Combinaison d’approches biochimique, moléculaire et biophysique afin d’étudier l’impact de la souche et du procédé industriel sur la composition et l’architecture moléculaire de la paroi cellulaire de la levure

Due to increasing yeast biomass production resulting from the expansion of the Biorefinery as an alternative to petrol-based energy, the yeast cell wall is receiving an increasing interest as an added value product targeting agro-nutrition markets, such as in animal nutrition and in wine for its probiotic and sorption properties. The purpose of this thesis was therefore to combine DNA microarrays, biochemical and biophysical approaches in order to investigate the relationships between these parameters as well as to highlight the impact of strains, growth conditions and processes on the cell wall composition and biophysical properties. To achieve this objective, an acido-enzymatic method was developed to specifically quantify each of the four components of the yeast cell wall, namely mannan, chitin, β-1,3-glucan and β-1,6-glucan. This method was validated on mutant strains and allowed highlighten various stresses effects. Then, the use of atomic force microscopy (AFM) has allowed investigating the same strains and four strains used in industrial fermentation. They demonstrated distinct nanomechanical and adhesive properties, due to differences in their cell wall structure and composition. In the last part, the effects of the autolysis and fluid-bed drying processes are presented. This industrial process does not change the composition of the cell wall but induces a modification in topography and surface properties of the cell. Moreover, using AFM we imaged on S. cerevisiae cell surface highly adhesive patches forming nanodomains.L’intérêt pour la paroi de la levure s’est accru récemment par l’explosion des activités de bioraffineries augmentant la production de biomasse, et par le besoin de valoriser cette biomasse dans d’autres débouchés comme en nutrition animale et en œnologie pour leurs propriétés probiotiques et de sorption. Le but de cette thèse était de combiner des approches biochimiques, biophysiques et les puces à ADN afin d'étudier les relations entre ces paramètres ainsi que de mettre en évidence l'impact des souches, des conditions de croissance et des procédés sur la composition et les propriétés biophysiques de la paroi cellulaire. Une méthode acido-enzymatique a été développée pour quantifier spécifiquement chacun des quatre composants de la paroi cellulaire de la levure, à savoir les mannanes, la chitine, les β-1,3-glucanes et les β-1,6-glucanes. Cette méthode a été validée sur des souches mutantes et a permis d’évaluer les effets de divers stress. Ultérieurement, l'utilisation de la microscopie à force atomique (AFM) a permis l'étude des mêmes souches et de quatre souches utilisées dans la fermentation industrielle. Ils ont démontré des propriétés nanomécaniques et adhésives distinctes, en raison de différences dans la composition et la structure de la paroi cellulaire. Dans la dernière partie, les effets du procédé d’autolyse et du séchage à lit fluidisé sont présentés. Ce procédé industriel ne modifie pas la composition de la paroi cellulaire, mais induit une modification de la topographie et des propriétés de surface de la cellule. En outre, en utilisant l'AFM nous avons imagés sur S. cerevisiae des patchs hautement adhésifs formant des nanodomaines à la surface de la cellule

FLO11, a Developmental Gene Conferring Impressive Adaptive Plasticity to the Yeast Saccharomyces cerevisiae

International audienceThe 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

The dark side of the wall: atomic force microscopy revelations on drug resistance and adhesion

Ce poster a été présenté à l'oral.International audienc

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

International audienceNon-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

Stress, Drug Resistance, Adhesion: the dark side of the wall.

International audienc

The Dark Side of the Wall: Atomic Force Microscopy Revelations on Drug Resistance and Adhesion

International audienceThe cell wall of yeast and fungi plays a crucial role in the way these cells sense, respond and adapt to environmental perturbations. Using recent Atomic Force Microscopy technological developments, the biophysical consequences of different stresses on the major human fungal pathogen, Candida albicans, were imaged and measured. Morphological changes were characterized at the nanoscale, including surface roughness, elasticity and adhesive properties. Exposure to the antifungal Caspofungin was shown to cause a deep cell wall remodeling with major modifications of chitin and beta-glucan content. Remarkably, a low dose of Caspofungin (i.e., 0.5 × MIC) provoked a strong expression of adhesive proteins on the cell surface of C. albicans, a side effect highly relevant considering its wide spread medical use. Moreover, Single Molecule Force Spectroscopy (SMFS) experiments by AFM allowed us to visualize the organization of these adhesins, to map them on the cell surface and to quantify the adhesion forces, including on cells undergoing mophogenetic differentiation. Combined with molecular biology tools, this approach enabled us to unravel the particular contribution of previously uncharacterized proteins (PGA22 and PGA59) to C. albicans adhesion mechanism. In addition, functionalizing the AFM tip with antibodies allows following the appearance of specific proteins, while precisely mapping them at the cell surface and even measuring the time scale of their progression through the cell wall. The example of Hwp1 appearance on geminating hyphal tubes will be illustrated

Drug Resistance and Adhesion: a Closer Look at the Dark Side of the Wall.

International audienc

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

International audienceThe 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

Drug resistance and adhesion: a closer look into the dark side of the wall.

International audienceDrug resistance and adhesion:a closer look into the dark side of the wall.Hélène Martin-Yken, Cécile Formosa, Marion Schiavone, François Jean-Marie and Etienne Dague. Stress conditions and presence of drugs or antifungal compounds induce significant changes in yeast and fungi cell wall composition. The molecular architecture of the cell wall is also modified in these conditions, particularly the nature, repartition and attachment of cell wall proteins to the cell surface. Atomic Force Microscopy (AFM) is a powerful tool for studying the morphology, nanomechanical and adhesive properties of live microorganisms under physiological conditions. We took advantage of the most recent AFM technological developments to image and measure the biophysical consequences of these various stresses on C. albicans cell morphology at the nanoscale, focusing on changes in cell surface aspect and characteristics: roughness, elasticity, and adhesive properties. We notably explored the effects of the antifungal drug caspofungin used in human health1. Our investigation revealed a deep cell wall remodeling induced by this drug, evidenced by a dramatic increase in chitin and decrease in beta-glucan content. Remarkably, a low dose of caspofungin (i.e., 0.5 × MIC) also leads to a characteristic expression of adhesins on C. albicans cell surface. Moreover, in order to get a better understanding of C. albicans adhesion mechanisms, we performed Single Molecule Force Spectroscopy (SMFS) experiments to visualize the organization of adhesins and quantify the adhesion forces. We were able to map the adhesins at the cell surface and to distinguish between hydrophobic and specific affinity interactions2. Combined with molecular biology tools, this approach also enabled us to further unravel the particular contribution of previously uncharacterized proteins (PGA22 and PGA59) to C. albicans adhesion mechanism3. In the future we will focus on new approaches using Single Cell Force Spectroscopy with AFM and Optical Tweezers as well as Sheer-Stress Flow Chamber to study adhesion from the molecule scale to the population scale. 1. Formosa C. et al., 2013. Nanoscale effects of caspofungin against two yeast species; Saccharomyces cerevisiae and Candida albicans, Antimicrobial Agents and Chemotherapy, 57. 3498-3506.2. Formosa C. et al., Multiparametric Imaging of Adhesive Nanodomains at the Surface of Candida Albicans by Atomic Force Microscopy. Nanomedicine NBM, 11, 57-65.3. Cabral V. et al., Targeted changes of the cell wall proteome influence Candida albicans ability to form single- and multi-strain biofilms. PloS Pathogens

Stress, Drug resistance and Adhesion: a closer look at the dark side of the wall

International audienceStress conditions and antifungal drugs induce significant changes in the cell wall composition of yeasts and fungi. They cause modifications of the cell wall molecular architecture, including nature, repartition and attachment of cell wall proteins to the cell surface. Atomic Force Microscopy (AFM) is a powerful tool for studying the morphology, nanomechanical and adhesive properties of live microorganisms under physiological conditions. We imaged and measured the biophysical consequences of various stresses on both S. cerevisiae and C. albicans cell morphology at the nanoscale, focusing on changes in cell surface aspect and characteristics: roughness, elasticity, and adhesive properties. AFM allowed us to unravel the morphologic effect on yeast cells of Heat Shock, Osmotic Shock, exposure to toxins and drugs. Moreover, using Single Molecule Force Spectroscopy (SMFS) we can now explore the organization of adhesins, map them on the cell surface and quantify their adhesion forces. Altogether, our studies establish the great interest of AFM to explore molecular mechanisms occurring on the cell surface of live fungal cells