Mapping HA-tagged protein at the surface of living cells by atomic force microscopy.

Single-molecule force spectroscopy using atomic force microscopy (AFM) is more and more used to detect and map receptors, enzymes, adhesins, or any other molecules at the surface of living cells. To be specific, this technique requires antibodies or ligands covalently attached to the AFM tip that can specifically interact with the protein of interest. Unfortunately, specific antibodies are usually lacking (low affinity and specificity) or are expensive to produce (monoclonal antibodies). An alternative strategy is to tag the protein of interest with a peptide that can be recognized with high specificity and affinity with commercially available antibodies. In this context, we chose to work with the human influenza hemagglutinin (HA) tag (YPYDVPDYA) and labeled two proteins: covalently linked cell wall protein 12 (Ccw12) involved in cell wall remodeling in the yeast Saccharomyces cerevisiae and the ß2-adrenergic receptor (ß2-AR), a G protein-coupled receptor (GPCR) in higher eukaryotes. We first described the interaction between HA antibodies, immobilized on AFM tips, and HA epitopes, immobilized on epoxy glass slides. Using our system, we then investigated the distribution of Ccw12 proteins over the cell surface of the yeast S. cerevisiae. We were able to find the tagged protein on the surface of mating yeasts, at the tip of the mating projections. Finally, we could unfold multimers of ß2-AR from the membrane of living transfected chinese hamster ovary cells. This result is in agreement with GPCR oligomerization in living cell membranes and opens the door to the study of the influence of GPCR ligands on the oligomerization process.

Unravelling of a mechanism of resistance to colistin in Klebsiella pneumoniae using atomic force microscopy.

OBJECTIVES: In this study we focused on the mechanism of colistin resistance in Klebsiella pneumoniae. METHODS: We used two strains of K. pneumoniae: a colistin-susceptible strain (K. pneumoniae ATCC 700603, KpATCC) and its colistin-resistant derivative (KpATCCm, MIC of colistin 16 mg/L). We performed a genotypic analysis based on the expression of genes involved in LPS synthesis and L-Ara4N moiety addition. We also explored the status of the mgrB gene. Then, a phenotypic analysis was performed using atomic force microscopy (AFM). The Young modulus was extracted from force curves fitted using the Hertz model, and stiffness values were extracted from force curves fitted using the Hooke model. RESULTS: We failed to observe any variation in the expression of genes implicated in LPS synthesis or L-Ara4N moiety addition in KpATCCm, in the absence of colistin or under colistin pressure (versus KpATCC). This led us to identify an insertional inactivation/mutation in the mgrB gene of KpATCCm. In addition, morphology results obtained by AFM showed that colistin removed the capsule from the susceptible strain, but not from the resistant strain. Nanomechanical data on the resistant strain showed that colistin increased the Young modulus of the capsule. Extend force curves recorded on top of the cells allowed us to make the following hypothesis about the nanoarchitecture of the capsule of the two strains: KpATCC has a soft capsule consisting of one layer, whereas the KpATCCm capsule is harder and organized in several layers. CONCLUSIONS: We hypothesize that capsular polysaccharides might be implicated in the mechanism of colistin resistance in K. pneumoniae, depending on its genotype.

Surface-anchored carbon nanomaterials for antimicrobial surfaces.

Carbon nanomaterials (CNMs) are known for their antimicrobial (antibacterial and antiviral) activity when dispersed in a liquid, but whether this can be transferred to the surface of common materials has rarely been investigated. We have compared two typical CNMs (double-walled carbon nanotubes and few-layer graphene) in their non-oxidised and oxidised forms in terms of their antibacterial (Pseudomonas aeruginosa and Staphylococcus aureus) and antiviral (SARS-CoV2) activities after anchoring them onto the surface of silicone. We propose a very simple and effective protocol using the air-brush spray deposition method to entrap CNMs on the surfaces of two different silicone materials and demonstrate that the nanomaterials are anchored within the polymer while still being in contact with bacteria. We also investigated their antiviral activity against SARS-COV2 after deposition on standard surgical respiratory masks. Our results show that while suspensions of double-walled carbon nanotubes had a moderate effect on P. aeruginosa, this was not transferred after anchoring them to the surface of silicone. In contrast, graphene oxide showed a very strong antibacterial effect on P. aeruginosa and oxidised double-walled carbon nanotubes on S. aureus only when anchored to the surface. No significant antiviral activity was observed. This work paves the way for new antibacterial surfaces based on CNMs.

Nanoscale effects of caspofungin against two yeast species, Saccharomyces cerevisiae and Candida albicans.

Saccharomyces cerevisiae and Candida albicans are model yeasts for biotechnology and human health, respectively. We used atomic force microscopy (AFM) to explore the effects of caspofungin, an antifungal drug used in hospitals, on these two species. Our nanoscale investigation revealed similar, but also different, behaviors of the two yeasts in response to treatment with the drug. While administration of caspofungin induced deep cell wall remodeling in both yeast species, as evidenced by a dramatic increase in chitin and decrease in ß-glucan content, changes in cell wall composition were more pronounced with C. albicans cells. Notably, the increase of chitin was proportional to the increase in the caspofungin dose. In addition, the Young modulus of the cell was three times lower for C. albicans cells than for S. cerevisiae cells and increased proportionally with the increase of chitin, suggesting differences in the molecular organization of the cell wall between the two yeast species. Also, at a low dose of caspofungin (i.e., 0.5× MIC), the cell surface of C. albicans exhibited a morphology that was reminiscent of cells expressing adhesion proteins. Interestingly, this morphology was lost at high doses of the drug (i.e., 4× MIC). However, the treatment of S. cerevisiae cells with high doses of caspofungin resulted in impairment of cytokinesis. Altogether, the use of AFM for investigating the effects of antifungal drugs is relevant in nanomedicine, as it should help in understanding their mechanisms of action on fungal cells, as well as unraveling unexpected effects on cell division and fungal adhesion.

Assembly of live micro-organisms on microstructured PDMS stamps by convective/capillary deposition for AFM bio-experiments.

Immobilization of live micro-organisms on solid substrates is an important prerequisite for atomic force microscopy (AFM) bio-experiments. The method employed must immobilize the cells firmly enough to enable them to withstand the lateral friction forces exerted by the tip during scanning but without denaturing the cell interface. In this work, a generic method for the assembly of living cells on specific areas of substrates is proposed. It consists in assembling the living cells within the patterns of microstructured, functionalized poly-dimethylsiloxane (PDMS) stamps using convective/capillary deposition. This versatile approach is validated by applying it to two systems of foremost importance in biotechnology and medicine: Saccharomyces cerevisiae yeasts and Aspergillus fumigatus fungal spores. We show that this method allows multiplexing AFM nanomechanical measurements by force spectroscopy on S. cerevisiae yeasts and high-resolution AFM imaging of germinated Aspergillus conidia in buffer medium. These two examples clearly demonstrate the immense potential of micro-organism assembly on functionalized, microstructured PDMS stamps by convective/capillary deposition for performing rigorous AFM bio-experiments on living cells.

Nanoscale structural mapping as a measure of maturation in the murine frontal cortex.

Atomic force microscopy (AFM) is emerging as an innovative tool to phenotype the brain. This study demonstrates the utility of AFM to determine nanomechanical and nanostructural features of the murine dorsolateral frontal cortex from weaning to adulthood. We found an increase in tissue stiffness of the primary somatosensory cortex with age, along with an increased cortical mechanical heterogeneity. To characterize the features potentially responsible for this heterogeneity, we applied AFM scan mode to directly image the topography of thin sections of the primary somatosensory cortical layers II/III, IV and V/VI. Topographical mapping of the cortical layers at successive ages showed progressive smoothing of the surface. Topographical images were also compared with histochemically derived morphological information, which demonstrated the deposition of perineuronal nets, important extracellular components and markers of maturity. Our work demonstrates that high-resolution AFM images can be used to determine the nanostructural properties of cortical maturation, well beyond embryonic and postnatal development. Furthermore, it may offer a new method for brain phenotyping and screening to uncover topographical changes in early stages of neurodegenerative diseases.

High speed indentation measures by FV, QI and QNM introduce a new understanding of bionanomechanical experiments.

Structural and mechanical mapping at the nanoscale by novel high-speed multiparametric Quantitative Imaging (QI) and PeakForce Quantitative Nanomechanical Mapping (PF-QNM) AFM modes was compared to the classical Force Volume (FV) mapping for the case of living Pseudomonas aeruginosa bacterial cells. QI and PF-QNM modes give results consistent with FV for the whole cells in terms of morphology and elastic modulus, while providing higher resolution and shorter acquisition time. As an important complement, the influence of scanning parameters on elastic modulus values was explored for small 0.2(2)µm(2) central area on top of cells. The modulus decreases with the indentation depth due to the effect of the hard cell wall, while it increases vs. tip oscillation frequency, displaying viscoelastic behaviour of the living bacterial cells. The ability of different AFM modes to follow correctly the bacteria viscoelastic behaviour at high oscillation frequency was tested.

Imaging living cells surface and quantifying its properties at high resolution using AFM in QI™ mode.

Since the last 10 years, AFM has become a powerful tool to study biological samples. However, the classical modes offered (imaging or tapping mode) often damage sample that are too soft or loosely immobilized. If imaging and mechanical properties are required, it requests long recording time as two different experiments must be conducted independently. In this study we compare the new QI™ mode against contact imaging mode and force volume mode, and we point out its benefit in the new challenges in biology on six different models: Escherichia coli, Candida albicans, Aspergillus fumigatus, Chinese hamster ovary cells and their isolated nuclei, and human colorectal tumor cells.

Nanoscale analysis of the effects of antibiotics and CX1 on a Pseudomonas aeruginosa multidrug-resistant strain.

Drug resistance is a challenge that can be addressed using nanotechnology. We focused on the resistance of the bacteria Pseudomonas aeruginosa and investigated, using Atomic Force Microscopy (AFM), the behavior of a reference strain and of a multidrug resistant clinical strain, submitted to two antibiotics and to an innovative antibacterial drug (CX1). We measured the morphology, surface roughness and elasticity of the bacteria under physiological conditions and exposed to the antibacterial molecules. To go further in the molecules action mechanism, we explored the bacterial cell wall nanoscale organization using functionalized AFM tips. We have demonstrated that affected cells have a molecularly disorganized cell wall; surprisingly long molecules being pulled off from the cell wall by a lectin probe. Finally, we have elucidated the mechanism of action of CX1: it destroys the outer membrane of the bacteria as demonstrated by the results on artificial phospholipidic membranes and on the resistant strain.

Combining convective/capillary deposition and AFM oxidation lithography for close-packed directed assembly of colloids.

We combine convective/capillary deposition and oxidation lithography by atomic force microscopy to direct the close-packed assembly of colloids on SiOx patterns fabricated on silicon substrates previously functionalized with a hydrophobic monolayer of octadecyltrimethoxysilane. The efficiency of this original generic method, which is well adapted to integrate colloids into silicon devices, is demonstrated for 100 nm colloidal latex nanoparticles and Escherichia coli bacteria in aqueous suspensions. A three-step mechanism involving convective flow and capillary forces appears to be responsible for these close-packed assemblies of colloids onto SiOx patterns.

[Microelectrophoresis and atomic force microscopy: new tools to explore mechanism of action of antibacterial compounds]. / Microélectrophorèse et microscopie à force atomique: deux nouveaux outils d'évaluation de l'effet pariétal d'antibactériens.

Microbial cell surface properties play a central role in controlling phenomena such as bacterial adhesion and biofilm formation (on stent or on prosthesis for example). The quantification of these properties and the understanding of interactions with antibacterial compounds remain difficult, in view of the complex and dynamic nature of the cell wall constituents. Various approaches, macroscopic, microscopic or molecular, have been developed. Two of them interest us today: (i) microelectrophoresis, which permits to evaluate surface modifications by measuring eletrophoretic mobility; and (ii) atomic force microscopy (AFM), a high resolution imaging device, which allows investigations at nanometric scale. After brief presentation of principles and instrumentations, the aim of this article is to present the different applications of these techniques in Microbiology, and to discuss interest of these tools in order to investigate mechanism of action of antibacterial compounds.