ABSTRACT
Functional organic nanomaterials are nowadays largely spread in the field of nanomedicine. In situ modulation of their morphology is thus expected to considerably impact their interactions with the surroundings. In this context, photoswitchable nanoparticles that are manufactured, amenable to extensive disassembling upon illumination in the visible, and reversible reshaping under UV exposure. Such reversibility turns to be strongly impaired for photochromic nanoparticles in close contact with a substrate. In situ atomic force microscopy investigations at the nanoscale actually reveal progressive disintegration of the organic nanoparticles under successive UV-vis cycles of irradiation, in the absence of intrinsic elastic forces. These results point out the dramatic interactions exerted by surfaces on the cohesion of non-covalently bonded organic nanoparticles. They invite to harness such systems, often used as biomarkers, to also serve as photoactivatable drug delivery nanocarriers.
ABSTRACT
Preclinical data acquired for human muscle stem (hMuStem) cells indicate their great repair capacity in the context of muscle injury. However, their clinical potential is limited by their moderate ability to survive after transplantation. To overcome these limitations, their encapsulation within protective environment would be beneficial. In this study, tunable calcium-alginate hydrogels obtained through molding method using external or internal gelation were investigated as a new strategy for hMuStem cell encapsulation. The mechanical properties of these hydrogels were characterized in their fully hydrated state by compression experiments using Atomic Force Microscopy. Measured elastic moduli strongly depended on the gelation mode and calcium/alginate concentrations. Values ranged from 1 to 12.5 kPa and 3.9 to 25 kPa were obtained for hydrogels prepared following internal and external gelation, respectively. Also, differences in mechanical properties of hydrogels resulted from their internal organization, with an isotropic structure for internal gelation, while external mode led to anisotropic one. It was further shown that viability, morphological and myogenic differentiation characteristics of hMuStem cells incorporated within alginate hydrogels were preserved after their release. These results highlight that hMuStem cells encapsulated in calcium-alginate hydrogels maintain their functionality, thus allowing to develop muscle regeneration protocols to improve their therapeutic efficacy.
Subject(s)
Alginates , Cell Differentiation , Hydrogels , Stem Cells , Stress, Mechanical , Alginates/chemistry , Humans , Hydrogels/chemistry , Stem Cells/cytology , Cell Differentiation/drug effects , Cell Survival/drug effects , Elastic Modulus , Tissue Scaffolds/chemistryABSTRACT
Diabolican is an exopolysaccharide (EPS) produced by Vibrio diabolicus HE800, a mesophilic bacterium firstly isolated from a deep-sea hydrothermal field. Its glycosaminoglycan (GAG)-like structure, consisting of a tetrasaccharide repeating unit composed of two aminosugars (N-acetyl-glucosamine and N-acetyl-galactosamine) and two glucuronic acid units, suggested to subject it to regioselective sulfation processes, in order to obtain some sulfated derivatives potentially acting as GAG mimics. To this aim, a multi-step semi-synthetic approach, relying upon tailored sequence of regioselective protection, sulfation and deprotection steps, was employed in this work. The chemical structure of the obtained sulfated diabolican derivatives was characterized by a multi-technique analytic approach, in order to define both degree of sulfation (DS) and sulfation pattern within the polysaccharide repeating unit, above all. Finally, binding affinity for some growth factors relevant for biomedical applications was measured for both starting diabolican and sulfated derivatives thereof. Collected data suggested that sulfation pattern could be a key structural element for the selective interaction with signaling proteins not only in the case of native GAGs, as already known, but also for GAG-like structures obtained by regioselective sulfation of naturally unsulfated polysaccharides.
Subject(s)
Polysaccharides , Sulfates , Sulfates/chemistry , Polysaccharides/chemistry , Glycosaminoglycans , Oligosaccharides , Intercellular Signaling Peptides and ProteinsABSTRACT
Glycosaminoglycans (GAGs) are essential constituents of the cell surface and extracellular matrix, where they are involved in several cellular processes through their interactions with various proteins. For successful tissue regeneration, developing an appropriate matrix supporting biological activities of cells in a similar manner than GAGs remains still challenging. In this context, this study aims to design a thermosensitive polysaccharide that could further be used as hydrogel for tissue engineering applications. For this purpose, infernan, a marine bacterial exopolysaccharide (EPS) endowed with GAG-mimetic properties was grafted with a thermosensitive polymer, poly(N-isopropylacrylamide) (pNIPAM). Eight grafted polysaccharides were obtained by varying EPS/pNIPAM molar ratio and the molecular weight of pNIPAM. Their physicochemical characteristics and their thermosensitive properties were determined using a multi-technique, experimental approach. In parallel, molecular dynamics and Monte Carlo simulations were applied at two different scales to elucidate, respectively, the molecular conformation of grafted infernan chain and their ability to form an infinite network undergoing a sol-gel transition near the percolation, a necessary condition in hydrogel formation. It comes out from this study that thermosensitive infernan was successfully developed and its potential use in tissue regeneration as a hydrogel scaffold will further be assessed.
Subject(s)
Glycosaminoglycans , Hydrogels , Temperature , Hydrogels/chemistry , PolysaccharidesABSTRACT
Sulfated glycosaminoglycans (GAGs) are fundamental constituents of both the cell surface and extracellular matrix. By playing a key role in cell-cell and cell-matrix interactions, GAGs are involved in many physiological and pathological processes. To design GAG mimetics with similar therapeutic potential as the natural ones, the specific structural features, among them sulfate content, sulfation pattern, and chain length, should be considered. In the present study, we describe a sulfation method based on microwave radiation to obtain highly sulfated derivatives as GAG mimetics. The starting low-molecular-weight (LMW) derivative was prepared from the infernan exopolysaccharide, a highly branched naturally slightly sulfated heteropolysaccharide synthesized by the deep-sea hydrothermal vent bacterium Alteromonas infernus. LMW highly sulfated infernan derivatives obtained by conventional heating sulfation have already been shown to display GAG-mimetic properties. Here, the potential of microwave-assisted sulfation versus that of the conventional method to obtain GAG mimetics was explored. Structural analysis by NMR revealed that highly sulfated derivatives from the two methods shared similar structural features, emphasizing that microwave-assisted sulfation with a 12-fold shorter reaction time is as efficient as the classical one.
Subject(s)
Glycosaminoglycans , Microwaves , Glycosaminoglycans/chemistry , Sulfates/chemistry , Magnetic Resonance Spectroscopy , Extracellular Matrix/metabolismABSTRACT
In the field of tissue engineering, in order to restore tissue functionality hydrogels that closely mimic biological and mechanical properties of the extracellular matrix are intensely developed. Mechanical properties including relaxation of the surrounding microenvironment regulate essential cellular processes. However, the mechanical properties of engineered hydrogels are particularly complex since they involve not only a nonlinear elastic behavior but also time-dependent responses. An accurate determination of these properties at microscale, i.e. as probed by cells, becomes an essential step to further design hydrogel-based biomaterials able to induce specific cellular responses. Atomic Force Microscopy (AFM) with contact sizes of the order of few micrometers constitutes an appropriate technique to determine the origin of relaxation mechanisms occurring in hydrogels. In the present study, AFM force relaxation experiments are conducted on chemically and physically crosslinked hydrogels respectively based on a synthetic polymer, polyacrylamide and a natural polymer, a bacterial exopolysaccharide infernan, produced by the deep-sea hydrothermal vent bacterium, Alteromonas infernus. Two distinct relaxation mechanisms are clearly evidenced depending on the nature of hydrogel network crosslinks. Chemically crosslinked hydrogel exhibits poroelastic relaxations, whereas physically crosslinked hydrogel shows time-dependent responses arising from viscoelastic effects. In addition, two relaxation processes are revealed in ionic physical hydrogel originating from chain rearrangement and breaking/reforming of the ionic crosslinks. The effect of the ionic strength on both the long-term elastic modulus and relaxation times of physical hydrogels was also shown. These findings highlight that physical hydrogels with well-defined time-dependent mechanical properties could be tuned for an optimized response of cells.
Subject(s)
Hydrogels , Tissue Engineering , Biocompatible Materials , Elastic Modulus , Extracellular Matrix , Hydrogels/chemistry , Tissue Engineering/methodsABSTRACT
With the increasing need for hydrogels with tunable properties for specific biomedical applications, a complete understanding of the structure-function relationship of polymers used for hydrogel development remains crucial for their optimal use. In the present study, by combining experimental and theoretical approaches, the structure-function relationship of a bacterial exopolysaccharide, infernan, displaying both glycosaminoglycan-mimetic and gelling properties, was investigated at molecular and microscopic levels. Atomic force microscopy (AFM) experiments and molecular dynamics simulations were applied to determine the persistence length of individual infernan chains before studying their association induced by calcium. Infernan-based microgels were then produced using microfluidics and their mechanical properties were characterized by AFM methods. The mechanical properties of EPS/calcium microgels were finely tuned by varying the crosslinking density of their network, either by calcium or EPS concentrations. The obtained set of viscoelastic microgels with different elastic modulus values opens several possibilities for their applications in tissue engineering.
Subject(s)
Microgels , Calcium , Hydrogels , Microscopy, Atomic Force , Tissue Engineering/methodsABSTRACT
Bone Morphogenetic Protein (BMP-2) is an osteoinductive growth factor clinically used for bone regeneration. Tuneable sustained strategies for BMP-2 delivery are intensely developed to avoid severe complications related to supraphysiological doses applied. To address this issue, we investigated the ability of the bacterial exopolysaccharide (EPS) called Infernan produced by the deep-sea hydrothermal vent bacterium Alteromonas infernus, exhibiting both glycosaminoglycan-mimetic and physical gelling properties, to efficiently bind and release the bioactive BMP-2. Two delivery systems were designed based on BMP-2 retention in either single or complex EPS-based microgels, both manufactured using a microfluidic approach. BMP-2 release kinetics were highly influenced by the ionic strength, affecting both microgel stability and growth factor/EPS binding, appearing essential for BMP-2 bioactivity. The osteogenic activity of human bone-marrow derived mesenchymal stem cells studied in vitro emphasized that Infernan microgels constitute a promising platform for BMP-2 delivery for further in vivo bone repair.
Subject(s)
Microgels , Bone Morphogenetic Protein 2/chemistry , Bone Morphogenetic Protein 2/pharmacology , Bone Morphogenetic Proteins , Bone Regeneration , Glycosaminoglycans , Humans , OsteogenesisABSTRACT
The exopolysaccharide Infernan, from the bacterial strain GY785, has a complex repeating unit of nine monosaccharides established on a double-layer of sidechains. A cluster of uronic and sulfated monosaccharides confers to Infernan functional and biological activities. We characterized the 3-dimensional structures and dynamics along Molecular Dynamics trajectories and clustered the conformations in extended two-fold and five-fold helical structures. The electrostatic potential distribution over all the structures revealed negatively charged cavities explored for Ca2+ binding through quantum chemistry computation. The transposition of the model of Ca2+complexation indicates that the five-fold helices are the most favourable for interactions. The ribbon-like shape of two-fold helices brings neighbouring chains in proximity without steric clashes. The cavity chelating the Ca2+ of one chain is completed throughout the interaction of a sulfate group from the neighbouring chain. The resulting is a 'junction zone' based on unique chain-chain interactions governed by a heterotypic binding mode.
Subject(s)
Alteromonas/chemistry , Calcium/chemistry , Polysaccharides, Bacterial/chemistry , Models, Molecular , Molecular Conformation , Molecular Dynamics Simulation , Molecular Structure , Monosaccharides/chemistry , Quantum Theory , Sulfates/chemistryABSTRACT
Secreted proteins are key players in fungal physiology and cell protection against external stressing agents and antifungals. Oak stress-induced protein 1 (OSIP1) is a fungal-specific protein with unknown function. By using Podospora anserina and Phanerochaete chrysosporium as models, we combined both in vivo functional approaches and biophysical characterization of OSIP1 recombinant protein. The P. anserina OSIP1Δ mutant showed an increased sensitivity to the antifungal caspofungin compared to the wild type. This correlated with the production of a weakened extracellular exopolysaccharide/protein matrix (ECM). Since the recombinant OSIP1 from P. chrysosporium self-assembled as fibers and was capable of gelation, it is likely that OSIP1 is linked to ECM formation that acts as a physical barrier preventing drug toxicity. Moreover, compared to the wild type, the OSIP1Δ mutant was more sensitive to oak extractives including chaotropic phenols and benzenes. It exhibited a strongly modified secretome pattern and an increased production of proteins associated to the cell-wall integrity signalling pathway, when grown on oak sawdust. This demonstrates that OSIP1 has also an important role in fungal resistance to extractive-induced stress.
Subject(s)
Phanerochaete , Podospora , Antifungal Agents/pharmacology , Fungal Proteins/genetics , Fungal Proteins/metabolism , Phanerochaete/metabolism , Signal TransductionABSTRACT
Highly concentrated dispersions of fluorescent organic nanoparticles (FONs), broadly used for optical tracking, bioimaging and drug delivery monitoring, are obtained using a newly designed micromixer chamber involving high impacting flows. Fine size tuning and narrow size distributions are easily obtained by varying independently the flow rates of the injected fluids and the concentration of the dye stock solution. The flash nanoprecipitation process employed herein is successfully applied to the fabrication of bicomposite FONs designed to allow energy transfer. Considerable enhancement of the emission signal of the energy acceptors is promoted and its origin is found to result from polarity rather than steric effects. Finally, we exploit the high spatial confinement encountered in FONs and their ability to encapsulate hydrophobic photosensitizers to induce photocrosslinking. An increase in the photocrosslinked FON stiffness is evidenced by measuring the elastic modulus at the nanoscale using atomic force microscopy. These results pave the way toward the straightforward fabrication of multifunctional and mechanically photoswitchable FONs, opening novel opportunities in sensing, multimodal imaging, and theranostics.
Subject(s)
Fluorescent Dyes/chemistry , Microfluidic Analytical Techniques , Nanoparticles/chemistry , Fluorescent Dyes/chemical synthesis , Molecular Structure , Particle Size , Photochemical Processes , Surface PropertiesABSTRACT
Polysaccharides of marine origin are gaining interest as biomaterial components. Bacteria derived from deep-sea hydrothermal vents can produce sulfated exopolysaccharides (EPS), which can influence cell behavior. The use of such polysaccharides as components of organic, collagen fibril-based coatings on biomaterial surfaces remains unexplored. In this study, collagen fibril coatings enriched with HE800 and GY785 EPS derivatives were deposited on titanium alloy (Ti6Al4V) scaffolds produced by rapid prototyping and subjected to physicochemical and cell biological characterization. Coatings were formed by a self-assembly process whereby polysaccharides were added to acidic collagen molecule solution, followed by neutralization to induced self-assembly of collagen fibrils. Fibril formation resulted in collagen hydrogel formation. Hydrogels formed directly on Ti6Al4V surfaces, and fibrils adsorbed onto the surface. Scanning electron microscopy (SEM) analysis of collagen fibril coatings revealed association of polysaccharides with fibrils. Cell biological characterization revealed good cell adhesion and growth on bare Ti6Al4V surfaces, as well as coatings of collagen fibrils only and collagen fibrils enhanced with HE800 and GY785 EPS derivatives. Hence, the use of both EPS derivatives as coating components is feasible. Further work should focus on cell differentiation.
ABSTRACT
Articular cartilage is an avascular, non-innervated connective tissue with limited ability to regenerate. Articular degenerative processes arising from trauma, inflammation or due to aging are thus irreversible and may induce the loss of the joint function. To repair cartilaginous defects, tissue engineering approaches are under intense development. Association of cells and signalling proteins, such as growth factors, with biocompatible hydrogel matrix may lead to the regeneration of the healthy tissue. One current strategy to enhance both growth factor bioactivity and bioavailability is based on the delivery of these signalling proteins in microcarriers. In this context, the aim of the present study was to develop microcarriers by encapsulating Transforming Growth Factor-ß1 (TGF-ß1) into microparticles based on marine exopolysaccharide (EPS), namely GY785 EPS, for further applications in cartilage engineering. Using a capillary microfluidic approach, two microcarriers were prepared. The growth factor was either encapsulated directly within the microparticles based on slightly sulphated derivative or complexed firstly with the highly sulphated derivative before being incorporated within the microparticles. TGF-ß1 release, studied under in vitro model conditions, revealed that the majority of the growth factor was retained inside the microparticles. Bioactivity of released TGF-ß1 was particularly enhanced in the presence of highly sulphated derivative. It comes out from this study that GY785 EPS based microcarriers may constitute TGF-ß1 reservoirs spatially retaining the growth factor for a variety of tissue engineering applications and in particular cartilage regeneration, where the growth factor needs to remain in the target location long enough to induce robust regenerative responses.
Subject(s)
Alteromonas/chemistry , Drug Carriers/chemistry , Polysaccharides/chemistry , Transforming Growth Factor beta1/administration & dosage , Biological Availability , Cartilage, Articular/drug effects , Cartilage, Articular/physiology , Cell Line , Delayed-Action Preparations/administration & dosage , Delayed-Action Preparations/pharmacokinetics , Drug Carriers/isolation & purification , Drug Compounding/methods , Drug Implants , Drug Liberation , Humans , Hydrothermal Vents/microbiology , Microfluidics , Polysaccharides/isolation & purification , Regeneration/drug effects , Tissue Scaffolds/chemistry , Transforming Growth Factor beta1/pharmacokineticsABSTRACT
Sulfated polysaccharides, such as glycosaminoglycans (GAG) regulate various biological activities through their interactions with growth factors. Investigating these interactions becomes the key to understand the structure-function relationship of GAG. Highly sulfated derivatives prepared from the marine GY785 exopolysaccharide (EPS) produced by the deep-sea hydrothermal vent bacterium Alteromonas infernus have previously shown to stimulate the chondrogenic differentiation of mesenchymal stem cells in the presence of Transforming Growth Factor-ß1 (TGF-ß1). Here, the interactions between the GAG-mimetic GY785 EPS derivatives and TGF-ß1 were investigated by Atomic Force Microscopy (AFM). The affinity between slightly sulfated or highly sulfated derivatives and TGF-ß1 was explored by AFM imaging and single-molecule force spectroscopy experiments. The number of measured interactions and the interaction strength were both higher for highly sulfated derivative compared to the slightly sulfated one. These results clearly emphasize the involvement of sulfate groups in the protein binding and open new ways to tune cellular processes by designing macromolecules with adjustable sulfate charge density.
ABSTRACT
During the past decades, atomic force microscopy (AFM) has emerged as a powerful tool in microbiology. Although most of the works concerned bacteria, AFM also permitted major breakthroughs in the understanding of physiology and pathogenic mechanisms of some fungal species associated with cystic fibrosis. Complementary to electron microscopies, AFM offers unprecedented insights to visualize the cell wall architecture and components through three-dimensional imaging with nanometer resolution and to follow their dynamic changes during cell growth and division or following the exposure to drugs and chemicals. Besides imaging, force spectroscopy with piconewton sensitivity provides a direct means to decipher the forces governing cell-cell and cell-substrate interactions, but also to quantify specific and non-specific interactions between cell surface components at the single-molecule level. This nanotool explores new ways for a better understanding of the structures and functions of the cell surface components and therefore may be useful to elucidate the role of these components in the host-pathogen interactions as well as in the complex interplay between bacteria and fungi in the lung microbiome.
Subject(s)
Cystic Fibrosis/microbiology , Fungi/pathogenicity , Lung Diseases, Fungal/microbiology , Microscopy, Atomic Force/methods , Cystic Fibrosis/complications , Host-Pathogen Interactions , HumansABSTRACT
BACKGROUND: Flavin-dependent monooxygenases are involved in key biological processes as they catalyze a wide variety of chemo-, regio- and enantioselective oxygenation reactions. Flavoprotein monooxygenases are frequently encountered in micro-organisms, most of which require further functional and biocatalytic assessment. Here we investigated the function of the AbMak1 gene, which encodes a group A flavin monooxygenase in the plant pathogenic fungus Alternaria brassicicola, by generating a deficient mutant and examining its phenotype. RESULTS: Functional analysis indicates that the AbMak1 protein is involved in cell wall biogenesis and influences the melanization process. We documented a significant decrease in melanin content in the Δabmak1 strain compared to the wild-type and complemented strains. We investigated the cell wall morphology and physical properties in the wild-type and transformants using electron and atomic force microscopy. These approaches confirmed the aberrant morphology of the conidial wall structure in the Δabmak1 strain which had an impact on hydrophilic adhesion and conidial surface stiffness. However, there was no significant impairment in growth, conidia formation, pathogenicity or susceptibility to various environmental stresses in the Δabmak1 strain. CONCLUSION: This study sheds new light on the function of a fungal flavin-dependent monooxygenase, which plays an important role in melanization.
ABSTRACT
Molecular recognition events driven by protein-carbohydrate interactions play fundamental roles in various physiological and pathological processes in living organisms, including cohesion inside tissues, innate immune response, cancer cell metastasis, and infections. Unlike widely investigated carbohydrates, detailed knowledge of both the spatial organization of specific lectins and their identification on cell surfaces remains an essential prerequisite for the understanding of pathogen adhesion to host tissues and subsequent infection prevention. In this study, the spatially resolved localization, identification, and quantification of multiple carbohydrate-binding sites are directly revealed on the surface of fungal pathogen Aspergillus fumigatus. Nanoscale reconstructed mapping from several recognition maps, corresponding each to a unique specific interaction probed by single-molecule force spectroscopy, shows the distribution of carbohydrate-binding sites on the pathogen surface. The identified binding sites are then blocked with the appropriate carbohydrate, attesting the possibility to control lectin-mediated host-pathogen interactions. Germination markedly affects both the spatial distribution of carbohydrate-binding sites, mostly expressed at the apex of hyphae, and the identity of the predominant ones, which depend on the location on germ tubes. These insights clearly open exciting avenues in nanomedicine to control host-pathogen interactions with the development of vaccines or inhibitory drugs that preferentially target the identified carbohydrate-binding sites.
ABSTRACT
Assembly of biopolymers into microgels is an elegant strategy for bioencapsulation with various potential biomedical applications. Such biocompatible and biodegradable microassemblies are developed not only to protect the encapsulated molecule but also to ensure its sustained local delivery. The present study describes the fabrication of microassemblies from a marine HE800 exopolysaccharide (EPS), which displays a glycosaminoglycan (GAG)-like structure and biological properties. HE800 EPS was assembled, through physical cross-linking with divalent ions, into microgel particles and microfibers using microfluidics. The microparticle morphology was highly affected by the polysaccharide concentration and its molecular weight. A model protein, namely Bovine Serum Albumin (BSA) was subsequently encapsulated within HE800 microparticles in one-step process using microfluidics. The protein release was tuned by the microparticle morphology with a lower protein amount released from the most homogeneous structures. Our findings demonstrate the high potential of HE800 EPS based microassemblies as innovative protein microcarriers for further biomedical applications.
Subject(s)
Drug Carriers/chemistry , Gels/chemistry , Glycosaminoglycans/chemistry , Polysaccharides, Bacterial/chemistry , Serum Albumin, Bovine/administration & dosage , Vibrio/chemistry , Animals , Cattle , Microfluidic Analytical Techniques , Polychaeta/microbiology , Serum Albumin, Bovine/chemistryABSTRACT
Alginate microgels are widely used as delivery systems in food, cosmetics, and pharmaceutical industries for encapsulation and sustained release of hydrophilic compounds and cells. However, the encapsulation of lipophilic molecules inside these microgels remains a great challenge because of the complex oil-core matrix required. The present study describes an original two-step approach allowing the easy encapsulation of several oil microdroplets within alginate microgels. In the first step, stable oil microdroplets were formed by preparing an oil-in-water (O/W) Pickering emulsion. To stabilize this emulsion, we used two solid particles, namely the cotton cellulose nanocrystals (CNC) and calcium carbonate (CaCO3). It was observed that the surface of the oil microdroplets formed was totally covered by a CNC layer, whereas CaCO3 particles were adsorbed onto the cellulose layer. This solid CNC shell efficiently stabilized the oil microdroplets, preventing them from undesired coalescence. In the second step, oil microdroplets resulting from the Pickering emulsion were encapsulated within alginate microgels using microfluidics. Precisely, the outermost layer of oil microdroplets composed of CaCO3 particles was used to initiate alginate gelation inside the microfluidic device, following the internal gelation mode. The released Ca2+ ions induced the gel formation through physical cross-linking with alginate molecules. This innovative and easy to carry out two-step approach was successfully developed to fabricate monodisperse alginate microgels of 85 µm in diameter containing around 12 oil microdroplets of 15 µm in diameter. These new oil-core alginate microgels represent an attractive system for encapsulation of lipophilic compounds such as vitamins, aroma compounds or anticancer drugs that could be applied in various domains including food, cosmetics, and medical applications.
ABSTRACT
Scedosporium boydii is a pathogenic filamentous fungus that causes a wide range of human infections, notably respiratory infections in patients with cystic fibrosis. The development of new therapeutic strategies targeting S. boydii necessitates a better understanding of the physiology of this fungus and the identification of new molecular targets. In this work, we studied the conidium-to-germ tube transition using a variety of techniques including scanning and transmission electron microscopy, atomic force microscopy, two-phase partitioning, microelectrophoresis and cationized ferritin labeling, chemical force spectroscopy, lectin labeling, and nanoLC-MS/MS for cell wall GPI-anchored protein analysis. We demonstrated that the cell wall undergoes structural changes with germination accompanied with a lower hydrophobicity, electrostatic charge and binding capacity to cationized ferritin. Changes during germination also included a higher accessibility of some cell wall polysaccharides to lectins and less CH3/CH3 interactions (hydrophobic adhesion forces mainly due to glycoproteins). We also extracted and identified 20 GPI-anchored proteins from the cell wall of S. boydii, among which one was detected only in the conidial wall extract and 12 only in the mycelial wall extract. The identified sequences belonged to protein families involved in virulence in other fungi like Gelp/Gasp, Crhp, Bglp/Bgtp families and a superoxide dismutase. These results highlighted the cell wall remodeling during germination in S. boydii with the identification of a substantial number of cell wall GPI-anchored conidial or hyphal specific proteins, which provides a basis to investigate the role of these molecules in the host-pathogen interaction and fungal virulence.