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1. A flow cytometry method for quantitative measurement and molecular investigation of the adhesion of bacteria to yeast cells

Author

Marion Schiavone, Adilya Dagkesamanskaya, Pierre-Gilles Vieu, Maëlle Duperray, Valérie Duplan-Eche, and Jean Marie François

Subjects
Yeast, Bacteria, Adhesion, Cell wall, Fimbriae, Flow cytometry, Medicine, Science
Abstract

Abstract The study of microorganism interactions is important for understanding the organization and functioning of microbial consortia. Additionally, the interaction between yeast and bacteria is of interest in the field of health and nutrition area for the development of probiotics. To investigate these microbial interactions at the cellular and molecular levels, a simple, reliable, and quantitative method is proposed. We demonstrated that flow cytometry enables the measurement of interactions at a single-cell level by detecting and counting yeast cells with bound fluorescent bacteria. Imaging flow cytometry revealed that the number of bacteria attached to yeast followed a Gaussian distribution whose maximum reached 14 bacterial cells using a clinical Escherichia coli strain E22 and the laboratory yeast strain BY4741. We found that the dynamics of adhesion resemble a Langmuir adsorption model, albeit it is a rapid and almost irreversible process. This adhesion is dependent on the mannose-specific type 1 fimbriae, as E. coli mutants lacking these appendages no longer adhere to yeast. However, this type 1 fimbriae-dependent adhesion could involve additional yeast cell wall factors, since the interaction between bacteria and yeast mutants with altered mannan content remained comparable to that of wild-type yeast. In summary, flow cytometry is an appropriate method for studying bacteria-yeast adhesion, as well as for the high-throughput screening of candidate molecules likely to promote or counteract this interaction.

Published
2024
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2. Yeast cell wall extracts from Saccharomyces cerevisiae varying in structure and composition differentially shape the innate immunity and mucosal tissue responses of the intestine of zebrafish (Danio rerio)

Author

Mark Rawling, Marion Schiavone, Emmanuelle Apper, Daniel L. Merrifield, Mathieu Castex, Eric Leclercq, and Andrew Foey

Subjects
α-mannans, yeast cell wall polysaccharides, Bioactivities, zebrafish, β-1,3-glucans, Immunologic diseases. Allergy, RC581-607
Abstract

With the rising awareness of antimicrobial resistance, the development and use of functional feed additives (FFAs) as an alternative prophylactic approach to improve animal health and performance is increasing. Although the FFAs from yeasts are widely used in animal and human pharma applications already, the success of future candidates resides in linking their structural functional properties to their efficacy in vivo. Herein, this study aimed to characterise the biochemical and molecular properties of four proprietary yeast cell wall extracts from S. cerevisiae in relation to their potential effect on the intestinal immune responses when given orally. Dietary supplementation of the YCW fractions identified that the α-mannan content was a potent driver of mucus cell and intraepithelial lymphocyte hyperplasia within the intestinal mucosal tissue. Furthermore, the differences in α-mannan and β-1,3-glucans chain lengths of each YCW fraction affected their capacity to be recognised by different PRRs. As a result, this affected the downstream signalling and shaping of the innate cytokine milieu to elicit the preferential mobilisation of effector T-helper cell subsets namely Th17, Th1, Tr1 and FoxP3+-Tregs. Together these findings demonstrate the importance of characterising the molecular and biochemical properties of YCW fractions when assessing and concluding their immune potential. Additionally, this study offers novel perspectives in the development specific YCW fractions derived from S. cerievisae for use in precision animal feeds.

Published
2023
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3. Modulation of Zebrafish (Danio rerio) Intestinal Mucosal Barrier Function Fed Different Postbiotics and a Probiotic from Lactobacilli

Author

Mark Rawling, Marion Schiavone, Amélie Mugnier, Eric Leclercq, Daniel Merrifield, Andrew Foey, and Emmanuelle Apper

Subjects
postbiotics, lactobacilli, intestinal barrier function, probiotics, zebrafish, Biology (General), QH301-705.5
Abstract

It is generally accepted that microbes play a critical role in maintaining gut barrier function, making them ideal to target in order to mitigate the effects of intestinal diseases such as inflammatory bowel disease with specialist supplementations such as probiotic or postbiotic preparations. In this study, specific strains of Lactobacillus helvictus both live and inactivated and Lactobacillus plantarum inactivated were fed to zebrafish at an inclusion level of 6 × 106 cells/g in order to assess the effects on gut barrier function and protection. Taken together, our results indicate that dietary administration of pro- or postbiotics strengthens the gut barrier function and innate immunity of healthy zebrafish in a strain-specific and process-dependent way. With some differences in the response intensity, the three treatments led to increased intestinal villi length and proportion of IELs, reinforcement of the GC population and up-regulated expression of biomarkers of AMP production and tight junction zona-occludin 2a (zo-2a). In addition, LPPost had an impact on the adaptive immune response, and we hypothesized that it conferred the potential to drive Th17/ILC3 immunity, as suggested by its effect on the gene expression of il22, of different AMPs, and the expression of zo2a. Moreover, LPPost showed the potential to drive Th1/ILC1-like immunity, with a higher percentage of CD8+ cells and higher ifnγ gene expression. In summary, the use of inactivated Lactobacilli species in this study represented a promising strategy for improving barrier function and regulating the immune fate of the intestinal mucosa in a strain-specific way.

Published
2023
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4. Emerging relevance of cell wall components from non-conventional yeasts as functional ingredients for the food and feed industry

Author

Marion Schiavone, Jean M. François, Didier Zerbib, and Jean-Pascal Capp

Subjects
Yeast, Non-conventional, Cell wall, β-glucan, Mannan, Prebiotic, Nutrition. Foods and food supply, TX341-641, Food processing and manufacture, TP368-456
Abstract

Non-conventional yeast species, or non-Saccharomyces yeasts, are increasingly recognized for their involvement in fermented foods. Many of them exhibit probiotic characteristics that are mainly due to direct contacts with other cell types through various molecular components of their cell wall. The biochemical composition and/or the molecular structure of the cell wall components are currently considered the primary determinant of their probiotic properties. Here we first present the techniques that are used to extract and analyze the cell wall components of food industry-related non-Saccharomyces yeasts. We then review the current understanding of the cell wall composition and structure of each polysaccharide from these yeasts. Finally, the data exploring the potential beneficial role of their cell wall components, which could be a source of innovative functional ingredients, are discussed. Such research would allow the development of high value-added products and provide the food industry with novel inputs beyond the well-established S. cerevisiae.

Published
2023
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5. Impact of down-stream processing on functional properties of yeasts and the implications on gut health of Atlantic salmon (Salmo salar)

Author

Jeleel Opeyemi Agboola, Marion Schiavone, Margareth Øverland, Byron Morales-Lange, Leidy Lagos, Magnus Øverlie Arntzen, David Lapeña, Vincent G. H. Eijsink, Svein Jarle Horn, Liv Torunn Mydland, Jean Marie François, Luis Mercado, and Jon Øvrum Hansen

Subjects
Medicine, Science
Abstract

Abstract Yeasts are becoming popular as novel ingredients in fish feeds because of their potential to support better growth and concomitantly ensure good fish health. Here, three species of yeasts (Cyberlindnera jadinii, Blastobotrys adeninivorans and Wickerhamomyces anomalus), grown on wood sugars and hydrolysates of chicken were subjected to two down-stream processes, either direct heat-inactivation or autolysis, and the feed potential of the resulting yeast preparations was assessed through a feeding trial with Atlantic salmon fry. Histological examination of distal intestine based on widening of lamina propria, showed that autolyzed W. anomalus was effective in alleviating mild intestinal enteritis, while only limited effects were observed for other yeasts. Our results showed that the functionality of yeast in counteracting intestinal enteritis in Atlantic salmon was dependent on both the type of yeast and the down-stream processing method, and demonstrated that C. jadinii and W. anomalus have promising effects on gut health of Atlantic salmon.

Published
2021
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6. The dual role of amyloid-β-sheet sequences in the cell surface properties of FLO11-encoded flocculins in Saccharomyces cerevisiae

Author

Clara Bouyx, Marion Schiavone, Marie-Ange Teste, Etienne Dague, Nathalie Sieczkowski, Anne Julien, and Jean Marie François

Subjects
cell surface, amyloids, cell adhesion, FLO11, AFM, Medicine, Science, Biology (General), QH301-705.5
Abstract

Fungal adhesins (Als) or flocculins are family of cell surface proteins that mediate adhesion to diverse biotic and abiotic surfaces. A striking characteristic of Als proteins originally identified in the pathogenic Candida albicans is to form functional amyloids that mediate cis-interaction leading to the formation of adhesin nanodomains and trans-interaction between amyloid sequences of opposing cells. In this report, we show that flocculins encoded by FLO11 in Saccharomyces cerevisiae behave like adhesins in C. albicans. To do so, we show that the formation of nanodomains under an external physical force requires a threshold number of amyloid-forming sequences in the Flo11 protein. Then, using a genome editing approach, we constructed strains expressing variants of the Flo11 protein under the endogenous FLO11 promoter, leading to the demonstration that the loss of amyloid-forming sequences strongly reduces cell-cell interaction but has no effect on either plastic adherence or invasive growth in agar, both phenotypes being dependent on the N- and C-terminal ends of Flo11p. Finally, we show that the location of Flo11 is not altered either by the absence of amyloid-forming sequences or by the removal of the N- or C-terminus of the protein.

Published
2021
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8. AFM dendritips functionalized with molecular probes specific to cell wall polysaccharides as a tool to investigate cell surface structure and organization

Author

Marion Schiavone, Nathalie Sieczkowski, Mathieu Castex, Emmanuelle Trevisiol, Etienne Dague, and Jean Marie François

Subjects
Cytology, QH573-671
Abstract

The yeast cell wall is composed of mannoproteins, β-1,3/β-1, 6-glucans and chitin. Each of these components has technological properties that are relevant for industrial and medical applications. To address issues related to cell wall structure and alteration in response to stress or conditioning processes, AFM dendritips were functionalized with biomolecules that are specific for each of the wall components, which was wheat germ agglutinin (WGA) for chitin, concanavalin A (ConA) for mannans and anti-β-1,3/anti-β-1,6-glucan antibodies for β-1,3/β-1,6-glucans. Binding specificity of these biomolecules were validated using penta-N-acetylchitopentaose, α-mannans, laminarin (short β-1,3-glucan chain) and gentiobiose (2 glucose units linked in β 1→6) immobilized on epoxy glass slides. Dynamic force spectroscopy was employed to obtain kinetic and thermodynamic information on the intermolecular interaction of the binary complexes using the model of Friddle-Noy-de Yoreo. Using this model, transition state distance xt, dissociate rate koff and the lowest force (feq) required to break the intermolecular bond of the complexes were approximated. These functionalized dendritips were then used to probe the yeast cell surface treated with a bacterial protease. As expected, this treatment, which removed the outer layer of the cell wall, gave accessibility to the inner layer composed of β-glucans. Likewise, bud scars were nicely localized using AFM dendritip bearing the WGA probe. To conclude, these functionalized AFM dendritips constitute a new toolbox that can be used to investigate cell surface structure and organization in response to a wide arrays of cultures and process conditions. Keywords: Cell surface, Cell wall, β-Glucan, Chitin, AFM dendrtips, Anti β-1, 3-glucan antibodies, Molecular interaction

Published
2019
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9. FLO11, a Developmental Gene Conferring Impressive Adaptive Plasticity to the Yeast Saccharomyces cerevisiae

Author

Clara Bouyx, Marion Schiavone, and Jean Marie François

Subjects
Saccharomyces cerevisiae, cell wall, flocculins, adhesins, FLO11, phenotypic variation, Medicine
Abstract

The yeast Saccharomyces cerevisiae has a remarkable ability to adapt its lifestyle to fluctuating or hostile environmental conditions. This adaptation most often involves morphological changes such as pseudofilaments, biofilm formation, or cell aggregation in the form of flocs. A prerequisite for these phenotypic changes is the ability to self-adhere and to adhere to abiotic surfaces. This ability is conferred by specialized surface proteins called flocculins, which are encoded by the FLO genes family in this yeast species. This mini-review focuses on the flocculin encoded by FLO11, which differs significantly from other flocculins in domain sequence and mode of genetic and epigenetic regulation, giving it an impressive plasticity that enables yeast cells to swiftly adapt to hostile environments or into new ecological niches. Furthermore, the common features of Flo11p with those of adhesins from pathogenic yeasts make FLO11 a good model to study the molecular mechanism underlying cell adhesion and biofilm formation, which are part of the initial step leading to fungal infections.

Published
2021
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10. Integration of Biochemical, Biophysical and Transcriptomics Data for Investigating the Structural and Nanomechanical Properties of the Yeast Cell Wall

Author

Marion Schiavone, Sébastien Déjean, Nathalie Sieczkowski, Mathieu Castex, Etienne Dague, and Jean M. François

Subjects
cell wall, β-glucans, mannans, chitin, yeast, atomic force microscopy, Microbiology, QR1-502
Abstract

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.

Published
2017
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11. Cell Wall Remodeling Enzymes Modulate Fungal Cell Wall Elasticity and Osmotic Stress Resistance

Author

Iuliana V. Ene, Louise A. Walker, Marion Schiavone, Keunsook K. Lee, Hélène Martin-Yken, Etienne Dague, Neil A. R. Gow, Carol A. Munro, and Alistair J. P. Brown

Subjects
Microbiology, QR1-502
Abstract

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.

Published
2015
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12. cis-Golgi phosphate transporters harboring an EXS domain are essential for plant growth and development

Author

Yi-Fang Hsieh, Dmitry Suslov, Luca Espen, Marion Schiavone, Carsten Rautengarten, Annika Griess-Osowski, Catalin Voiniciuc, and Yves Poirier

Subjects
Physiology, Genetics, Plant Science
Abstract

Cell wall synthesis and protein glycosylation require the import of nucleotide diphosphate–sugar conjugates into the Golgi that must be counterbalanced by phosphate (Pi) export. Numerous Golgi nucleotide-sugar transporters have been characterized, but transporters mediating Golgi Pi export remain poorly understood. We used plant and yeast genetics to characterize the role of 2 Arabidopsis (Arabidopsis thaliana) proteins possessing an EXS domain, namely ERD1A and ERD1B, in Golgi Pi homeostasis. ERD1A and ERD1B localized in cis-Golgi and were broadly expressed in vegetative and reproductive tissues. We identified ERD1 putative orthologs in algae, bryophytes, and vascular plants. Expressing ERD1A and ERD1B in yeast complemented the erd1 mutant phenotype of cellular Pi loss via exocytosis associated with reduced Golgi Pi export. The Arabidopsis erd1a mutant had a similar phenotype of apoplastic Pi loss dependent on exocytosis. ERD1A overexpression in Nicotiana benthamiana and Arabidopsis led to partial mislocalization of ERD1A to the plasma membrane and specific Pi export to the apoplastic space. Arabidopsis erd1a had defects in cell wall biosynthesis, which were associated with reduced shoot development, hypocotyl growth, cell wall extensibility, root elongation, pollen germination, pollen tube elongation, and fertility. We identified ERD1 proteins as Golgi Pi exporters that are essential for optimal plant growth and fertility.

Published
2023

13. Combining AFM, XPS and chemical hydrolysis to understand the complexity and dynamics of C. vulgaris cell wall composition and architecture

Author

Irem Demir-Yilmaz, Marion Schiavone, Jérôme Esvan, Pascal Guiraud, Cécile Formosa-Dague, Toulouse Biotechnology Institute (TBI), Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Université de Toulouse (UT)-Institut National des Sciences Appliquées (INSA)-Université de Toulouse (UT)-Centre National de la Recherche Scientifique (CNRS)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Équipe Ingénierie pour les sciences du vivant (LAAS-ELIA), Laboratoire d'analyse et d'architecture des systèmes (LAAS), Université Toulouse Capitole (UT Capitole), Université de Toulouse (UT)-Université de Toulouse (UT)-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Université de Toulouse (UT)-Institut National des Sciences Appliquées (INSA)-Université Toulouse - Jean Jaurès (UT2J), Université de Toulouse (UT)-Université Toulouse III - Paul Sabatier (UT3), Université de Toulouse (UT)-Centre National de la Recherche Scientifique (CNRS)-Institut National Polytechnique (Toulouse) (Toulouse INP), Université de Toulouse (UT)-Université Toulouse Capitole (UT Capitole), Université de Toulouse (UT), Lallemand SAS, Centre interuniversitaire de recherche et d'ingenierie des matériaux (CIRIMAT), Université Toulouse III - Paul Sabatier (UT3), Université de Toulouse (UT)-Université de Toulouse (UT)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Institut National Polytechnique (Toulouse) (Toulouse INP), Fédération de Recherche Fluides, Energie, Réacteurs, Matériaux et Transferts (FERMAT), Institut National des Sciences Appliquées (INSA)-Université de Toulouse (UT)-Institut National des Sciences Appliquées (INSA)-Université de Toulouse (UT)-Université Toulouse III - Paul Sabatier (UT3), and ANR-18-CE43-0001,FLOTALG,Biophysique de la flottation des microalgues(2018)

Subjects
[SPI]Engineering Sciences [physics], chemical hydrolysis, Cell wall, [SDE]Environmental Sciences, XPS, Microalgae, AFM, Agronomy and Crop Science
Abstract

The microalgae cell wall represents its interface with its environment and a strong barrier to disrupt in order to extract the cell’s products. Understanding its composition and architecture is a challenge that if overcome, could lead to substantial advancements in optimizing microalgae-production systems. However, the cell wall is a dynamic and complex structure that evolves depending on the growth phase or culture conditions. To apprehend this complexity, an experimental approach combining AFM, XPS, and chemical hydrolysis followed by HPAEC-PAD was developed to understand the cell wall of Chlorella vulgaris, a biotechnologically-relevant green microalgae species. Exponential and stationary growth stages were investigated, as well as saline stress condition inducing lipid production. Results showed that both the cell wall composition and architecture changes in stationary phase, with an increase of the lipidic fraction at the expanse of the proteic fraction, changes in the polysaccharidic composition, and a significant increase of its rigidity. Under saline stress, cell wall architecture seems to be affected as its rigidity decreases importantly. Altogether, this study demonstrates the power of combining these three techniques to give new insights into C. vulgaris cell wall, in terms of composition and architecture, and of its dynamics in different conditions.

Published
2022

14. Author Correction: Impact of down-stream processing on functional properties of yeasts and the implications on gut health of Atlantic salmon (Salmo salar)

Author

Vincent G. H. Eijsink, Luis Mercado, Byron Morales-Lange, Liv Torunn Mydland, Magnus Ø. Arntzen, Leidy Lagos, Jeleel Opeyemi Agboola, David Lapeña, Svein Jarle Horn, Marion Schiavone, Jon Øvrum Hansen, Margareth Øverland, and Jean Marie François

Subjects
Multidisciplinary, Science, Salmo salar, Zoology, Aquaculture, Biology, biology.organism_classification, Animal Feed, Enteritis, Yeasts, Animals, Medicine, Intestinal Mucosa, Salmo, Author Correction, Chickens
Abstract

Yeasts are becoming popular as novel ingredients in fish feeds because of their potential to support better growth and concomitantly ensure good fish health. Here, three species of yeasts (Cyberlindnera jadinii, Blastobotrys adeninivorans and Wickerhamomyces anomalus), grown on wood sugars and hydrolysates of chicken were subjected to two down-stream processes, either direct heat-inactivation or autolysis, and the feed potential of the resulting yeast preparations was assessed through a feeding trial with Atlantic salmon fry. Histological examination of distal intestine based on widening of lamina propria, showed that autolyzed W. anomalus was effective in alleviating mild intestinal enteritis, while only limited effects were observed for other yeasts. Our results showed that the functionality of yeast in counteracting intestinal enteritis in Atlantic salmon was dependent on both the type of yeast and the down-stream processing method, and demonstrated that C. jadinii and W. anomalus have promising effects on gut health of Atlantic salmon.

Published
2021

15. The dual role of amyloid-β-sheet sequences in the cell surface properties of FLO11-encoded flocculins in Saccharomyces cerevisiae

Author

Etienne Dague, Marie-Ange Teste, Jean Marie François, Clara Bouyx, Nathalie Sieczkowski, Anne Ortiz Julien, Marion Schiavone, Toulouse Biotechnology Institute (TBI), Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Université de Toulouse (UT)-Institut National des Sciences Appliquées (INSA)-Université de Toulouse (UT)-Centre National de la Recherche Scientifique (CNRS)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Équipe Ingénierie pour les sciences du vivant (LAAS-ELIA), Laboratoire d'analyse et d'architecture des systèmes (LAAS), Université Toulouse Capitole (UT Capitole), Université de Toulouse (UT)-Université de Toulouse (UT)-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Université de Toulouse (UT)-Institut National des Sciences Appliquées (INSA)-Université Toulouse - Jean Jaurès (UT2J), Université de Toulouse (UT)-Université Toulouse III - Paul Sabatier (UT3), Université de Toulouse (UT)-Centre National de la Recherche Scientifique (CNRS)-Institut National Polytechnique (Toulouse) (Toulouse INP), Université de Toulouse (UT)-Université Toulouse Capitole (UT Capitole), Université de Toulouse (UT), Lallemand SAS, Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Centre National de la Recherche Scientifique (CNRS)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Université Toulouse - Jean Jaurès (UT2J)-Université Toulouse 1 Capitole (UT1), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Centre National de la Recherche Scientifique (CNRS)-Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Institut National Polytechnique (Toulouse) (Toulouse INP), Université Fédérale Toulouse Midi-Pyrénées-Université Toulouse - Jean Jaurès (UT2J)-Université Toulouse 1 Capitole (UT1), and Université Fédérale Toulouse Midi-Pyrénées

Subjects
Amyloid, QH301-705.5, Science, [PHYS.PHYS.PHYS-BIO-PH]Physics [physics]/Physics [physics]/Biological Physics [physics.bio-ph], Cell, Saccharomyces cerevisiae, General Biochemistry, Genetics and Molecular Biology, 03 medical and health sciences, [SDV.BC.IC]Life Sciences [q-bio]/Cellular Biology/Cell Behavior [q-bio.CB], medicine, amyloids, Biology (General), [PHYS.MECA.BIOM]Physics [physics]/Mechanics [physics]/Biomechanics [physics.med-ph], Candida albicans, Cell adhesion, [SDV.MP.MYC]Life Sciences [q-bio]/Microbiology and Parasitology/Mycology, 030304 developmental biology, 0303 health sciences, General Immunology and Microbiology, biology, 030306 microbiology, Chemistry, General Neuroscience, cell adhesion, General Medicine, FLO11, biology.organism_classification, Phenotype, Corpus albicans, Cell biology, Bacterial adhesin, cell surface, medicine.anatomical_structure, Medicine, AFM
Abstract

Fungal adhesins (Als) or flocculins are family of cell surface proteins that mediate adhesion to diverse biotic and abiotic surfaces. A striking characteristic of Als proteins originally identified in the pathogenic Candida albicans is to form functional amyloids that mediate cis-interaction leading to the formation of adhesin nanodomains and trans-interaction between amyloid sequences of opposing cells. In this report, we show that flocculins encoded by FLO11 in Saccharomyces cerevisiae behave like adhesins in C. albicans. To do so, we show that the formation of nanodomains under an external physical force requires a threshold number of amyloid-forming sequences in the Flo11 protein. Then, using a genome editing approach, we constructed strains expressing variants of the Flo11 protein under the endogenous FLO11 promoter, leading to the demonstration that the loss of amyloid-forming sequences strongly reduces cell-cell interaction but has no effect on either plastic adherence or invasive growth in agar, both phenotypes being dependent on the N- and C-terminal ends of Flo11p. Finally, we show that the location of Flo11 is not altered either by the absence of amyloid-forming sequences or by the removal of the N- or C-terminus of the protein.

Published
2021

17. Physiological function of Flo11p domains and the particular role of amyloid core sequences of this adhesin in Saccharomyces cerevisiae

Author

Marie-Ange Teste, C. Bouyx, Etienne Dague, Nathalie Sieczkowski, Marion Schiavone, A. Julien, and Jean Marie François

Subjects
Cell wall, biology, Amyloid, Chemistry, Saccharomyces cerevisiae, Biofilm, Adhesion, biology.organism_classification, Phenotype, Yeast, Function (biology), Cell biology
Abstract

Flocculins are a family of glycosylated proteins that provide yeast cells with several properties such as biofilm formation, flocculation, invasive growth or formation of velum. These proteins are similarly organised with a N-terminal (adhesion) domain, a stalk-like central B-domain with several repeats and a C-terminal sequence carrying a cell wall anchor site. They also contain amyloid β-aggregation-prone sequences whose functional role is still unclear. In this work, we show that Flo11p differs from other flocculins by the presence of unique amyloid-forming sequences, whose the number is critical in the formation of adhesion nanodomains under a physical shear force. Using a genome editing approach to identify the function of domains in Flo11p phenotypes, we show that the formation of cellular aggregates whose density increases with the number of amyloid sequences cannot be attributed to a specific domain of Flo11p. The same is true for plastic adhesion and surface hydrophobicity the intensity of which depends mainly on the abundance of Flo11p on the cell surface. In contrast, the N and C domains of Flo11p are essential for invasive growth in agar, whereas a reduction in the number of repeats of the B domain weakens this phenotype. However, expression of FLO11 alone is not sufficient to trigger this invasion phenotype. Finally, we show that this flocculin contributes to the integrity of the cell wall.

Published
2021

18. Impact of down-stream processing on functional properties of yeasts and the implications on gut health of Atlantic salmon (Salmo salar)

Author

Magnus Ø. Arntzen, Marion Schiavone, David Lapeña, Jeleel Opeyemi Agboola, Svein Jarle Horn, Byron Morales-Lange, Jon Øvrum Hansen, Liv Torunn Mydland, Leidy Lagos, Margareth Øverland, Luis Mercado, Vincent G. H. Eijsink, Jean Marie François, Norwegian University of Life Sciences (NMBU), Toulouse Biotechnology Institute (TBI), Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Centre National de la Recherche Scientifique (CNRS)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Lallemand SAS, Équipe Ingénierie pour les sciences du vivant (LAAS-ELIA), Laboratoire d'analyse et d'architecture des systèmes (LAAS), Université Toulouse - Jean Jaurès (UT2J)-Université Toulouse 1 Capitole (UT1), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Centre National de la Recherche Scientifique (CNRS)-Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Institut National Polytechnique (Toulouse) (Toulouse INP), Université Fédérale Toulouse Midi-Pyrénées-Université Toulouse - Jean Jaurès (UT2J)-Université Toulouse 1 Capitole (UT1), Université Fédérale Toulouse Midi-Pyrénées, University of South-Eastern Norway (USN), Pontificia Universidad Católica de Valparaíso (PUCV), Foods of Norway, a Centre for Research-based Innovation (the Research Council of Norway) 237841/030, Postdoctoral program from the National Research and Development Agency of Chile 74200139, Institut National des Sciences Appliquées (INSA)-Université de Toulouse (UT)-Institut National des Sciences Appliquées (INSA)-Université de Toulouse (UT)-Centre National de la Recherche Scientifique (CNRS)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Université Toulouse Capitole (UT Capitole), Université de Toulouse (UT)-Université de Toulouse (UT)-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Université de Toulouse (UT)-Institut National des Sciences Appliquées (INSA)-Université Toulouse - Jean Jaurès (UT2J), Université de Toulouse (UT)-Université Toulouse III - Paul Sabatier (UT3), Université de Toulouse (UT)-Centre National de la Recherche Scientifique (CNRS)-Institut National Polytechnique (Toulouse) (Toulouse INP), Université de Toulouse (UT)-Université Toulouse Capitole (UT Capitole), and Université de Toulouse (UT)

Subjects
0301 basic medicine, Autolysis (biology), Wickerhamomyces anomalus, Science, [SDV]Life Sciences [q-bio], Biology, Blastobotrys adeninivorans, Hydrolysate, Article, Enteritis, 03 medical and health sciences, Atomic force microscopy, medicine, 14. Life underwater, Food science, Salmo, Lamina propria, Multidisciplinary, Immunochemistry, Tumour-necrosis factors, 04 agricultural and veterinary sciences, medicine.disease, biology.organism_classification, Immunohistochemistry, Yeast, 030104 developmental biology, medicine.anatomical_structure, Ulcerative colitis, 040102 fisheries, Medicine, 0401 agriculture, forestry, and fisheries, ELISA
Abstract

Yeasts are becoming popular as novel ingredients in fish feeds because of their potential to support better growth and concomitantly ensure good fish health. Here, three species of yeasts (Cyberlindnera jadinii, Blastobotrys adeninivorans and Wickerhamomyces anomalus), grown on wood sugars and hydrolysates of chicken were subjected to two down-stream processes, either direct heat-inactivation or autolysis, and the feed potential of the resulting yeast preparations was assessed through a feeding trial with Atlantic salmon fry. Histological examination of distal intestine based on widening of lamina propria, showed that autolyzed W. anomalus was effective in alleviating mild intestinal enteritis, while only limited effects were observed for other yeasts. Our results showed that the functionality of yeast in counteracting intestinal enteritis in Atlantic salmon was dependent on both the type of yeast and the down-stream processing method, and demonstrated that C. jadinii and W. anomalus have promising effects on gut health of Atlantic salmon.

Published
2021

19. Down-stream processing of baker's yeast (Saccharomyces cerevisiae) – Effect on nutrient digestibility and immune response in Atlantic salmon (Salmo salar)

Author

Luis Mercado, Byron Morales-Lange, Jon Øvrum Hansen, Ricardo Tavares Benicio, Liv Torunn Mydland, Line Degn Hansen, Margareth Øverland, Peng Lei, Leidy Lagos, Marion Schiavone, Magnus Ø. Arntzen, and Felipe Eduardo Reveco-Urzua

Subjects
0303 health sciences, Autolysis (biology), biology, medicine.diagnostic_test, Saccharomyces cerevisiae, 04 agricultural and veterinary sciences, Aquatic Science, biology.organism_classification, Yeast, Flow cytometry, 03 medical and health sciences, Fish meal, 040102 fisheries, medicine, 0401 agriculture, forestry, and fisheries, Yeast extract, Secretion, Food science, Salmo, 030304 developmental biology
Abstract

The increased demand for high-quality feed ingredients for the aquaculture industry has led to an increased focus on microbial ingredients as nutrient sources. Limited information exists, however, on the effect of down-stream processing of yeast on nutrient digestibility and health in Atlantic salmon (Salmo salar). In the present study, three laboratory-scale down-stream processing methods were used: direct inactivation by spray-drying, autolysis, or cell crushing. All yeast was inactivated with sray-drying post treatment. The yeast products were evaluated for their nutritional value and health effects in diets for Atlantic salmon. The experimental diets consisted of a high-quality fishmeal-based reference diet (FM) and six experimental diets were 300 g kg−1 of each of the processed yeast candidates were added to the FM control mixture. Fish were fed the diets for 21 days, and at the end of the experiment, feces were collected, and spleen, head kidney, distal intestine (DI) and plasma were sampled for assessment of immune response. Processing of yeast resulted in increased level of soluble protein in the yeast cream, with the highest level for yeast disrupted by cell crushing (20 K PSI). This was confirmed with scanning electron microscopy, where disrupted and cracked cells could be observed. Significant lower protein digestibility of the directly inactivated yeast coincided with the increased detection of whole yeast cells by flow cytometry in the DI content of fish fed the directly inactivated yeast. Autolysis for 16 h resulted in the highest protein digestibility, which was similar to the FM control. Interestingly, 16 h autolyzed yeast induced the secretion of IL-8, while cell crushed yeast induced the secretion of TNFα in the DI as analyzed by ELISA. To conclude, different down-stream processing of S. cerevisiae led to increased protein and β-glucan solubility that further increased protein digestibility in Atlantic salmon. Furthermore, different processing of the yeast triggered different immune stimulatory effects in Atlantic salmon.

Published
2021

20. Differential homotypic and heterotypic interactions of antigen 43 (Ag43) variants in autotransporter-mediated bacterial autoaggregation

Author

Grégory Jubelin, Begoña Heras, Ingrid Chafsey, Frédérique Chaucheyras-Durand, Xavier Bailly, Amanda E. Rossiter, Valentin Ageorges, Marion Schiavone, Ian R. Henderson, Mickaël Desvaux, Jason J. Paxman, Philippe Ruiz, Etienne Dague, Sabine Leroy, Nelly Caccia, Microbiologie Environnement Digestif Santé (MEDIS), Institut National de la Recherche Agronomique (INRA)-Université Clermont Auvergne [2017-2020] (UCA [2017-2020]), Lallemand SAS, Unité Mixte de Recherche d'Épidémiologie des maladies Animales et zoonotiques (UMR EPIA), Institut National de la Recherche Agronomique (INRA)-VetAgro Sup - Institut national d'enseignement supérieur et de recherche en alimentation, santé animale, sciences agronomiques et de l'environnement (VAS), Équipe Ingénierie pour les sciences du vivant (LAAS-ELIA), Laboratoire d'analyse et d'architecture des systèmes (LAAS), Université Toulouse Capitole (UT Capitole), Université de Toulouse (UT)-Université de Toulouse (UT)-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Université de Toulouse (UT)-Institut National des Sciences Appliquées (INSA)-Université Toulouse - Jean Jaurès (UT2J), Université de Toulouse (UT)-Université Toulouse III - Paul Sabatier (UT3), Université de Toulouse (UT)-Centre National de la Recherche Scientifique (CNRS)-Institut National Polytechnique (Toulouse) (Toulouse INP), Université de Toulouse (UT)-Université Toulouse Capitole (UT Capitole), Université de Toulouse (UT), La Trobe University [Melbourne], Institute of Microbiology and Infection, University of Birmingham [Birmingham], INRA (Institut National de la Recherche Agronomique), CoMBa ('Colonisation de la Matrice extracellulaire Bacterienne') project - 'Region Auvergne' FRI ('Fond Regional Innovation') cluster IRP ('Institut de Recherche Pharmabiotique'), Australian Research Council (ARC) DP150102287 DP180102987 FT130100580, Conseil Regional d'Auvergne - FEDER (Fonds Europeen de Developpement Regional), INRA Clermont-Ferrand-Theix-Université Clermont Auvergne [2017-2020] (UCA [2017-2020]), Université Toulouse - Jean Jaurès (UT2J)-Université Toulouse 1 Capitole (UT1), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Centre National de la Recherche Scientifique (CNRS)-Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Institut National Polytechnique (Toulouse) (Toulouse INP), Université Fédérale Toulouse Midi-Pyrénées-Université Toulouse - Jean Jaurès (UT2J)-Université Toulouse 1 Capitole (UT1), Université Fédérale Toulouse Midi-Pyrénées, Microbiologie Environnement Digestif Santé - Clermont Auvergne (MEDIS), INRA Clermont-Ferrand-Theix-Université Clermont Auvergne (UCA), Unité de recherche d'Épidémiologie Animale (UEA), Institut National de la Recherche Agronomique (INRA), Institut National Polytechnique (Toulouse) (Toulouse INP), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Centre National de la Recherche Scientifique (CNRS)-Université Toulouse 1 Capitole (UT1)-Université Toulouse - Jean Jaurès (UT2J)-Institut National Polytechnique (Toulouse) (Toulouse INP), and Université Fédérale Toulouse Midi-Pyrénées-Centre National de la Recherche Scientifique (CNRS)-Université Toulouse 1 Capitole (UT1)-Université Toulouse - Jean Jaurès (UT2J)

Subjects
0301 basic medicine, Signal peptide, Type V Secretion Systems, Molecular biology, [SDV]Life Sciences [q-bio], Structural alignment, lcsh:Medicine, Microbiology, Bacterial Adhesion, Article, spectroscopie, Bacterial genetics, sécrétion, 03 medical and health sciences, Bacterial secretion, 0302 clinical medicine, Phylogenetics, Secretion, lcsh:Science, Phylogeny, antigène, Adhesins, Escherichia coli, Antigens, Bacterial, Multidisciplinary, Chemistry, Escherichia coli Proteins, lcsh:R, Microbiology and Parasitology, Biological Transport, Antigenic Variation, Microbiologie et Parasitologie, Cell biology, Bacterial adhesin, 030104 developmental biology, Biofilms, lcsh:Q, escherichia coli, Bacterial outer membrane, 030217 neurology & neurosurgery, Bacterial Outer Membrane Proteins, Autotransporters
Abstract

Antigen 43 (Ag43) is a cell-surface exposed protein of Escherichia coli secreted by the Type V, subtype a, secretion system (T5aSS) and belonging to the family of self-associating autotransporters (SAATs). These modular proteins, comprising a cleavable N-terminal signal peptide, a surface-exposed central passenger and an outer membrane C-terminal translocator, self-recognise in a Velcro-like handshake mechanism. A phylogenetic network analysis focusing on the passenger revealed for the first time that they actually distribute into four distinct classes, namely C1, C2, C3 and C4. Structural alignment and modelling analyses demonstrated these classes arose from shuffling of two different subdomains within the Ag43 passengers. Functional analyses revealed that homotypic interactions occur for all Ag43 classes but significant differences in the sedimentation kinetics and aggregation state were present when Ag43C3 was expressed. In contrast, heterotypic interaction occurred in a very limited number of cases. Single cell-force spectroscopy demonstrated the importance of specific as well as nonspecific interactions in mediating Ag43-Ag43 recognition. We propose that structural differences in the subdomains of the Ag43 classes account for different autoaggregation dynamics and propensities to co-interact.

Published
2019

21. Biosynthesis of β-d-glucan‑gold nanoparticles, cytotoxicity and oxidative stress in mouse splenocytes

Author

Marion Schiavone, Luis Hernandez-Adame, Gabriela Palestino, Lourdes Betancourt-Mendiola, Mathieu Castex, Karen Delgado, Martha Reyes-Becerril, and Carlos Angulo

Subjects
Cytotoxicity, Immunologic, Male, Scanning electron microscope, Cell Survival, Gene Expression, Metal Nanoparticles, 02 engineering and technology, medicine.disease_cause, Nitric Oxide, Biochemistry, Antioxidants, 03 medical and health sciences, chemistry.chemical_compound, Mice, Biosynthesis, Dynamic light scattering, Phagocytosis, Structural Biology, medicine, Leukocytes, Animals, Cytotoxicity, Molecular Biology, Glucans, 030304 developmental biology, Respiratory Burst, 0303 health sciences, biology, Yarrowia, Green Chemistry Technology, General Medicine, 021001 nanoscience & nanotechnology, biology.organism_classification, Catalase, Oxidative Stress, chemistry, Colloidal gold, Biophysics, Cytokines, Gold, Nanocarriers, 0210 nano-technology, Oxidative stress, Biomarkers, Spleen
Abstract

This study reports biosynthesis of gold-nanoparticles (AuNPs) by using β-d-glucans isolated from the yeast Yarrowia lypolitica D1. β-d-glucans serve as reducing and stabilizing mediators that induce the formation of AuNPs on the outer surface of the own β-d-glucan. The systems were physicochemically characterized by ultraviolet visible (UV-Vis) spectroscopy, high-resolution transmission electron microscopy (HR-TEM), scanning electron microscope (SEM), energy-dispersive X-ray spectroscopy (EDS), and dynamic light scattering (DLS) analyses. The results revealed the generation of AuNPs with quasi-spherical shape or large one dimension (1D) gold-nanostructures (AuNSs) depending on the HAuCl4 concentration. A cytotoxic study was assessed in mouse splenocytes. Contrary to that expected, important cytotoxicity was found in all β-d-gluc+AuNPs systems by an oxidative stress increase. This study discusses the cytotoxic mechanism, suggesting that the resulting β-d-gluc+AuNPs systems may not be candidates for the formulation of immunostimulants or nanocarriers for biomedical applications.

Published
2019

22. AFM dendritips functionalized with molecular probes specific to cell wall polysaccharides as a tool to investigate cell surface structure and organization

Author

Jean Marie François, Mathieu Castex, Etienne Dague, Nathalie Sieczkowski, Marion Schiavone, Emmanuelle Trévisiol, Laboratoire d'Ingénierie des Systèmes Biologiques et des Procédés (LISBP), Centre National de la Recherche Scientifique (CNRS)-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Institut National de la Recherche Agronomique (INRA), Lallemand S.A.S., Équipe Ingénierie pour les sciences du vivant (LAAS-ELIA), Laboratoire d'analyse et d'architecture des systèmes (LAAS), Institut National Polytechnique (Toulouse) (Toulouse INP), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Centre National de la Recherche Scientifique (CNRS)-Université Toulouse 1 Capitole (UT1)-Université Toulouse - Jean Jaurès (UT2J)-Institut National Polytechnique (Toulouse) (Toulouse INP), Université Fédérale Toulouse Midi-Pyrénées-Centre National de la Recherche Scientifique (CNRS)-Université Toulouse 1 Capitole (UT1)-Université Toulouse - Jean Jaurès (UT2J), Institut National de la Recherche Agronomique (INRA)-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Centre National de la Recherche Scientifique (CNRS), Université Toulouse - Jean Jaurès (UT2J)-Université Toulouse 1 Capitole (UT1), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Centre National de la Recherche Scientifique (CNRS)-Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Institut National Polytechnique (Toulouse) (Toulouse INP), Université Fédérale Toulouse Midi-Pyrénées-Université Toulouse - Jean Jaurès (UT2J)-Université Toulouse 1 Capitole (UT1), Université Fédérale Toulouse Midi-Pyrénées, Institut National des Sciences Appliquées (INSA)-Université de Toulouse (UT)-Institut National des Sciences Appliquées (INSA)-Université de Toulouse (UT)-Centre National de la Recherche Scientifique (CNRS), Université Toulouse Capitole (UT Capitole), Université de Toulouse (UT)-Université de Toulouse (UT)-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Université de Toulouse (UT)-Institut National des Sciences Appliquées (INSA)-Université Toulouse - Jean Jaurès (UT2J), Université de Toulouse (UT)-Université Toulouse III - Paul Sabatier (UT3), Université de Toulouse (UT)-Centre National de la Recherche Scientifique (CNRS)-Institut National Polytechnique (Toulouse) (Toulouse INP), Université de Toulouse (UT)-Université Toulouse Capitole (UT Capitole), and Université de Toulouse (UT)

Subjects
[SDV.BIO]Life Sciences [q-bio]/Biotechnology, [SDV]Life Sciences [q-bio], Chitin, macromolecular substances, Polysaccharide, Applied Microbiology and Biotechnology, Microbiology, Cell surface, Article, Cell wall, 03 medical and health sciences, chemistry.chemical_compound, β-Glucan, Gentiobiose, lcsh:QH573-671, Molecular Biology, 030304 developmental biology, chemistry.chemical_classification, Anti β-1, 0303 health sciences, biology, lcsh:Cytology, 030306 microbiology, Biomolecule, Molecular interaction, Cell Biology, Wheat germ agglutinin, 3. Good health, carbohydrates (lipids), 3-glucan antibodies, chemistry, Concanavalin A, biology.protein, Biophysics, Molecular probe, AFM dendrtips
Abstract

Highlights • Functionalisation of AFM dendritips with conA, WGA and anti-β-1,3/β-1, 6-glucan antibodies. • Cell wall polysaccharides were immobilized on epoxy-activated glass slides. • Specific binding of immobilized polysaccharides to functionalized dendritips. • Functionalized dendritips used as a new tool to probe yeast cell surface., The yeast cell wall is composed of mannoproteins, β-1,3/β-1, 6-glucans and chitin. Each of these components has technological properties that are relevant for industrial and medical applications. To address issues related to cell wall structure and alteration in response to stress or conditioning processes, AFM dendritips were functionalized with biomolecules that are specific for each of the wall components, which was wheat germ agglutinin (WGA) for chitin, concanavalin A (ConA) for mannans and anti-β-1,3/anti-β-1,6-glucan antibodies for β-1,3/β-1,6-glucans. Binding specificity of these biomolecules were validated using penta-N-acetylchitopentaose, α-mannans, laminarin (short β-1,3-glucan chain) and gentiobiose (2 glucose units linked in β 1→6) immobilized on epoxy glass slides. Dynamic force spectroscopy was employed to obtain kinetic and thermodynamic information on the intermolecular interaction of the binary complexes using the model of Friddle-Noy-de Yoreo. Using this model, transition state distance xt, dissociate rate koff and the lowest force (feq) required to break the intermolecular bond of the complexes were approximated. These functionalized dendritips were then used to probe the yeast cell surface treated with a bacterial protease. As expected, this treatment, which removed the outer layer of the cell wall, gave accessibility to the inner layer composed of β-glucans. Likewise, bud scars were nicely localized using AFM dendritip bearing the WGA probe. To conclude, these functionalized AFM dendritips constitute a new toolbox that can be used to investigate cell surface structure and organization in response to a wide arrays of cultures and process conditions.

Published
2019

23. Nanoscale Effects of Caspofungin against Two Yeast Species, Saccharomyces cerevisiae and Candida albicans

Author

Jean Marie François, Raphaël E. Duval, Cécile Formosa, Marion Schiavone, Hélène Martin-Yken, Etienne Dague, Laboratoire d'analyse et d'architecture des systèmes (LAAS), Université Toulouse - Jean Jaurès (UT2J)-Université Toulouse 1 Capitole (UT1), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Centre National de la Recherche Scientifique (CNRS)-Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Institut National Polytechnique (Toulouse) (Toulouse INP), Université Fédérale Toulouse Midi-Pyrénées, Structure et Réactivité des Systèmes Moléculaires Complexes (SRSMC), Institut de Chimie du CNRS (INC)-Université de Lorraine (UL)-Centre National de la Recherche Scientifique (CNRS), Laboratoire d'Ingénierie des Systèmes Biologiques et des Procédés (LISBP), Centre National de la Recherche Scientifique (CNRS)-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Institut National de la Recherche Agronomique (INRA), ABC Platform, Équipe NanoBioSystèmes (LAAS-NBS), Université Toulouse 1 Capitole (UT1)-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Université Toulouse - Jean Jaurès (UT2J)-Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Centre National de la Recherche Scientifique (CNRS)-Institut National Polytechnique (Toulouse) (Toulouse INP), Université Fédérale Toulouse Midi-Pyrénées-Université Toulouse 1 Capitole (UT1)-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National de la Recherche Agronomique (INRA)-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Centre National de la Recherche Scientifique (CNRS), Laboratoire de Chimie Physique Organique et Colloı̈dale, Centre National de la Recherche Scientifique (CNRS)-Université Henri Poincaré - Nancy 1 (UHP), Institut National Polytechnique (Toulouse) (Toulouse INP), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Centre National de la Recherche Scientifique (CNRS)-Université Toulouse 1 Capitole (UT1)-Université Toulouse - Jean Jaurès (UT2J), Université de Lorraine (UL)-Centre National de la Recherche Scientifique (CNRS), Université Toulouse Capitole (UT Capitole), Université de Toulouse (UT)-Université de Toulouse (UT)-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Université de Toulouse (UT)-Institut National des Sciences Appliquées (INSA)-Université Toulouse - Jean Jaurès (UT2J), Université de Toulouse (UT)-Université Toulouse III - Paul Sabatier (UT3), Université de Toulouse (UT)-Centre National de la Recherche Scientifique (CNRS)-Institut National Polytechnique (Toulouse) (Toulouse INP), Université de Toulouse (UT), and Institut National des Sciences Appliquées (INSA)-Université de Toulouse (UT)-Institut National des Sciences Appliquées (INSA)-Université de Toulouse (UT)-Centre National de la Recherche Scientifique (CNRS)

Subjects
Antifungal Agents, beta-Glucans, BUDDING YEAST, [SDV]Life Sciences [q-bio], Drug Evaluation, Preclinical, Antifungal drug, Microscopy, Atomic Force, Echinocandins, chemistry.chemical_compound, Caspofungin, Cell Wall, Candida albicans, Nanotechnology, Pharmacology (medical), 0303 health sciences, Corpus albicans, 3. Good health, ANTIFUNGAL DRUGS, [SDV.MP]Life Sciences [q-bio]/Microbiology and Parasitology, Infectious Diseases, Cell Division, SURFACE, Saccharomyces cerevisiae, Microbial Sensitivity Tests, Biology, Microbiology, POLYSACCHARIDES, Cell wall, Lipopeptides, 03 medical and health sciences, QUANTITATIVE-DETERMINATION, [SDV.SP.MED]Life Sciences [q-bio]/Pharmaceutical sciences/Medication, Elastic Modulus, Cell Adhesion, MOTHER-BUD NECK, Mechanisms of Action: Physiological Effects, ATOMIC-FORCE MICROSCOPY, 030304 developmental biology, Pharmacology, 030306 microbiology, CELL-WALL ARCHITECTURE, ALS GENE FAMILY, biology.organism_classification, Yeast, chemistry, CHITIN, Cytokinesis
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.

Published
2013

24. 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 cerevisiaeto Ethanol Stress

Author

Hélène Martin-Yken, Carolina Elsztein, Jean Marie François, Marion Schiavone, Marie-Ange Teste, Etienne Dague, Cécile Formosa-Dague, Marcos de Morais, Laboratoire d'Ingénierie des Systèmes Biologiques et des Procédés (LISBP), Institut National de la Recherche Agronomique (INRA)-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Université de Toulouse (UT)-Institut National des Sciences Appliquées (INSA)-Université de Toulouse (UT)-Centre National de la Recherche Scientifique (CNRS), Équipe Ingénierie pour les sciences du vivant (LAAS-ELIA), Laboratoire d'analyse et d'architecture des systèmes (LAAS), Université Toulouse Capitole (UT Capitole), Université de Toulouse (UT)-Université de Toulouse (UT)-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Université de Toulouse (UT)-Institut National des Sciences Appliquées (INSA)-Université Toulouse - Jean Jaurès (UT2J), Université de Toulouse (UT)-Université Toulouse III - Paul Sabatier (UT3), Université de Toulouse (UT)-Centre National de la Recherche Scientifique (CNRS)-Institut National Polytechnique (Toulouse) (Toulouse INP), Université de Toulouse (UT)-Université Toulouse Capitole (UT Capitole), Université de Toulouse (UT), Department of Genetics, Universidade Federal de Pernambuco [Recife] (UFPE), Region Midi Pyrenees [10051296], Direction Generale de l'Armement (DGA), CIFRE grant from ANRT, CAPES-FACEPE/PNPD program [681/2011, APQ-0201-2.02/10], Lallemand Inc., ANR-11-JSV5-0001,AFMYST,Etude Biophysique, biochimique et biomoléculaire de la paroi des levures.(2011), Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Centre National de la Recherche Scientifique (CNRS), Université Toulouse - Jean Jaurès (UT2J)-Université Toulouse 1 Capitole (UT1), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Centre National de la Recherche Scientifique (CNRS)-Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Institut National Polytechnique (Toulouse) (Toulouse INP), Université Fédérale Toulouse Midi-Pyrénées-Université Toulouse - Jean Jaurès (UT2J)-Université Toulouse 1 Capitole (UT1), Université Fédérale Toulouse Midi-Pyrénées, Centre National de la Recherche Scientifique (CNRS)-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Institut National de la Recherche Agronomique (INRA), Institut National Polytechnique (Toulouse) (Toulouse INP), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Centre National de la Recherche Scientifique (CNRS)-Université Toulouse 1 Capitole (UT1)-Université Toulouse - Jean Jaurès (UT2J)-Institut National Polytechnique (Toulouse) (Toulouse INP), and Université Fédérale Toulouse Midi-Pyrénées-Centre National de la Recherche Scientifique (CNRS)-Université Toulouse 1 Capitole (UT1)-Université Toulouse - Jean Jaurès (UT2J)

Subjects
0301 basic medicine, Physiology, Saccharomyces cerevisiae, Microscopy, Atomic Force, Applied Microbiology and Biotechnology, Cell membrane, Cell wall, 03 medical and health sciences, chemistry.chemical_compound, Cell Wall, medicine, Transcription factor, Ethanol, Ecology, biology, Cell Membrane, Wild type, biology.organism_classification, Yeast, 030104 developmental biology, Membrane, medicine.anatomical_structure, [SDV.MP]Life Sciences [q-bio]/Microbiology and Parasitology, Biochemistry, chemistry, Biophysics, Food Science, Biotechnology
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 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.

Published
2016

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

Author

Louise A. Walker, Marion Schiavone, Alistair J. P. Brown, Hélène Martin-Yken, Iuliana V. Ene, Etienne Dague, Neil A. R. Gow, Keunsook K. Lee, Carol A. Munro, School of Medical Sciences, University of Aberdeen, Laboratoire d'Ingénierie des Systèmes Biologiques et des Procédés (LISBP), Centre National de la Recherche Scientifique (CNRS)-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Institut National de la Recherche Agronomique (INRA), Équipe NanoBioSystèmes (LAAS-NBS), Laboratoire d'analyse et d'architecture des systèmes (LAAS), Université Toulouse - Jean Jaurès (UT2J)-Université Toulouse 1 Capitole (UT1), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Centre National de la Recherche Scientifique (CNRS)-Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Institut National Polytechnique (Toulouse) (Toulouse INP), Université Fédérale Toulouse Midi-Pyrénées-Université Toulouse - Jean Jaurès (UT2J)-Université Toulouse 1 Capitole (UT1), Université Fédérale Toulouse Midi-Pyrénées, UK Biotechnology and Biological Research Council (BB/F00513X/1, BB/K017365/1), the UK Medical Research Council (G0400284), the Wellcome Trust (080088, 088858/Z/09/Z 097377, 101873). ANR (AFMYST project ANR-11-JSV5-001-01 n° SD 30024331), ANR-11-JSV5-0001,AFMYST,Etude Biophysique, biochimique et biomoléculaire de la paroi des levures.(2011), Institut National Polytechnique (Toulouse) (Toulouse INP), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Centre National de la Recherche Scientifique (CNRS)-Université Toulouse 1 Capitole (UT1)-Université Toulouse - Jean Jaurès (UT2J)-Institut National Polytechnique (Toulouse) (Toulouse INP), Université Fédérale Toulouse Midi-Pyrénées-Centre National de la Recherche Scientifique (CNRS)-Université Toulouse 1 Capitole (UT1)-Université Toulouse - Jean Jaurès (UT2J), School of Medical Sciences - Institute of Medical Sciences, Laboratoire d'Ingénierie des Systèmes Biologiques et des Procédés ( LISBP ), Institut National de la Recherche Agronomique ( INRA ) -Institut National des Sciences Appliquées - Toulouse ( INSA Toulouse ), Institut National des Sciences Appliquées ( INSA ) -Institut National des Sciences Appliquées ( INSA ) -Centre National de la Recherche Scientifique ( CNRS ), Équipe NanoBioSystèmes ( LAAS-NBS ), Laboratoire d'analyse et d'architecture des systèmes [Toulouse] ( LAAS ), Centre National de la Recherche Scientifique ( CNRS ) -Université Toulouse III - Paul Sabatier ( UPS ), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Institut National des Sciences Appliquées - Toulouse ( INSA Toulouse ), Institut National des Sciences Appliquées ( INSA ) -Institut National des Sciences Appliquées ( INSA ) -Institut National Polytechnique [Toulouse] ( INP ) -Centre National de la Recherche Scientifique ( CNRS ) -Université Toulouse III - Paul Sabatier ( UPS ), Institut National des Sciences Appliquées ( INSA ) -Institut National des Sciences Appliquées ( INSA ) -Institut National Polytechnique [Toulouse] ( INP ), Institut National Polytechnique [Toulouse] ( INP ) -Institut National des Sciences Appliquées - Toulouse ( INSA Toulouse ), Institut National des Sciences Appliquées ( INSA ) -Institut National des Sciences Appliquées ( INSA ) -Université Paul Sabatier - Toulouse 3 ( UPS ) -Centre National de la Recherche Scientifique ( CNRS ) -Institut National Polytechnique [Toulouse] ( INP ) -Institut National des Sciences Appliquées - Toulouse ( INSA Toulouse ), Institut National des Sciences Appliquées ( INSA ) -Institut National des Sciences Appliquées ( INSA ) -Université Paul Sabatier - Toulouse 3 ( UPS ) -Centre National de la Recherche Scientifique ( CNRS ), Martin-Yken, Helene, Brown, Alistair J. P., Institut National de la Recherche Agronomique (INRA)-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Université de Toulouse (UT)-Institut National des Sciences Appliquées (INSA)-Université de Toulouse (UT)-Centre National de la Recherche Scientifique (CNRS), Université Toulouse Capitole (UT Capitole), Université de Toulouse (UT)-Université de Toulouse (UT)-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Université de Toulouse (UT)-Institut National des Sciences Appliquées (INSA)-Université Toulouse - Jean Jaurès (UT2J), Université de Toulouse (UT)-Université Toulouse III - Paul Sabatier (UT3), Université de Toulouse (UT)-Centre National de la Recherche Scientifique (CNRS)-Institut National Polytechnique (Toulouse) (Toulouse INP), Université de Toulouse (UT)-Université Toulouse Capitole (UT Capitole), and Université de Toulouse (UT)

Subjects
MAPK/ERK pathway, Programmed cell death, Osmotic shock, Mycology, Biology, Cell morphology, Microbiology, Cell wall, 03 medical and health sciences, Cell Wall, Osmotic Pressure, Virology, Candida albicans, medicine, Lactic Acid, Protein kinase A, [ SDV.MP.MYC ] Life Sciences [q-bio]/Microbiology and Parasitology/Mycology, [SDV.MP.MYC]Life Sciences [q-bio]/Microbiology and Parasitology/Mycology, 030304 developmental biology, 0303 health sciences, Osmotic concentration, 030306 microbiology, Elasticity, QR1-502, Culture Media, Enzymes, Cell biology, Mycologie, Glucose, Biochemistry, Shock (circulatory), medicine.symptom, Research Article
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.

Published
2015

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

Author

Nathalie Sieczkowski, Marion Schiavone, Etienne Dague, Mathieu Castex, Jean Marie François, Équipe Ingénierie pour les sciences du vivant (LAAS-ELIA), Laboratoire d'analyse et d'architecture des systèmes (LAAS), Université Toulouse - Jean Jaurès (UT2J)-Université Toulouse 1 Capitole (UT1), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Centre National de la Recherche Scientifique (CNRS)-Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Institut National Polytechnique (Toulouse) (Toulouse INP), Université Fédérale Toulouse Midi-Pyrénées-Université Toulouse - Jean Jaurès (UT2J)-Université Toulouse 1 Capitole (UT1), Université Fédérale Toulouse Midi-Pyrénées, Laboratoire d'Ingénierie des Systèmes Biologiques et des Procédés (LISBP), Institut National de la Recherche Agronomique (INRA)-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Centre National de la Recherche Scientifique (CNRS), Lallemand S.A.S., Region Midi Pyrenees [10051296], Lallemand SAS (ANRT), ANR-11-JSV5-0001,AFMYST,Etude Biophysique, biochimique et biomoléculaire de la paroi des levures.(2011), Institut National Polytechnique (Toulouse) (Toulouse INP), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Centre National de la Recherche Scientifique (CNRS)-Université Toulouse 1 Capitole (UT1)-Université Toulouse - Jean Jaurès (UT2J)-Institut National Polytechnique (Toulouse) (Toulouse INP), Université Fédérale Toulouse Midi-Pyrénées-Centre National de la Recherche Scientifique (CNRS)-Université Toulouse 1 Capitole (UT1)-Université Toulouse - Jean Jaurès (UT2J), Centre National de la Recherche Scientifique (CNRS)-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Institut National de la Recherche Agronomique (INRA), Université Toulouse Capitole (UT Capitole), Université de Toulouse (UT)-Université de Toulouse (UT)-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Université de Toulouse (UT)-Institut National des Sciences Appliquées (INSA)-Université Toulouse - Jean Jaurès (UT2J), Université de Toulouse (UT)-Université Toulouse III - Paul Sabatier (UT3), Université de Toulouse (UT)-Centre National de la Recherche Scientifique (CNRS)-Institut National Polytechnique (Toulouse) (Toulouse INP), Université de Toulouse (UT)-Université Toulouse Capitole (UT Capitole), Université de Toulouse (UT), and Institut National des Sciences Appliquées (INSA)-Université de Toulouse (UT)-Institut National des Sciences Appliquées (INSA)-Université de Toulouse (UT)-Centre National de la Recherche Scientifique (CNRS)

Subjects
Autolysis (biology), Surface Properties, Saccharomyces cerevisiae, Cell, chitin, Microscopy, Atomic Force, Applied Microbiology and Biotechnology, Microbiology, Biophysical Phenomena, Cell wall, autolysis process, 03 medical and health sciences, chemistry.chemical_compound, Industrial Microbiology, Chitin, Cell Wall, medicine, Cell Adhesion, [SDV.MP.MYC]Life Sciences [q-bio]/Microbiology and Parasitology/Mycology, 030304 developmental biology, Mannan, 0303 health sciences, atomic force microscopy, biology, 030306 microbiology, General Medicine, biology.organism_classification, Yeast, Yeast in winemaking, medicine.anatomical_structure, cell surface, chemistry, Biochemistry, β-glucans, Autolysis
Abstract

International audience; 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.

Published
2015

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

Author

Raphaël E. Duval, Anita Boisrame, Marion Schiavone, Cécile Formosa, Mathias L. Richard, Etienne Dague, Structure et Réactivité des Systèmes Moléculaires Complexes (SRSMC), Université de Lorraine (UL)-Centre National de la Recherche Scientifique (CNRS)-Institut de Chimie du CNRS (INC), Équipe Ingénierie pour les sciences du vivant (LAAS-ELIA), Laboratoire d'analyse et d'architecture des systèmes (LAAS), Université Toulouse - Jean Jaurès (UT2J)-Université Toulouse 1 Capitole (UT1), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Centre National de la Recherche Scientifique (CNRS)-Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Institut National Polytechnique (Toulouse) (Toulouse INP), Université Fédérale Toulouse Midi-Pyrénées-Université Toulouse - Jean Jaurès (UT2J)-Université Toulouse 1 Capitole (UT1), Université Fédérale Toulouse Midi-Pyrénées, Laboratoire d'Ingénierie des Systèmes Biologiques et des Procédés (LISBP), Institut National de la Recherche Agronomique (INRA)-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Centre National de la Recherche Scientifique (CNRS), MICrobiologie de l'ALImentation au Service de la Santé (MICALIS), Institut National de la Recherche Agronomique (INRA)-AgroParisTech, ANR [ANR-11-JSV5-001-01, SD 30024331], 'Direction Generale de l'Armement' (DGA), Lallemand SAS, Association Lorraine pour la Recherche et le Developpement de Composes Bioactifs, Université de Lorraine (UL)-Centre National de la Recherche Scientifique (CNRS), Institut National Polytechnique (Toulouse) (Toulouse INP), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Centre National de la Recherche Scientifique (CNRS)-Université Toulouse 1 Capitole (UT1)-Université Toulouse - Jean Jaurès (UT2J)-Institut National Polytechnique (Toulouse) (Toulouse INP), Université Fédérale Toulouse Midi-Pyrénées-Centre National de la Recherche Scientifique (CNRS)-Université Toulouse 1 Capitole (UT1)-Université Toulouse - Jean Jaurès (UT2J), Centre National de la Recherche Scientifique (CNRS)-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Institut National de la Recherche Agronomique (INRA), Institut de Chimie du CNRS (INC)-Université de Lorraine (UL)-Centre National de la Recherche Scientifique (CNRS), Université Toulouse Capitole (UT Capitole), Université de Toulouse (UT)-Université de Toulouse (UT)-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Université de Toulouse (UT)-Institut National des Sciences Appliquées (INSA)-Université Toulouse - Jean Jaurès (UT2J), Université de Toulouse (UT)-Université Toulouse III - Paul Sabatier (UT3), Université de Toulouse (UT)-Centre National de la Recherche Scientifique (CNRS)-Institut National Polytechnique (Toulouse) (Toulouse INP), Université de Toulouse (UT)-Université Toulouse Capitole (UT Capitole), Université de Toulouse (UT), and Institut National des Sciences Appliquées (INSA)-Université de Toulouse (UT)-Institut National des Sciences Appliquées (INSA)-Université de Toulouse (UT)-Centre National de la Recherche Scientifique (CNRS)

Subjects
Amyloid, Materials science, [PHYS.PHYS.PHYS-BIO-PH]Physics [physics]/Physics [physics]/Biological Physics [physics.bio-ph], Biomedical Engineering, Pharmaceutical Science, Medicine (miscellaneous), Virulence, Bioengineering, Context (language use), Ligands, Microscopy, Atomic Force, Opportunistic pathogen, Atomic force microscopy, Cell Wall, Candida albicans, Cell Adhesion, Concanavalin A, General Materials Science, Dimethylpolysiloxanes, [SDV.MP.MYC]Life Sciences [q-bio]/Microbiology and Parasitology/Mycology, biology, Cell Membrane, Temperature, Multiparametric AFM, biology.organism_classification, Adhesins, Corpus albicans, Fibronectins, Protein Structure, Tertiary, Cell biology, Bacterial adhesin, Biofilms, Nanoparticles, Molecular Medicine, [SDV.IB]Life Sciences [q-bio]/Bioengineering, Adhesive, Nanodomains, Peptides, Hydrophobic and Hydrophilic Interactions, Protein Binding
Abstract

International audience; 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. From the Clinical Editor: The present study reports on the successful atomic force microscopy-based mapping of the adhesive properties of C. albicans cells, an accomplishment that highlights the utility of AFM.

Published
2015

28. Generation of living cell arrays for atomic force microscopy studies

Author

Cécile Formosa, Etienne Dague, Raphaël E. Duval, Marion Schiavone, Flavien Pillet, Laurence Ressier, Équipe NanoBioSystèmes (LAAS-NBS), Laboratoire d'analyse et d'architecture des systèmes (LAAS), Institut National Polytechnique (Toulouse) (Toulouse INP), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Centre National de la Recherche Scientifique (CNRS)-Université Toulouse 1 Capitole (UT1)-Université Toulouse - Jean Jaurès (UT2J)-Institut National Polytechnique (Toulouse) (Toulouse INP), Université Fédérale Toulouse Midi-Pyrénées-Centre National de la Recherche Scientifique (CNRS)-Université Toulouse 1 Capitole (UT1)-Université Toulouse - Jean Jaurès (UT2J), Structure et Réactivité des Systèmes Moléculaires Complexes (SRSMC), Université de Lorraine (UL)-Centre National de la Recherche Scientifique (CNRS), Institut de pharmacologie et de biologie structurale (IPBS), Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Centre National de la Recherche Scientifique (CNRS), Laboratoire d'Ingénierie des Systèmes Biologiques et des Procédés (LISBP), Centre National de la Recherche Scientifique (CNRS)-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Institut National de la Recherche Agronomique (INRA), ABC Platform, Laboratoire de physique et chimie des nano-objets (LPCNO), Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Institut de Chimie de Toulouse (ICT-FR 2599), Institut de Recherche pour le Développement (IRD)-Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Centre National de la Recherche Scientifique (CNRS)-Institut National Polytechnique (Toulouse) (Toulouse INP), Université Fédérale Toulouse Midi-Pyrénées-Institut de Recherche pour le Développement (IRD)-Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Institut de Recherche sur les Systèmes Atomiques et Moléculaires Complexes (IRSAMC), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS), Structure et Réactivité des Systèmes Moléculaires Complexes ( SRSMC ), Université de Lorraine ( UL ) -Centre National de la Recherche Scientifique ( CNRS ), Laboratoire d'Ingénierie des Systèmes Biologiques et des Procédés ( LISBP ), Institut National de la Recherche Agronomique ( INRA ) -Institut National des Sciences Appliquées - Toulouse ( INSA Toulouse ), Institut National des Sciences Appliquées ( INSA ) -Institut National des Sciences Appliquées ( INSA ) -Centre National de la Recherche Scientifique ( CNRS ), Laboratoire de physique et chimie des nano-objets ( LPCNO ), Institut National des Sciences Appliquées - Toulouse ( INSA Toulouse ), Institut National des Sciences Appliquées ( INSA ) -Institut National des Sciences Appliquées ( INSA ) -Université Toulouse III - Paul Sabatier ( UPS ), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Centre National de la Recherche Scientifique ( CNRS ), Institut des Technologies Avancées en sciences du Vivant ( ITAV ), Université Toulouse III - Paul Sabatier ( UPS ), Université Toulouse - Jean Jaurès (UT2J)-Université Toulouse 1 Capitole (UT1), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Centre National de la Recherche Scientifique (CNRS)-Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Institut National Polytechnique (Toulouse) (Toulouse INP), Université Fédérale Toulouse Midi-Pyrénées-Université Toulouse - Jean Jaurès (UT2J)-Université Toulouse 1 Capitole (UT1), Université Fédérale Toulouse Midi-Pyrénées, Université de Lorraine (UL)-Centre National de la Recherche Scientifique (CNRS)-Institut de Chimie du CNRS (INC), Centre National de la Recherche Scientifique (CNRS)-Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées, Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche pour le Développement (IRD)-Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Institut de Chimie du CNRS (INC)-Institut National Polytechnique (Toulouse) (Toulouse INP), Université Fédérale Toulouse Midi-Pyrénées-Institut de Chimie du CNRS (INC)-Institut de Recherche sur les Systèmes Atomiques et Moléculaires Complexes (IRSAMC), Cancer Research UK (CRUK) [C20510/A12401], CRUK [C6946/A14492], Wellcome Trust [WT092096], CRUK program [C6/A11224], European Research Council, European Community Seventh Framework Programme [HEALTH-F2-2010-259893], University of Cambridge, UK, Université Toulouse Capitole (UT Capitole), Université de Toulouse (UT)-Université de Toulouse (UT)-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Université de Toulouse (UT)-Institut National des Sciences Appliquées (INSA)-Université Toulouse - Jean Jaurès (UT2J), Université de Toulouse (UT)-Université Toulouse III - Paul Sabatier (UT3), Université de Toulouse (UT)-Centre National de la Recherche Scientifique (CNRS)-Institut National Polytechnique (Toulouse) (Toulouse INP), Université de Toulouse (UT)-Université Toulouse Capitole (UT Capitole), Université de Toulouse (UT), Institut de Chimie du CNRS (INC)-Université de Lorraine (UL)-Centre National de la Recherche Scientifique (CNRS), Université de Toulouse (UT)-Université de Toulouse (UT)-Centre National de la Recherche Scientifique (CNRS), Institut National de la Recherche Agronomique (INRA)-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Université de Toulouse (UT)-Institut National des Sciences Appliquées (INSA)-Université de Toulouse (UT)-Centre National de la Recherche Scientifique (CNRS), Institut National des Sciences Appliquées (INSA)-Université de Toulouse (UT)-Institut National des Sciences Appliquées (INSA)-Université de Toulouse (UT)-Institut de Chimie de Toulouse (ICT), Université de Toulouse (UT)-Université de Toulouse (UT)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Institut National Polytechnique (Toulouse) (Toulouse INP), Université de Toulouse (UT)-Institut de Recherche pour le Développement (IRD)-Université Toulouse III - Paul Sabatier (UT3), Université de Toulouse (UT)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Institut National Polytechnique (Toulouse) (Toulouse INP), Université de Toulouse (UT)-Institut de Recherche sur les Systèmes Atomiques et Moléculaires Complexes (IRSAMC), and Université de Toulouse (UT)-Université de Toulouse (UT)-Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS)

Subjects
Materials science, Silicon, Capillary action, chemistry.chemical_element, Nanotechnology, macromolecular substances, Living cell, [ SDV.MP.BAC ] Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, Microscopy, Atomic Force, [ SDV.MP.VIR ] Life Sciences [q-bio]/Microbiology and Parasitology/Virology, General Biochemistry, Genetics and Molecular Biology, Soft lithography, chemistry.chemical_compound, [SDV.SP.MED]Life Sciences [q-bio]/Pharmaceutical sciences/Medication, Dimethylpolysiloxanes, ComputingMilieux_MISCELLANEOUS, Polydimethylsiloxane, Atomic force microscopy, [ SDV.SP.MED ] Life Sciences [q-bio]/Pharmaceutical sciences/Medication, fungi, PDMS stamp, technology, industry, and agriculture, Cells, Immobilized, [SDV.MP]Life Sciences [q-bio]/Microbiology and Parasitology, chemistry, Tissue Array Analysis, Biophysics, Adhesive
Abstract

International audience; 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.

Published
2015

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

Author

Hélène Martin-Yken, Etienne Dague, Cécile Formosa, Amélie Vax, Marion Schiavone, Jean Marie François, Laboratoire d'Ingénierie des Systèmes Biologiques et des Procédés (LISBP), Centre National de la Recherche Scientifique (CNRS)-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Institut National de la Recherche Agronomique (INRA), Lallemand S.A.S., Laboratoire d'analyse et d'architecture des systèmes (LAAS), Université Toulouse - Jean Jaurès (UT2J)-Université Toulouse 1 Capitole (UT1), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Centre National de la Recherche Scientifique (CNRS)-Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Institut National Polytechnique (Toulouse) (Toulouse INP), Université Fédérale Toulouse Midi-Pyrénées, Équipe NanoBioSystèmes (LAAS-NBS), Université Fédérale Toulouse Midi-Pyrénées-Université Toulouse - Jean Jaurès (UT2J)-Université Toulouse 1 Capitole (UT1), Institut National de la Recherche Agronomique (INRA)-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Université de Toulouse (UT)-Institut National des Sciences Appliquées (INSA)-Université de Toulouse (UT)-Centre National de la Recherche Scientifique (CNRS), Université Toulouse Capitole (UT Capitole), Université de Toulouse (UT)-Université de Toulouse (UT)-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Université de Toulouse (UT)-Institut National des Sciences Appliquées (INSA)-Université Toulouse - Jean Jaurès (UT2J), Université de Toulouse (UT)-Université Toulouse III - Paul Sabatier (UT3), Université de Toulouse (UT)-Centre National de la Recherche Scientifique (CNRS)-Institut National Polytechnique (Toulouse) (Toulouse INP), Université de Toulouse (UT), and Université de Toulouse (UT)-Université Toulouse Capitole (UT Capitole)

Subjects
[SDV]Life Sciences [q-bio], 6)-glucan, Antifungal drug, 3)-glucan, macromolecular substances, Polysaccharide, chitin, Applied Microbiology and Biotechnology, Microbiology, glucanases, Cell wall, SACCHAROMYCES-CEREVISIAE, chemistry.chemical_compound, Chitin, Stress, Physiological, Yeasts, Enzymatic hydrolysis, [SDV.IDA]Life Sciences [q-bio]/Food engineering, BIOSYNTHESIS, [SPI.GPROC]Engineering Sciences [physics]/Chemical and Process Engineering, Glucans, ALKALI-SOLUBLE GLUCAN, chemistry.chemical_classification, CANDIDA-ALBICANS, FUNGAL, ARCHITECTURE, biology, MEMBRANE-PROTEIN, Hydrolysis, Aspergillus niger, Reproducibility of Results, Fungal Polysaccharides, (1, General Medicine, biology.organism_classification, Yeast, INTEGRITY, carbohydrates (lipids), chemistry, Biochemistry, chitinase, Mutation, Chitinase, BETA-GLUCANS, biology.protein, cell wall
Abstract

International audience; 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 -(1,3) and -(1,6)-glucan in the total -glucan of the cell wall. In the first step, chitin and -(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--(1,3)-glucanase from Trichoderma species. This step was followed by addition of recombinant endo--(1,6)-glucanase from Trichoderma harzianum with -glucosidase from Aspergillus niger to hydrolyse the remaining -glucan. This latter component corresponded to a highly branched -(1,6)-glucan that contained about 75-80% of linear -(1,6)-glucose linked units as deduced from periodate oxidation. We validated this novel method by showing that the content of -(1,3), -(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 degrees 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 -(1,3)-glucan accompanied by an increase of -(1,6)-glucan, whereas ethanol stress had apparently no effect on yeast cell wall composition.

Published
2014

30. Uncovering by Atomic Force Microscopy of an original circular structure at the yeast cell surface in response to heat shock

Author

Cécile Formosa, Marion Schiavone, Hélène Martin-Yken, Jean Marie François, Etienne Dague, Flavien Pillet, Stéphane Lemonier, Laboratoire d'analyse et d'architecture des systèmes (LAAS), Université Toulouse - Jean Jaurès (UT2J)-Université Toulouse 1 Capitole (UT1), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Centre National de la Recherche Scientifique (CNRS)-Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Institut National Polytechnique (Toulouse) (Toulouse INP), Université Fédérale Toulouse Midi-Pyrénées, Institut des Technologies Avancées en sciences du Vivant (ITAV), Centre National de la Recherche Scientifique (CNRS)-Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées, Laboratoire d'Ingénierie des Systèmes Biologiques et des Procédés (LISBP), Centre National de la Recherche Scientifique (CNRS)-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Institut National de la Recherche Agronomique (INRA), Laboratoire de Chimie Physique Organique et Colloı̈dale, Centre National de la Recherche Scientifique (CNRS)-Université Henri Poincaré - Nancy 1 (UHP), Équipe NanoBioSystèmes (LAAS-NBS), Université Fédérale Toulouse Midi-Pyrénées-Université Toulouse - Jean Jaurès (UT2J)-Université Toulouse 1 Capitole (UT1), ANR young scientist program (AFMYST project) [ANR-11-JSV5-001-01, SD 30024331], Region Midi Pyrenees [10051296], 'Direction Generale de l'Armement' (DGA), Lallemand SAS, Université Toulouse Capitole (UT Capitole), Université de Toulouse (UT)-Université de Toulouse (UT)-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Université de Toulouse (UT)-Institut National des Sciences Appliquées (INSA)-Université Toulouse - Jean Jaurès (UT2J), Université de Toulouse (UT)-Université Toulouse III - Paul Sabatier (UT3), Université de Toulouse (UT)-Centre National de la Recherche Scientifique (CNRS)-Institut National Polytechnique (Toulouse) (Toulouse INP), Université de Toulouse (UT), Université Toulouse III - Paul Sabatier (UT3), Université de Toulouse (UT)-Université de Toulouse (UT)-Centre National de la Recherche Scientifique (CNRS), Institut National de la Recherche Agronomique (INRA)-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Université de Toulouse (UT)-Institut National des Sciences Appliquées (INSA)-Université de Toulouse (UT)-Centre National de la Recherche Scientifique (CNRS), Université Henri Poincaré - Nancy 1 (UHP)-Centre National de la Recherche Scientifique (CNRS), Université de Toulouse (UT)-Université Toulouse Capitole (UT Capitole), Lemonier, Stéphane, Francois, Jean-Marie, and Dague, Etienne

Subjects
Physiology, [SDV]Life Sciences [q-bio], Mutant, Chitin, Plant Science, Microscopy, Atomic Force, Structural Biology, [SDV.IDA]Life Sciences [q-bio]/Food engineering, bourgeonnement, 0303 health sciences, Budding, Agricultural and Biological Sciences(all), Cell wall, Atomic Force Microscopy (AFM), Heat-shock, Biomechanical Phenomena, Cell biology, paroi cellulaire, General Agricultural and Biological Sciences, Research Article, Signal Transduction, Biotechnology, choc, Saccharomyces cerevisiae, macromolecular substances, Biology, Cell Membrane Structures, General Biochemistry, Genetics and Molecular Biology, 03 medical and health sciences, Elastic Modulus, [SPI.GPROC]Engineering Sciences [physics]/Chemical and Process Engineering, Heat shock, Ecology, Evolution, Behavior and Systematics, Actin, 030304 developmental biology, Microbial Viability, 030306 microbiology, Biochemistry, Genetics and Molecular Biology(all), Trehalose, Cell Biology, Actin cytoskeleton, biology.organism_classification, Actins, Yeast, Microscopy, Fluorescence, Mutation, chitine, Heat-Shock Response, Developmental Biology
Abstract

International audience; 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 degrees C to 42 degrees 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 mu m 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 wsc1 Delta or bck1 Delta mutants that are defective in the CWI signaling pathway, nor in chs1 Delta, chs3 Delta and bni1 Delta 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.

Published
2014

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

Author

Cécile Formosa, Flavien Pillet, Marion Schiavone, Hélène Martin-Yken, Jean Marie François, Etienne Dague, Laboratoire d'Ingénierie des Systèmes Biologiques et des Procédés (LISBP), Institut National de la Recherche Agronomique (INRA)-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Centre National de la Recherche Scientifique (CNRS), Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées, Équipe NanoBioSystèmes (LAAS-NBS), Laboratoire d'analyse et d'architecture des systèmes (LAAS), Université Toulouse - Jean Jaurès (UT2J)-Université Toulouse 1 Capitole (UT1), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Centre National de la Recherche Scientifique (CNRS)-Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Institut National Polytechnique (Toulouse) (Toulouse INP), Université Fédérale Toulouse Midi-Pyrénées-Université Toulouse - Jean Jaurès (UT2J)-Université Toulouse 1 Capitole (UT1), Centre National de la Recherche Scientifique (CNRS)-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Institut National de la Recherche Agronomique (INRA), Institut National Polytechnique (Toulouse) (Toulouse INP), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Centre National de la Recherche Scientifique (CNRS)-Université Toulouse 1 Capitole (UT1)-Université Toulouse - Jean Jaurès (UT2J)-Institut National Polytechnique (Toulouse) (Toulouse INP), Université Fédérale Toulouse Midi-Pyrénées-Centre National de la Recherche Scientifique (CNRS)-Université Toulouse 1 Capitole (UT1)-Université Toulouse - Jean Jaurès (UT2J), Institut National des Sciences Appliquées (INSA)-Université de Toulouse (UT)-Institut National des Sciences Appliquées (INSA)-Université de Toulouse (UT)-Centre National de la Recherche Scientifique (CNRS), Université de Toulouse (UT), Université Toulouse Capitole (UT Capitole), Université de Toulouse (UT)-Université de Toulouse (UT)-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Université de Toulouse (UT)-Institut National des Sciences Appliquées (INSA)-Université Toulouse - Jean Jaurès (UT2J), Université de Toulouse (UT)-Université Toulouse III - Paul Sabatier (UT3), Université de Toulouse (UT)-Centre National de la Recherche Scientifique (CNRS)-Institut National Polytechnique (Toulouse) (Toulouse INP), and Université de Toulouse (UT)-Université Toulouse Capitole (UT Capitole)

Subjects
integrity pathway, architecture, actin cytoskeleton, [SDV]Life Sciences [q-bio], Saccharomyces cerevisiae, Cell, cellular sensor, Microscopy, Atomic Force, Models, Biological, Cell wall, 03 medical and health sciences, stress, Stress, Physiological, Organelle, Genetics, medicine, saccharomyces cerevisiae, antifungal agent, caspofungin, 030304 developmental biology, protein kinase c, chemistry.chemical_classification, 0303 health sciences, atomic force microscopy, biology, 030306 microbiology, Biomolecule, General Medicine, bakers yeast, Cells, Immobilized, biology.organism_classification, Actin cytoskeleton, Yeast, Cell biology, Biomechanical Phenomena, surface sensor, adhesion, medicine.anatomical_structure, cell surface, chemistry, cell wall, candida albicans, microbial cell, Biogenesis
Abstract

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.

Published
2013

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