Low-density lipoprotein receptor-related protein 1 (LRP-1) can internalize proteases involved in cancer progression and is thus considered a promising therapeutic target. However, it has been ...demonstrated that LRP-1 is also able to regulate the endocytosis of membrane-anchored proteins. Thus, strategies that target LRP-1 to modulate proteolysis could also affect adhesion and cytoskeleton dynamics. Here, we investigated the effect of LRP-1 silencing on parameters reflecting cancer cells' invasiveness by atomic force microscopy (AFM). The results show that LRP-1 silencing induces changes in the cells' adhesion behavior, particularly the dynamics of cell attachment. Clear alterations in morphology, such as more pronounced stress fibers and increased spreading, leading to increased area and circularity, were also observed. The determination of the cells' mechanical properties by AFM showed that these differences are correlated with an increase in Young's modulus. Moreover, the measurements show an overall decrease in cell motility and modifications of directional persistence. An overall increase in the adhesion force between the LRP-1-silenced cells and a gelatin-coated bead was also observed. Ultimately, our AFM-based force spectroscopy data, recorded using an antibody directed against the β1 integrin subunit, provide evidence that LRP-1 silencing modifies the rupture force distribution. Together, our results show that techniques traditionally used for the investigation of cancer cells can be coupled with AFM to gain access to complementary phenotypic parameters that can help discriminate between specific phenotypes associated with different degrees of invasiveness.
Green microalgae are a natural source of oil for commercial biodiesel production. However, their cell-wall barrier remains a major obstacle for effective intracellular lipid extraction. Solution to ...this issue includes the use of surface-digesting enzymes and the control of the so-far poorly understood changes in structural and mechanical properties of the microalgal envelops during growth and upon action of enzymes. Here, we used a combination of atomic force microscopy (AFM) and confocal microscopy to decipher the variation of cell-wall ultrastructure, composition and nanomechanics of Chlorella vulgaris upon culture ageing and lysozyme treatment. AFM imaging revealed the presence of a fibrillated mesh at the surface of cells harvested in the stationary phase, hardly distinguishable on cells from younger culture (mid-log phase). The fibrils form a chitin-like network containing the N-acetyl-d-glucosamine unit, and this structured network is severely damaged upon lysozyme treatment, a property we identified at the cellular and molecular scales by fluorescent-lectin staining and AFM-based force spectroscopy using lectin-modified tips, respectively. The enzyme was also found to act on algal physiology, to trigger oxidative stress and changes in cell lipid content. Detailed single-cell mapping of the nanomechanical properties of C. vulgaris further indicated that microalgal cells soften upon ageing and it confirmed that lysozyme affects both their surface and intracellular compartments. Altogether, our results emphasize that AFM-based multi-parametric analysis in combination with confocal microscopy allows accurate evaluation of physico-chemical surface properties of microalgae and offers an exciting perspective on cell culture condition optimization for facilitated oil extraction.
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•Chlorella vulgaris surface morphology and mechanics depend on growth phase.•A fibrillated network appears on cells harvested in stationary phase.•The fibrils are composed of N-acetyl-d-glucosamine.•Enzymatic treatment (lysozyme) modifies physical chemistry of the algal cell wall.•Lysozyme acts on cell physiology via oxidative-stress and changes in lipid content.
The major human pathogen Streptococcus pneumoniae is a leading cause of disease and death worldwide. Pneumococcal biofilm formation within the nasopharynx leads to long-term colonization and ...persistence within the host. We have previously demonstrated that the capsular surface-associated pneumococcal serine rich repeat protein (PsrP), key factor for biofilm formation, binds to keratin-10 (KRT10) through its microbial surface component recognizing adhesive matrix molecule (MSCRAMM)-related globular binding region domain (BR187-385). Here, we show that BR187-385 also binds to DNA, as demonstrated by electrophoretic mobility shift assays and size exclusion chromatography. Further, heterologous expression of BR187-378 or the longer BR120-378 construct on the surface of a Gram-positive model host bacterium resulted in the formation of cellular aggregates that was significantly enhanced in the presence of DNA. Crystal structure analyses revealed the formation of BR187-385 homo-dimers via an intermolecular β-sheet, resulting in a positively charged concave surface, shaped to accommodate the acidic helical DNA structure. Furthermore, small angle X-ray scattering and circular dichroism studies indicate that the aggregate-enhancing N-terminal region of BR120-166 adopts an extended, non-globular structure. Altogether, our results suggest that PsrP adheres to extracellular DNA in the biofilm matrix and thus promotes pneumococcal biofilm formation.
The adsorption of two dextrin-based polymers, a regular wheat dextrin (TY) and a carboxymethyl-substituted (CM) dextrin, onto an anatase TiO2 particle film has been studied using in situ attenuated ...total reflection (ATR) FTIR spectroscopy. Infrared spectra of the polymer solutions and the polymer adsorbed at the anatase surface were acquired for two solution conditions: pH 3 and pH 9; below and above the isoelectric point (IEP) of anatase, respectively. Comparison of the polymer solution spectra and the adsorbed layer spectra highlighted a number of spectral differences that were attributed to involvement of the carboxyl group of CM Dextrin interacting with the anatase surface directly and the adsorption of oxidized dextrin chains in the case of regular dextrin (TY) at high pH. The adsorption/desorption kinetics were determined by monitoring spectral peaks of the pyranose ring of both polymers. Adsorption equilibrium was not established for Dextrin TY for many hours, whereas CM Dextrin reached equilibrium in its adsorption within 60 min. The extent of desorption of Dextrin TY (observed by flowing a background electrolyte dextrin-free solution) was extensive at both pH values, which reflects the poor affinity and binding of the polymer on anatase. In contrast, CM Dextrin underwent almost no desorption, indicating a high affinity between the carboxyl groups of the polymer and the anatase surface.
The adsorption of dextrin on talc, molybdenite, and graphite (three naturally hydrophobic minerals) has been compared. Adsorption isotherms and in situ tapping mode atomic force microscope (TMAFM) ...imaging have enabled polymer adsorbed amount and morphology of the adsorbed layer (area coverage and polymer domain size) to be determined and also the amount of hydration water in the structure of the adsorbed layer. The effect of the polymer on the mineral contact angles, measured by the captive bubble method on cleaved mineral surfaces, indicates clear correlations between the hydrophobicity reduction of the minerals, the adsorbed amount, and the surface coverage of the adsorbed polymer. Predictions of the flotation recovery of the treated mineral phases have been confirmed by performing batch flotation experiments. The influence of the polymer surface coverage on flotation recovery has highlighted the importance of this key parameter in the predictions of depressant efficiency. The roles of the initial hydrophobicity and the surface structure of the mineral basal plane in determining adsorption parameters and flotation response of the polymer-treated minerals are also discussed.
The advent of fungal pathogens that are resistant to the classic repertoire of antifungal drugs has increased the need for new therapeutic agents. A prominent example of such a novel compound is ...caspofungin, known to alter cell wall biogenesis by inhibiting β-1,3-D-glucan synthesis. Although much progress has been made in understanding the mechanism of action of caspofungin, little is known about its influence on the biophysical properties of the fungal cells. Here, we use atomic force microscopy (AFM) to demonstrate that caspofungin induces major remodelling of the cell surface properties of Candida albicans. Caspofungin causes major morphological and structural alterations of the cells, which correlate with a decrease of the cell wall mechanical strength. Moreover, we find that the drug induces the massive exposure of the cell adhesion protein Als1 on the cell surface and leads to increased cell surface hydrophobicity, two features that trigger cell aggregation. This behaviour is not observed in yeast species lacking Als1, demonstrating the key role that the protein plays in determining the aggregation phenotype of C. albicans. The results show that AFM opens up new avenues for understanding the molecular bases of microbe-drug interactions and for developing new therapeutic agents.
Many fungal pathogens produce cell surface polysaccharides that play essential roles in host-pathogen interactions. In Aspergillus fumigatus, the newly discovered polysaccharide galactosaminogalactan ...(GAG) mediates adherence to a variety of substrates through molecular mechanisms that are poorly understood. Here we use atomic force microscopy to unravel the localization and adhesion of GAG on living fungal cells. Using single-molecule imaging with tips bearing anti-GAG antibodies, we found that GAG is massively exposed on wild-type (WT) germ tubes, consistent with the notion that this glycopolymer is secreted by the mycelium of A. fumigatus, while it is lacking on WT resting conidia and on germ tubes from a mutant (Δuge3) deficient in GAG. Imaging germ tubes with tips bearing anti-β-glucan antibodies shows that exposure of β-glucan is strongly increased in the Δuge3 mutant, indicating that this polysaccharide is masked by GAG during hyphal growth. Single-cell force measurements show that expression of GAG on germ tubes promotes specific adhesion to pneumocytes and non-specific adhesion to hydrophobic substrates. These results provide a molecular foundation for the multifunctional adhesion properties of GAG, thus suggesting it could be used as a potential target in anti-adhesion therapy and immunotherapy. Our methodology represents a powerful approach for characterizing the nanoscale organization and adhesion of cell wall polysaccharides during fungal morphogenesis, thereby contributing to increase our understanding of their role in biofilm formation and immune responses.
The surface of many bacterial pathogens is covered with polysaccharides that play important roles in mediating pathogen-host interactions. In Streptococcus agalactiae, the capsular polysaccharide ...(CPS) is recognized as a major virulence factor while the group B carbohydrate (GBC) is crucial for peptidoglycan biosynthesis and cell division. Despite the important roles of CPS and GBC, there is little information available on the molecular organization of these glycopolymers on the cell surface. Here, we use atomic force microscopy (AFM) and transmission electron microscopy (TEM) to analyze the nanoscale distribution of CPS and GBC in wild-type (WT) and mutant strains of S. agalactiae. TEM analyses reveal that in WT bacteria, peptidoglycan is covered with a very thin (few nm) layer of GBC (the "pellicle") overlaid by a 15-45 nm thick layer of CPS (the "capsule"). AFM-based single-molecule mapping with specific antibody probes shows that CPS is exposed on WT cells, while it is hardly detected on mutant cells impaired in CPS production (ΔcpsE mutant). By contrast, both TEM and AFM show that CPS is over-expressed in mutant cells altered in GBC expression (ΔgbcO mutant), indicating that the production of the two surface glycopolymers is coordinated in WT cells. In addition, AFM topographic imaging and molecular mapping with specific lectin probes demonstrate that removal of CPS (ΔcpsE), but not of GBC (ΔgbcO), leads to the exposure of peptidoglycan, organized into 25 nm wide bands running parallel to the septum. These results indicate that CPS forms a homogeneous barrier protecting the underlying peptidoglycan from environmental exposure, while the presence of GBC does not prevent peptidoglycan detection. This work shows that single-molecule AFM, combined with high-resolution TEM, represents a powerful platform for analysing the molecular arrangement of the cell wall polymers of bacterial pathogens.
P1 (antigen I/II) is a sucrose-independent adhesin of Streptococcus mutans whose functional architecture on the cell surface is not fully understood. S. mutans cells subjected to mechanical ...extraction were significantly diminished in adherence to immobilized salivary agglutinin but remained immunoreactive and were readily aggregated by fluid-phase salivary agglutinin. Bacterial adherence was restored by incubation of postextracted cells with P1 fragments that contain each of the two known adhesive domains. In contrast to untreated cells, glutaraldehyde-treated bacteria gained reactivity with anti-C-terminal monoclonal antibodies (mAbs), whereas epitopes recognized by mAbs against other portions of the molecule were masked. Surface plasmon resonance experiments demonstrated the ability of apical and C-terminal fragments of P1 to interact. Binding of several different anti-P1 mAbs to unfixed cells triggered release of a C-terminal fragment from the bacterial surface, suggesting a novel mechanism of action of certain adherence-inhibiting antibodies. We also used atomic force microscopy-based single molecule force spectroscopy with tips bearing various mAbs to elucidate the spatial organization and orientation of P1 on living bacteria. The similar rupture lengths detected using mAbs against the head and C-terminal regions, which are widely separated in the tertiary structure, suggest a higher order architecture in which these domains are in close proximity on the cell surface. Taken together, our results suggest a supramolecular organization in which additional P1 polypeptides, including the C-terminal segment originally identified as antigen II, associate with covalently attached P1 to form the functional adhesive layer.
Background: P1 is an adhesin on the surface of Streptococcus mutans.
Results: Adhesive P1 on the surface of S. mutans exhibits a macromolecular ultrastructure.
Conclusion:The architecture of P1 on the surface of S. mutans plays a critical role in adherence.
Significance: Recognizing the macromolecular assembly of P1 on the surface of S. mutans is critical to understanding the adhesive function of the molecule.
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