Marion Schiavone

Cell Wall Remodeling Enzymes Modulate Fungal Cell Wall Elasticity and Osmotic Stress Resistance

ABSTRACT The fungal cell wall confers cell morphology and protection against environmental insults. For fungal pathogens, the cell wall is a key immunological modulator and an ideal therapeutic target. Yeast cell walls possess an inner matrix of interlinked β-glucan and chitin that is thought to provide tensile strength and rigidity. Yeast cells remodel their walls over time in response to environmental change, a process controlled by evolutionarily conserved stress (Hog1) and cell integrity (Mkc1, Cek1) signaling pathways. These mitogen-activated protein kinase (MAPK) pathways modulate cell wall gene expression, leading to the construction of a new, modified cell wall. We show that the cell wall is not rigid but elastic, displaying rapid structural realignments that impact survival following osmotic shock. Lactate-grown Candida albicans cells are more resistant to hyperosmotic shock than glucose-grown cells. We show that this elevated resistance is not dependent on Hog1 or Mkc1 signaling and that most cell death occurs within 10 min of osmotic shock. Sudden decreases in cell volume drive rapid increases in cell wall thickness. The elevated stress resistance of lactate-grown cells correlates with reduced cell wall elasticity, reflected in slower changes in cell volume following hyperosmotic shock. The cell wall elasticity of lactate-grown cells is increased by a triple mutation that inactivates the Crh family of cell wall cross-linking enzymes, leading to increased sensitivity to hyperosmotic shock. Overexpressing Crh family members in glucose-grown cells reduces cell wall elasticity, providing partial protection against hyperosmotic shock. These changes correlate with structural realignment of the cell wall and with the ability of cells to withstand osmotic shock. IMPORTANCE The C. albicans cell wall is the first line of defense against external insults, the site of immune recognition by the host, and an attractive target for antifungal therapy. Its tensile strength is conferred by a network of cell wall polysaccharides, which are remodeled in response to growth conditions and environmental stress. However, little is known about how cell wall elasticity is regulated and how it affects adaptation to stresses such as sudden changes in osmolarity. We show that elasticity is critical for survival under conditions of osmotic shock, before stress signaling pathways have time to induce gene expression and drive glycerol accumulation. Critical cell wall remodeling enzymes control cell wall flexibility, and its regulation is strongly dependent on host nutritional inputs. We also demonstrate an entirely new level of cell wall dynamism, where significant architectural changes and structural realignment occur within seconds of an osmotic shock. The C. albicans cell wall is the first line of defense against external insults, the site of immune recognition by the host, and an attractive target for antifungal therapy. Its tensile strength is conferred by a network of cell wall polysaccharides, which are remodeled in response to growth conditions and environmental stress. However, little is known about how cell wall elasticity is regulated and how it affects adaptation to stresses such as sudden changes in osmolarity. We show that elasticity is critical for survival under conditions of osmotic shock, before stress signaling pathways have time to induce gene expression and drive glycerol accumulation. Critical cell wall remodeling enzymes control cell wall flexibility, and its regulation is strongly dependent on host nutritional inputs. We also demonstrate an entirely new level of cell wall dynamism, where significant architectural changes and structural realignment occur within seconds of an osmotic shock.

Nanoscale effects of Caspofungin against two yeast species; Saccharomyces cerevisiae and Candida albicans

ABSTRACT 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 β-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.

Generation of living cell arrays for atomic force microscopy studies.

Atomic force microscopy (AFM) is a useful tool for studying the morphology or the nanomechanical and adhesive properties of live microorganisms under physiological conditions. However, to perform AFM imaging, living cells must be immobilized firmly enough to withstand the lateral forces exerted by the scanning tip, but without denaturing them. This protocol describes how to immobilize living cells, ranging from spores of bacteria to yeast cells, into polydimethylsiloxane (PDMS) stamps, with no chemical or physical denaturation. This protocol generates arrays of living cells, allowing statistically relevant measurements to be obtained from AFM measurements, which can increase the relevance of results. The first step of the protocol is to generate a microstructured silicon master, from which many microstructured PDMS stamps can be replicated. Living cells are finally assembled into the microstructures of these PDMS stamps using a convective and capillary assembly. The complete procedure can be performed in 1 week, although the first step is done only once, and thus repeats can be completed within 1 d.

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.

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

ABSTRACT 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 Youngs 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.

Integration of Biochemical, Biophysical and Transcriptomics Data for Investigating the Structural and Nanomechanical Properties of the Yeast Cell Wall

The yeast cell is surrounded by a cell wall conferring protection and resistance to environmental conditions that can be harmful. Identify the molecular cues (genes) which shape the biochemical composition and the nanomechanical properties of the cell wall and the links between these two parameters represent a major issue in the understanding of the biogenesis and the molecular assembly of this essential cellular structure, which may have consequences in diverse biotechnological applications. We addressed this question in two ways. Firstly, we compared the biochemical and biophysical properties using atomic force microscopy (AFM) methods of 4 industrial strains with the laboratory sequenced strain BY4743 and used transcriptome data of these strains to infer biological hypothesis about differences of these properties between strains. This comparative approach showed a 4–6-fold higher hydrophobicity of industrial strains that was correlated to higher expression of genes encoding adhesin and adhesin-like proteins and not to their higher mannans content. The second approach was to employ a multivariate statistical analysis to identify highly correlated variables among biochemical, biophysical and genes expression data. Accordingly, we found a tight association between hydrophobicity and adhesion events that positively correlated with a set of 22 genes in which the main enriched GO function was the sterol metabolic process. We also identified a strong association of β-1,3-glucans with contour length that corresponds to the extension of mannans chains upon pulling the mannosyl units with the lectin-coated AFM tips. This association was positively correlated with a group of 27 genes in which the seripauperin multigene family was highly documented and negatively connected with a set of 23 genes whose main GO biological process was sulfur assimilation/cysteine biosynthetic process. On the other hand, the elasticity modulus was found weakly associated with levels of β-1,6-glucans, and this biophysical variable was positively correlated with a set of genes implicated in microtubules polymerization, tubulin folding and mitotic organization.