Grégory Francius

Automated Force Volume Image Processing for Biological Samples

Atomic force microscopy (AFM) has now become a powerful technique for investigating on a molecular level, surface forces, nanomechanical properties of deformable particles, biomolecular interactions, kinetics, and dynamic processes. This paper specifically focuses on the analysis of AFM force curves collected on biological systems, in particular, bacteria. The goal is to provide fully automated tools to achieve theoretical interpretation of force curves on the basis of adequate, available physical models. In this respect, we propose two algorithms, one for the processing of approach force curves and another for the quantitative analysis of retraction force curves. In the former, electrostatic interactions prior to contact between AFM probe and bacterium are accounted for and mechanical interactions operating after contact are described in terms of Hertz-Hooke formalism. Retraction force curves are analyzed on the basis of the Freely Jointed Chain model. For both algorithms, the quantitative reconstruction of force curves is based on the robust detection of critical points (jumps, changes of slope or changes of curvature) which mark the transitions between the various relevant interactions taking place between the AFM tip and the studied sample during approach and retraction. Once the key regions of separation distance and indentation are detected, the physical parameters describing the relevant interactions operating in these regions are extracted making use of regression procedure for fitting experiments to theory. The flexibility, accuracy and strength of the algorithms are illustrated with the processing of two force-volume images, which collect a large set of approach and retraction curves measured on a single biological surface. For each force-volume image, several maps are generated, representing the spatial distribution of the searched physical parameters as estimated for each pixel of the force-volume image.

Elasticity, biodegradability and cell adhesive properties of chitosan/hyaluronan multilayer films

In the bioengineering field, a recent and promising approach to modifying biomaterial surfaces is the layer-by-layer (LbL) technique used to build thin polyelectrolyte multilayer films. In this work, we focused on polyelectrolyte multilayer films made of two polysaccharides, chitosan (CHI) and hyaluronan (HA), and on the control of their physico-chemical and cell adhesive properties by chemical cross-linking. CHI/HA films were cross-linked using a water soluble carbodiimide and observed by confocal laser scanning microscopy (CLSM) with a fluorescently labeled CHI. Film thicknesses were similar for native and cross-linked films. The film nanometer roughness was measured by atomic force microscopy and was found to be higher for cross-linked films. Cross-linking the films also leads to a drastic change in film stiffness. The elastic modulus of the films (Youngs modulus) as measured by AFM nano-indentation was about tenfold increased for cross-linked films as compared to native ones. From a biological point of view, cross-liked films are more resistant to enzymatic degradation by hyaluronidase. Furthermore, the increase in film stiffness has a favorable effect on the adhesion and spreading of chondrosarcoma cells. Thus, the CHI/HA cross-linked films could be used for various applications due to their adhesive properties and to their mechanical properties (including stability in enzymatic media).

Bacterial Surface Appendages Strongly Impact Nanomechanical and Electrokinetic Properties of Escherichia coli Cells Subjected to Osmotic Stress

The physicochemical properties and dynamics of bacterial envelope, play a major role in bacterial activity. In this study, the morphological, nanomechanical and electrohydrodynamic properties of Escherichia coli K-12 mutant cells were thoroughly investigated as a function of bulk medium ionic strength using atomic force microscopy (AFM) and electrokinetics (electrophoresis). Bacteria were differing according to genetic alterations controlling the production of different surface appendages (short and rigid Ag43 adhesins, longer and more flexible type 1 fimbriae and F pilus). From the analysis of the spatially resolved force curves, it is shown that cells elasticity and turgor pressure are not only depending on bulk salt concentration but also on the presence/absence and nature of surface appendage. In 1 mM KNO3, cells without appendages or cells surrounded by Ag43 exhibit large Young moduli and turgor pressures (∼700–900 kPa and ∼100–300 kPa respectively). Under similar ionic strength condition, a dramatic ∼50% to ∼70% decrease of these nanomechanical parameters was evidenced for cells with appendages. Qualitatively, such dependence of nanomechanical behavior on surface organization remains when increasing medium salt content to 100 mM, even though, quantitatively, differences are marked to a much smaller extent. Additionally, for a given surface appendage, the magnitude of the nanomechanical parameters decreases significantly when increasing bulk salt concentration. This effect is ascribed to a bacterial exoosmotic water loss resulting in a combined contraction of bacterial cytoplasm together with an electrostatically-driven shrinkage of the surface appendages. The former process is demonstrated upon AFM analysis, while the latter, inaccessible upon AFM imaging, is inferred from electrophoretic data interpreted according to advanced soft particle electrokinetic theory. Altogether, AFM and electrokinetic results clearly demonstrate the intimate relationship between structure/flexibility and charge of bacterial envelope and propensity of bacterium and surface appendages to contract under hypertonic conditions.

Cohesiveness and hydrodynamic properties of young drinking water biofilms

Drinking water biofilms are complex microbial systems mainly composed of clusters of different size and age. Atomic force microscopy (AFM) measurements were performed on 4, 8 and 12 weeks old biofilms in order to quantify the mechanical detachment shear stress of the clusters, to estimate the biofilm entanglement rate ξ. This AFM approach showed that the removal of the clusters occurred generally for mechanical shear stress of about 100 kPa only for clusters volumes greater than 200 μm3. This value appears 1000 times higher than hydrodynamic shear stress technically available meaning that the cleaning of pipe surfaces by water flushing remains always incomplete. To predict hydrodynamic detachment of biofilm clusters, a theoretical model has been developed regarding the averaging of elastic and viscous stresses in the cluster and by including the entanglement rate ξ. The results highlighted a slight increase of the detachment shear stress with age and also the dependence between the posting of clusters and their volume. Indeed, the experimental values of ξ allow predicting biofilm hydrodynamic detachment with same order of magnitude than was what reported in the literature. The apparent discrepancy between the mechanical and the hydrodynamic detachment is mainly due to the fact that AFM mechanical experiments are related to the clusters local properties whereas hydrodynamic measurements reflected the global properties of the whole biofilm.

In vitro interactions between probiotic bacteria and milk proteins probed by atomic force microscopy.

Interactions between microbial cells and milk proteins are important for cell location into dairy matrices. In this study, interactions between two probiotic strains, Lactobacillus rhamnosus GG and Lactobacillus rhamnosus GR-1, and milk proteins (micellar casein, native and denatured whey proteins) were studied. The bacterial surface characterization was realized with X-ray photoelectron spectroscopy (XPS) to evaluate surface composition (in terms of proteins, polysaccharides and lipid-like compounds) and electrophoretic mobility that provide information on surface charge of both bacteria and proteins along the 3-7 pH range. In addition, atomic force microscopy (AFM) enabled the identification of specific interactions between bacteria and whey proteins, in contrast to the observed nonspecific interactions with micellar casein. These specific events appeared to be more important for the GG strain than for the GR-1 strain, showing that matrix interaction is strain-specific. Furthermore, our study highlighted that in addition to the nature of the strains, many other factors influence the bacterial interaction with dairy matrix including the nature of the proteins and the pH of the media.

Elasticity and physico-chemical properties during drinking water biofilm formation

Atomic force microscope techniques and multi-staining fluorescence microscopy were employed to study the steps in drinking water biofilm formation. During the formation of a conditioning layer, surface hydrophobic forces increased and the range of characteristic hydrophobic forces diversified with time, becoming progressively complex in macromolecular composition, which in return triggered irreversible cellular adhesion. AFM visualization of 1 to 8 week drinking water biofilms showed a spatially discontinuous and heterogeneous distribution comprising an extensive network of filamentous fungi in which biofilm aggregates were embedded. The elastic modulus of 40-day-old biofilms ranged from 200 to 9000 kPa, and the biofilm deposits with a height >0.5 μm had an elastic modulus <600 kPa, suggesting that the drinking water biofilms were composed of a soft top layer and a basal layer with significantly higher elastic modulus values falling in the range of fungal elasticity.

Lactic acid bacteria in dairy food: surface characterization and interactions with food matrix components.

This review gives an overview of the importance of interactions occurring in dairy matrices between Lactic Acid Bacteria and milk components. Dairy products are important sources of biological active compounds of particular relevance to human health. These compounds include immunoglobulins, whey proteins and peptides, polar lipids, and lactic acid bacteria including probiotics. A better understanding of interactions between bioactive components and their delivery matrix may successfully improve their transport to their target site of action. Pioneering research on probiotic lactic acid bacteria has mainly focused on their host effects. However, very little is known about their interaction with dairy ingredients. Such knowledge could contribute to designing new and more efficient dairy food, and to better understand relationships between milk constituents. The purpose of this review is first to provide an overview of the current knowledge about the biomolecules produced on bacterial surface and the composition of the dairy matter. In order to understand how bacteria interact with dairy molecules, adhesion mechanisms are subsequently reviewed with a special focus on the environmental conditions affecting bacterial adhesion. Methods dedicated to investigate the bacterial surface and to decipher interactions between bacteria and abiotic dairy components are also detailed. Finally, relevant industrial implications of these interactions are presented and discussed.

Double entrapment of growth factors by nanoparticles loaded into polyelectrolyte multilayer films

Delivery of growth factors and control of vascularization are prominent problems in regenerative medicine. Vascular endothelial growth factor (VEGF) has been used both in vitro and in vivo to promote angiogenesis but due to its short half-life its controlled delivery is a sought after method. In this study we present a new concept of degradable drug loaded nanoparticles entrapped into exponentially growing multilayer films. Through hydrolysis of the nanoparticles, the drug can be delivered over long periods in a controlled manner. Poly(ε-caprolactone) nanoparticles were loaded with VEGF and in turn the release of VEGF from a surface is controlled by a thick layer-by-layer polyelectrolyte film. Direct loading of VEGF inside the film was not efficient for long-term applications. When VEGF loaded nanoparticles were introduced into the film, the particles were equally distributed inside and were stable after several washes. Moreover, the presence of the film sustained the release of VEGF for 7 days. Addition of the nanoparticles to the film promoted endothelial cell proliferation, mainly due to the presence of VEGF. Mechanical properties of the film (Youngs moduli) were also improved by the presence of nanoparticles. However, in the presence of the film loaded with nanoparticles and without any direct contact with this film, endothelial cell growth was also enhanced on polystyrene and on Transwell insert surfaces which demonstrates the effectiveness of the nanoparticles not only to improve the mechanical properties of the film but also to deliver active VEGF. An increase in nitric oxide levels as an indicator of endothelial cell activity was monitored and was correlated with the release of VEGF from the nanoparticle/film platform. Finally, such a system can be used as an auxiliary delivery body within implants to finely control the release of bioactive agent containing nanoparticles.

Antibacterial activity of class IIa bacteriocin Cbn BM1 depends on the physiological state of the target bacteria.

Carnobacteriocin BM1 (Cbn BM1) is a class IIa bacteriocin produced by Carnobacterium maltaromaticum CP5 isolated from a French mold ripened cheese. Numerous studies highlight variations in numerous parameters, such as bacterial membrane composition and potential, according to physiological changes. In this work, the mechanism of action of an oxidized form of Cbn BM1 was studied on C. maltaromaticum DSM20730 in log and stationary growth phases. Membrane integrity assessment and high resolution imaging by atomic force microscopy confirmed the link between physiological state and bacterial sensitivity to Cbn BM1. Indeed, these approaches enable visualizing morphological damage of C. maltaromaticum DSM20730 only in an active dividing state. To specifically address the interaction between peptide and bacterial membrane, fluorescence anisotropy measurements were conducted. Results revealed strong modifications in membrane fluidity by Cbn BM1 only for C. maltaromaticum DSM20730 in log growth phase. In a similar way, the Δψ component, but not the ΔpH component of the proton-motive force, was perturbed only for bacteria in log growth phase. These results clearly show that a class IIa bacteriocin antimicrobial mechanism of action can be modulated by the physiological state of its target bacteria.

Production of extracellular glycogen by Pseudomonas fluorescens: spectroscopic evidence and conformational analysis by biomolecular recognition.

Glycogen is mainly found as the principal storage form of glucose in cells. Many bacteria are able to synthesize large amounts of glycogen under unfavorable life conditions. By combining infrared spectroscopy, single molecule force spectroscopy (SMFS) and immuno-staining technique, we evidenced that planktonic P. fluorescens (Pf) cells are also able to produce glycogen as an extracellular polymeric substance. For this purpose, Pf suspensions were examined at 3 and 21 h of growth in nutritive medium (LB, 0.5 g/L). The conformation of the extracellular glycogen, revealed through its infrared spectral signature, has been investigated by SMFS measurements using Freely Jointed Chain model. The analysis of force versus distance curves showed over growth time that the increase of glycogen production was accompanied by an increase in glycogen contour lengths and ramifications. These results demonstrated that the production of extracellular bacterial glycogen can occur even if the cells are not subjected to unfavorable life conditions.