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.

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.

Cell biology of microbes and pharmacology of antimicrobial drugs explored by Atomic Force Microscopy.

Antimicrobial molecules have been used for more than 50 years now and are the basis of modern medicine. No surgery can nowdays be imagined to be performed without antibiotics; dreadful diseases like tuberculosis, leprosis, siphilys, and more broadly all microbial induced diseases, can be cured only through the use of antimicrobial treatments. However, the situation is becoming more and more complex because of the ability of microbes to adapt, develop, acquire, and share mechanisms of resistance to antimicrobial agents. We choose to introduce this review by briefly drawing the panorama of antimicrobial discovery and development, but also of the emergence of microbial resistance. Then we describe how Atomic Force Microscopy (AFM) can be used to provide a better understanding of the mechanisms of action of these drugs at the nanoscale level on microbial interfaces. In this section, we will address these questions: (1) how does drug treatment affect the morphology of single microbes?; (2) do antimicrobial molecules modify the nanomechanical properties of microbes, or do the nanomechanical properties of microbes play a role in antimicrobial activity and efficiency?; and (3) how are the adhesive abilitites of microbes affected by antimicrobial drugs treatment? Finally, in a second part of this review we focus on recent studies aimed at changing the paradigm of the single molecule/cell technology that AFM typically represents. Recent work dealing with the creation of a microbe array which can be explored by AFM will be presented, as these developments constitute the first steps toward transforming AFM into a higher throughput technology. We also discuss papers using AFM as NanoMechnanicalSensors (NEMS), and demonstrate the interest of such approaches in clinical microbiology to detect quickly and with high accuracy microbial resistance.

Biophysical properties of cardiomyocyte surface explored by multiparametric AFM.

PeakForce Quantitative Nanomechanical Mapping (PeakForce QNM) multiparametric AFM mode was adapted to qualitative and quantitative study of the lateral membrane of cardiomyocytes (CMs), extending this powerful mode to the study of soft cells. On living CM, PeakForce QNM depicted the crests and hollows periodic alternation of cell surface architecture previously described using AFM Force Volume (FV) mode. PeakForce QNM analysis provided better resolution in terms of pixel number compared to FV mode and reduced acquisition time, thus limiting the consequences of spontaneous living adult CM dedifferentiation once isolated from the cardiac tissue. PeakForce QNM mode on fixed CMs clearly visualized subsarcolemmal mitochondria (SSM) and their loss following formamide treatment, concomitant with the interfibrillar mitochondria climbing up and forming heaps at the cell surface. Interestingly, formamide-promoted SSM loss allowed visualization of the sarcomeric apparatus ultrastructure below the plasma membrane. High PeakForce QNM resolution led to better contrasted mechanical maps than FV mode and provided correlation between adhesion, dissipation, mechanical and topographical maps. Modified hydrophobic AFM tip enhanced contrast on adhesion and dissipation maps and suggested that CM surface crests and hollows exhibit distinct chemical properties. Finally, two-dimensional Fast Fourier Transform to objectively quantify AFM maps allowed characterization of periodicity of both sarcomeric Z-line and M-band. Overall, this study validated PeakForce QNM as a valuable and innovative mode for the exploration of living and fixed CMs. In the future, it could be applied to depict cell membrane architectural, mechanical and chemical defects as well as sarcomeric abnormalities associated with cardiac diseases.

Targeted changes of the cell wall proteome influence Candida albicans ability to form single- and multi-strain biofilms.

Biofilm formation is an important virulence trait of the pathogenic yeast Candida albicans. We have combined gene overexpression, strain barcoding and microarray profiling to screen a library of 531 C. albicans conditional overexpression strains (∼10% of the genome) for genes affecting biofilm development in mixed-population experiments. The overexpression of 16 genes increased strain occupancy within a multi-strain biofilm, whereas overexpression of 4 genes decreased it. The set of 16 genes was significantly enriched for those encoding predicted glycosylphosphatidylinositol (GPI)-modified proteins, namely Ihd1/Pga36, Phr2, Pga15, Pga19, Pga22, Pga32, Pga37, Pga42 and Pga59; eight of which have been classified as pathogen-specific. Validation experiments using either individually- or competitively-grown overexpression strains revealed that the contribution of these genes to biofilm formation was variable and stage-specific. Deeper functional analysis of PGA59 and PGA22 at a single-cell resolution using atomic force microscopy showed that overexpression of either gene increased C. albicans ability to adhere to an abiotic substrate. However, unlike PGA59, PGA22 overexpression led to cell cluster formation that resulted in increased sensitivity to shear forces and decreased ability to form a single-strain biofilm. Within the multi-strain environment provided by the PGA22-non overexpressing cells, PGA22-overexpressing cells were protected from shear forces and fitter for biofilm development. Ultrastructural analysis, genome-wide transcript profiling and phenotypic analyses in a heterologous context suggested that PGA22 affects cell adherence through alteration of cell wall structure and/or function. Taken together, our findings reveal that several novel predicted GPI-modified proteins contribute to the cooperative behaviour between biofilm cells and are important participants during C. albicans biofilm formation. Moreover, they illustrate the power of using signature tagging in conjunction with gene overexpression for the identification of novel genes involved in processes pertaining to C. albicans virulence.

Evidence for a Role for the Plasma Membrane in the Nanomechanical Properties of the Cell Wall as Revealed by an Atomic Force Microscopy Study of the Response of Saccharomyces cerevisiae to Ethanol Stress.

UNLABELLED: A wealth of biochemical and molecular data have been reported regarding ethanol toxicity in the yeast Saccharomyces cerevisiae However, direct physical data on the effects of ethanol stress on yeast cells are almost nonexistent. This lack of information can now be addressed by using atomic force microscopy (AFM) technology. In this report, we show that the stiffness of glucose-grown yeast cells challenged with 9% (vol/vol) ethanol for 5 h was dramatically reduced, as shown by a 5-fold drop of Young's modulus. Quite unexpectedly, a mutant deficient in the Msn2/Msn4 transcription factor, which is known to mediate the ethanol stress response, exhibited a low level of stiffness similar to that of ethanol-treated wild-type cells. Reciprocally, the stiffness of yeast cells overexpressing MSN2 was about 35% higher than that of the wild type but was nevertheless reduced 3- to 4-fold upon exposure to ethanol. Based on these and other data presented herein, we postulated that the effect of ethanol on cell stiffness may not be mediated through Msn2/Msn4, even though this transcription factor appears to be a determinant in the nanomechanical properties of the cell wall. On the other hand, we found that as with ethanol, the treatment of yeast with the antifungal amphotericin B caused a significant reduction of cell wall stiffness. Since both this drug and ethanol are known to alter, albeit by different means, the fluidity and structure of the plasma membrane, these data led to the proposition that the cell membrane contributes to the biophysical properties of yeast cells. IMPORTANCE: Ethanol is the main product of yeast fermentation but is also a toxic compound for this process. Understanding the mechanism of this toxicity is of great importance for industrial applications. While most research has focused on genomic studies of ethanol tolerance, we investigated the effects of ethanol at the biophysical level and found that ethanol causes a strong reduction of the cell wall rigidity (or stiffness). We ascribed this effect to the action of ethanol perturbing the cell membrane integrity and hence proposed that the cell membrane contributes to the cell wall nanomechanical properties.

Atomic Force Microscopy and pharmacology: from microbiology to cancerology.

BACKGROUND: Atomic Force Microscopy (AFM) has been extensively used to study biological samples. Researchers take advantage of its ability to image living samples to increase our fundamental knowledge (biophysical properties/biochemical behavior) on living cell surface properties, at the nano-scale. SCOPE OF REVIEW: AFM, in the imaging modes, can probe cells morphological modifications induced by drugs. In the force spectroscopy mode, it is possible to follow the nanomechanical properties of a cell and to probe the mechanical modifications induced by drugs. AFM can be used to map single molecule distribution at the cell surface. We will focus on a collection of results aiming at evaluating the nano-scale effects of drugs, by AFM. Studies on yeast, bacteria and mammal cells will illustrate our discussion. Especially, we will show how AFM can help in getting a better understanding of drug mechanism of action. MAJOR CONCLUSIONS: This review demonstrates that AFM is a versatile tool, useful in pharmacology. In microbiology, it has been used to study the drugs fighting Candida albicans or Pseudomonas aeruginosa. The major conclusions are a better understanding of the microbes' cell wall and of the drugs mechanism of action. In cancerology, AFM has been used to explore the effects of cytotoxic drugs or as an innovative diagnostic technology. AFM has provided original results on cultured cells, cells extracted from patient and directly on patient biopsies. GENERAL SIGNIFICANCE: This review enhances the interest of AFM technologies for pharmacology. The applications reviewed range from microbiology to cancerology.

Deletion of the α-(1,3)-glucan synthase genes induces a restructuring of the conidial cell wall responsible for the avirulence of Aspergillus fumigatus.

α-(1,3)-Glucan is a major component of the cell wall of Aspergillus fumigatus, an opportunistic human fungal pathogen. There are three genes (AGS1, AGS2 and AGS3) controlling the biosynthesis of α-(1,3)-glucan in this fungal species. Deletion of all the three AGS genes resulted in a triple mutant that was devoid of α-(1,3)-glucan in its cell wall; however, its growth and germination was identical to that of the parental strain in vitro. In the experimental murine aspergillosis model, this mutant was less pathogenic than the parental strain. The AGS deletion resulted in an extensive structural modification of the conidial cell wall, especially conidial surface where the rodlet layer was covered by an amorphous glycoprotein matrix. This surface modification was responsible for viability reduction of conidia in vivo, which explains decrease in the virulence of triple agsΔ mutant.

Effects of the strain background and autolysis process on the composition and biophysical properties of the cell wall from two different industrial yeasts.

The Saccharomyces cerevisiae cell surface is endowed with some relevant technological properties, notably antimicrobial and biosorption activities. For these purposes, yeasts are usually processed and packaged in an 'autolysed/dried' formula, which may have some impacts on cell surface properties. In this report, we showed using a combination of biochemical, biophysical and molecular methods that the composition of the cell wall of two wine yeast strains was not altered by the autolysis process. In contrast, this process altered the nanomechanical properties as shown by a 2- to 4-fold increased surface roughness and to a higher adhesion to the atomic force microscope tips of the autolysed cells as compared to live yeast cells. Besides, we found that the two strains harboured differences in biomechanical properties that could be due in part to higher levels of mannan in one of them, and to the fact that the surface of this mannan-enriched strain is decorated with highly adhesive patches forming nanodomains. The presence of these nanodomains could be correlated with the upregulation of flocculin encoding FLO11 as well as to higher expression of few other genes encoding cell wall mannoproteins in this mannan-enriched strain as compared to the other strain.

Multiparametric imaging of adhesive nanodomains at the surface of Candida albicans by atomic force microscopy.

Candida albicans is an opportunistic pathogen. It adheres to mammalian cells through a variety of adhesins that interact with host ligands. The spatial organization of these adhesins on the cellular interface is however poorly understood, mainly because of the lack of an instrument able to track single molecules on single cells. In this context, the atomic force microscope (AFM) makes it possible to analyze the force signature of single proteins on single cells. The present study is dedicated to the mapping of the adhesive properties of C. albicans cells. We observed that the adhesins at the cell surface were organized in nanodomains composed of free or aggregated mannoproteins. This was demonstrated by the use of functionalized AFM tips and synthetic amyloid forming/disrupting peptides. This direct visualization of amyloids nanodomains will help in understanding the virulence factors of C. albicans.

Uncovering by atomic force microscopy of an original circular structure at the yeast cell surface in response to heat shock.

BACKGROUND: Atomic Force Microscopy (AFM) is a polyvalent tool that allows biological and mechanical studies of full living microorganisms, and therefore the comprehension of molecular mechanisms at the nanoscale level. By combining AFM with genetical and biochemical methods, we explored the biophysical response of the yeast Saccharomyces cerevisiae to a temperature stress from 30°C to 42°C during 1 h. RESULTS: We report for the first time the formation of an unprecedented circular structure at the cell surface that takes its origin at a single punctuate source and propagates in a concentric manner to reach a diameter of 2-3 µm at least, thus significantly greater than a bud scar. Concomitantly, the cell wall stiffness determined by the Young's Modulus of heat stressed cells increased two fold with a concurrent increase of chitin. This heat-induced circular structure was not found either in wsc1Δ or bck1Δ mutants that are defective in the CWI signaling pathway, nor in chs1Δ, chs3Δ and bni1Δ mutant cells, reported to be deficient in the proper budding process. It was also abolished in the presence of latrunculin A, a toxin known to destabilize actin cytoskeleton. CONCLUSIONS: Our results suggest that this singular morphological event occurring at the cell surface is due to a dysfunction in the budding machinery caused by the heat shock and that this phenomenon is under the control of the CWI pathway.

Atomic force and electron microscopic-based study of sarcolemmal surface of living cardiomyocytes unveils unexpected mitochondrial shift in heart failure.

Loss of T-tubules (TT), sarcolemmal invaginations of cardiomyocytes (CMs), was recently identified as a general heart failure (HF) hallmark. However, whether TT per se or the overall sarcolemma is altered during HF process is still unknown. In this study, we directly examined sarcolemmal surface topography and physical properties using Atomic Force Microscopy (AFM) in living CMs from healthy and failing mice hearts. We confirmed the presence of highly organized crests and hollows along myofilaments in isolated healthy CMs. Sarcolemma topography was tightly correlated with elasticity, with crests stiffer than hollows and related to the presence of few packed subsarcolemmal mitochondria (SSM) as evidenced by electron microscopy. Three days after myocardial infarction (MI), CMs already exhibit an overall sarcolemma disorganization with general loss of crests topography thus becoming smooth and correlating with a decreased elasticity while interfibrillar mitochondria (IFM), myofilaments alignment and TT network were unaltered. End-stage post-ischemic condition (15days post-MI) exacerbates overall sarcolemma disorganization with, in addition to general loss of crest/hollow periodicity, a significant increase of cell surface stiffness. Strikingly, electron microscopy revealed the total depletion of SSM while some IFM heaps could be visualized beneath the membrane. Accordingly, mitochondrial Ca(2+) studies showed a heterogeneous pattern between SSM and IFM in healthy CMs which disappeared in HF. In vitro, formamide-induced sarcolemmal stress on healthy CMs phenocopied post-ischemic kinetics abnormalities and revealed initial SSM death and crest/hollow disorganization followed by IFM later disarray which moved toward the cell surface and structured heaps correlating with TT loss. This study demonstrates that the loss of crest/hollow organization of CM surface in HF occurs early and precedes disruption of the TT network. It also highlights a general stiffness increased of the CM surface most likely related to atypical IFM heaps while SSM died during HF process. Overall, these results indicate that initial sarcolemmal stress leading to SSM death could underlie subsequent TT disarray and HF setting.

Destabilization induced by electropermeabilization analyzed by atomic force microscopy.

Electropermeabilization is a physical method that uses electric field pulses to deliver molecules into cells and tissues. Despite its increasing interest in clinics, little is known about plasma membrane destabilization process occurring during electropermeabilization. In this work, we took advantage of atomic force microscopy to directly visualize the consequences of electropermeabilization in terms of membrane reorganization and to locally measure the membrane elasticity. We visualized transient rippling of membrane surface and measured a decrease in membrane elasticity by 40%. Our results obtained both on fixed and living CHO cells give evidence of an inner effect affecting the entire cell surface that may be related to cytoskeleton destabilization. Thus, AFM appears as a useful tool to investigate basic process of electroporation on living cells in absence of any staining or cell preparation.

Stress disrupts intestinal mucus barrier in rats via mucin O-glycosylation shift: prevention by a probiotic treatment.

Despite well-known intestinal epithelial barrier impairment and visceral hypersensitivity in irritable bowel syndrome (IBS) patients and IBS-like models, structural and physical changes in the mucus layer remain poorly understood. Using a water avoidance stress (WAS) model, we aimed at evaluating whether 1) WAS modified gut permeability, visceral sensitivity, mucin expression, biochemical structure of O-glycans, and related mucus physical properties, and 2) whether Lactobacillus farciminis treatment prevented these alterations. Wistar rats received orally L. farciminis or vehicle for 14 days; at day 10, they were submitted to either sham or 4-day WAS. Intestinal paracellular permeability and visceral sensitivity were measured in vivo. The number of goblet cells and Muc2 expression were evaluated by histology and immunohistochemistry, respectively. Mucosal adhesion of L. farciminis was determined ex situ. The mucin O-glycosylation profile was obtained by mass spectrometry. Surface imaging of intestinal mucus was performed at nanoscale by atomic force microscopy. WAS induced gut hyperpermeability and visceral hypersensitivity but did not modify either the number of intestinal goblet cells or Muc2 expression. In contrast, O-glycosylation of mucins was strongly affected, with the appearance of elongated polylactosaminic chain containing O-glycan structures, associated with flattening and loss of the mucus layer cohesive properties. L. farciminis bound to intestinal Muc2 and prevented WAS-induced functional alterations and changes in mucin O-glycosylation and mucus physical properties. WAS-induced functional changes were associated with mucus alterations resulting from a shift in O-glycosylation rather than from changes in mucin expression. L. farciminis treatment prevented these alterations, conferring epithelial and mucus barrier strengthening.

A combined chemical and enzymatic method to determine quantitatively the polysaccharide components in the cell wall of yeasts.

A reliable method to determine cell wall polysaccharides composition in yeast is presented, which combines acid and enzymatic hydrolysis. Sulphuric acid treatment is used to determine mannans, whereas specific hydrolytic enzymes are employed in a two sequential steps to quantify chitin and the proportion of ß-(1,3) and ß-(1,6)-glucan in the total ß-glucan of the cell wall. In the first step, chitin and ß-(1,3)-glucan were hydrolysed into their corresponding monomers N-acetylglucosamine and glucose, respectively, by the combined action of a chitinase from Streptomyces griseus and a pure preparation of endo/exo-ß-(1,3)-glucanase from Trichoderma species. This step was followed by addition of recombinant endo-ß-(1,6)-glucanase from Trichoderma harzianum with ß-glucosidase from Aspergillus niger to hydrolyse the remaining ß-glucan. This latter component corresponded to a highly branched ß-(1,6)-glucan that contained about 75-80% of linear ß-(1,6)-glucose linked units as deduced from periodate oxidation. We validated this novel method by showing that the content of ß-(1,3), ß-(1,6)-glucan or chitin was dramatically decreased in yeast mutants defective in the biosynthesis of these cell wall components. Moreover, we found that heat shock at 42 °C in Saccharomyces cerevisiae and treatment of this yeast species and Candida albicans with the antifungal drug caspofungin resulted in 2- to 3-fold increase of chitin and in a reduction of ß-(1,3)-glucan accompanied by an increase of ß-(1,6)-glucan, whereas ethanol stress had apparently no effect on yeast cell wall composition.

Ephrin-B1 is a novel specific component of the lateral membrane of the cardiomyocyte and is essential for the stability of cardiac tissue architecture cohesion.

RATIONALE: Cardiac tissue cohesion relying on highly ordered cardiomyocytes (CM) interactions is critical because most cardiomyopathies are associated with tissue remodeling and architecture alterations. OBJECTIVE: Eph/ephrin system constitutes a ubiquitous system coordinating cellular communications which recently emerged as a major regulator in adult organs. We examined if eph/ephrin could participate in cardiac tissue cyto-organization. METHODS AND RESULTS: We reported the expression of cardiac ephrin-B1 in both endothelial cells and for the first time in CMs where ephrin-B1 localized specifically at the lateral membrane. Ephrin-B1 knock-out (KO) mice progressively developed cardiac tissue disorganization with loss of adult CM rod-shape and sarcomeric and intercalated disk structural disorganization confirmed in CM-specific ephrin-B1 KO mice. CMs lateral membrane exhibited abnormal structure by electron microscopy and notably increased stiffness by atomic force microscopy. In wild-type CMs, ephrin-B1 interacted with claudin-5/ZO-1 complex at the lateral membrane, whereas the complex disappeared in KO/CM-specific ephrin-B1 KO mice. Ephrin-B1 deficiency resulted in decreased mRNA expression of CM basement membrane components and disorganized fibrillar collagen matrix, independently of classical integrin/dystroglycan system. KO/CM-specific ephrin-B1 KO mice exhibited increased left ventricle diameter and delayed atrioventricular conduction. Under pressure overload stress, KO mice were prone to death and exhibited striking tissue disorganization. Finally, failing CMs displayed downregulated ephrin-B1/claudin-5 gene expression linearly related to the ejection fraction. CONCLUSIONS: Ephrin-B1 is necessary for cardiac tissue architecture cohesion by stabilizing the adult CM morphology through regulation of its lateral membrane. Because decreased ephrin-B1 is associated with molecular/functional cardiac defects, it could represent a new actor in the transition toward heart failure.

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.

Use of atomic force microscopy (AFM) to explore cell wall properties and response to stress in the yeast Saccharomyces cerevisiae.

Over the past 20 years, the yeast cell wall has been thoroughly investigated by genetic and biochemical methods, leading to remarkable advances in the understanding of its biogenesis and molecular architecture as well as to the mechanisms by which this organelle is remodeled in response to environmental stresses. Being a dynamic structure that constitutes the frontier between the cell interior and its immediate surroundings, imaging cell surface, measuring mechanical properties of cell wall or probing cell surface proteins for localization or interaction with external biomolecules are among the most burning questions that biologists wished to address in order to better understand the structure-function relationships of yeast cell wall in adhesion, flocculation, aggregation, biofilm formation, interaction with antifungal drugs or toxins, as well as response to environmental stresses, such as temperature changes, osmotic pressure, shearing stress, etc. The atomic force microscopy (AFM) is nowadays the most qualified and developed technique that offers the possibilities to address these questions since it allows working directly on living cells to explore and manipulate cell surface properties at nanometer resolution and to analyze cell wall proteins at the single molecule level. In this minireview, we will summarize the most recent contributions made by AFM in the analysis of the biomechanical and biochemical properties of the yeast cell wall and illustrate the power of this tool to unravel unexpected effects caused by environmental stresses and antifungal agents on the surface of living yeast cells.

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!

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.