Biophysical insights into sugar-dependent medium acidification promoting YfaL protein-mediated Escherichia coli self-aggregation, biofilm formation and acid stress resistance.

The ability of bacteria to interact with their environment is crucial to form aggregates and biofilms, and develop a collective stress resistance behavior. Despite its environmental and medical importance, bacterial aggregation is poorly understood and mediated by few known adhesion structures. Here, we identified a new role for a surface-exposed Escherichia coli protein, YfaL, which can self-recognize and induce bacterial autoaggregation. This process occurs only under acidic conditions generated during E. coli growth in the presence of fermentable sugars. These findings were supported by electrokinetic and atomic force spectroscopy measurements, which revealed changes in the electrostatic, hydrophobic, and structural properties of YfaL-decorated cell surface upon sugar consumption. Furthermore, YfaL-mediated autoaggregation promotes biofilm formation and enhances E. coli resistance to acid stress. The prevalence and conservation of YfaL in environmental and clinical E. coli suggest strong evolutionary selection for its function inside or outside the host. Overall, our results emphasize the importance of environmental parameters such as low pH as physicochemical cues influencing bacterial adhesion and aggregation, affecting E. coli and potentially other bacteria's resistance to environmental stress.

Multimodal imaging analysis in silver fir reveals coordination in cellulose and lignin deposition.

Despite lignin being a key component of wood, the dynamics of tracheid lignification are generally overlooked in xylogenesis studies, which hampers our understanding of environmental drivers and blurs the interpretation of isotopic and anatomical signals stored in tree rings. Here, we analyzed cell wall formation in silver fir (Abies alba Mill.) tracheids to determine if cell wall lignification lags behind secondary wall deposition. For this purpose, we applied a multimodal imaging approach combining transmitted light microscopy (TLM), confocal laser scanning microscopy (CLSM), and confocal Raman microspectroscopy (RMS) on anatomical sections of wood microcores collected in northeast France on 11 dates during the 2010 growing season. Wood autofluorescence after laser excitation at 405 and 488 nm associated with the RMS scattering of lignin and cellulose, respectively, which allowed identification of lignifying cells (cells showing lignified and nonlignified wall fractions at the same time) in CLSM images. The number of lignifying cells in CLSM images mirrored the number of wall-thickening birefringent cells in polarized TLM images, revealing highly synchronized kinetics for wall thickening and lignification (similar timings and durations at the cell level). CLSM images and RMS chemical maps revealed a substantial incorporation of lignin into the wall at early stages of secondary wall deposition. Our results show that most of the cellulose and lignin contained in the cell wall undergo concurrent periods of deposition. This suggests a strong synchronization between cellulose and lignin-related features in conifer tree-ring records, as they originated over highly overlapped time frames.

Molecular Insights for Alzheimer's Disease: An Unexplored Storyline on the Nanoscale Impact of Nascent Aß1-42 toward the Lipid Membrane.

Deciphering the mechanism of Alzheimer's disease is a key element for designing an efficient therapeutic strategy. Molecular dynamics (MD) calculations, atomic force microscopy, and infrared spectroscopy were combined to investigate ß-amyloid (Aß1-42) peptide interactions with supported lipid bilayers (SLBs). The MD simulations showed that nascent Aß1-42 monomers remain anchored within a model phospholipid bilayer's hydrophobic core, which suggests their stability in their native environment. We tested this prediction experimentally by studying the behavior of Aß1-42 monomers and oligomers when interacting with SLBs. When Aß1-42 monomers and oligomers were self-assembled with a lipid bilayer and deposited as an SLB, they remain within the bilayers. Their presence in the bilayers induces destabilization of the model membranes. No specific interactions between Aß1-42 and the SLBs were detected when SLBs free of Aß1-42 were exposed to Aß1-42. This study suggests that Aß can remain in the membrane after cleavage by γ-secretase and cause severe damage to the membrane.

Supported lysozyme for improved antimicrobial surface protection.

Surface protection against biofilms is still an open challenge. Current strategies rely on coatings that are meant to guarantee antiadhesive or antimicrobial effects. While it seems difficult to ensure antiadhesion in complex media and against all the adhesive arsenal of microbes, strategies based on antimicrobials lack from sustainable functionalization methodologies to allow the perfect efficiency of the grafted molecules. Here we used the high affinity ligand-receptor interaction between biotin and streptavidin to functionalize surfaces with lysozyme, an enzyme that degrades the bacterial peptidoglycan cell wall. Biotinylated lysozyme was grafted on surfaces coated with streptavidin receptors. Using atomic force microscopy (AFM)-based single molecule force spectroscopy, we showed that grafting through ligand-receptor interaction allows the correct orientation of the enzyme on the substrate for enhanced activity towards the microbial target. The antibacterial efficiency was tested against Micrococcus luteus and revealed that surface protection was improved when lysozyme was grafted through the ligand-receptor interaction. These results suggest that bio-molecular interactions are promising for a sustainable grafting of antimicrobial agents on surfaces.

Parietal Structures of Escherichia coli Can Impact the D-Cateslytin Antibacterial Activity.

Bacterial resistance to conventional antibiotics is of major concern. Antimicrobial peptides (AMPs) are considered excellent alternatives. Among them, D-cateslytin (D-Ctl, derivative of a host defense peptide) has shown high efficiency against a broad spectrum of bacteria. The first target of AMPs is the outer membrane of the bacterium. However, the role of bacterial cell-wall structures on D-Ctl's mechanism of action has not yet been understood. In this study, we investigated the activity of D-Ctl on two isogenic strains of E. coli: one is devoid of any parietal structures; the other constitutively overexpresses only type 1 fimbriae. We studied the damage caused by D-Ctl at several initial concentrations of bacteria and D-Ctl, and exposure times to D-Ctl were examined using a combination of epifluorescence microscopy, atomic force microscopy (AFM), and Fourier transform infrared spectroscopy in attenuated total reflectance mode (ATR-FTIR). The analysis of nanomechanical and spectrochemical properties related to the antibacterial mechanism showed a concentration dependent activity. Whereas the membrane permeabilization was evidenced for all concentrations of D-Ctl and both mutants, no pore formation was observed. The bacterial stiffness is modified dramatically concomitantly to major membrane damage and changes in the spectral fingerprints of the bacteria. In the case of the occurrence of type 1 fimbriae only, an intracellular activity was additionally detected. Our results evidenced that D-Ctl activity is highly impacted by the cell-wall external structures and surface properties of the bacteria.

Mechanobiologically induced bone-like nodules: Matrix characterization from micro to nanoscale.

In bone tissue engineering, stem cells are known to form inhomogeneous bone-like nodules on a micrometric scale. Herein, micro- and nano-infrared (IR) micro-spectroscopies were used to decipher the chemical composition of the bone-like nodule. Histological and immunohistochemical analyses revealed a cohesive tissue with bone-markers positive cells surrounded by dense mineralized type-I collagen. Micro-IR gathered complementary information indicating a non-mature collagen at the top and periphery and a mature collagen within the nodule. Atomic force microscopy combined to IR (AFM-IR) analyses showed distinct spectra of "cell" and "collagen" rich areas. In contrast to the "cell" area, spectra of "collagen" area revealed the presence of carbohydrate moieties of collagen and/or the presence of glycoproteins. However, it was not possible to determine the collagen maturity, due to strong bands overlapping and/or possible protein orientation effects. Such findings could help developing protocols to allow a reliable characterization of in vitro generated complex bone tissues.

Chemical Functionalization of the Zinc Selenide Surface and Its Impact on Lactobacillus rhamnosus GG Biofilms.

Bacteria grow on surfaces and form communities called biofilms. Bacterial adhesion and properties of the derived biofilms depend on, among others, the nature of the supporting substrate. Here, we report how the surface properties of the substrate affect the biofilm growth of probiotic Lactobacillus rhamnosus GG (LGG). Hydrophilic (OH), hydrophobic (CH3), and positively charged (NH3+) surfaces were obtained by the functionalization of a ZnSe crystal with alkanethiol self-assembled monolayers (SAM). The self-assembly of alkanethiols onto ZnSe was studied in situ using infrared spectroscopy in attenuated total reflection mode (ATR-FTIR). The organization of grafted SAMs was analyzed based on the results of ATR-FTIR, high-energy elastic backscattering spectrometry, and contact angle measurements. The kinetics and adhesion strength of LGG initial attachment as well as its physiological state on surfaces terminated by the different functional groups were assessed by the combination of ATR-FTIR, force measurements based on atomic force microscopy, and fluorescent staining of bacteria. The strength of interactions between LGG and the surface was strongly affected by the terminal group of the alkanethiol chain. The -NH3+ groups displayed the highest affinity with LGG at the first stage of interaction. The surface properties also played an important role when LGG biofilms were further grown in a nutritive medium for 24 h under flow conditions. Notably, the analysis of the infrared spectra recorded during the biofilm cultivation revealed differences in the kinetics of growth and in the polysaccharide features of the biofilm depending on the substrate functionality. LGG biofilm was stable only on the positively charged surface upon rinsing. Findings of this work clearly show that the adhesion features and the growth of LGG biofilms are substrate-dependent.

Probing the Adhesion of the Common Freshwater Diatom Nitzschia palea at Nanoscale.

Freshwater biofilms play an essential ecological role, but they also adversely affect human activities through undesirable biofouling of artificial submerged structures. They form complex aggregates of microorganisms that colonize any type of substratum. In phototrophic biofilms, diatoms dominate in biomass and produce copious amount of extracellular polymeric substances (EPSs), making them efficient early colonizers. Therefore, a better understanding of diatoms adhesive properties is essential to develop new anti-biofouling strategies. In this context, we used atomic force microscopy (AFM) to decipher the topography and adhesive mechanisms of the common freshwater diatom Nitzschia palea. Images taken in physiological conditions revealed typical ultrastructural features with a few nanometers resolution. Using single-cell force spectroscopy, we showed that N. palea strongly adheres to hydrophobic surfaces as compared to hydrophilic ones. Chemical force spectroscopy with hydrophobic tips further confirmed that the adhesion is governed by surface-associated hydrophobic EPS distributed in clusters at the frustule surface, and mostly composed of (glyco)-lipids as revealed by Raman spectroscopy. Collectively, our results demonstrate that AFM-based nanoscopy, combined with Raman spectroscopy, is a powerful tool to provide new insights into the adhesion mechanisms of diatoms.

Doxorubicin-Loaded Thermoresponsive Superparamagnetic Nanocarriers for Controlled Drug Delivery and Magnetic Hyperthermia Applications.

This study reports on the development of thermoresponsive core/shell magnetic nanoparticles (MNPs) based on an iron oxide core and a thermoresponsive copolymer shell composed of 2-(2-methoxy)ethyl methacrylate (MEO2MA) and oligo(ethylene glycol)methacrylate (OEGMA) moieties. These smart nano-objects combine the magnetic properties of the core and the drug carrier properties of the polymeric shell. Loading the anticancer drug doxorubicin (DOX) in the thermoresponsive MNPs via supramolecular interactions provides advanced features to the delivery of DOX with spatial and temporal controls. The so coated iron oxide MNPs exhibit superparamagnetic behavior with a saturation magnetization of around 30 emu g-1. Drug release experiments confirmed that only a small amount of DOX was released at room temperature, while almost 100% drug release was achieved after 52 h at 42 °C with Fe3-δO4@P(MEO2MA60OEGMA40), which grafted polymer chains displaying a low critical solution temperature of 41 °C. Moreover, the MNPs exhibit magnetic hyperthermia properties as shown by specific absorption rate measurements. Finally, the cytotoxicity of the core/shell MNPs toward human ovary cancer SKOV-3 cells was tested. The results showed that the polymer-capped MNPs exhibited almost no toxicity at concentrations up to 12 µg mL-1, whereas when loaded with DOX, an increase in cytotoxicity and a decrease of SKOV-3 cell viability were observed. From these results, we conclude that these smart superparamagnetic nanocarriers with stealth properties are able to deliver drugs to tumor and are promising for applications in multimodal cancer therapy.

Boosting mesenchymal stem cells regenerative activities on biopolymers-calcium phosphate functionalized collagen membrane.

The regeneration of bone-soft tissue interface, using functional membranes, remains challenging and can be promoted by improving mesenchymal stem cells (MSCs) paracrine function. Herein, a collagen membrane, used as guided bone regeneration membrane, was functionalized by calcium phosphate, chitosan and hyaluronic acid hybrid coating by simultaneous spray of interacting species process. Composed of brushite, octacalcium phosphate and hydroxyapatite, the hybrid coating increased the membrane stiffness by 50%. After 7 days of MSCs culture on the hybrid coated polymeric membrane, biological studies were marked by a lack of osteoblastic commitment. However, MSCs showed an enhanced proliferation along with the secretion of cytokines and growth factors that could block bone resorption and favour endothelial cell recruitment without exacerbating polynuclear neutrophils infiltration. These data shed light on the great potential of inorganic/organic coated collagen membranes as an alternative bioactive factor-like platform to improve MSCs regenerative capacity, in particular to support bone tissue vascularization and to modulate inflammatory infiltrates.

In situ spectroscopic analysis of Lactobacillus rhamnosus GG flow on an abiotic surface reveals a role for nutrients in biofilm development.

In this work, infrared spectroscopy was used to monitor the changes in the biochemical composition of biofilms of the probiotic bacterium Lactobacillus rhamnosus GG (LGG) in three nutritive media (10-fold diluted MRS, AOAC, and mTSB), in situ and under flow conditions. Epifluorescence microscopy was used to observe the shape of LGG cells and their distribution on the surface. Spectroscopic fingerprints recorded as a function of time revealed a medium-dependent content of nucleic acids, phospholipids and polysaccharides in the biofilms. In addition, time-dependent synthesis of lactic acid was observed in MRS/10 and AOAC/10. Polysaccharides were produced to the highest extent in mTSB/10, and the biofilms obtained were the densest in this medium. The rod shape of the cells was preserved in MRS/10, whereas acidic stress induced in AOAC/10 and the nutritional quality of mTSB/10 led to strong morphological changes. These alterations due to the nutritive environment are important to consider in research and use of LGG biofilms.

D-Cateslytin, a new antimicrobial peptide with therapeutic potential.

The rise of antimicrobial resistant microorganisms constitutes an increasingly serious threat to global public health. As a consequence, the efficacy of conventional antimicrobials is rapidly declining, threatening the ability of healthcare professionals to cure common infections. Over the last two decades host defense peptides have been identified as an attractive source of new antimicrobials. In the present study, we characterized the antibacterial and mechanistic properties of D-Cateslytin (D-Ctl), a new epipeptide derived from L-Cateslytin, where all L-amino acids were replaced by D-amino acids. We demonstrated that D-Ctl emerges as a potent, safe and robust peptide antimicrobial with undetectable susceptibility to resistance. Using Escherichia coli as a model, we reveal that D-Ctl targets the bacterial cell wall leading to the permeabilization of the membrane and the death of the bacteria. Overall, D-Ctl offers many assets that make it an attractive candidate for the biopharmaceutical development of new antimicrobials either as a single therapy or as a combination therapy as D-Ctl also has the remarkable property to potentiate several antimicrobials of reference such as cefotaxime, amoxicillin and methicillin.

AFM combined to ATR-FTIR reveals Candida cell wall changes under caspofungin treatment.

Fungal pathogens from Candida genus are responsible for severe life-threatening infections and the antifungal arsenal is still limited. Caspofungin, an antifungal drug used for human therapy, acts as a blocking agent of the cell wall synthesis by inhibiting the ß-1,3-glucan-synthase encoded by FKS genes. Despite its efficiency, the number of genetic mutants that are resistant to caspofungin is increasing. An important challenge to improve antifungal therapy is to understand cellular phenomenon that are associated with drug resistance. Here we used atomic force microscopy (AFM) combined to Fourier transform infrared spectroscopy in attenuated total reflection mode (ATR-FTIR) to decipher the effect of low and high drug concentration on the morphology, mechanics and cell wall composition of two Candida strains, one susceptible and one resistant to caspofungin. Our results confirm that caspofungin induces a dramatic cell wall remodelling via activation of stress responses, even at high drug concentration. Additionally, we highlighted unexpected changes related to drug resistance, suggesting that caspofungin resistance associated with FKS gene mutations comes from a combination of effects: (i) an overall remodelling of yeast cell wall composition; and (ii) cell wall stiffening through chitin synthesis. This work demonstrates that AFM combined to ATR-FTIR is a valuable approach to understand at the molecular scale the biological mechanisms associated with drug resistance.

Discrepancies between Cyclic and Linear Antimicrobial Peptide Actions on the Spectrochemical and Nanomechanical Fingerprints of a Young Biofilm.

Antimicrobial peptides (AMPs) are currently known for their potential as an alternative to conventional antibiotics and new weapons against drug-resistant bacteria and biofilms. In the present work, the mechanism of action of a cyclic (colistin) and a linear (catestatin) AMP on a young E. coli biofilm was deciphered from the molecular to the cellular scale. To this end, infrared spectroscopy (attenuated total reflection-Fourier transform infrared) assisted by chemometric analysis was combined with fluorescence and atomic force microscopies to address the very different behaviors of both AMPs. Indeed, the colistin dramatically damaged the bacterial cell wall and the metabolism even though its action was not homogeneous over the whole bacterial population and repopulation can be observed after peptide removal. Conversely, catestatin did not lead to major damages in the bacterial morphology but its action was homogeneous over the whole bacterial population and the cells were unable to regrow after the peptide treatment. Our results strongly suggested that contrary to the cyclic molecule, the linear one is able to cause irreversible damages in the bacterial membrane concomitantly to a strong impact on the bacterial metabolism.

Nanoscale investigation of the interaction of colistin with model phospholipid membranes by Langmuir technique, and combined infrared and force spectroscopies.

Colistin (Polymyxin E), an antimicrobial peptide, is increasingly put forward as salvage for severe multidrug-resistant infections. Unfortunately, colistin is potentially toxic to mammalian cells. A better understanding of the interaction with specific components of the cell membranes may be helpful in controlling the factors that may enhance toxicity. Here, we report a physico-chemical study of model phospholipid (PL) mono- and bilayers exposed to colistin at different concentrations by Langmuir technique, atomic force microscopy (AFM) and attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR). The effect of colistin on chosen PL monolayers was examined. Insights into the topographical and elastic changes in the PL bilayers within time after peptide injection are presented via AFM imaging and force spectra. Finally, changes in the PL bilayers' ATR-FTIR spectra as a function of time within three bilayer compositions, and the influence of colistin on their spectral fingerprint are examined together with the time-evolution of the Amide II and νCO band integrated intensity ratios. Our study reveals a great importance in the role of the PL composition as well as the peptide concentration on the action of colistin on PL model membranes.

In situ and real time investigation of the evolution of a Pseudomonas fluorescens nascent biofilm in the presence of an antimicrobial peptide.

Against the increase of bacterial resistance to traditional antibiotics, antimicrobial peptides (AMP) are considered as promising alternatives. Bacterial biofilms are more resistant to antibiotics that their planktonic counterpart. The purpose of this study was to investigate the action of an AMP against a nascent bacterial biofilm. The activity of dermaseptin S4 derivative S4(1-16)M4Ka against 6 h-old Pseudomonas fluorescens biofilms was assessed by using a combination of Attenuated Total Reflectance-Fourier Transform InfraRed (ATR-FTIR) spectroscopy in situ and in real time, fluorescence microscopy using the Baclight™ kit, and Atomic Force Microscopy (AFM, imaging and force spectroscopy). After exposure to the peptide at three concentrations, different dramatic and fast changes over time were observed in the ATR-FTIR fingerprints reflecting a concentration-dependent action of the AMP. The ATR-FTIR spectra revealed major biochemical and physiological changes, adsorption/accumulation of the AMP on the bacteria, loss of membrane lipids, bacterial detachment, bacterial regrowth, or inhibition of biofilm growth. AFM allowed estimating at the nanoscale the effect of the AMP on the nanomechanical properties of the sessile bacteria. The bacterial membrane elasticity data measured by force spectroscopy were consistent with ATR-FTIR spectra, and they allowed suggesting a mechanism of action of this AMP on sessile P. fluorescens. The combination of these three techniques is a powerful tool for in situ and in real time monitoring the activity of AMPs against bacteria in a biofilm.

On the production of glycogen by Pseudomonas fluorescens during biofilm development: an in situ study by attenuated total reflection-infrared with chemometrics.

Attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy was used to monitor Pseudomonas fluorescens biofilms in situ, non-destructively, in real time, and under fully hydrated conditions. Changes accompanying the metabolic evolution of the sessile bacterial cells from the nascent biofilm monolayer to the beginning of the multi-layered structure in the presence of nutrients were identified via the ATR-FTIR fingerprints of the young biofilm on the ATR crystal. The ATR-FTIR spectra were analysed by classical methods (time evolution of integrated intensities and profile evolution of specific bands), and also by a multivariate curve resolution, Bayesian positive source separation, to extract the pure component spectra and their change of concentration over time occurring during biofilm settlement. This work showed clearly the overproduction of glycogen by sessile P. fluorescens, which had not previously been described by other research groups.

In situ analysis of bacterial extracellular polymeric substances from a Pseudomonas fluorescens biofilm by combined vibrational and single molecule force spectroscopies.

Extracellular polymeric substances (EPS) play an important role in biofilm cohesion and adhesion to surfaces. EPS of a P. fluorescens biofilm were characterized through their vibrational spectra (infrared and Raman) and their conformational properties using single molecule force spectroscopy with specific probes for glucose, galactose, and N-acetyl glucosamine-rich EPS. Vibrational spectra evidenced the overproduction of glycogen and other carbohydrates in the biofilm. The conformational analysis was performed from both the freely jointed chain (FJC) and worm like chain (WLC) models. The results of the FJC fittings showed highly ramified and/or folded structures for all the detected EPS with molecular elongations up to 1000-2500 nm, and typical Kuhn lengths of glycogen macromolecules. The characteristics of galactose-rich EPS have been found to be significantly different from those of glucose- and N-acetyl glucosamine-rich EPS. On the basis of the theoretical fittings with the WLC model, our results suggested that carbohydrates may be associated with peptide domains.

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.

Durable cofactor immobilization in sol-gel bio-composite thin films for reagentless biosensors and bioreactors using dehydrogenases.

A new strategy directed to the durable immobilization of NAD(+)/NADH cofactors has been tested, along with a suitable redox mediator (ferrocene), in biocompatible sol-gel matrices encapsulating a bi-enzymatic system (a dehydrogenase and a diaphorase, this latter being useful to the safe regeneration of the cofactor), which were deposited as thin films onto glassy carbon electrode surfaces. It involves the chemical attachment of NAD(+) to the silica matrix using glycidoxypropylsilane in the course of the sol-gel process (in smooth chemical conditions). This approach based on chemical bonding of the cofactor (which was checked by infrared spectroscopy) led to good performances in terms of long-term stability of the electrochemical response. The possibility to integrate all components (proteins, cofactor, mediator) in the sol-gel layer in an active and durable form gave rise to reagentless devices with extended operational stability (i.e. high amperometric response maintained for more than 12h of continuous use under constant potential, whereas the signal completely vanished within the first few minutes of working with non-covalently bonded NAD(+)). To confirm the wide applicability of the proposed approach, the same strategy has been applied to the elaboration of biosensors for D-sorbitol, D-glucose and L-lactate with using D-sorbitol dehydrogenase, D-glucose dehydrogenase and L-lactate dehydrogenase respectively. The analytical characteristics of the glucose sensors are given and compared to previous approaches described in the literature for the elaboration of reagentless biosensors.