Bacterial eradication by a low-energy pulsed electron beam generator.

Low-energy electron beams (LEEB) are a safe and practical sterilization solution for in-line industrial applications, such as sterilizing medical products. However, their low dose rate induces product degradation, and the limited maximal energy prohibits high-throughput applications. To address this, we developed a low-energy 'pulsed' electron beam generator (LEPEB) and evaluated its efficacy and mechanism of action. Bacillus pumilus vegetative cells and spores were irradiated with a 250 keV LEPEB system at a 100 Hz pulse repetition frequency and a pulse duration of only 10 ns. This produced highly efficient bacterial inactivation at a rate of >6 log10, the level required for sterilization in industrial applications, with only two pulses for vegetative bacteria (20 ms) and eight pulses for spores (80 ms). LEPEB induced no morphological or structural defects, but decreased cell wall hydrophobicity in vegetative cells, which may inhibit biofilm formation. Single- and double-strand DNA breaks and pyrimidine dimer formation were also observed, likely causing cell death. Together, the unique combination of high dose rate and nanosecond delivery of LEPEB enable effective and high-throughput bacterial eradication for direct integration into production lines in a wide range of industrial applications.

Host Cell Geometry and Cytoskeletal Organization Governs Candida-Host Cell Interactions at the Nanoscale.

Candida is one of the most common opportunistic fungal pathogens in humans. Its adhesion to the host cell is required in parasitic states and is important for pathogenesis. Many studies have shown that there is an increased risk of developing candidiasis when normal tissue barriers are weakened or when immune defenses are compromised, for example, during cancer treatment that induces immunosuppression. The mechanical properties of malignant cells, such as adhesiveness and viscoelasticity, which contribute to cellular invasion and migration are different from those of noncancerous cells. To understand host invasion and its relationship with host cell health, we probed the interaction of Candida spp. with cancerous and noncancerous human cell lines using atomic force microscopy in the single-cell force spectroscopy mode. There was significant adhesion between Candida and human cells, with more adhesion to cancerous versus noncancerous cell lines. This increase in adhesion is related to the mechanobiological properties of cancer cells, which have a disorganized cytoskeleton and lower rigidity. Altered geometry and cytoskeletal disruption of the human cells impacted adhesion parameters, underscoring the role of cytoskeletal organization in Candida-human cell adhesion and implicating the manipulation of cell properties as a potential future therapeutic strategy.

Indentation of living cells by AFM tips may not be what we thought!

The models used to calculate Young's moduli from atomic force microscopy (AFM) force curves consider the shape of the indentation. It is then assumed that the geometry of the indentation is identical to the geometry of the indenter, which has been verified for hard materials (E > 1 MPa). Based on this assumption, the force curves calculated by these models, for the same object with a given Young's modulus, are different if the indenter geometry is different. On the contrary, we observe experimentally that the force curves recorded on soft living cells, with pyramidal, spherical, or tipless indenters, are almost similar. This indicates that this basic assumption on the indentation geometry does not work for soft materials (E of the order of 5 kPa or less). This means that, in this case, the shape of the indentation is therefore different from the shape of the indenter. Indentation of living cells by AFM is not what we thought!

Development and Use of a Monoclonal Antibody Specific for the Candida albicans Cell-Surface Protein Hwp1.

The Candida albicans cell-surface protein Hwp1 functions in adhesion to the host and in biofilm formation. A peptide from the Gln-Pro-rich adhesive domain of Hwp1 was used to raise monoclonal antibody (MAb) 2-E8. MAb 2-E8 specificity for Hwp1 was demonstrated using a hwp1/hwp1 C. albicans isolate and strains that expressed at least one HWP1 allele. Immunofluorescence and atomic force microscopy experiments using MAb 2-E8 confirmed C. albicans germ-tube-specific detection of the Hwp1 protein. MAb 2-E8 also immunolabeled the tips of some Candida dubliniensis germ tubes grown under conditions that maximized HWP1 expression. The phylogeny of HWP1 and closely related genes suggested that the Gln-Pro-rich adhesive domain was unique to C. albicans and C. dubliniensis focusing the utility of MAb 2-E8 on these species. This new reagent can be used to address unanswered questions about Hwp1 and its interactions with other proteins in the context of C. albicans biology and pathogenesis.

Atomic force microscopy-single-molecule force spectroscopy unveils GPCR cell surface architecture.

G protein-coupled receptors (GPCRs) form the largest family of cell surface receptors. Despite considerable insights into their pharmacology, the GPCR architecture at the cell surface still remains largely unexplored. Herein, we present the specific unfolding of different GPCRs at the surface of living mammalian cells by atomic force microscopy-based single molecule force spectroscopy (AFM-SMFS). Mathematical analysis of the GPCR unfolding distances at resting state revealed the presence of different receptor populations relying on distinct oligomeric states which are receptor-specific and receptor expression-dependent. Moreover, we show that the oligomer size dictates the receptor spatial organization with nanoclusters of high-order oligomers while lower-order complexes spread over the whole cell surface. Finally, the receptor activity reshapes both the oligomeric populations and their spatial arrangement. These results add an additional level of complexity to the GPCR pharmacology until now considered to arise from a single receptor population at the cell surface.

Macromolecular crowding limits growth under pressure.

Cells that grow in confined spaces eventually build up mechanical compressive stress. This growth-induced pressure (GIP) decreases cell growth. GIP is important in a multitude of contexts from cancer, to microbial infections, to biofouling, yet our understanding of its origin and molecular consequences remains limited. Here, we combine microfluidic confinement of the yeast Saccharomyces cerevisiae, with rheological measurements using genetically encoded multimeric nanoparticles (GEMs) to reveal that growth-induced pressure is accompanied with an increase in a key cellular physical property: macromolecular crowding. We develop a fully calibrated model that predicts how increased macromolecular crowding hinders protein expression and thus diminishes cell growth. This model is sufficient to explain the coupling of growth rate to pressure without the need for specific molecular sensors or signaling cascades. As molecular crowding is similar across all domains of life, this could be a deeply conserved mechanism of biomechanical feedback that allows environmental sensing originating from the fundamental physical properties of cells.

Elucidating bacterial adhesion to mucosal surface by an original AFM approach.

BACKGROUND: Fish skin represents an ancient vertebrate mucosal surface, sharing characteristics with other mucosal surfaces including those of the intestine. The skin mucosa is continuously exposed to microbes in the surrounding water and is therefore important in the first line defense against environmental pathogens by preventing bacteria from accessing the underlying surfaces. Understanding the microbe-host interactions at the fish skin mucosa is highly relevant in order to understand and control infection, commensalism, colonization, persistence, infection, and disease. Here we investigate the interactions between the pathogenic bacteria Aeromonas salmonicida (A. salmonicida) and Yersinia ruckeri (Y. ruckeri), respectively, and the skin mucosal surface of Atlantic salmon fry using AFM force spectroscopy. RESULTS: The results obtained revealed that when retracting probes functionalized with bacteria from surfaces coated with immobilized mucins, isolated from salmon mucosal surfaces, rupture events reflecting the disruption of adhesive interactions were observed, with rupture strengths centered around 200 pN. However, when retracting probes functionalized with bacteria from the intact mucosal surface of salmon fish fry no adhesive interactions could be detected. Furthermore, rheological measurements revealed a near fluid-like behavior for the fish fry skin mucus. Taken together, the experimental data indicate that the adhesion between the mucin molecules within the mucous layer may be significantly weaker than the interaction between the bacteria and the mucin molecules. The bacteria, immobilized on the AFM probe, do bind to individual mucins in the mucosal layer, but are released from the near fluid mucus with little resistance upon retraction of the AFM probe, to which they are immobilized. CONCLUSION: The data provided in the current paper reveal that A. salmonicida and Y. ruckeri do bind to the immobilized mucins. However, when retracting the bacteria from intact mucosal surfaces, no adhesive interactions are detected. These observations suggest a mechanism underlying the protective function of the mucosal surface based on the clearing of potential threats by adhering them to loosely attached mucus that is subsequently released from the fish skin.

The dual role of amyloid-ß-sheet sequences in the cell surface properties of FLO11-encoded flocculins in Saccharomyces cerevisiae.

Fungal adhesins (Als) or flocculins are family of cell surface proteins that mediate adhesion to diverse biotic and abiotic surfaces. A striking characteristic of Als proteins originally identified in the pathogenic Candida albicans is to form functional amyloids that mediate cis-interaction leading to the formation of adhesin nanodomains and trans-interaction between amyloid sequences of opposing cells. In this report, we show that flocculins encoded by FLO11 in Saccharomyces cerevisiae behave like adhesins in C. albicans. To do so, we show that the formation of nanodomains under an external physical force requires a threshold number of amyloid-forming sequences in the Flo11 protein. Then, using a genome editing approach, we constructed strains expressing variants of the Flo11 protein under the endogenous FLO11 promoter, leading to the demonstration that the loss of amyloid-forming sequences strongly reduces cell-cell interaction but has no effect on either plastic adherence or invasive growth in agar, both phenotypes being dependent on the N- and C-terminal ends of Flo11p. Finally, we show that the location of Flo11 is not altered either by the absence of amyloid-forming sequences or by the removal of the N- or C-terminus of the protein.

Probing the interactions between air bubbles and (bio)interfaces at the nanoscale using FluidFM technology.

Understanding the molecular mechanisms underlying bubble-(bio)surfaces interactions is currently a challenge that if overcame, would allow to understand and control the various processes in which they are involved. Atomic force microscopy is a useful technique to measure such interactions, but it is limited by the large size and instability of the bubbles that it can use, attached either on cantilevers or on surfaces. We here present new developments where microsized and stable bubbles are produced using FluidFM technology, which combines AFM and microfluidics. The air bubbles produced were used to probe the interactions with hydrophobic samples, showing that bubbles in water behave like hydrophobic surfaces. They thus could be used to measure the hydrophobic properties of microorganisms' surfaces, but in this case the interactions are also influenced by electrostatic forces. Finally a strategy was developed to functionalize their surface, thereby modulating their interactions with microorganism interfaces. This new method provides a valuable tool to understand bubble-(bio)surfaces interactions but also to engineer them.

From the first touch to biofilm establishment by the human pathogen Candida glabrata: a genome-wide to nanoscale view.

Candida glabrata is an opportunistic pathogen that adheres to human epithelial mucosa and forms biofilm to cause persistent infections. In this work, Single-cell Force Spectroscopy (SCFS) was used to glimpse at the adhesive properties of C. glabrata as it interacts with clinically relevant surfaces, the first step towards biofilm formation. Following a genetic screening, RNA-sequencing revealed that half of the entire transcriptome of C. glabrata is remodeled upon biofilm formation, around 40% of which under the control of the transcription factors CgEfg1 and CgTec1. Using SCFS, it was possible to observe that CgEfg1, but not CgTec1, is necessary for the initial interaction of C. glabrata cells with both abiotic surfaces and epithelial cells, while both transcription factors orchestrate biofilm maturation. Overall, this study characterizes the network of transcription factors controlling massive transcriptional remodelling occurring from the initial cell-surface interaction to mature biofilm formation.

Nanobiomechanical behavior of Fe3O4@SiO2and Fe3O4@SiO2-NH2nanoparticles over HeLa cells interfaces.

In this work, we studied the impact of magnetic nanoparticles (MNPs) interactions with HeLa cells when they are exposed to high frequency alternating magnetic field (AMF). Specifically, we measured the nanobiomechanical properties of cell interfaces by using atomic force microscopy (AFM). Magnetite (Fe3O4) MNPs were synthesized by coprecipitation and encapsulated with silica (SiO2): Fe3O4@SiO2and functionalized with amino groups (-NH2): Fe3O4@SiO2-NH2, by sonochemical processing. HeLa cells were incubated with or without MNPs, and then exposed to AMF at 37 °C. A biomechanical analysis was then performed through AFM, providing the Young's modulus and stiffness of the cells. The statistical analysis (p < 0.001) showed that AMF application or MNPs interaction modified the biomechanical behavior of the cell interfaces. Interestingly, the most significant difference was found for HeLa cells incubated with Fe3O4@SiO2-NH2and exposed to AMF, showing that the local heat of these MNPs modified their elasticity and stiffness.

Automation of Bio-Atomic Force Microscope Measurements on Hundreds of C. albicans Cells.

The method presented in this paper aims to automate Bio-AFM experiments and the recording of force curves. Using this method, it is possible to record forces curves on 1000 cells in 4 hours automatically. To maintain a 4 hour analysis time, the number of force curves per cell is reduced to 9 or 16. The method combines a Jython based program and a strategy for assembling cells on defined patterns. The program, implemented on a commercial Bio-AFM, can center the tip on the first cell of the array and then move, automatically, from cell to cell while recording force curves on each cell. Using this methodology, it is possible to access the biophysical parameters of the cells such as their rigidity, their adhesive properties, etc. With the automation and the large number of cells analyzed, one can access the behavior of the cell population. This is a breakthrough in the Bio-AFM field where data have, so far, been recorded on only a few tens of cells.

Pili and other surface proteins influence the structure and the nanomechanical properties of Lactococcus lactis biofilms.

Lactic acid bacteria, in particular Lactococcus lactis, are widely used in the food industry, for the control and/or the protection of the manufacturing processes of fermented food. While L. lactis has been reported to form compact and uniform biofilms it was recently shown that certain strains able to display pili at their surface form more complex biofilms exhibiting heterogeneous and aerial structures. As the impact of those biofilm structures on the biomechanical properties of the biofilms is poorly understood, these were investigated using AFM force spectroscopy and imaging. Three types of strains were used i.e., a control strain devoid of pili and surface mucus-binding protein, a strain displaying pili but no mucus-binding proteins and a strain displaying both pili and a mucus-binding protein. To identify potential correlations between the nanomechanical measurements and the biofilm architecture, 24-h old biofilms were characterized by confocal laser scanning microscopy. Globally the strains devoid of pili displayed smoother and stiffer biofilms (Young Modulus of 4-100 kPa) than those of piliated strains (Young Modulus around 0.04-0.1 kPa). Additional display of a mucus-binding protein did not affect the biofilm stiffness but made the biofilm smoother and more compact. Finally, we demonstrated the role of pili in the biofilm cohesiveness by monitoring the homotypic adhesion of bacteria to the biofilm surface. These results will help to understand the role of pili and mucus-binding proteins withstanding external forces.

Development of Polythiourethane/ZnO-Based Anti-Fouling Materials and Evaluation of the Adhesion of Staphylococcus aureus and Candida glabrata Using Single-Cell Force Spectroscopy.

The attachment of bacteria and other microbes to natural and artificial surfaces leads to the development of biofilms, which can further cause nosocomial infections. Thus, an important field of research is the development of new materials capable of preventing the initial adhesion of pathogenic microorganisms. In this work, novel polymer/particle composite materials, based on a polythiourethane (PTU) matrix and either spherical (s-ZnO) or tetrapodal (t-ZnO) shaped ZnO fillers, were developed and characterized with respect to their mechanical, chemical and surface properties. To then evaluate their potential as anti-fouling surfaces, the adhesion of two different pathogenic microorganism species, Staphylococcus aureus and Candida glabrata, was studied using atomic force microscopy (AFM). Our results show that the adhesion of both S. aureus and C. glabrata to PTU and PTU/ZnO is decreased compared to a model surface polydimethylsiloxane (PDMS). It was furthermore found that the amount of both s-ZnO and t-ZnO filler had a direct influence on the adhesion of S. aureus, as increasing amounts of ZnO particles resulted in reduced adhesion of the cells. For both microorganisms, material composites with 5 wt.% of t-ZnO particles showed the greatest potential for anti-fouling with significantly decreased adhesion of cells. Altogether, both pathogens exhibit a reduced capacity to adhere to the newly developed nanomaterials used in this study, thus showing their potential for bio-medical applications.

Analysis of Homotypic Interactions of Lactococcus lactis Pili Using Single-Cell Force Spectroscopy.

Cell surface proteins of Gram-positive bacteria play crucial roles in their adhesion to abiotic and biotic surfaces. Pili are long and flexible proteinaceous filaments known to enhance bacterial initial adhesion. They promote surface colonization and are thus considered as essential factors in biofilm cohesion. Our hypothesis is that pili mediate interactions between cells and may thereby directly affect biofilm formation. In this study, we use single-cell force spectroscopy (SCFS) to quantify the force of the homotypic pili interactions between individual bacterial cells, using different Lactococcus lactis strains producing pili or not as model bacteria. Moreover the force-distance curves were analyzed to determine the physical and nanomechanical properties of L. lactis pili. The results for pili-devoided strains showed a weak adhesion between cells (adhesion forces and work in the range of 100 pN and 7 × 10-18 J, respectively). On the contrary, the piliated strains showed high adhesion levels with adhesion forces and adhesion work over 200 pN and 50 × 10-18 J, respectively. The force-extension curves showed multiple adhesion events, typical of the unfolding of macromolecules. These unfolding force peaks were fitted using the physical worm-like chain model to get fundamental knowledge on the pili nanomechanical properties. In addition, SCFS applied to a L. lactis isolate expressing both pili and mucus-binding protein at its surface and two derivative mutants revealed the capacity of pili to interact with other surface proteins including mucus-binding proteins. This study demonstrates that pili are involved in L. lactis homotypic interactions and thus can influence biofilm structuring.

Nanoscale Evidence Unravels Microalgae Flocculation Mechanism Induced by Chitosan.

Microalgae are a promising resource for biofuel production, although their industrial use is limited by the lack of effective harvesting techniques. Flocculation consists in the aggregation and adhesion of cells into flocs that can be more easily removed from water than individual cells. Although it is an efficient harvesting technique, contamination is a major issue as chemical flocculants are often used. An alternative is to use natural biopolymers flocculants such as chitosan. Chitosan is a biobased nontoxic polymer that has been effectively used to harvest Chlorella vulgaris cells at a pH lower than its pKa (6.5). While the reported flocculation mechanism is said to rely on electrostatic interactions between chitosan and the negative cell surface, no molecular evidence has yet confirmed this mechanism. In this study, we performed force spectroscopy atomic force microscopy (AFM) experiments to probe the interactions between C. vulgaris cells and chitosan at the molecular scale to decipher its flocculation mechanism. Our results showed that at pH 6, chitosan interacts with C. vulgaris cell wall through biological interactions rather than electrostatic interactions. These observations were confirmed by comparing the data with cationically modified cellulose nanocrystals, for which the flocculation mechanism, relying on an electrostatic patch mechanism, has already been described for C. vulgaris. Further AFM experiments also showed that a different mechanism was at play at higher pH, based on chitosan precipitation. Thus, this AFM-based approach highlights the complexity of chitosan-induced flocculation mechanisms for C. vulgaris.

Differential homotypic and heterotypic interactions of antigen 43 (Ag43) variants in autotransporter-mediated bacterial autoaggregation.

Antigen 43 (Ag43) is a cell-surface exposed protein of Escherichia coli secreted by the Type V, subtype a, secretion system (T5aSS) and belonging to the family of self-associating autotransporters (SAATs). These modular proteins, comprising a cleavable N-terminal signal peptide, a surface-exposed central passenger and an outer membrane C-terminal translocator, self-recognise in a Velcro-like handshake mechanism. A phylogenetic network analysis focusing on the passenger revealed for the first time that they actually distribute into four distinct classes, namely C1, C2, C3 and C4. Structural alignment and modelling analyses demonstrated these classes arose from shuffling of two different subdomains within the Ag43 passengers. Functional analyses revealed that homotypic interactions occur for all Ag43 classes but significant differences in the sedimentation kinetics and aggregation state were present when Ag43C3 was expressed. In contrast, heterotypic interaction occurred in a very limited number of cases. Single cell-force spectroscopy demonstrated the importance of specific as well as nonspecific interactions in mediating Ag43-Ag43 recognition. We propose that structural differences in the subdomains of the Ag43 classes account for different autoaggregation dynamics and propensities to co-interact.

Changes in nanomechanical properties and adhesion dynamics of algal cells during their growth.

Nanomechanical and structural characterisations of algal cells are of key importance for understanding their adhesion behaviour at interfaces in the aquatic environment. We examine here the nanomechanical properties and adhesion dynamics of the algal cells during two phases of their growth using complementary surface methods and the mathematical modelling. Mechanical properties of motile cells are hard to assess while keeping cells viable, and studies to date have been limited. Immobilisation of negatively charged cells to a positively charged substrate enables high-resolution AFM imaging and nanomechanical measurements. Cells were stiffer and more hydrophobic in the exponential than in the stationary phase, suggesting molecular modification of the cell envelope during aging. The corresponding properties of algal cells were in agreement with the increase of critical interfacial tensions of adhesion, determined amperometrically. Cells in exponential phase possessed a larger cell volume, in agreement with the large amount of amperometrically measured displaced charge at the interface. Differences in the kinetics of adhesion and spreading of cells at the interface were attributed to their various volumes and nanomechanical properties that varied during cell aging. Our findings contribute to the present body of knowledge on the biophysics of algal cells on a fundamental level.

AFM dendritips functionalized with molecular probes specific to cell wall polysaccharides as a tool to investigate cell surface structure and organization.

The yeast cell wall is composed of mannoproteins, ß-1,3/ß-1, 6-glucans and chitin. Each of these components has technological properties that are relevant for industrial and medical applications. To address issues related to cell wall structure and alteration in response to stress or conditioning processes, AFM dendritips were functionalized with biomolecules that are specific for each of the wall components, which was wheat germ agglutinin (WGA) for chitin, concanavalin A (ConA) for mannans and anti-ß-1,3/anti-ß-1,6-glucan antibodies for ß-1,3/ß-1,6-glucans. Binding specificity of these biomolecules were validated using penta-N-acetylchitopentaose, α-mannans, laminarin (short ß-1,3-glucan chain) and gentiobiose (2 glucose units linked in ß 1→6) immobilized on epoxy glass slides. Dynamic force spectroscopy was employed to obtain kinetic and thermodynamic information on the intermolecular interaction of the binary complexes using the model of Friddle-Noy-de Yoreo. Using this model, transition state distance xt, dissociate rate koff and the lowest force (feq ) required to break the intermolecular bond of the complexes were approximated. These functionalized dendritips were then used to probe the yeast cell surface treated with a bacterial protease. As expected, this treatment, which removed the outer layer of the cell wall, gave accessibility to the inner layer composed of ß-glucans. Likewise, bud scars were nicely localized using AFM dendritip bearing the WGA probe. To conclude, these functionalized AFM dendritips constitute a new toolbox that can be used to investigate cell surface structure and organization in response to a wide arrays of cultures and process conditions.

Structural evidence for a new elaborate 3D-organization of the cardiomyocyte lateral membrane in adult mammalian cardiac tissues.

AIMS: This study explored the lateral crest structures of adult cardiomyocytes (CMs) within healthy and diseased cardiac tissue. METHODS AND RESULTS: Using high-resolution electron and atomic force microscopy, we performed an exhaustive quantitative analysis of the three-dimensional (3D) structure of the CM lateral surface in different cardiac compartments from various mammalian species (mouse, rat, cow, and human) and determined the technical pitfalls that limit its observation. Although crests were observed in nearly all CMs from all heart compartments in all species, we showed that their heights, dictated by the subsarcolemmal mitochondria number, substantially differ between compartments from one species to another and tightly correlate with the sarcomere length. Differences in crest heights also exist between species; for example, the similar cardiac compartments in cows and humans exhibit higher crests than rodents. Unexpectedly, we found that lateral surface crests establish tight junctional contacts with crests from neighbouring CMs. Consistently, super-resolution SIM or STED-based immunofluorescence imaging of the cardiac tissue revealed intermittent claudin-5-claudin-5 interactions in trans via their extracellular part and crossing the basement membrane. Finally, we found a loss of crest structures and crest-crest contacts in diseased human CMs and in an experimental mouse model of left ventricle barometric overload. CONCLUSION: Overall, these results provide the first evidence for the existence of differential CM surface crests in the cardiac tissue as well as the existence of CM-CM direct physical contacts at their lateral face through crest-crest interactions. We propose a model in which this specific 3D organization of the CM lateral membrane ensures the myofibril/myofiber alignment and the overall cardiac tissue cohesion. A potential role in the control of sarcomere relaxation and of diastolic ventricular dysfunction is also discussed. Whether the loss of CM surface crests constitutes an initial and common event leading to the CM degeneration and the setting of heart failure will need further investigation.