate-directed ordering process. The relevant mechanism is discussed.

ORGANIC SOLVENT-ASSISTED TRANSFER PRINTING ON HYDROPHOBIC POLYMER SUBSTRATE WITH HIGH EFFICIENCY

Patterning materials on hydrophobic polymer substrates have extensive applications in fabricating flexible devices at low cost, while the low transfer efficiency encumbers its advance. This paper provides a facile route to transfer materials onto hydrophobic polymer substrates with high efficiency by operating in organic solvent atmosphere. Under the assistance of condensed organic solvent layer on the substrate, bovine serum albumin (BSA) are desirably transferred onto untreated hydrophobic polymer substrates, such as poly(dimethylsiloxane) (PDMS), polystyrene (PS), and poly(ethylene terephthalate) (PET) as a proof-of-concept experiment. Moreover, the combination of this method with stepwise contraction and adsorption nanolithography (SCAN) was also demonstrated as an alternative way to further miniaturize patterns prepared by other methods.

Stigmergy co-ordinates multicellular collective behaviours during Myxococcus xanthus surface migration.

Surface translocation by the soil bacterium Myxococcus xanthus is a complex multicellular phenomenon that entails two motility systems. However, the mechanisms by which the activities of individual cells are coordinated to manifest this collective behaviour are currently unclear. Here we have developed a novel assay that enables detailed microscopic examination of M. xanthus motility at the interstitial interface between solidified nutrient medium and a glass coverslip. Under these conditions, M. xanthus motility is characterised by extensive micro-morphological patterning that is considerably more elaborate than occurs at an air-surface interface. We have found that during motility on solidified nutrient medium, M. xanthus forges an interconnected furrow network that is lined with an extracellular matrix comprised of exopolysaccharides, extracellular lipids, membrane vesicles and an unidentified slime. Our observations have revealed that M. xanthus motility on solidified nutrient medium is a stigmergic phenomenon in which multi-cellular collective behaviours are co-ordinated through trail-following that is guided by physical furrows and extracellular matrix materials.