Dairy powders are usually subjected to environmental variations during storage and/or shipment that strongly impact their chemical, nutritional and structural features. Nevertheless, these ...modifications are rarely investigated at the particle surface level, which represents the interface in contact with air, water, materials or other powders and directly influences powder functionalities. In this paper, atomic force microscopy (AFM) is used in imaging, nanoindentation and force spectroscopy modes to investigate the evolution of the surface properties such as the hydrophobicity and stiffness of whey protein powders at the nanoscale after controlled storage conditions. Our results evidenced that surface modifications are more enhanced by high storage temperature than storage time (for the same storage energy) and correspond to an increase of both surface hydrophobicity and heterogeneity. The strong impact of residual lactose in the powder (around 1.5% lactose) is also highlighted on these phenomena by performing surface comparisons with a reference powder (β-lactoglobulin) without lactose. This reference powder permitted the discrimination between surface protein denaturation and surface lactosylation.
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•Surface properties of whey protein particles at the nanoscale were dramatically impacted by high temperature storage.•Storage at high temperature led to smoother particles surfaces without size modifications.•Upon storage, particle morphological changes were correlated to the formation of strongly hydrophobic domains.•Nanoindentation measurements revealed that storage at high temperature induced particle surface mechanical heterogeneities by formation of stiff domains.•Physicochemical modifications at the particle surface could be attributed to protein denaturation and lactosylation together with compounds rearrangement and migration.
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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.
Single-cell force spectroscopy is a powerful atomic force microscopy modality in which a single living cell is attached to the atomic force microscopy cantilever to quantify the forces that drive ...cell-cell and cell-substrate interactions. Although various single-cell force spectroscopy protocols are well established for animal cells, application of the method to individual bacterial cells remains challenging, mainly owing to the lack of appropriate methods for the controlled attachment of single live cells on cantilevers. We present a nondestructive protocol for single-bacterial cell force spectroscopy, which combines the use of colloidal probe cantilevers and of a bioinspired polydopamine wet adhesive. Living cells from the probiotic species Lactobacillus plantarum are picked up with a polydopamine-coated colloidal probe, enabling us to quantify the adhesion forces between single bacteria and biotic (lectin monolayer) or abiotic (hydrophobic monolayer) surfaces. These minimally invasive single-cell experiments provide novel, to our knowledge, insight into the specific and nonspecific forces driving the adhesion of L. plantarum, and represent a generic platform for studying the molecular mechanisms of cell adhesion in probiotic and pathogenic bacteria.
A variety of bacterial pathogens use nanoscale protein fibers called type IV pili to mediate cell adhesion, a primary step leading to infection. Currently, how these nanofibers respond to mechanical ...stimuli and how this response is used to control adhesion is poorly understood. Here, we use atomic force microscopy techniques to quantify the forces guiding the adhesion of Pseudomonas aeruginosa type IV pili to surfaces. Using chemical force microscopy and single-cell force spectroscopy, we show that pili strongly bind to hydrophobic surfaces in a time-dependent manner, while they weakly bind to hydrophilic surfaces. Individual nanofibers are capable of withstanding forces up to 250 pN, thereby explaining how they can resist mechanical stress. Pulling on individual pili yields constant force plateaus, presumably reflecting conformational changes, as well as nanospring properties that may help bacteria to withstand physiological shear forces. Analysis of mutant strains demonstrates that these mechanical responses originate solely from type IV pili, while flagella and the cell surface localized and proposed pili-associated adhesin PilY1 play no direct role. We also demonstrate that bacterial–host interactions involve constant force plateaus, the extension of bacterial pili, and the formation of membrane tethers from host cells. We postulate that the unique mechanical responses of type IV pili unravelled here enable the bacteria to firmly attach to biotic and abiotic surfaces and thus maintain attachment when subjected to high shear forces under physiological conditions, helping to explain why pili play a critical role in colonization of the host.
The Gram-positive bacterium Staphylococcus epidermidis is responsible for important nosocomial infections. With the continuous emergence of antibiotic-resistant strains, the search for new treatments ...has been amplified in the last decades. A potential candidate against multidrug-resistant bacteria is squalamine, a natural aminosterol discovered in dogfish sharks. Despite its broad-spectrum efficiency, little is known about squalamine mode of action. Here, we used atomic force microscopy (AFM) imaging to decipher the effect of squalamine on S. epidermidis morphology, revealing the peptidoglycan structure at the bacterial surface after the drug action. Single-molecule force spectroscopy with squalamine-decorated tips shows that squalamine binds to the cell surface via the spermidine motif, most likely through electrostatic interactions between the amine groups of the molecule and the negatively-charged bacterial cell wall. We demonstrated that - although spermidine is sufficient for the initial attachment of squalamine to S. epidermidis – the integrity of the molecule needs to be conserved for its antimicrobial action. A deeper analysis of the AFM force-distance signatures suggests the implication of the accumulation-associated protein (Aap), one of the main adhesins of S. epidermidis, in the initial binding of squalamine to the bacterial cell wall. This work highlights that AFM -combined with microbiological assays at the bacterial suspension scale- is a valuable approach to better understand the molecular mechanisms behind the efficiency of squalamine antibacterial activity.
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•Squalamine has an antimicrobial action against Staphylococcus epidermidis.•The minimal inhibitory concentration (MIC) against S. epidermidis is 2 µM.•Squalamine binds to the bacterial cell wall via the spermidine motif.•Unfolding signatures suggest that adhesins Aap are involved in the initial binding.
Micro- and Nano-Fibrillated Cellulose (MNFC) have gained increasing attention due to their remarkable properties, but their production usually requires an intensive multi-step process. This study ...proposes to find a novel approach involving steam explosion for the production of lignin-containing micro- and nano-fibrillated cellulose (L-MNFC) using
Eucalyptus globulus
bark as a new lignocellulosic feedstock.
Eucalyptus globulus
bark was first pre-treated by steam explosion in alkaline conditions (200 °C, 8 min) or by soda cooking in a rotating autoclave (170 °C, 60 min), refined and then ground until gels formed. The chemical composition of the pulps was studied with ion chromatography and FTIR-ATR. The morphology of the products was studied with measurements of suspension turbidity and Morfi Neo, optical and atomic force microscopies. Nanopapers were produced from L-MNFC to investigate mechanical properties. Results obtained showed that steam explosion produced pulps with slightly higher lignin content (≈ 9%), containing shorter fibers (≈ 400 µm) and higher amounts of fines (≈ 86%) compared to soda cooking (≈ 5%, ≈ 560 µm and 66%, respectively). AFM images of SteamEx L-MNFC gels showed a web-like structure containing lignin nanoparticles.
Graphical abstract
Lactic Acid Bacteria (LAB) are not homogeneously located in the dairy matrix and their spatial distribution seems to be controlled by the establishment of adhesive interactions between matrix ...components and bacterial surface biomolecules. However the mechanisms of interaction remain unknown although they constitute an interesting way of study to appreciate the interactions. The aim of this work was to understand the role of surface biomolecules in the adhesion of Lactobacillus rhamnosus GG - the most used LAB strain in food products for their health benefits to the consumer - to milk proteins. Adhesions were probed using atomic force microscopy based force spectroscopy. To this end, the wild type strain and three of its surface mutants were employed. The wild type strain interacts with the β-lactoglobulin through the pili SpaCBA. The use of LGG surface mutants revealed that other surface biomolecules as long/small exopolysaccharides and proteins are involved in adhesion with milk proteins, in a less pronounced way than pili and in absence of pili, as all other surface biomolecules are masked in presence of pili. Altogether, this study demonstrates that adhesive interactions between LGG and milk proteins are governed by the surface composition of the bacteria.
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•Use of AFM to study interaction between L. rhamnosus GG and milk proteins.•Bacterial biomolecules surface governed interactions between LGG and milk proteins.•LGG wild type strain interacts with the β-lactoglobulin through the pili SpaCBA.•In absence of pili, exopolysaccharides are involved in adhesion with milk proteins.•Key role of LGG biomolecules surface environment in adhesion with milk proteins.
The importance of force in microbial cell adhesion El-Kirat-Chatel, Sofiane; Beaussart, Audrey; Mathelié-Guinlet, Marion ...
Current opinion in colloid & interface science,
June 2020, 2020-06-00, 2020-06, Letnik:
47
Journal Article
Recenzirano
Odprti dostop
Microbes have evolved sophisticated strategies to colonize biotic and abiotic surfaces. Forces play a central role in microbial cell adhesion processes, yet until recently these were not accessible ...to study at the molecular scale. Unlike traditional assays, atomic force microscopy (AFM) is capable to study forces in single cell surface molecules and appendages, in their biologically relevant conformation and environment. Recent AFM investigations have demonstrated that bacterial pili exhibit a variety of mechanical responses upon contact with surfaces and that cell surface adhesion proteins behave as force-sensitive switches, two phenomena that play critical roles in cell adhesion and biofilm formation. AFM has also enabled to assess the efficiency of sugars, peptides, and antibodies in blocking cell adhesion, opening up new avenues for the development of antiadhesion therapies against pathogens.
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•Atomic force microscopy revealed an interaction between LGG SpaCBA pili and MFGM.•SpaCBA pili play a key role in the adhesion of LGG to intestinal epithelial cells.•The addition of ...MFGM decreased the bacterial attachment ability to Caco-2 cells.•A competition is possible between LGG SpaCBA adhesion to MFGM and Caco-2 cells.
Milk is the most popular matrix for the delivery of lactic acid bacteria, but little is known about how milk impacts bacterial functionality. Here, the adhesion mechanisms of Lactobacillus rhamnosus GG (LGG) surface mutants to a milk component, the milk fat globule membrane (MFGM), were compared using atomic force microscopy (AFM). AFM results revealed the key adhesive role of the LGG SpaCBA pilus in relation to MFGM. A LGG mutant without exopolysaccharides but with highly exposed pili improved the number of adhesive events between LGG and MFGM compared to LGG wild type (WT). In contrast, the number of adhesive events decreased significantly for a LGG mutant without SpaCBA pili. Moreover, the presence of MFGM in the dairy matrix was found to decrease significantly the bacterial attachment ability to Caco-2 TC7 cells. This work thus demonstrated a possible competition between LGG adhesion to MFGM and to epithelial intestinal cells. This competition could negatively impact the adhesion capacity of LGG to intestinal cells in vivo, but requires further substantiation.
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 β-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.
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