Les Erick Drs

Matthieu Dufourc

Sabine Chavent

Derrick Hediger

Vishu Robinson

Iñaki Aimanianda

Guijarro

Isabelle Marcotte

Merci D'

Merci Aux

Drs Isabel Valsecchi

Iñaki Guijarro

Drs Alexandre Alanio

Guilhem Janbon

Sarah Wong

Thierry Fontaine

Anne Beauvais

Prs Françoise Dromer

ainsi que le Pr

Titre : Caractérisation structurale de l'architecture des parois fongiques par RMN du solide

Résumé :

Il existe une grande variété de champignons pathogènes humains qui sont à l'origine de maladies bénignes à mortelles. La plupart du temps, ces infections sont associées à d'autres pathologies ou traitements médicaux comme l'asthmes, les leucémies, les transplantations d'organes, le SIDA ou les traitement immunosuppresseurs à base de corticostéroïdes. Malgré le nombre important de décès et le nombre grandissant d'occurrences des mycoses sévères à travers le monde, les infections fongiques sont encore négligées par les autorités sanitaires.

Parmi ces pathogènes fongiques, le champignon filamenteux Aspergillus fumigatus est un des pathogènes principaux du système respiratoire. L'aspergillose, dont les taux de d'infection et de mortalité demeurent élevés, devient un enjeu de santé publique. Les spores d'A. fumigatus sont entourés d'une paroi, essentielle pour leur croissance et leur permettant de résister face au système immunitaire de l'hôte. Cette paroi est composée d'un réseau de polysaccharides recouvert d'un pigment appelé DHN-mélanine et d'une couche de protéines appelées hydrophobines. Ce projet a pour but d'établir l'architecture structurale de la paroi des spores d'A. fumigatus à l'échelle atomique en utilisant la RMN du solide (SSNMR) en rotation à l'angle magique (MAS). D'un autre côté, Cryptococcus neoformans est l'agent pathogène responsable de la cryptococcose ; une mycose affectant le système nerveux central. Cette maladie fongique est, encore de nos jours, une cause significative de mortalité à travers le monde puisqu'elle entraîne de graves symptômes tels que la méningo-encéphalite ; particulièrement fréquente chez les patients déjà infectés par le VIH. C. neoformans se présente sous la forme d'une cellule encapsulée de 5 à 7 µm de diamètre entourée d'une paroi et d'une capsule. Cette paroi, rigide, est liée à la membrane plasmique et composée de polymères d'α-glucan, de β-glucan, de chitine et de chitosan. De plus, la capsule de C. neoformans est majoritairement composée de carbohydrates tels que le glucuronoxylomannan (GXM) (jusqu'à 90%) ou le glucuronoxylomannogalactan (GXMGal) mais aussi de mannoprotéines et de lipides. Le but de ce projet de thèse est d'identifier les différents composants de la paroi mais aussi de la capsule de C. neoformans par SSNMR et d'établir l'architecture de ces deux entités. Un des aspects de ce projet est aussi d'explorer les possibilités et les limitations des méthodes de détection proton en RMN couplée à un MAS élevé (100 kHz) comme outil d'analyse des parois fongiques.

En résumé, puisque la RMN du solide est une méthode de spectroscopie non invasive, nous avons appliqué ce type d'analyses dans le cadre de l'étude de l'architecture moléculaire de systèmes complexes (parois fongiques, capsules, …) dans des conditions aussi proches que possible de l'état natif des cellules. Pendant ces trois années de thèse, nous avons mis en place une méthodologie robuste et rapide permettant d'étudier la composition complexe des structures externes présentes dans les cellules fongiques ainsi que leur architecture au sein des cellules entières. De plus, puisque dans le cadre des infections microbiennes la pathogénicité du microbe repose souvent sur les structures externes des cellules infectieuses, les résultats obtenus au court de cette thèse, apportant une meilleure compréhension de l'organisation cellulaire d'A. fumigatus et C. neoformans, pourraient ainsi être utilisés dans le cadre du développement et de la mise en place de nouvelles stratégies thérapeutiques afin de combattre plus efficacement ces infections fongiques.

Mots clés : Aspergillus fumigatus, Cryptococcus neoformans, paroi cellulaire, RMN

Title: Structural characterization of fungal cell walls architecture by solid-state NMR

Abstract:

There is a broad range of fungal pathogen infecting humans and causing diseases that can be from mild to lethal.

Severe fungal infections are due to opportunistic pathogens that infect immunosuppressed individuals and are most of the time associated with other diseases or medical conditions such as asthma, leukemia, organ transplants, AIDS or immunosuppressive corticosteroid therapies. Despite the number of deaths and the increase in severe mycosis, fungal infections remain neglected by public health authorities.

Among fungal pathogens, the filamentous fungus Aspergillus fumigatus is one of the major pathogen of the respiratory system. Aspergillosis, displaying both high incidence and mortality rates, is becoming a massive public health issue. The spores of Aspergillus fumigatus are surrounded by a cell wall, essential for their growth and allowing them to resist against host defense mechanisms. The cell wall is composed of a set of polysaccharides covered by the DHN-melanin pigment and a layer of proteins called hydrophobins. In this project, we aimed at investigated the structural architecture of Aspergillus fumigatus cell wall at atomic resolution using MAS SSNMR spectroscopy.

In another hand, Cryptococcus neoformans is the etiological agent of cryptococcosis; which consists in mycosis affecting the central nervous system. This fungal disease remains a significant cause of mortality worldwide by leading to severe symptoms such as meningoencephalitis -especially for immunocompromised individuals suffering from AIDS. C. neoformans results in encapsulated particles with a size of 5-7µm with a two-layers external structure composed of a cell wall and a capsule. The cell wall, rigid, is bounded to the plasma membrane and composed of polymers of α-glucan, β-glucan, chitin and chitosan [START_REF] O'meara | The Cryptococcus Neoformans Capsule: A Sword and a Shield[END_REF] . Then, the capsule of C. neoformans is mainly composed of carbohydrates such as glucuronoxylomannan (GXM) (up to 90%), glucuronoxylomannogalactan (GXMGal), mannoproteins and lipids. During this thesis project, we aimed at identifying the different components of C.neoformans cell wall and capsule by SSNMR and to investigate the architecture of these two layers. Part of this project was also the exploration of possibilities and limits of 1H detection methods at fast MAS regime (100 kHz) as the tool to analyze intact cell walls.

To sum up, as the solid-state NMR is a non-destructive spectroscopy, we applied this method to the study of the molecular architecture of complex systems (cell wall, capsule…) in cellular conditions -as close as possible to the native state. During these three years, we set up a robust methodology allowing studying the complex composition of fungal external structures as well as their architecture in the cell context. Finally, because in microbial infections, the pathogenesis often relies on the external structures of the pathogen, all these results could give a better comprehension of the A. fumigatus and C. neoformans cell organization that may help to find new therapeutic strategies to fight, more efficiently, against fungal infections. [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C/ [START_REF] Fisher | Emerging Fungal Threats to Animal, Plant and Ecosystem Health[END_REF] N-labeled PFA-fixed dormant, swollen, germinating and Δags conidia analysis 1.2.2 [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C/ [START_REF] Fisher | Emerging Fungal Threats to Animal, Plant and Ecosystem Health[END_REF] N-labeled PFA-fixed dormant conidia 1.2.3 [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C/ [START_REF] Fisher | Emerging Fungal Threats to Animal, Plant and Ecosystem Health[END_REF] N-labeled ΔrodA and Δpksp dormant conidia 1.2.4 [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C/ [START_REF] Fisher | Emerging Fungal Threats to Animal, Plant and Ecosystem Health[END_REF] N-labeled dormant conidia 1.2.5 Unlabeled PFA-fixed dormant conidia 1.2.6 [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C/ [START_REF] Fisher | Emerging Fungal Threats to Animal, Plant and Ecosystem Health[END_REF] N-labeled PFA-fixed white dormant conidia 1.2.7 [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C/ [START_REF] Fisher | Emerging Fungal Threats to Animal, Plant and Ecosystem Health[END_REF] N-labeled melanin ghost 1.2.8 DHN-melanin 1.2.9 Hydrophobins analyses 1.3 Wide-angle X-ray scattering (WAXS) experiments. [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C/ [START_REF] Fisher | Emerging Fungal Threats to Animal, Plant and Ecosystem Health[END_REF] N-labeled capsular extract 1.2 Solid-state NMR analysis 1.2.1 The cell wall of [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C/ [START_REF] Fisher | Emerging Fungal Threats to Animal, Plant and Ecosystem Health[END_REF] N-labeled C. neoformans H99, Δuxs1, Δugd1 and Δuge1Δcap59 mutants 1.2.2 [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C/ [START_REF] Fisher | Emerging Fungal Threats to Animal, Plant and Ecosystem Health[END_REF] N acapsular C. neoformans Δcap59 mutants 1.2.3 [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C/ [START_REF] Fisher | Emerging Fungal Threats to Animal, Plant and Ecosystem Health[END_REF] N clinical isolates 1.2.4 1 H-detected spectra of C.neoformans H99 The fungal kingdom is one of the largest and most complex groups of eukaryotic organisms on Earth. In 1991, Hawksworth et al. estimated the total number of fungal species to be around 1.5 million with only 7% fully described [START_REF] Hawksworth | The Fungal Dimension of Biodiversity: Magnitude, Significance, and Conservation[END_REF] . This estimation was wildly accepted for the past decades until the publication of new statistics in 2017 showing the number of fungal species up to more than 3 millions [START_REF] Hawksworth | Fungal Diversity Revisited: 2.2 to 3.8 Million Species[END_REF] . More recently, based on new estimation methods, Wu et al. hypothesized the number of fungal species to be about 12 millions [START_REF] Wu | Current Insights into Fungal Species Diversity and Perspective on Naming the Environmental DNA Sequences of Fungi[END_REF] .

Key words: Aspergillus fumigatus, Cryptococcus neoformans, cell wall, NMR

Unité de Recherche

ASPERGILLUS FUMIGATUS CONIDIAL CELL WALL

CRYPTOCOCCUS NEOFORMANS EXTERNAL STRUCTURES

INDEX OF TABLES

This widespread "Third Kingdom" includes species that cannot be incorporated into the dichotomy vegetal versus animal, and represents thus a diverse group of heterotrophic eukaryotes characterized by their chitinous cell wall, their absence of phagotrophic mechanisms and their cellular organization encompassing unicellular organisms as well as macroscopic filamentous structures [START_REF] Naranjo-Ortiz | Fungal Evolution: Diversity, Taxonomy and Phylogeny of the Fungi[END_REF] . Despite a wide variety of morphologies, reproduction types, metabolisms and interactions with living organisms (e.g., mutualism, parasitism…), fungi are divided into nine lineages: Opisthosporidia, Chytridiomycota, Neocallimastigomycota, Blastocladiomycota, Zoopagomycota, Mucoromycota, Glomeromycota, Ascomycota and Basidiomycota according, essentially, to their genetic characteristics.

Fungal species are omnipresent on Earth: they can be found from stratosphere [START_REF] Wainwright | Microorganisms Cultured from Stratospheric Air Samples Obtained at 41 Km[END_REF] to deep ocean floors [START_REF] Oren | Fungal Life in the Dead Sea[END_REF] , from Atacama rocks desert [START_REF] Gonçalves | Fungi Associated with Rocks of the Atacama Desert: Taxonomy, Distribution, Diversity, Ecology and Bioprospection for Bioactive Compounds[END_REF] to Antarctic glaciers [START_REF] Freeman | Evidence That Chytrids Dominate Fungal Communities in High-Elevation Soils[END_REF] , from insects guts [START_REF] Blackwell | Made for Each Other: Ascomycete Yeasts and Insects[END_REF] to inorganic marine sediments [START_REF] Nagahama | Molecular Evidence That Deep-Branching Fungi Are Major Fungal Components in Deep-Sea Methane Cold-Seep Sediments[END_REF] and they are basically shaping the world. Furthermore, these organisms are one of the most important actors of bio-geochemistry by regulating carbon and nitrogen cycles as well as other bio-elements (e.g., phosphorus…).

Added to their crucial role in nutrients cycling, fungi also exhibit numerous other qualities: they help to maintain the plant biomass through symbiosis mechanisms (i.e., mycorrhizae), they have medical interests as the first antibiotic was isolated by A. Fleming from Penicillium spp. in 1929 [START_REF] Fleming | On the Antibacterial Action of Cultures of a Penicillium, with Special Reference to Their Use in the Isolation of B. Influenzae[END_REF][START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] , they were used since ages in fermentation processes applied to food and beverages and they are now about to be used in biotechnology field.

Indeed, in 2019, Hyde et al., proposed fifty new ways of using the amazing potential of fungi in industry, ranging from new strategies to fight against human and plant diseases to solving ecological issues by improving the production of biofuels from fungi [START_REF] Hyde | The Amazing Potential of Fungi: 50 Ways We Can Exploit Fungi Industrially[END_REF] .

On the other hand, even though fungi exhibit many benefic applications, they also play infectious roles for many living organisms. Fungal infections of plants by Magnaporthe oryzae, Botrytis cinerea or Puccinia spp. are responsible for the loss of one third of the five most important global crops annually (i.e., rice, wheat, corn, potatoes, and soybean) leading to economical loss and impacting global economy [START_REF] Fisher | Emerging Fungal Threats to Animal, Plant and Ecosystem Health[END_REF] . Additionally, fungal diseases lead to strong impact into the biodiversity when they infect animals such as bats [START_REF] Lorch | Experimental Infection of Bats with Geomyces Destructans Causes White-Nose Syndrome[END_REF] (infected by Geomyces destructans), frogs species [START_REF] Blaustein | Interspecific Variation in Susceptibility of Frog Tadpoles to the Pathogenic Fungus Batrachochytrium Dendrobatidis[END_REF] (infected by Batrachochytrium dendrobatidis) or even coral reef (infected by Aspergillus spp.) [START_REF] Soler-Hurtado | Aspergillus Sydowii and Other Potential Fungal Pathogens in Gorgonian Octocorals of the Ecuadorian Pacific[END_REF] . Fungal infections also impact humans: in the past decade, these diseases caused approximately 1.6 millions deaths annually out of one billion people infected [START_REF] Brown | Hidden Killers: Human Fungal Infections[END_REF] . On the cause of difficulties for accessing to anti-fungal drugs and to the inequalities in health-care systems worldwide, the three most common fungal diseases (candidiasis, cryptococcosis and aspergillosis) kill more people than the death caused by HIV [START_REF] Tudela | Recovery from Serious Fungal Infections Should Be Realisable for Everyone[END_REF] .

Human fungal infections

Fungal infections affecting humans worldwide encompass benign nail, skin or hair infections (over 1 billion people) [START_REF] Havlickova | Epidemiological Trends in Skin Mycoses Worldwide (Mycoses[END_REF] and mucosal candidiasis (10's of millions) [START_REF] Bongomin | Global and Multi-National Prevalence of Fungal Diseases-Estimate Precision[END_REF] (Table 1). On the other hand, mycosis also lead to severe symptoms (150 million people) -such as meningitis -often strongly impacting the life, sometimes lethal [START_REF] Fausto | The Still Underestimated Problem of Fungal Diseases Worldwide[END_REF] .

Severe fungal infections occur mainly in critically ill patients (i.e., patients suffering from burns or immunosuppressive diseases such as AIDS [START_REF] Bongomin | Global and Multi-National Prevalence of Fungal Diseases-Estimate Precision[END_REF] ): they are majorly caused by two yeasts (Candida spp. and Cryptococcus spp.) and one filamentous fungus (Aspergillus spp).

This increased rate of invasive fungal infections is often associated with the use of immunosuppressive agents, the poor knowledge on fungal pathobiology, the lack of rapid and sensitive diagnostic tests or the development of fungal resistance towards current clinically inuse antifungals [START_REF] Brown | Hidden Killers: Human Fungal Infections[END_REF] .

Candidiasis

Caused by Candida spp, a major cause of morbidity and mortality worldwide, and the main causative agent in systemic fungal infections. Among the Candida species, Candida albicans is the most common to cause infections in humans.

Coccidioidomycosis

Endemic to Southwestern United States and Central and South America, caused by

Coccidioides immitis and Coccidioides posadasii.

Cryptococcosis

Worldwide occurrence, caused by Cryptococcus neoformans. Affects immunocompromised hosts, and is a major cause of HIV-related deaths. One noticeable outbreak in Vancouver Islands of infection by C. gattii in immunocompetent patients.

Dermatophytosis

Worldwide, Trichophyton rubrum and Trychophyton interdigitale have been described as the most common to cause dermatophytosis, but many other species contribute. The most frequent type of superficial mycosis in humans, attacking skin and nails.

Histoplasmosis

Mostly endemic; Histoplasma capsulatum is the etiologic agent, seen mostly in immunocompromised individuals

Paracoccidiodomycosis

Endemic to Latin America, caused by thermodimorphic fungi of the Paracoccidioides species, is the most prevalent systemic mycosis in Latin America.

Pneumocystis pneumonia

Worldwide, caused by Pneumocystis jiroveci, affects immunosuppressed patients, such as cancer patients receiving chemotherapy or HIV patients Despite this wide range of diseases and their high infection rate, antifungal drugs approved for humans remains quite insufficient: clinically in-use antifungal drugs for human treatment were characterized 18-years ago. Moreover, probably due to the imbalance between both infection and mortality rates in the context of fungal infections, the development of new therapy is still neglected [START_REF] Coelho | Cryptococcal Therapies and Drug Targets: The Old, the New and the Promising[END_REF] . Indeed, as compared to 1.6 million annual deaths associated with tuberculosis worldwide, cryptococcal meningitis represent "only" 429,000 deaths annually (Fig. 1) resulting in an unequal distribution of global research and development funds: 17%

for tuberculosis versus less than 0.5% for cryptococcosis [START_REF] Moran | Neglected Disease Research and Development : The Ebola Effect[END_REF] . Investing more into antifungal drug development appears as a crucial point for the coming years and to do so, new structural insights about fungal architecture are required in order to develop new therapeutic strategies.

Fig. 1:

The global burden of fungal diseases.

(extracted from https://www.aspergillus.org.uk/)

Fungal cell wall

In the context of microbial infections, the pathogenesis often relies on the external structures of the pathogen such as its cell wall [START_REF] Doering | How Sweet It Is ! Cell Wall Biogenesis and Polysaccharide Capsule Formation in Cryptococcus Neoformans[END_REF] . This organelle located outside of the plasma membrane plays a crucial role into the relationships between the fungus and its environment.

Cell walls are a common determinant of the fungal kingdom and they display a global conserved organization of a set of polysaccharides, glycoproteins and often of a pigment called melanin (Fig. 2). The fungal cell wall is majorly composed of glucans (accounting for 50-60% of the cell wall dry weight). Among the glucans, the β-1,3-D-glucans are the most abundant ones and act as a scaffold to which other components of the cell wall are linked. Chitin, a polymer of β-1,4-N-acetylglucosamine, is produced according to the morphological state of the fungus and varies with the species: 4-5% in yeast and asexual spores and 10-15% in filamentous fungi. Moreover, the cell wall is composed of a strong proportion of proteins (20-50 % of the cell wall dry weight) often associated to carbohydrates (O-or N-glycosylation) resulting in glycoproteins [START_REF] Pardo | A Proteomic Approach for the Study of Saccharomyces Cerevisiae Cell Wall Biogenesis[END_REF] . Furthermore, the cell wall of some fungi such as C. neoformans and A.

fumigatus can be composed of a pigment called melanin. This pigment, negatively charged and hydrophobic, is produced through two different pathways resulting from the monomers 1,8-dihydroxynaphthalene (DHN) or from L-3,4-dihydroxyphenylalanine (L-DOPA), which polymerize to form pigments of high molecular weights.

The cell wall is then a structurally complex organelle among which each component plays a role: glucans and chitin act as an exoskeleton that maintains the integrity of the cell by resisting against the internal hydrostatic pressure. Then, the cell wall associated glycoproteins display many roles such as the protection of the fungus against external components or the adhesion of the fungus onto external surfaces. Such a wide diversity of key functions makes the cell wall a crucial target in the context of developing therapeutic strategies.

Aspergillus fumigatus and invasive aspergillosis

Aspergillosis encompasses a broad range of diseases ranging from non-invasive allergic bronchopulmonary aspergillosis (ABPA) to invasive aspergillosis (IA). Despite new advances in the diagnosis and the treatment of IA, morbidity rate due to invasive aspergillosis remains significantly high -especially for immunocompromised patients: more than 250 000 cases of invasive aspergillosis per year associated with a 30-80% mortality rate [START_REF] Bongomin | Global and Multi-National Prevalence of Fungal Diseases-Estimate Precision[END_REF]28 .

Among 250 species of the genus Aspergillus about 20 species are pathogenic [START_REF] Balajee | Aspergillus Species Identification in the Clinical Setting[END_REF] ; A.

fumigatus is the most commonly isolated from patients suffering from IA (57 %), followed by A. flavus (12 %), A. terreus (12 %) and A. niger (10 %) [START_REF] Cadena | Invasive Aspergillosis: Current Strategies for Diagnosis and Management[END_REF] . Thus, getting structural insights about A. fumigatus architecture seems to be a good starting point for a better understanding of IA and its potential new treatments.

Aspergillus fumigatus is a saprophytic filamentous fungus belonging to the lineage of Ascomycota that is characterized by the presence of a reproductive structure called ascus containing asexual spores called conidia [START_REF] Sugui | Aspergillus Fumigatus and Related Species[END_REF] (Fig. 3). Due to its clinical relevance, this mold, found in different ecological niches worldwide, has been widely studied over the past years in order to better understand how its cell wall, the first component to interact with the host system, mediates host/pathogen interactions. The composition of the cell wall of A. fumigatus varies through the germination process (Fig. 4) but the core remains mainly composed of polysaccharides such as β-1,3glucan cross-linked to α-1,3-glucan [START_REF] Beauvais | Aspergillus Cell Wall and Biofilm[END_REF] , galactomannan (β-1,5-galacto-α-1,2/-α-1,6-mannan) [START_REF] Latge | Galactofuranose Containing Molecules in Aspergillus Fumigatus[END_REF] , galactosaminogalactan (α-1-4 linked galactose and α-1-4 linked N-acetylgalactosamine residues, which is germination specific polysaccharide) [START_REF] Fontaine | Galactosaminogalactan, a New Immunosuppressive Polysaccharide of Aspergillus Fumigatus[END_REF] and β-1,3-1,4-glucans -which is a unique feature and was described for the first time in fungi by Fontaine et al. in 2000 [START_REF] Fontaine | Molecular Organization of the Alkali-Insoluble Fraction of Aspergillus Fumigatus Cell Wall[END_REF] . Then, chitin -which can represent up to 20% of the cell wall dry weight -is a polymer of β-1,4-Nacetylglucosamine produced extracellularly by plasma membrane-bound chitin synthases, and was shown to fold back on itself to form anti-parallel chains with intra-chain hydrogen bonds [START_REF] Fernandes | The Importance of Subclasses of Chitin Synthase Enzymes with Myosin-like Domains for the Fitness of Fungi[END_REF] . Moreover, chitin and β-1,3-glucan are responsible for the rigidity of the cell wall.

Furthermore, the conidial cell wall surface exhibits two specific features: the DHN-melanin and proteins assembled as rodlets structures that prevent the spore to be recognized by the host immune system [START_REF] Thau | Rodletless Mutants of Aspergillus Fumigatus[END_REF] . These two external layers are disintegrated once the conidia starts to swell and germinate [START_REF] Valsecchi | Assembly and Disassembly of Aspergillus Fumigatus Conidial Rodlets[END_REF] (Fig. 4).

As they are involved in the pathogenicity of A. fumigatus, the DHN-melanin and the rodlets structures seem to be one of the most interesting features of the dormant spore. First, the DHN-melanin is synthetized from 1,8-dihydroxynapthalen, which might be condensed via intracellular pathways into a polymer; this structure remains unknown.

Then, the external layer is made up from the self-assembly of the protein RodA. This amyloid protein belongs to the hydrophobin family (7 members identified in A. fumigatus) characterized by their low molecular weight (< 20k Da), hydrophobicity, variable primary sequences and their common and highly conserved tridimensional structure with a βstructured core stabilized by four disulfide bridges [START_REF] Sunde | Structural Analysis of Hydrophobins[END_REF] . This rodlets layer displays high surfactant activity through its self-assembly as an amphipathic monolayer at hydrophilichydrophobic interfaces. This rodlets layer allows the spores to breach the air/water interface and facilitates the conidial dissemination into the air [START_REF] Wessels | Unique Fungal Proteins[END_REF] . On the other hand, these structures are also involved into the host/pathogen interactions as they act as a shield preventing the recognition of β-1,3-glucan by the human immune receptor Dectin-1 [START_REF] Aimanianda | Surface Hydrophobin Prevents Immune Recognition of Airborne Fungal Spores[END_REF] . Thus, these rodlets confer immunological inertness to the conidia and are an interesting object for structural study.

Fig. 4:

The cell wall of Aspergillus fumigatus in conidia and hyphae.

(extracted from Garcia-Rubio et al., 2020).

Cryptococcus neoformans and cryptococcosis

Cryptococcal infections occurs primarily by inhalation of the propagules leading to pneumonia-like symptoms, and in the worst case, meningitis when they affect the central nervous system [START_REF] Kwon-Chung | Cryptococcus Neoformans and Cryptococcus Gattii, the Etiologic Agents of Cryptococcosis[END_REF] . Cryptococcosis is closely related to the AIDS pandemic that occurred in the mid-1980s as 80% of cryptococcosis cases worldwide occurred in HIV-positive individuals [START_REF] Hajjeh | Cryptococcosis: Population-Based Multistate Active Surveillance and Risk Factors in Human Immunodeficiency Virus-Infected Persons[END_REF] .

Recent estimations show cryptococcal mycosis to infects 278,000 individuals, among which 223,100 patients developing cryptococcal meningitis [START_REF] Rajasingham | Global Burden of Disease of HIV-Associated Cryptococcal Meningitis: An Updated Analysis[END_REF] . Worldwide, cryptococcal meningitis account for 181,100 deaths annually, including 15% of AIDS-related deaths making cryptococcosis the second most common cause of AIDS-related death after tuberculosis [START_REF] Rajasingham | Global Burden of Disease of HIV-Associated Cryptococcal Meningitis: An Updated Analysis[END_REF] .

This high mortality rate among HIV-positive patients could be explained by a partial inefficiency of current cryptococcal treatments -such as amphotericin B-based strategies [START_REF] Shourian | Resistance and Tolerance to Cryptococcal Infection: An Intricate Balance That Controls the Development of Disease[END_REF] .

The commonly known etiologic agents of cryptococcosis are Cryptococcus neoformans and

Cryptococcus gattii causing, respectively, 95% and 5% of infection [START_REF] Kwon-Chung | Cryptococcus Neoformans and Cryptococcus Gattii, the Etiologic Agents of Cryptococcosis[END_REF] .

The global architecture of C. neoformans, the most commonly found infectious species, was characterized by electron-microscopy-based studies, revealing this yeast to forms encapsulated particles with a size of 5-7 µm [START_REF] Reese | Loss of Cell Wall Alpha(1-3) Glucan Affects Cryptococcus Neoformans from Ultrastructure to Virulence[END_REF] and with an external structure consisting of two layers: the cell wall and the capsule (Fig. 5).

Fig. 5:

The cell wall of Cryptococcus neoformans in conidia and hyphae.

(extracted from Garcia-Rubio et al., 2020)

First, using GC/MS and HPLC analysis, the cell wall of C. neoformans was shown to be mostly composed of polymers of α-1,3-glucan, β-1,3-glucan, chitin and chitosan (deacetylated chitin) [START_REF] O'meara | The Cryptococcus Neoformans Capsule: A Sword and a Shield[END_REF][START_REF] Doering | How Sweet It Is ! Cell Wall Biogenesis and Polysaccharide Capsule Formation in Cryptococcus Neoformans[END_REF] . Architecturally, β-1,3-glucan and chitin fold as fibers parallel to the plasma membrane and the outer layer is described to be composed of β-and α-glucans [START_REF] O'meara | The Cryptococcus Neoformans Capsule: A Sword and a Shield[END_REF][START_REF] Doering | How Sweet It Is ! Cell Wall Biogenesis and Polysaccharide Capsule Formation in Cryptococcus Neoformans[END_REF][START_REF] Sakaguchi | Ultrastructural Study of Cryptococcus Neoformans by Quick-Freezing and Deep-Etching Method[END_REF] .

Additionally, glycoproteins, lipids and melanin pigments have also been described in C.

neoformans, in the outer parts of the cell wall [START_REF] Doering | How Sweet It Is ! Cell Wall Biogenesis and Polysaccharide Capsule Formation in Cryptococcus Neoformans[END_REF] . However, it was established that the composition of the external layers of C. neoformans as well as and the abundance of structural components may strongly vary with the isolates, or the culture method used [START_REF] Doering | How Sweet It Is ! Cell Wall Biogenesis and Polysaccharide Capsule Formation in Cryptococcus Neoformans[END_REF][START_REF] Mukaremera | Titan Cell Production in Cryptococcus Neoformans Reshapes the Cell Wall and Capsule Composition during Infection[END_REF] .

The capsule of C. neoformans is anchored to the cell wall via α-1,3-glucan [START_REF] Reese | Cell Wall α-1,3-Glucan Is Required to Anchor the Cryptococcus Neoformans Capsule[END_REF] and forms a mesh of long fibers. By interfering with the synthesis of the capsule, the whole cell cycle and infectivity of the pathogen can be abolished; thus, this layer seems to play an essential role into the virulence of the yeast, making it a potential target for drug development [START_REF] Doering | How Sweet It Is ! Cell Wall Biogenesis and Polysaccharide Capsule Formation in Cryptococcus Neoformans[END_REF][START_REF] Gates | Molecular Architecture of the Cryptococcus Neoformans Capsule[END_REF] . The capsule of C. neoformans is mainly composed of carbohydrates, among which the glucuronoxylomannan (GXM) is the most abundant one (up to 90%). This polymer is made of differently branched xylose, mannose and glucuronic acid residues [START_REF] Heiss | The Structure of Cryptococcus Neoformans Galactoxylomannan Contains β-d-Glucuronic Acid[END_REF] . Moreover, depending on the serotype of the yeast, at least six different types of GXM have been reported [START_REF] Doering | How Sweet It Is ! Cell Wall Biogenesis and Polysaccharide Capsule Formation in Cryptococcus Neoformans[END_REF] . The other components of the fungal capsule are glucuronoxylomannogalactan (GXMGal), mannoproteins and lipids. The latter ones are forming vesicles such as lipid droplets, and are part of the plasma membrane [START_REF] Rodrigues | Extracellular Vesicles Produced by Cryptococcus Neoformans Contain Protein Components Associated with Virulence[END_REF] .

NUCLEAR MAGNETIC RESONANCE

History

The history of the Nuclear Magnetic Resonance (NMR) started in 1902 when Pieter

Zeeman and Hendrik Lorentz were awarded the Nobel Prize in Physics for their researchs on the influence of magnetism upon radiation phenomena [START_REF] Zeeman | The Effect of Magnetisation on the Nature of Light Emitted by a Substance[END_REF][START_REF] Lorentz | La Théorie Electromagnétique de Maxwell et Son Application Aux Corps Mouvants[END_REF] [START_REF] Nagayama | Two-Dimensional J-Resolved 1H n.m.r. Spectroscopy for Studies of Biological Macromolecules[END_REF] but also extended its usefulness to other scientific fields, including physics, biology, and medicine [START_REF] Ernst | Nuclear Magnetic Resonance Fourier Transform Spectroscopy (Nobel Lecture)[END_REF] .

Finally, Kurt Wüthrich shared the Nobel Prize in chemistry in 2002 for his work on the application of NMR techniques for the elucidation of three-dimensional structures of biologic macromolecules in solution [START_REF] Riek | Solution NMR Techniques for Large Molecular and Supramolecular Structures[END_REF] ; paving the way for the emergence of NMR spectroscopy as powerful tool in the field of the structural biology.

NMR principle

As the detailed theory of NMR is well described in excellent books -from Keeler [START_REF] Keeler | Understanding NMR Spectroscopy[END_REF] for instance-in the following sections, we will only give an overview of the basis of NMR in the context of the structural biology research related to this thesis.

NMR is a non-invasive analytical spectroscopic technique based on the absorption of a radiofrequency radiation by magnetically active nuclei (e.g., [START_REF] O'meara | The Cryptococcus Neoformans Capsule: A Sword and a Shield[END_REF] H, 31 P, 2 H, [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C, [START_REF] Fisher | Emerging Fungal Threats to Animal, Plant and Ecosystem Health[END_REF] N, etc.) when placed in a strong and constant magnetic field. To be observable by NMR, these nuclei need to be in resonance. This phenomenon is possible only for nuclei characterized by a non-zero intrinsic angular momentum spin s and a magnetic moment µ. The ratio between these two moments defines the gyromagnetic ratio γ as γ = ! ! and the sensitivity of a nucleus in NMR spectroscopy depends on it (high γ, high sensitivity).

An NMR experiment follows three steps: (1) the spins are affected by the continuous field B 0 induced by a strong magnet and they get oriented according to this field; (2) the orientation of the spins are briefly perturbed by radiofrequency pulses with the field B 1 induced upon the selective activation of coils placed around the sample; (3) during the relaxation, the spins go back to the initial state creating an electrical signal oscillating at the NMR frequency. The response signal from all excited spins is recorded as a time-domain signal known as the free induction decay (FID) and then converted to frequency domain by applying a Fourier transformation. The resulting spectrum exhibits peaks characterized by: their position on the horizontal axis (chemical shift in ppm scale), their shape and their intensity.

Moreover, for ground-state spin of ½, the signal recorded is influenced by a sum of both external and internal interactions. First, external interactions encompass: the Zeeman interactions due to the static magnetic field B 0 and the effects of the magnetic radiofrequency B 1 . Then, internal (i.e., between spins) interactions are: (1) the dipolar (through space) couplings describing interactions between magnetic dipoles of spins; (2) the scalar (through chemical bonds) couplings originate from hyperfine couplings between two chemically bonded spins and (3) the chemical shift anisotropy (CSA). The CSA is linked to the electronic environment surrounding the nucleus, generally anisotropic (i.e., varying in magnitude according to the direction of measurement). Thus, as the NMR signal is directly dependent upon the electromagnetic environment of each nucleus, it can be used to recover the atomic structure of molecules.

Multidimensional NMR

For conventional NMR, the one-dimensional (1D) signal is recorded as a function of one time variable and, after Fourier transformation, the signal appears in the frequency domain. Typically for large systems (which included most of the biological systems), with many different spins, the spectral overlap is inevitable in 1D spectra. This can be overcome by applying multidimensional NMR experiments.

The higher number of dimensions can help to reduce the spectral overlapping. In practice, the spectra are still analyzed as 2D planes with additional resolution from other planes in multidimensional (3D or even higher dimensions) spectra. For example, for 2D spectra the signal is recorded as a function of two-time variables, t 1 and t 2 , and Fourier transformed twice to be plotted as a function of two frequency domains in order to spread the signals over a surface, usually called F 1 and F 2 . Depending on the experiment, this signal results from the interaction between two nuclei through chemical bonds (which are coupled through J couplings) or through space (through dipolar couplings) and result in a cross peak at the intersection of the frequencies F 1 and F 2 of those nuclei (Fig. 6). The multidimensional spectra are very crucial to conduct the assignment of biological samples; however, the addition of each dimension decreases the overall signal to noise ratio (SNR) by square of 2 factor at least. These experiments can be recorded as homo or hetero-nuclear spectra. In a homonuclear spectrum, there is usually a diagonal that represents the correlation of peaks to themselves and is not in itself very informative. The signals away from the diagonal represent correlations between two nuclei and are used for the assignment (Fig. 6A). In a heteronuclear spectrum there are no diagonal signals and all the signals represent correlations (Fig. 6B). Fig. 6: Schematic 2D NMR spectra.

Frequency F 1 (ppm)
Frequency F 2 (ppm)

ω 1 ω 2 Frequency F 1 (ppm)
Frequency F 2 (ppm)

ω 1 ω 2 A B
A) Homonuclear spectrum representing the off-diagonal cross-peaks correlating the signals at ω 1 and ω 2 frequencies. B) Heteronuclear spectrum in which each signal is a cross-peak between two frequencies.

Solid-state Nuclear Magnetic Resonance

Based on the presence or absence of molecular tumbling, NMR spectroscopy for biological systems can be separated in solution-state or solid-state NMR. The first one is dedicated to molecules solubilized in solvents that could range from small inorganic molecules to cell-extracted compounds such as proteins; whereas the second one does not require the solubilization of molecules and it could be applied to large and complex objects such as supramolecular complexes or even intact cells.

For solid-state NMR (SSNMR) experiments, the molecules targeted are relatively immobile as compared to the solution NMR. The spectra obtained are mainly dominated by anisotropic interactions, such as the CSA (average out by the fast tumbling of molecules in solution) and dipolar couplings, leading to broaden signals. To overcome this phenomenon, the sample can be spun at the 54.74° (historically called the magic angle) with respect to B 0 with a high spinning frequencies (thousands of Hertz) [START_REF] Andrew | Nuclear Magnetic Resonance Spectra from a Crystal Rotated at High Speed[END_REF][START_REF] Kessemeier | Pulsed Nuclear Magnetic Resonance in Rotating Solids[END_REF][START_REF] Hennel | Magic-Angle Spinning: A Historical Perspective[END_REF] .

The sample is packed into the rotor then placed into a stator, which is oriented at the magic angle with respect to the magnetic field. The spinning is initiated using two gas flows:

the bearing (making the rotor levitate) and the drive (creating a spinning movement via the wings of the cap) (Fig. 7A).

Fig. 7: Magic Angle Spinning in SSNMR.

A B

A) Schema of a rotor in a stator placed into a magnetic field B 0 and spun at the magic angle along the rotor axis. B) Different examples of SSNMR rotors with corresponding spinning frequencies and volume of sample required.

(The B panel is from https://warwick.ac.uk/).

The MAS technique allows averaging out anisotropic interactions and dipolar couplings making signals narrower and easier to analyze. However, the MAS is sometimes not efficient enough to fully average out these interactions and additional decoupling techniques have to be applied during the acquisition of spectra. The faster spinning frequency can additionally average out the dipolar couplings and requires rotors with smaller diameters that can be spun with higher spinning frequencies to overcome the speed of sound barrier (around 320 m/s in air at 20 °C) (Fig. 7B). This feature makes the lower power decoupling techniques at fast MAS sufficient, reducing the sample heating. So far, 170 kHz is the fastest one being reported 67 . Additionally, high MAS frequencies are important to average out dipolar couplings between 1 H nuclei, since these nuclei possess a strong network of these couplings. As the result, the high-resolution 1 H detected spectra can be acquired.

Furthermore, as the SSNMR is a non-destructive method, it can be also applied to the study of the molecular architecture of complex systems in cellular conditions -as close as possible to the native state -as it was already done for fungi [START_REF] Kang | Molecular Architecture of Fungal Cell Walls Revealed by Solid-State NMR[END_REF] , bacteria [START_REF] Arnold | Whole Cell Solid-State NMR Study of Chlamydomonas Reinhardtii Microalgae[END_REF][START_REF] Romaniuk | Bacterial Cell Wall Composition and the Influence of Antibiotics by Cell-Wall and Whole-Cell NMR[END_REF] , biofilms [START_REF] Quinn | NMR of Macromolecular Assemblies and Machines at 1 GHz and Beyond: New Transformative Opportunities for[END_REF] , plants [START_REF] Li | The Molecular Structure of Plant Sporopollenin[END_REF] and biomass of wood [START_REF] Terrett | Molecular Architecture of Softwood Revealed by Solid-State NMR[END_REF] . The SSNMR can also be applied as analytical tool to decipher the structural composition of subcellular structures. Typically, these samples are characterized by [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C detection SSNMR methods using a relatively large sample amount (around 20 mg). Moreover, the samples with limited production amounts would result in lower signal to noise ratio (SNR). This can be partially compensated without losing a resolution by applying [START_REF] O'meara | The Cryptococcus Neoformans Capsule: A Sword and a Shield[END_REF] H detection methods at high spinning frequencies. For example, the sub-milligram amounts of fully protonated sample characterized at 100 kHz MAS could be sufficient to determine the structure of proteins with 1 H detected methods [START_REF] Vasa | Assessment of a Large Enzyme-Drug Complex by Proton-Detected Solid-State NMR Spectroscopy without Deuteration[END_REF][START_REF] Andreas | Structure of Fully Protonated Proteins by Proton-Detected Magic-Angle Spinning NMR[END_REF]

The fungal cell samples preparation

The analysis of fungal constituents by SSNMR in intact cells requires many steps.

First, the growing of cells has to be carried in [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C/ [START_REF] Fisher | Emerging Fungal Threats to Animal, Plant and Ecosystem Health[END_REF] N-labeled medium to fully label the cell wall components. As the [START_REF] Fleming | On the Antibacterial Action of Cultures of a Penicillium, with Special Reference to Their Use in the Isolation of B. Influenzae[END_REF] C isotope of carbon cannot be used in NMR because its spin s = 0, carbon-detected NMR experiments are performed using [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C-enriched molecules ( 13 C natural abundance is only of 1.1 %). Then, the [START_REF] Fisher | Emerging Fungal Threats to Animal, Plant and Ecosystem Health[END_REF] N labeling is widely used for studying biological molecules as the [START_REF] Hyde | The Amazing Potential of Fungi: 50 Ways We Can Exploit Fungi Industrially[END_REF] N isotope of nitrogen (99.64 % abundance) has an integer nuclear spin: its NMR signal is influenced by quadrupolar interactions leading to broad line widths; on the contrary, [START_REF] Fisher | Emerging Fungal Threats to Animal, Plant and Ecosystem Health[END_REF] N has a ground-state spin of ½, which offers advantages for NMR (i.e., narrower line width).

Whole cells are can be fully [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C/ 15 N-labeled when the growth is carried in minimal medium containing [U- [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C]-Glucose and [START_REF] Fisher | Emerging Fungal Threats to Animal, Plant and Ecosystem Health[END_REF] N salts as sole sources of carbon and nitrogen.

Then, recombinant proteins -such as hydrophobins -can be uniformly [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C/ 15 N-labeled using a recombinant expression system, such as E. coli, grown using a minimal medium containing a [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C source of carbon ([U- [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C]-glucose, [1,3-13 C]-glycerol or [2- [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C]-glycerol) and a [START_REF] Fisher | Emerging Fungal Threats to Animal, Plant and Ecosystem Health[END_REF] N source of nitrogen (ammonium salts) [START_REF] Hong | Selective and Extensive 13C Labeling of a Membrane Protein for Solid-State NMR Investigations[END_REF][START_REF] Lemaster | Dynamical Mapping of E. Coli Thioredoxin via 13C NMR Relaxation Analysis[END_REF][START_REF] Higman | Assigning Large Proteins in the Solid State: A MAS NMR Resonance Assignment Strategy Using Selectively and Extensively 13C-Labelled Proteins[END_REF] . This uniform labeling maximizes the number of information available from a single sample but as the complexity of the sample increases, it may lead to spectral overlap.

The addition of deuterium to the labeling is important for 1 H-detected experiments in SSNMR. The water used for production of media can be substituted with D 2 O: the produced samples in D 2 O media will have lower 1 H density. Consequentially, the network of dipole couplings will be reduced. As the result the non-exchanged protons will exhibit improved resolution. [START_REF] Hologne | Deuterated Peptides and Proteins in MAS Solid-State NMR[END_REF] .

Dynamic-based experiments by SSNMR

The versatility of SSNMR pulse sequences allows to target populations of molecules according to their dynamics [START_REF] Matlahov | Hidden Motions and Motion-Induced Invisibility: Dynamics-Based Spectral Editing in Solid-State NMR[END_REF] . For instance, within the same sample, dynamic-based experiments can selectively target rigid or mobile molecules. In this thesis we will classify the analyzed molecules as being rigid or mobile.

Cross-polarization based experiments

Most rigid molecules (molecular motions > ms) are targeted using cross-polarization (CP)-based experiments. This type of polarization transfer relies on heteronuclear dipolar interactions. The CP will improve the sensitivity for heteronuclear detected spectra and additionally will allow to take advantage of shorter T 1 relaxation times of protons compared to spins with lower γ values (e.g., [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C, 15 N). The CP experiment is based on the magnetization transfer from 1 H to the other nuclei by applying simultaneous spin locks on both channels.

The power of spin lock frequencies should be set according to the sum or difference of the MAS spinning frequency. Depending on the specificity of the systems the spin lock τ CP time can be varied (typically from hundreds of µs to several ms). The efficiency of this transfer is dependent on the strength of the dipolar interaction.

There are plenty of SSNMR pulse sequences using CP transfers; we propose to defined below only the pulse sequences used during this thesis as well as the category of molecules they target.

First, one-dimension [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C-detected or [START_REF] Fisher | Emerging Fungal Threats to Animal, Plant and Ecosystem Health[END_REF] N-detected CP spectra allow identifying the different categories of rigid molecules present in the sample by giving a quick fingerprint of the system. In the simple pulse sequence presented Fig. 8A, the magnetization is transferred from the 1 H to the other nucleus ( 13 C or 15 N) and the resulting signal is recorded as a onedimension spectrum.

A B

Because of the complexity of biological samples such as intact cells (containing a wide range of biomolecules), the resolution in 1D spectra is not sufficient to do a precise assignment. Depending on their electronic environment, atoms in different molecules will exhibit different chemical shift values. Thus, different regions of one-dimension spectrum can be used to discriminate between different families of molecules such as lipids, carbohydrates, proteins, etc. This strategy was used for the structural characterization of the extracellular matrix of the fully [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C-labeled Bacillus subtilis both WT and extracellular matrix-deficient mutant [START_REF] Molina-Santiago | The Extracellular Matrix Protects Bacillus Subtilis Colonies from Pseudomonas Invasion and Modulates Plant Co-Colonization[END_REF] (Fig. 8B).

To overcome the spectral overlapping occurring in 1D [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C CP experiments, multidimensional CP-based experiments can be recorded. In order to transfer the magnetization between different [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C nuclei, various types of techniques can be used. The most common one is called Proton-Driven Spin Diffusion (PDSD) experiment. Spin diffusion occurs between carbons during the mixing time t mix (from ms to hundreds of ms range) (Fig. 9A). The longer is the mixing time, the further the magnetization will be transferred through the system. The resulting homonuclear spectrum shows through-space connectivities between two or more carbons. In 2017, Arnold et al., used this technique to investigate, in vivo, the structure of starch accumulated into grains within the chloroplast of the microalgae Chlamydomonas reinhardtii [START_REF] Arnold | Whole Cell Solid-State NMR Study of Chlamydomonas Reinhardtii Microalgae[END_REF] (Fig. 9B).

. A) The 2D 13 C- [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C PDSD pulse sequence where the magnetization transfer pathway is highlighted in light pink and the mixing time t mix used for the 13 C- [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C transfer in pink. B) An example of the use of 2D 13 C- [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C PDSD to establish the structure of both the fully [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C-labeled starch (red) and the not labeled starch (blue) from Chlamydomonas reinhardtii (extracted from Arnold et al., 2018) [START_REF] Arnold | Whole Cell Solid-State NMR Study of Chlamydomonas Reinhardtii Microalgae[END_REF] .

The 1 H-13 C Heteronuclear Correlation (HETCOR) is a 2D NMR technique that reveals spin-spin dipolar couplings between 1 H and nuclei other than 1 H such as [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C. In these

A B
experiments, the magnetization of 1 H is transferred, using a CP transfer, to [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C via dipolar interactions resulting in a cross peak between 1 H and 13 C close in space. By adding a 1 H-1 H spin diffusion time before the CP block, the initial magnetization of 1 H is essentially created on bulk water 1 H and then transferred preferentially to nuclei accessible to water (Fig. 10A).

The resulting intensity of 1 H- [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C cross peaks give an information about the accessibility of molecules to the bulk water and then to the surface of the sample (an intense peak meaning a molecule highly accessible to the bulk water). For instance, the surface of the extracellular matrix of Bacillus subtilis extracellular matrix was investigated using this technique [START_REF] Molina-Santiago | The Extracellular Matrix Protects Bacillus Subtilis Colonies from Pseudomonas Invasion and Modulates Plant Co-Colonization[END_REF] to highlight the proximity of polysaccharides to the exterior medium (Fig. 10B).

Fig. 10:

The 2D 1 H- [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C HETCOR with 1 H-1 H spin diffusion mixing time experiment.

A) The 2D 1D DP-based experiments can be used to discriminate between quantitative signals and mobility-based information. Indeed, after the radio-frequency pulse, the nuclear spins of the sample return to the equilibrium during a T 1 relaxation time dependent on many factors including nuclear environment and temperature. As all the molecules are located in different electronic environments, they relax with different relaxation rates. This relaxation time is taken into account during the "recycle time" of the pulse sequence (i.e., the time a system needs to come back to the equilibrium state) (Fig. 11A). It is placed at the beginning of the pulse sequence and aims at allowing the system to relax enough between scans. Indeed, the duration of the relaxation delay will strongly impact the signal recorded after each scan: with a long recycle delay (dozens of seconds for [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C in a biological system), all the spins have time to relax and the signal recorded results in a quantitative spectrum. On the contrary, if the recycle delay is short enough (few seconds), the resulting spectrum reflects only mobile molecules (ms < motion < µs).

Direct-polarization based experiments

In

A B

INEPT-based experiments

INEPT stands for Insensitive Nuclei Enhanced by Polarization Transfer and is nonselective polarization transfer from protons to X nucleus by using the appropriate 1 H-X coupling. These pulse sequences are used to transfer the magnetization from a high-γ nucleus (such as 1 H) to a low-γ nucleus (such as 13 C) [START_REF] Elena | Proton to Carbon-13 INEPT in Solid-State NMR Spectroscopy[END_REF][START_REF] Morris | Enhancement of Nuclear Magnetic Resonance Signals by Polarization Transfer[END_REF] .

In an INEPT-based experiment, the magnetization is first created on all 1 H, and then transferred to chemically bonded [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C nucleus through J HC -coupling (Fig. 12A). In NMR,

A B

Water-edited experiments

Water-edited experiments were extensively used to study, for instance, the hydration of polysaccharides located at the plants cell wall [START_REF] White | Water-Polysaccharide Interactions in the Primary Cell Wall of Arabidopsis Thaliana from Polarization Transfer Solid-State NMR[END_REF] . By transferring bulk water 1 H polarization to [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C present in these polysaccharides, this approach provides molecular insights about the amount of water-exposed surface areas of polysaccharides in non-grass primary cell walls.

Practically, this experiment results in a series of one-dimension [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C-detected CP spectra (targeting only rigid compounds) recorded with a 1 H T 2 filter and varying 1 H spin diffusion mixing time (t SD ). The magnetization of rigid components is first destroyed using a 1 H T 2 filter; then an initial magnetization is created on the bulk water and starts to diffuse according to the 1 H spin diffusion mixing time selected; finally, this magnetization is transferred to rigid molecule according to their water accessibility (Fig. 13A).

The resulting build-up curves obtained by plotting the intensity of NMR signal as function of the mixing time highlights the differences in water accessibility between all the components within the same system or to compare the water accessibility of the same compounds between different samples as it was illustrated for the crystalline cellulose present in the cell wall in four different samples of Arabidopsis thaliana by White et al., in 2014 (Fig. 13B) [START_REF] White | Water-Polysaccharide Interactions in the Primary Cell Wall of Arabidopsis Thaliana from Polarization Transfer Solid-State NMR[END_REF] . A) The pulse sequence used to record 1D water-edited [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C CP with the T 2 filter block in red, the varying 1 H spin diffusion mixing time (t SD ) in green; the magnetization transfer pathway is highlighted in light pink. B) Build-up curves plotted from a series of water-edited [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C CP and representing the intensities of crystalline cellulose peaks as function of the t SD for four different samples of Arabidopsis thaliana (extracted from White et al., in 2014) [START_REF] White | Water-Polysaccharide Interactions in the Primary Cell Wall of Arabidopsis Thaliana from Polarization Transfer Solid-State NMR[END_REF] .

Double-quantum experiments

To enhance the resolution and to reduce spectral overlaps occurring for highly complex systems, one of the two dimensions is encoded as double quantum dimension (i.e.,

A B

the sum of the two correlating frequencies). This technique can help to split all the NMR signals and then to facilitate the assignment of the carbon skeleton of complex structures, in case of close chemical shift values. Such informative spectra can be recorded using the [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C- [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C INADEQUATE (Incredible Natural Abundance DoublE QUAntum Transfer Experiment) pulse sequence. It employs a transfer (using the J CC coupling constant) between two neighboring carbons. Here, the initial magnetization of these experiments is created on mobile or rigid molecules. It can be used in combination with CP (Fig. 14A) and DP excitation techniques (Fig. 15A).

As these experiments allow connecting two neighboring carbons through chemical bonds, and then ease the spectral assignment, they are helpful for accessing the molecular composition of complex samples such as whole-cell subcompartments (e.g., cell wall, lipid droplets…). In 2011, Dick-Pérez et al., successfully used 13 C-13 C DP-INADEQUATE to identify the main mobile polysaccharides components of the cell wall of the uniformly [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] Clabeled Arabidopsis thaliana (Fig. 14B) [START_REF] Dick-Pérez | Structure and Interactions of Plant Cell-Wall Polysaccharides by Two-and Three-Dimensional Magic-Angle-Spinning Solid-State NMR[END_REF] . Then, in 2019, Kang et al. revealed the rigid cell wall composition of Aspergillus fumigatus mycelium using [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C-13 C CP-INADEQUATE (Fig. 15B) [START_REF] Kang | Molecular Architecture of Fungal Cell Walls Revealed by Solid-State NMR[END_REF] . In these two examples, the double quantum (DQ) dimension was required to discriminate peaks belonging to different molecules but exhibiting pretty similar [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C chemical shifts as they all belong to the carbohydrates family.

The assignment using proton-detection SSNMR methods

Due to its high gyromagnetic ratio and natural abundance, 1 H is the optimal choice for sensitive detection in solution. In solids, however, these same properties are responsible for a network of strong homonuclear 1 H dipolar couplings that dramatically lower the signal resolution. To observe high-resolution 1 H signals in solids, the homogeneous broadening characteristic of this network needs to be overcome, by dilution of the proton content in the sample (deuteration of the sample) and/or by fast sample spinning in conjunction with high magnetic fields.

In our study, we also employed SSNMR (H)CANH to connect NH of the i-residue with its own CA (CA i N i H i ),or with the CO in i-1

(CO i-1 N i H i ) as shown

THESIS PROJECT

As previously described, there is a broad range of fungal pathogens infecting humans and causing diseases that can be from mild to lethal. Severe fungal infections are due to opportunistic pathogens that infect immunosuppressed individuals and are most of the time associated with other diseases or medical conditions such as asthma, leukemia, organ transplants, AIDS or immunosuppressive corticosteroid therapies. Despite the number of deaths and the increase in severe mycosis, fungal infections remain neglected by public health authorities.

Among the fungal pathogens, the filamentous fungus Aspergillus fumigatus is one of the major pathogens of the respiratory system. Aspergillosis displaying both high incidence and mortality rates is becoming an important public health issue. The spores of Aspergillus fumigatus are surrounded by a cell wall, allowing them to resist against host defense mechanisms. The cell wall is composed of a set of polysaccharides covered by the DHNmelanin pigment and a layer of protein belonging to the family of hydrophobins [START_REF] Beauvais | Aspergillus Cell Wall and Biofilm[END_REF][START_REF] Aimanianda | Surface Hydrophobin Prevents Immune Recognition of Airborne Fungal Spores[END_REF] . In this project, we aimed at investigating the structural architecture of Aspergillus fumigatus conidial cell wall at atomic resolution using MAS SSNMR spectroscopy. We used multidimensional SSNMR to identify the cell wall components, decipher their rigidity/mobility and understand their interaction with bulk water molecules. By comparing dormant, swollen and germinating conidia, we studied the composition of the cell wall during the germination process as well as its molecular organization. Moreover, using different mutants, we explored the effect of the

A B

deletion of a specific cell wall component on the whole organization. These results will be presented in Chapter 1.

Cryptococcus neoformans is the etiological agent of cryptococcosis, and cerebral meningitis [START_REF] Kwon-Chung | Cryptococcus Neoformans and Cryptococcus Gattii, the Etiologic Agents of Cryptococcosis[END_REF] . This fungal disease remains a significant cause of mortality worldwide causing meningoencephalitis, especially in immunocompromised individuals suffering from AIDS [START_REF] Rajasingham | Global Burden of Disease of HIV-Associated Cryptococcal Meningitis: An Updated Analysis[END_REF] .

C. neoformans is an encapsulated yeast having a size of 5-7µm [START_REF] Reese | Loss of Cell Wall Alpha(1-3) Glucan Affects Cryptococcus Neoformans from Ultrastructure to Virulence[END_REF] with a two-layer external structure composed of a cell wall and a capsule. The rigid cell wall is bound to the plasma membrane and composed of polymers of α-glucan, β-glucan, chitin and chitosan [START_REF] O'meara | The Cryptococcus Neoformans Capsule: A Sword and a Shield[END_REF] . The capsule of C. neoformans is mainly composed of glucuronoxylomannan (GXM) (up to 90%) [START_REF] Heiss | The Structure of Cryptococcus Neoformans Galactoxylomannan Contains β-d-Glucuronic Acid[END_REF] , glucuronoxylomannogalactan (GXMGal), mannoproteins and lipids. In this thesis work, we aimed at identifying the different components of C. neoformans cell wall and capsule by SSNMR and investigating the architecture of these two layers. Moreover, we explored different mutants of C. neoformans to study the effects of the mutation onto the structure of both the cell wall and capsule. Furthermore, we also analyzed different clinical isolates (isolated from patients and grown in vitro) in order to identify the differences between isolates that could be correlated with their virulence potential. Part of this project was also the exploration of possibilities and limits of 1 H detection methods at fast MAS regime (100 kHz)

as the tool to analyze intact cell walls. These results will be presented in Chapter 2.

CHAPTER 1: ASPERGILLUS FUMIGATUS CHAPTER 1: ASPERGILLUS FUMIGATUS

MATERIAL AND METHODS

Biological samples

All the Aspergillus fumigatus conidial samples as well as the hydrophobin proteins RodA, B and C were kindly provided by our collaborators from the Pasteur Institute, Paris: Drs. Vishu Aimanianda, Iñaki Guijarro, Isabel Valsecchi and Sarah Wong.

Conidial samples

Cell culture -13 C/ 15 N-labeled dormant conidial samples were obtained by growing Aspergillus fumigatus on 2 % malt-agar slants containing 1% [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C-glucose and 20 mM 15 NH 4 Cl as carbon and nitrogen sources, respectively, supplemented with salt solution and trace elements [START_REF] Hill | Improved Protocols for Aspergillus Minimal Medium: Trace Element and Minimal Medium Salt Stock Solutions[END_REF] . Dormant conidia were harvested after 10-12 days growth at ambient temperature, using 0.05 % Tween-80, washed twice. To obtain [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C/ [START_REF] Fisher | Emerging Fungal Threats to Animal, Plant and Ecosystem Health[END_REF] N swollen and germinating conidia, harvested conidia were inoculated into Aspergillus minimal medium [START_REF] Hill | Improved Protocols for Aspergillus Minimal Medium: Trace Element and Minimal Medium Salt Stock Solutions[END_REF] (containing 1% [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C-glucose and 20 mM 15 NH 4 Cl, supplemented with salt solution and trace elements; 1 x 10 8 conidia per 50 mL) and incubated in a shaking-incubator (150 rpm) maintained at 37°C. For swollen conidia, they were harvested after 5 h and for germinating conidia, after 8 h. Both swollen and germinated conidia were washed twice with MilliQ water. Unlabeled conidia were prepared as presented above but using unlabeled glucose and ammonium chloride.

PFA fixation -Different conidial morphotypes (dormant, swollen and germinating) were fixed by adding 2.5% para-formaldehyde (PFA) in PBS, kept at ambient temperature for an hour and then at 4°C overnight. Then, the PFA-fixed conidial suspension was centrifuged (1600 g, 10 min), the supernatant was discarded and the reaction is quenched with 0.1 M ammonium chloride by repeated addition and centrifugation (thrice; 1600 g, 10 min). Finally, conidia were washed twice with MilliQ water.

The 13 C/ 15 N-labeled mutants Δags (α-glucan deficient), ΔrodA (RodA deficient) and Δpksp (DHN-melanin deficient) dormant conidia [START_REF] Valsecchi | The Puzzling Construction of the Conidial Outer Layer of Aspergillus Fumigatus[END_REF] as well as the [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C/ 15 N-labeled white dormant conidia were prepared as presented above (ΔrodA and Δpksp dormant conidia were not PFA-fixed). Conidial rodlet layer was extracted by incubating dry conidia with 48% hydrofluoric acid for 72 h at 4 °C41 .

Melanin samples

The 13 C/ 15 N-labeled melanin ghosts were prepared from dormant conidia (strain CEA10, a clinical isolate of A. fumigatus [START_REF] Thau | Rodletless Mutants of Aspergillus Fumigatus[END_REF] ) grown on [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C/ 15 N-labeled minimal medium as described by Bayry et al., 2009 [START_REF] Bayry | Surface Structure Characterization of Aspergillus Fumigatus Conidia Mutated in the Melanin Synthesis Pathway and Their Human Cellular Immune Response[END_REF] . Briefly, dormant conidia were submitted to proteolytic and glycohydrolytic enzymes, denaturating agent and hot, concentrated HCl (6 M). This treatment destroys the majority of the cellular constituents resulting in an electron-dense layer similar in size and shape to the original conidial melanin layer called "melanin ghosts". Then, the melanin ghosts were subjected to boiling in Tris ( 50

Production and purification of hydrophobins

Both 13 C/ 15 N-labeled RodA and RodB were produced using the protocol described by Valsecchi et al.; 2019 [START_REF] Valsecchi | Assembly and Disassembly of Aspergillus Fumigatus Conidial Rodlets[END_REF] . Briefly, Escherichia coli BL21 (DE3) were transformed using a pET15b-HisTag-ubiquitin-rRodA (Uniprot ID: P41746) or pET15b-HisTag-ubiquitin-rRodB (Uniprot ID: B0XPA4) vector. Bacteria were grown at 37°C in minimal medium containing [START_REF] Fisher | Emerging Fungal Threats to Animal, Plant and Ecosystem Health[END_REF] NH 4 Cl and [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C-glucose (Eurisotop, Saclay) as sole sources of nitrogen and carbon, respectively. The expression of the recombinant protein was induced by adding isopropyl β-D-1-thiogalactopyranoside (IPTG) (final concentration, 1 mM) to the culture medium. After 4 h of induction at 37°C, the cell culture was centrifuged (10 000g -10min), the pellet was collected and stored at -20°C.

Recombinant 1 H/ 13 C/ 15 N-labeled RodA was produced as described above by substituting minimal medium with [START_REF] Fisher | Emerging Fungal Threats to Animal, Plant and Ecosystem Health[END_REF] NH 4 Cl and U-D 7 -13 C 6 glucose (Eurisotop, Saclay) as sole sources of nitrogen and carbon, respectively. The recombinant protein was recovered from inclusion body by adding guanidine hydrochloride (6 M) buffer (pH 8.0) to the pellet and purified on a nickel affinity column (Histrap, GE Healthcare) under denaturing conditions.

Then, the oxidative folding of the hydrophobin was performed by dialysis against a refolding buffer containing 10 mM of reduced and 1 mM of oxidized glutathione (pH 5). His-ubiquitintag was cleaved using TEV-His 6 -tag protease and both Ubiquitin-His 6 -tag and TEV-His 6 -tag protease were removed using nickel affinity columns (Histrap, GE Healthcare). To separate hydrophobin monomers with correct disulfide bridges from inter-monomeric disulfide linkages, the recombinant protein was subjected to reverse-phase chromatography on hydrophobic C 18 (Waters) with acetonitrile gradients. Finally, to separate monomers from aggregates, the recombinant protein mix was loaded on size exclusion chromatography.

Proteins were dialyzed against H 2 O and lyophilized until further use.

Solid-state NMR experiments

Experimental data were processed using TopSpin and analyzed using CCPNMR.

13 C/ 15 N-labeled PFA-fixed dormant, swollen, germinating and

Δags conidia analysis

All experiments were performed on a 600 MHz 1 H Larmor frequency spectrometer (Bruker Biospin) using 3.2 mm MAS rotors filled with PFA-fixed [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C/ 15 N-labeled dormant, swollen, germinating or Δags conidia. The MAS frequency was set at 11 kHz and the sample temperature was set to 276 K with the internal reference DSS [START_REF] Carole | Characterization of Different Water Pools in Solid-State NMR Protein Samples[END_REF][START_REF] Markley | Recommendations for the Presentation of NMR Structures of Proteins and Nucleic Acids IUPAC-IUBMB-IUPAB Inter-Union Task Group on the Standardization of Data Bases of Protein and Nucleic Acid Structures Determined by NMR Spectroscopy[END_REF] .

One-dimension [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C cross-polarization (CP) experiments (512 scans) were recorded using a 1 ms contact time for the 1 H- [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C polarization transfer to detect rigid molecules. Onedimension [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] Two-dimension 13 C- [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C PDSD experiments (48 scans) with 50 ms mixing time and a 1 ms contact time for the 1 H- [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C polarization transfer were used to identify the polymorphism of the carbohydrates. Proton decoupling with a frequency of 90 kHz was applied during acquisition times, using the SPINAL-64 decoupling sequence.

To determine the water accessibility of cell wall components, two dimension 13 C-13 C water-edited PDSD experiments (80 scans) with 1 H-T 2 filter (by mean of a spin echo sequence placed before the CP contact time) of 1.5 ms and a variable 1 H-1 H spin diffusion of 5 ms or 20 ms were performed using a 1 ms contact time for the 1 H-13 C polarization transfer. Onedimension [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C CP spectra (256 scans) using a 1 ms contact time for the 1 H- [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C polarization transfer with a 1 H-T 2 filter of 1.5 ms and a variable 1 H-1 H mixing time (from 0.25 to 150 ms) were used to obtain build-up curves using the intensities of the peaks assigned as the C2 of the chitin, the C6 of the β-Glucans and the C4 of the α-Glucan. Results were plotted as the square root of the 1 H-1 H mixing time for reflecting the relayed nature of the spin diffusion. Two-dimension 1 H- [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C HETCOR experiments (128 scans) were recorded with and without a 5 ms 1 H-1 H spin diffusion and using a 1 ms contact time for the 1 H-13 C polarization transfer. A one-dimension [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C trace was extracted at the chemical shift of the bulk water (4.97 ppm) and peaks intensities were used to estimate the proportion of sugar highly accessible to water.

The mobile parts of the cell wall were investigated using 2D 1 H- [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C INEPT experiments (192 scans) recorded using 90 kHz proton decoupling SPINAL-64.

The nitrogen content of the cell wall was investigated using 15 N- [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C NCO (576 scans) and NCA (576 scans) were recorded using a CP transfer from 1 H to [START_REF] Fisher | Emerging Fungal Threats to Animal, Plant and Ecosystem Health[END_REF] N of 1 ms and a double CP transfer 15 N- [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C and 1 H- [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C with a contact time of 3 ms.

13 C/ 15 N-labeled PFA-fixed dormant conidia

The HMQC 31 P/ [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C was recorded on a 400MHz 1 H Larmor frequency spectrometer (Bruker Biospin) on a 4 mm rotor at 276 K.

13 C/ 15 N-labeled ΔrodA and Δpksp dormant conidia

All experiments were performed on a 600 MHz 1 H Larmor frequency spectrometer (Bruker Biospin) using 4 mm MAS rotors filled with [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C/ 15 N-labeled ΔrodA and Δpksp dormant conidia. The MAS frequency was set at 11 kHz and the sample temperature was set to 276 K with the internal reference DSS [START_REF] Carole | Characterization of Different Water Pools in Solid-State NMR Protein Samples[END_REF][START_REF] Markley | Recommendations for the Presentation of NMR Structures of Proteins and Nucleic Acids IUPAC-IUBMB-IUPAB Inter-Union Task Group on the Standardization of Data Bases of Protein and Nucleic Acid Structures Determined by NMR Spectroscopy[END_REF] One-dimension [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C CP experiments (64 scans) recorded using a 1 ms contact time for the 1 H-13 C polarization transfer. One-dimension [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C DP spectra (64 scans) were recorded with a 3 s recycle delay. Two-dimension 13 C- [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C PDSD experiments (80 scans) were recorded with a 50 ms mixing time and a 1 ms contact time for the 1 H-13 C polarization transfer. Proton decoupling with a frequency of 90 kHz was applied during acquisition time, using the SPINAL-64 decoupling sequence. Two-dimension 1 H- [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C HETCOR experiments (224 scans) were recorded using a 1 ms contact time for the 1 H-13 C polarization transfer. Two-dimension 1 H- [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C INEPT experiments (192 scans) recorded using 90 kHz proton decoupling SPINAL-64.

13 C/ 15 N-labeled dormant conidia

All experiments were performed on a 600 MHz 1 H Larmor frequency spectrometer (Bruker Biospin) using a 4 mm MAS rotor filled with [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C/ 15 N-labeled dormant conidia. The MAS frequency was set at 11 kHz and the sample temperature was set to 276 K with the internal reference DSS [START_REF] Carole | Characterization of Different Water Pools in Solid-State NMR Protein Samples[END_REF][START_REF] Markley | Recommendations for the Presentation of NMR Structures of Proteins and Nucleic Acids IUPAC-IUBMB-IUPAB Inter-Union Task Group on the Standardization of Data Bases of Protein and Nucleic Acid Structures Determined by NMR Spectroscopy[END_REF] One-dimension [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C CP experiments (64 scans) recorded using a 1 ms contact time for the 1 H-13 C polarization transfer. One-dimension [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C DP spectra (64 scans) were recorded with a 3 s recycle delay. Two-dimension 13 C- [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C PDSD experiments (128 scans) were recorded with 50 ms mixing time and a 1 ms contact time for the 1 H-13 C polarization transfer. Proton decoupling with a frequency of 90 kHz was applied during acquisition times, using the SPINAL-64 decoupling sequence. Two-dimension 1 H- [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C HETCOR experiments (224 scans) were recorded using a 1 ms contact time for the 1 H-13 C polarization transfer. Two-dimension 1 H- [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C INEPT experiments (192 scans) were recorded using 90 kHz proton decoupling SPINAL-64.

Unlabeled PFA-fixed dormant conidia

One-dimension CP experiment (112640 scans, 11 kHz MAS) was recorded at 276 K on a 300 MHz spectrometer (Bruker Biospin) using a 4 mm MAS rotor filled with unlabeled PFA-fixed dormant conidia. The contact time for the 1 H-13 C polarization transfer was 1 ms.

13 C/ 15 N-labeled PFA-fixed white dormant conidia

All experiments were performed on a 600 MHz 1 H Larmor frequency spectrometer (Bruker Biospin) using a 4 mm MAS rotor filled with [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C/ 15 N-labeled dormant conidia. The MAS frequency was set at 11kHz and the sample temperature was set to 276 K with the internal reference DSS [START_REF] Carole | Characterization of Different Water Pools in Solid-State NMR Protein Samples[END_REF][START_REF] Markley | Recommendations for the Presentation of NMR Structures of Proteins and Nucleic Acids IUPAC-IUBMB-IUPAB Inter-Union Task Group on the Standardization of Data Bases of Protein and Nucleic Acid Structures Determined by NMR Spectroscopy[END_REF] Two-dimension 13 C- [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C DP INADEQUATE (32 scans) were performed with a recycle delay of 2 s and 13 C- [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C CP-INADEQUATE (32 scans) with a 0.5 ms contact time for the 1 H-13 C polarization transfer. Two-dimension [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C- [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C PDSD experiments (64 scans) were recorded with 50 ms mixing time and a 0.6 ms contact time for the 1 H-13 C polarization transfer. Proton decoupling with a frequency of 90 kHz was applied during the acquisition time, using the SPINAL-64 decoupling sequence. Two-dimension 1 H- [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C INEPT experiments (64 scans) recorded using 90 kHz proton decoupling SPINAL-64.

13 C/ 15 N-labeled melanin ghost

All experiments were performed on a 600 MHz 1 H Larmor frequency spectrometer (Bruker Biospin) using a 3.2 mm MAS rotor filled with [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C/ 15 N-labeled melanin ghost. The MAS frequency was set at 11 kHz and the sample temperature was set to 276 K with the internal reference DSS [START_REF] Carole | Characterization of Different Water Pools in Solid-State NMR Protein Samples[END_REF][START_REF] Markley | Recommendations for the Presentation of NMR Structures of Proteins and Nucleic Acids IUPAC-IUBMB-IUPAB Inter-Union Task Group on the Standardization of Data Bases of Protein and Nucleic Acid Structures Determined by NMR Spectroscopy[END_REF] One-dimension [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C CP experiments (8 scans) was recorded using a 1 ms contact time for the 1 H-13 C polarization transfer. One-dimension [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C DP spectra (8 scans) were recorded with a 2 s recycle delay. Two-dimension [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C- [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C PDSD experiments (48 scans) were recorded with 50 ms mixing time and a 1 ms contact time for the 1 H-13 C polarization transfer. Proton decoupling with a frequency of 90 kHz was applied during acquisition times, using the SPINAL-64 decoupling sequence. Two-dimension [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C- [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C DP INADEQUATE (256 scans) were performed with a recycle delay of 2 s. Two-dimension 1 H- [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C INEPT experiments (128 scans) recorded using 90 kHz proton decoupling SPINAL-64.

DHN-melanin

Experiments were performed on an 800 MHz 1 H Larmor frequency spectrometer (Bruker Biospin) using a 3.2 mm MAS rotor filled with DHN-melanin. The MAS frequency was set at 11kHz and the sample temperature was set to 276 K with the internal reference DSS [START_REF] Carole | Characterization of Different Water Pools in Solid-State NMR Protein Samples[END_REF][START_REF] Markley | Recommendations for the Presentation of NMR Structures of Proteins and Nucleic Acids IUPAC-IUBMB-IUPAB Inter-Union Task Group on the Standardization of Data Bases of Protein and Nucleic Acid Structures Determined by NMR Spectroscopy[END_REF] One-dimension [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C CP experiments (16384 scans) was recorded using a 1 ms contact time for the 1 H-13 C polarization transfer.

1.2.9 Hydrophobins analyses [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C/ [START_REF] Fisher | Emerging Fungal Threats to Animal, Plant and Ecosystem Health[END_REF] N-labeled RodA, RodB, RodC -All experiments were performed on a 600 MHz 1 H Larmor frequency spectrometer (Bruker Biospin), at 276 K with a 11 kHz MAS and using 3.2 mm MAS rotors filled with [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C/ 15 N-labeled RodA, RodB or RodC.

The comparison of the three hydrophobins were compared by recording onedimension [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C CP experiments (64 scans) using a 1 ms contact time for the 1 H-13 C polarization transfer. Two dimensions [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C- [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C PDSD experiments (128 scans) fingerprints were recorded with 50 ms mixing time and a 1 ms contact time for the 1 H-13 C polarization transfer. Proton decoupling with a frequency of 90 kHz was applied during acquisition times, using the SPINAL-64 decoupling sequence.

13 C/ [START_REF] Fisher | Emerging Fungal Threats to Animal, Plant and Ecosystem Health[END_REF] N-labeled RodA-All experiments were performed on a 600 MHz 1 H Larmor frequency spectrometer (Bruker Biospin), at 276 K with a 11kHz MAS and using a 3.2 mm MAS rotor filled with [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C/ 15 N-labeled RodA. The assignment of the hydrophobin RodA was done using 2D [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C- [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C PDSD data recorded using 1 ms contact time for the 1 H- [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C polarization transfer and 50 ms (to obtain intra-residue correlations) or 150 ms (to obtain inter-residues correlations) mixing time.

Two dimensions 15 N- [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C NCO (416 scans) and NCA (416 scans) were recorded using a CP transfer from 1 H to [START_REF] Fisher | Emerging Fungal Threats to Animal, Plant and Ecosystem Health[END_REF] N of 0,6 ms and a double cross-polarization transfer [START_REF] Fisher | Emerging Fungal Threats to Animal, Plant and Ecosystem Health[END_REF] N- [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C and 1 H- [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C with a contact time of 4 ms.

Two dimensions 1 H- [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C INEPT experiments (80 scans) recorded using 90 kHz proton decoupling SPINAL-64.

2 H/ [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C/ [START_REF] Fisher | Emerging Fungal Threats to Animal, Plant and Ecosystem Health[END_REF] N-labeled RodA-All experiments were performed on a 800 MHz 1 H Larmor frequency spectrometer (Bruker Biospin), at 298 K with a 110 kHz MAS and using a 0.7 mm MAS rotor filled with 2 H/ 13 C/ 15 N-labeled RodA.

Wide-angle X-ray scattering (WAXS) experiments.

Fiber diffraction patterns of RodA, RodB and RodC were measured at 4 °C on a Rigaku FR-X rotating anode X-ray generator (Rigaku, Tokyo, Japan) equipped with a Pilatus 200 K hybrid pixel (Dectris, Baden, Switzerland) at the copper wavelength. The concentrated hydrated samples were mounted in a MicroLoops from Mitegen (Ithaca, NY, USA) on a goniometer head under cold nitrogen flow. Each diffraction pattern corresponds to a 360° rotation along the w axis with an exposure time of 720 s after subtraction of a blank image of the same exposure time with only the loop on the goniometer head.

ASPERGILLUS FUMIGATUS CONIDIAL CELL WALL

Conidia are small airborne asexual spores designed to be widespread in order to find an appropriated medium -such as lung alveoli -to start germinating. They are among the most dominant fungal cells in the air and can travel on both short and long distances [START_REF] Bennett | An Overview of the Genus Aspergillus[END_REF] . These conidia are thus made to resist external parameters such as UV-light, extreme heat or dehydration 98 .

For A. fumigatus, the dormant conidia are surrounded by a cell wall, essential for their growth and allowing them to resist against host defense mechanisms. This cell wall, rigid but permeable, accounts for 20 to 40% of the dry cell weight and it is mainly composed of polysaccharides (> 90% of the cell wall content) [START_REF] Latgé | The Cell Wall: A Carbohydrate Armour for the Fungal Cell[END_REF] . These polysaccharides are usually separated between two categories: alkali-insoluble polymers (i.e., the fibrillar core) and alkalisoluble polymers (i.e., amorphous part) intercalating between fibrils. The conidial fibrillar core is composed of β-1,3-glucan, chitin and chitosan whereas the amorphous part of the cell wall contains α-1,3-glucan and mannan. This complex polysaccharides network is then covered by the DHN-melanin pigment and a rodlets layer of hydrophobins.

In this part, we aim at investigating the cell wall of the dormant conidia -especially the carbohydrates network -using multidimensional SSNMR to characterize its composition as well as its dynamic (rigidity/mobility of the polysaccharides).

Effect of the PFA-fixation on A. fumigatus conidia

The duration of NMR experimentation ranges from hours to days-scale. This large time-scale could be problematic when applied to the study of living organism because their structural organization could evolve during the acquisition time (e.g., growth, differentiation…). Indeed, the sample homogeneity remains a crucial issue in NMR analysis 100 : by preventing the differentiation of spores (hours) during the NMR experiments (days), fixation processes could help in reducing the sample heterogeneity and will increase the NMR spectral resolution. Here, we chose to prevent the conidial germination by fixing cells in one define state (i.e., dormant, swollen or germinating) using paraformaldehyde-based fixation process.

Fixation was used in biology for decades to preserve cellular architecture and composition of cells in the tissues. This process also preserves the proteins, carbohydrates and other bio-active moieties in their spatial relationship to the cell; making fixation processes suitable for NMR structural analysis of whole cells 101 . It is a gradual and complex physico-chemical process, involving diffusion of fixative agent into the tissue and a variety of potential physical phenomenon and chemical reactions.

Among all the fixatives, formaldehyde is one of the most commonly used.

Formaldehyde is a gas; this small molecule (HCHO, of which the -CHO is the aldehyde group) dissolves rapidly in water, and combines chemically with it to form methylene hydrate (HO-CH 2 -OH) (Fig. 17A). The aldehyde group can combine with -NH or -OH from proteins or carbohydrates, if they are very close together, to form a cross-link -CH 2 -bridge called a methylene bridge. Briefly, formaldehyde reacts with primary amines to form Schiff bases or with amides to form hydroxymethyl compounds. Hydroxymethyl groups condense with another amide moiety to form methyl diamides (Fig. 17B). Alcoholic hydroxyl forms acetals while sulfhydro groups form sulfhydral acetal analogues with formaldehyde.

In this study, we chose to fixed cells using para-formaldehyde (PFA), a polymer of formaldehyde, insoluble in water and requiring heat and high pH to depolymerize (Fig. 17C).

Depolymerization of PFA produces formaldehyde molecules in solution that will be involved in the creation of chemical bonds between molecules in the sample. PFA also dissolves some lipids in cellular membranes which slightly damages the cell membrane integrity 101-104 . The impact of the PFA-fixation process on A. fumigatus cells was investigated by SSNMR to decipher whether it modifies the conidial cell wall composition and/or structure.

We recorded 1D [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C CP spectra on both unfixed and PFA-fixed dormant conidia to obtain comparable [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C fingerprints (Fig. 18). To do so, we performed our experiments using two full As the PFA covalently cross-link polysaccharides and proteins, it was suspected to globally rigidify the complete cell organization by preventing the movement of molecules within the cell wall. We chose to use CP-based experiments aiming at targeting rigid molecules (motion < ms) to probe a potential rigidification effect of the PFA onto the cell wall. As expected, the PFA-fixed sample exhibits a higher signal/noise ratio reflecting a higher rigidity of the molecules in this sample. Nevertheless, no chemical shift perturbations were observed in the carbohydrates or aliphatic spectral area as the fingerprint of both fixed and unfixed conidia remain superimposable. Thus, the fixation of conidia using PFA seems to be a good alternative for stopping cell growth without influencing the cell wall composition but it increases the global cell wall rigidity. Assuming that the PFA impacts all the conidial compounds in the same way, the potential differences in dynamic between all the compounds will be conserved and a dynamic-based study of these cell wall components remains relevant.

Moreover, as the PFA reactant is not [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C-labeled, its use is compatible with NMR spectroscopy. To conclude, all the following experiments of this chapter will be performed using PFA-fixed conidial samples.

The dormant conidial cell wall

The rigid part of the conidial cell wall

In this section, we first investigated the dormant conidial external structures using 2D [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C- [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C PDSD experiments (Fig. 19A). This experiment relies on 1 H- [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C CP-based transfers and thus targets rigid molecules. The magnetization is propagated through dipolar couplings (i.e., through space) between all the carbons of a spin system (in this case 'spin system' is employed to describe all nuclei of a single molecule or a molecular subunit in case of polymeric carbohydrates). For instance, all the carbons belonging to one pyranose sugar ring result in one spin system of at least six carbons. Based on the literature, we were able to assign the [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C carbohydrates chemical shifts corresponding to the main carbohydrates composing the conidial cell wall of A. fumigatus (Fig. 19B) [START_REF] Kang | Molecular Architecture of Fungal Cell Walls Revealed by Solid-State NMR[END_REF][START_REF] White | Water-Polysaccharide Interactions in the Primary Cell Wall of Arabidopsis Thaliana from Polarization Transfer Solid-State NMR[END_REF]105 . Surprisingly, despite the complex composition of the conidial cell wall, the spectrum was not over-crowded and allowed assigning unambiguously the different carbons of each

A B

polysaccharide. Moreover, this experiment probes preferentially the rigid part of the cell wall:

as expected, the network of polysaccharides covalently linked together (i.e., α-glucan, βglucan and chitin), highly constrained, stabilized and rigid, is strongly represented [START_REF] Beauvais | Aspergillus Cell Wall and Biofilm[END_REF] .

The spin system assigned to β-1,3-glucan is the most intense: this could be due either to the huge amount of β-glucan within the cell wall and/or the rigidity of the carbohydrate.

Indeed, the fibrillar β-glucan is one of the main components of the conidial cell wall.

Moreover, it is involved in the rigidity of the cell wall as it is covalently linked with the chitin to form the fibrillar core [START_REF] Beauvais | Aspergillus Cell Wall and Biofilm[END_REF] . As the β-glucan glucan do not exhibit any polymorphism, we could here hypothesize that this polysaccharide is mainly synthetized as rigid fibrils located in only one electronic environment.

Despite its well-described role in the cell wall rigidity, the chitin does not exhibit intense signals when analyzed by SSNMR techniques. Indeed, chitin accounts for only 4 -15% of the polysaccharides within the cell wall 106 . This low amount of N-acetylglucosamine as compared with glucans (more than 50 % of the cell wall) could be a sufficient explanation for the low intensity of the chitin spin system. Moreover, the location of the chitin could also impact the magnetization transfer efficiency: the chitin is described to be embedded into the cell wall (i.e., close to the plasma membrane); this could prevent an efficient magnetization of the polysaccharide during the 1 H-13 C CP transfer. Finally, we can notice here the impossibility for deciphering chitin from chitosan (partially deacetylated chitin) as the [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C chemical shifts associated with the C2 of the glucose unit will be too close to be distinguish.

Furthermore, the α-1,3-glucan exhibits two close spin systems. As no significant chemical shift perturbation indicates different branching mode, we assumed the α-glucan to be polymorph: the dormant conidial cell may contains α-glucan in, at least, two different chemical environments. As the α-glucan is an amorphous polysaccharide accounting for approximately 25 % of the total cell wall 107 , it is not surprising to find it present at different location within the cell wall. That way, this polysaccharide could achieve many biological functions. The role and the impact of the α-1,3-glucan will be further discussed in the section "4. The cell wall of α-1,3-glucan-deficiant Δags mutant".

At the same time, we recorded 1D [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C CP spectrum and, because of its sufficient spectral resolution, we were able to unambiguously assign it. We first assigned carbohydrates and we noticed the presence of signals in the aliphatic (0-40 ppm) and C=C area (around 130 ppm) that could correspond to unsaturated lipid (Fig. 20). However, the intensities of these signals are quite low and no cross peaks corresponding to these putative lipids were identified on the 13 C-13 C PDSD spectrum. This indicates that the lipid is not rigid enough and/or in sufficient amount to be detected with a high intensity by CP-based experiment. We will further investigate this lipid content in further sections of this work. 1D [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C CP spectrum of the 13 C/ 15 N-labeled dormant conidia assigned with α-glucan (pink), βglucan (red), chitin (black) and putative lipid (green).

The 2D 13 C-13 C PDSD presented above was informative to assign the three main carbohydrates composing the cell wall. However, spin systems corresponding to other polysaccharides such as mannan or galactan -also described in the literature to be present in the dormant conidial cell wall-remained absent from this experiment. Moreover, [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C- [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C PDSD experiment contains information about both the fibrillar core and the amorphous part of the cell wall. Thus, the alkali-soluble and alkali-insoluble fraction of cell wall polysaccharides is rigid enough to be probed by CP-based experiments.

Furthermore, the [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C chemical shift similarities of all the polysaccharides spin systems lead to signal overlapping. Indeed, all the carbohydrates present in the cell wall exhibit similar structures (i.e., hexose in pyranose conformation except the galactose from galactomannan which is in furanose conformation). To achieve a better resolution and to reduce the spectral overlapping we recorded a 2D [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] The gain in spectral resolution was sufficient to identify three new spin systems as compared with [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C- [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C PDSD. We newly assigned α-1,6-mannan 108 (coming from the galactomannan) and two other incomplete carbohydrates spin systems (Y and X, Fig. 21). The spin system name Y in the Fig. 21 could correspond to galactose, as its C 1 , C 2 , C 3 and C 4 are coherent with the 13 C chemical shift of hexose. Unfortunately, we were not able to assign the C 5 and C 6 of this carbohydrate; absolutely required to identify its conformation (i.e., pyranose or furanose). Considering the X spin system, its C 1 indicates a reducing end of a polysaccharide or a free monomer, rigid enough to be detected by CP-based experiment. Once again, because of the uncompleted characterization of these two spin systems, an unambiguous assignment of these polysaccharides is not possible.

The mobile part of the conidial cell wall

Despite the wide range of rigid carbohydrates probed by CP-based experiments, we were also interested in the mobile content of the cell wall. Thus, we used 2D [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C-13 C DP-INADEQUATE to inquire about its mobile part (Fig. 22A). The 13 C are directly excited (on the opposite to the CP where the [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C are indirectly excited via the 1 H magnetization) and the magnetization is then transferred to neighboring [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C through chemical bonds.

Surprisingly, this experiment reveals at least two other spin systems corresponding to carbohydrates that were not represented in CP-based experiments; this highlights the significant contribution of mobile carbohydrates within the cell wall. Unfortunately, as these spin systems are incomplete, the unambiguous assignment of those polysaccharides remains impossible. Nevertheless, the low chemical shift of C 1 (around 95 ppm) of the Z molecule suggests a reducing sugar with a free C 1 . In this case, this could correspond to a reducing end of the polysaccharides, more flexible than the core of the structure. Potentially, these unassigned spin system could also correspond to galactomannan components as this polysaccharides was described to be both in the flexible and rigid conformations. Indeed, this polysaccharide can be extracted with both the alkali-soluble and alkali-insoluble fractions of the cell wall [START_REF] Fontaine | Molecular Organization of the Alkali-Insoluble Fraction of Aspergillus Fumigatus Cell Wall[END_REF] . Moreover, this β-1,5-galactof-α-1,2-/α-1,6-mannanp polymer was described to be linked to either the amorphous α-1,3-glucan or to the fibrillar β-1,3-glucan giving a clue for a potential duality of the dynamic of the galactomannan. Chitin is exclusively detected in experiments revealing rigid molecules. This potentially confirms its supramolecular organization by folding on itself and its stabilization by intra-chains hydrogens-bonds [START_REF] Fernandes | The Importance of Subclasses of Chitin Synthase Enzymes with Myosin-like Domains for the Fitness of Fungi[END_REF] that make it highly rigid. Moreover, it was already described that chitin, covalently linked to β-1,3-glucan via β-1,4-bonds, composes the conidial fibrillar core [START_REF] Beauvais | Aspergillus Cell Wall and Biofilm[END_REF] . Our results correlate the hypothesis that chitin acts as an exoskeleton for the cell, responsible for its rigidity as it is only found in the alkali-insoluble fraction of the cell wall extraction in A. fumigatus cell wall [START_REF] Fontaine | Molecular Organization of the Alkali-Insoluble Fraction of Aspergillus Fumigatus Cell Wall[END_REF] .

Identification of cell wall lipids

The INADEQUATE experiment reveals an unexpected amount of lipids trapped into the cell wall and probed only with mobile-targeted NMR sequences (Fig. 22B). Our main hypothesis relies on the identification of fatty acid fingerprints (in the spectral region of aliphatic signals) as well as glycerol moieties (-O-CH 2 -) reflecting the presence of a potential Tri-, Di-or Monoacyl glycerol (TAG, DAG or MAG respectively). These lipids were not assigned to phospholipids because no evidence for phosphate content was identified by [START_REF] Sugui | Aspergillus Fumigatus and Related Species[END_REF] P NMR investigations (data not shown). Then, our hypothesis relies on the strong importance of such neutral lipids in many biological functions and in the cell wall structuration 109 . Indeed, glycerol, presumably acting as an osmoregulator, is accumulated in spores of several Aspergillus fungi, and its content is also changing considerably during conidial germination.

Furthermore, recently, TAGs were demonstrated as a suitable scaffold associated with melanin formation in the yeast Cryptococcus neoformans 110 leading here to the hypothesis that these lipids could play the same role in the cell wall of A. fumigatus.

A B

The fatty acids esterifying the glycerol skeleton can be of different length and with or without unsaturations. In Aspergillus spp., the most commonly found fatty acids are C 16:0 , C 18:0 , C 18:1 , and C 18:2 111 . In our case, the 13 C-13 C DP-INADEQUATE, which uses throughbond magnetization transfer, did not allow us to probe the entire acyl chain because of the over crowdedness of the aliphatic area and the magnetization loss (i.e., too low SNR).

Moreover, as an oxygen atom prevents the 13 C- [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C magnetization transfer between the glycerol and the fatty acid, no evidence can be established as regard to the acylation level of the glycerolipid (i.e., TAG, DAG, MAG). For these reasons and based on our assignment, we hypothesized the structure of a TAG as presented in Fig. 23.

Fig. 23: Structure of a putative TAG identified with the conidial cell wall. [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C chemical shifts obtained from 2D [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C- [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C DP-INADEQUATE are presented in green.

Discussion on the dormant conidia cell wall

The investigation of the composition of the dormant conidial cell wall of A. fumigatus by SSNMR proves it to be mainly composed of a strong network of polysaccharides with a predominance of α-glucan, β-glucan and chitin. The dynamic-based study of the cell wall components showed these three carbohydrates to be mainly produced as rigid carbohydrates (probed by CP-based experiments). However, a part of both α-glucan and β-glucan is also present in a mobile state (probed by DP-based experiments). This could be consistent with the expression of different pools of sugar exported at different locations within the cell wall and exhibiting different mobility states.

To conclude, despite some analytical drawbacks, such as the impossibility of distinguishing between chitin and chitosan by [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C-detected analysis or to assign unambiguously all the carbohydrates spin systems, our analysis of the conidial cell wall is robust enough to start studying complex phenomenon such as the germination process.

INVESTIGATION OF THE GERMINATION PROCESS

As Aspergillus fumigatus belongs to the Ascomycota, it exhibits two types of life cycles: an asexual and a sexual one. The asexual reproduction involves specialized cells called conidia formed exogenously on specialized hyphal branches called conidiophores.

These conidia are not only useful for the reproduction of the fungus but also for A. fumigatus to survive in hostile conditions. Moreover, in immunocompromised individuals, inhaled conidia can germinate inside the respiratory system, which may result in invasive pulmonary aspergillosis. The proliferation of conidia is then threatening the patient survival and the development of alternative strategies is stressed by the fungal drug resistance burden.

Thus, the germination of dormant conidia appears crucial for infection; the prevention of this process might be a new therapeutic target. In this context, a broader understanding of this biologic phenomenon through the atomic remodeling of the conidial cell wall during the process could open up the way to the discovery of anti-germination targets.

The germination of A. fumigatus

The germination of the fungus starts with the activation of the resting spores. Once the dormancy is broken, the intracellular osmotic pressure strongly increases and leads to a quick uptake of water. The resulting swollen conidia doubles in cellular diameter (isotropic growth)

as compared to the dormant conidia and both morphotypes can then be distinguished by conventional microscopy. Then, a germ tube is formed on a polarized fashion (polarized growth) resulting in a germinating conidia (Fig. 24). To sum up, the germination of A. fumigatus can be divided in three events: breaking of dormancy, isotropic swelling and establishment of the cell polarity/formation of a germ tube.

In order to get new insights into the evolution of the cell wall architecture during this process, we focused on three samples representing the three main steps of the germination: (1) the dormant conidia; (2) the swollen conidia; (3) the germinating conidia.

These conidia exhibit morphological differences as listed below but share also many common components. For instance, the conidial fibrillar core is exclusively composed of polysaccharides: a β-glucan scaffold to which chitin/chitosan and galactomannan are covalently linked. Then, amorphous linear chains of α-glucan have adhesive properties and stabilize the cell wall. This core (i.e., fibrillar and amorphous polysaccharides) evolves with the germination as the fungus produces new carbohydrates and regulates differentially the amount of others. Added to all the morphological specificities of the three different conidia, this carbohydrates regulation suggests a complex cell wall remodeling throughout the germination process.

Dormant conidia

Dormant conidia are the resting form of A. fumigatus conidia. These cells are of 2.8-3 µm diameter and are extremely resistant to external conditions: for instance, they can survive in liquid nitrogen up to eighteen years 112 .

Structurally, the fibrillar core of the dormant conidial cell wall is composed of the polysaccharides presented above. Two external dense layers surround this core: the superficial one is composed of rodlets covering a layer of DHN-melanin. These two structures confer hydrophobicity properties to the conidia and prevent its recognition by the host immune system. They do not uniformly cover the surface of the dormant conidia but forms a patchy network [START_REF] Valsecchi | The Puzzling Construction of the Conidial Outer Layer of Aspergillus Fumigatus[END_REF] . The role and the structure of both RodA rodlets and DHN-melanin will be further discussed in following sections.

Swollen conidia

Generally, as soon as the dormancy is broken (in vivo and in vitro), the rodlet and the melanin layers disappear, exposing α-glucans at the surface of the swollen conidia [START_REF] Bayry | Surface Structure Characterization of Aspergillus Fumigatus Conidia Mutated in the Melanin Synthesis Pathway and Their Human Cellular Immune Response[END_REF] . Then swollen conidia increase their diameter up to 5-6 µm. During the conidial swelling in liquid medium, these α-glucans interact together and lead to the conidial agglutination.

Nevertheless, even though the rodlets layer is always degraded by aspartic proteases, the melanin layer can be still present as the pksp gene involved in the melanin synthesis pathway remains active in some particular cases (both in vivo or in vitro) 113 .

Germinating conidia

Once the polarized growth starts, a germ tube appears at one side of the conidia making the germinating conidia easily identifiable. The diameter of the conidial head remains 5-6 µm and the germ tube has to be 5-10 µm long. These morphological changes are accompanied with the production of a new polysaccharide, the galactosaminogalactan (GAG), arranged as an outer layer. GAG is a heterogeneous linear polymer composed of galactopyranose (in contrast to galactofuranose found in the galactomannan) and N-acetylgalactosamine linked with α-1,4 linkages. This polymer is involved in the pathogenesis of aspergillosis [114][115][116] : GAG suppresses host inflammatory responses partly through the masking of α-glucans from their recognition by Dectin-1.

The conidial cell wall composition

Remodeling of the rigid conidial cell wall

In this section, all the experiments presented rely on 1 H- [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C CP-based transfer probing only the rigid part of the cell wall. In order to get a first [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C fingerprint of the conidial cell wall in the three stages of the germination, we recorded 1D [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C CP experiments in comparable experimental conditions (i.e., size of the rotor, rotor filling, experimental parameters…) (Fig. 25).

First, these three fingerprints exhibit significant differences in signals intensities. For instance, important variations are observed for sugar spin systems, especially visible in the [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C spectral region corresponding to anomeric carbons (from 95 to 110 ppm). Based on these preliminaries results, we observed the composition of the cell wall to remain globally identical throughout the germination: during the three steps, the core of β-glucan, α-glucan and chitin seems to be conserved. Nevertheless, we noticed the strong diminution of the βglucan spin system intensity in the swollen conidia as compared to the dormant and the germinating ones; suggesting a potential regulation of conidial carbohydrates content in a germination state-dependent way. To increase the spectral resolution and to reduce the peaks overlap, inevitable in 1D experiments, we then recorded 2D 13 C- [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C PDSD spectra on the three samples (Fig. 26A).

First, the intensity of both chitin and α-glucan spin systems increases during the swelling step and is maximal during the polarized growth (i.e., germinating conidia). These variations could be due to the over-expression of such carbohydrates at the end of the process and/or to the rigidification of these polysaccharides making them more sensitive to CP-based polarization.

These carbohydrates also exhibit a higher degree of polymorphism in the swollen and germinating state as compared with the dormant state. Anyway, these 2D spectra clearly highlight a carbohydrate-dependent regulation of the cell wall components during the germination. Second, during the swelling, important modifications of the cell envelope are required to adapt to this new state. This involves cell wall remodelling 117 . As observed earlier, the intensity of the rigid β-glucan spin system strongly deceases during the swollen step (Fig. 26B). This observation is coherent with the production of β-glucanase 118 during the isotropic growth of the dormant conidia. Indeed, the β-glucan is known to be responsible for the rigidity of the dormant spores as it can adopt tertiary structures in simple or triple stranded helices 119 ; this rigidity playing a role in the resistance against exterior stresses. On the opposite, during the growth, the cell wall has to be softened and then β-glucan need to be enzymatically degraded. These data are coherent with the strong decrease of NMR signals recorded for the β-glucan (Fig. 26C).

Such kinds of precise carbohydrates regulations were not as well described for the other components of the cell wall. To inquire further about this phenomenon, we used the A B C quantitative 1D [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C DP spectra (independent from the dynamic of the molecule) to decipher the relative amount of each component of the cell wall throughout the germination process (Fig. 26C). First, the total relative amount of β-glucan varies as described earlier for the rigid part of the polysaccharide: the amount of β-glucan strongly decreases during the isotropic growth due to the action of β-glucanases but it is overexpressed during the polarized growth; reaching a higher amount than in the dormant conidia. This observation could support the hypothesis that a rigid conidial cell wall is required during the emergence of the germ tube to preserve the conidial integrity while forming hyphae.

Then, the relative amount of α-glucan continuously increases throughout the conidial germination. This overproduction of α-glucan is positively correlated with its polymorphism pattern. The breaking of the dormancy apparently triggers the production of α-glucan taking place in different locations within the cell wall. These different locations would induce different electronic environments, explaining the polymorphism displayed by the carbohydrate.

Finally, the relative quantity of chitin does not exhibit strong variations throughout the germination process. However, its polymorphism increases from the swelling step. We hypothesize here that the location of the chitin could be modified during the conidial growth.

First linked to the β-glucan in the dormant conidia, the chitin network would be remodelled following the action of β-glucanases onto the fibrillar core. The newly less-constrained chitin would be found in different locations within the cell wall, increasing its polymorphism.

Moreover, the increased intensity of CP-detected signals could be linked either to higher amounts and/or increased rigidity of the carbohydrates and/or better magnetization accessibility. For this last point, we could notice here the disappearance of both rodlets and DHN-melanin layers from the starting of the isotropic growth, potentially allowing a better polarization of carbohydrates.

Surprisingly, the 2D 13 C-13 C PDSD does not reveal spin systems that corresponds to galactosaminogalactan. Its absence in CP-based experiments led us to the hypothesis that this polysaccharide is only produced in a mobile state. To investigate its presence in the mobile part of the cell wall, we recorded a 2D 13 C-13 C DP-INADEQUATE (probing only mobile molecules) on the germinating conidia. We then compared the resulting spectrum to the one obtained for the dormant conidia (Fig. 27). The superimposition of both spectra highlights the presence of a new and intense spin system in the germinating sample. Its intensity strongly suggests that this carbohydrate is produced in comparable amount with the mobile glucan; making it quantitatively significant within the carbohydrates network. Based on the literature, we assigned this spin system to the N-acetylgalactosamine composing the GAG; one of the main carbohydrates composing the cell wall of the germinating conidia. This polysaccharide mediates the adhesion of germinating conidia to other materials 120 and it is thus supposed to be expressed at the surface of the conidia. Moreover, this polysaccharide is extracted with the alkali-soluble fraction of the conidial carbohydrates confirming that this polysaccharide only weakly interacts with the fibrillar core of the cell wall. All together, these arguments correlate the global mobility of the polysaccharide.

Dynamic-based regulation of polysaccharides

This dichotomy between the mobile and the rigid contributions of each polysaccharide raises many questions. One of them concerns the potential impact of the germination on these dynamic states. To investigate whether the conidial growth impacts the dynamics of the different conidial components, we recorded 1D [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C DP spectra with a long recycle delay (35 s) to obtain quantitative spectra on the three samples. These spectra were compared with their corresponding [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C DP spectra recorded with a short recycle delay (2 s) reflecting only the mobile part of the cell wall (Fig. 28). First, during the dormancy, both quantitative and mobile spectra are well superimposed proving the cell wall to be globally mobile. Then, once the dormancy is broken, the intensity of the mobile spectrum decreases continuously. Thus, the proportion of mobile compounds within the cell wall decreases during the germination reflecting a global rigidification of the complete cell wall.

Nevertheless, we observed that all the compounds of the cell wall do not behave the same way. To go further in our analysis and to probe the different molecules according to their dynamic states, we then compared the quantitative 1D [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C DP spectra to [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C-detected spectra probing only rigid (Fig. 25) or mobile (Fig. 29A) molecules. By plotting the relative intensity of both mobile and rigid contributions of each component as function of the germination state, we investigated how the dynamic of each component evolves during the germination (Fig. 29B). We first observed that all the main conidial components exhibit different behavior throughout the isotropic and polarize growth. First, the rigid contribution of chitin increases during the swelling state (Fig. 29B). This correlates with its higher sensitivity to CP-based polarization transfer. However, hypothesis explaining the putative chitin rigidification will be discussed later. Moreover, even though the potential regulation of β-glucan during the isotropic growth was already discussed above, the dynamics-based study of this carbohydrate provides new insights. We speculated that only the rigid part of β-glucan is degraded by βglucanases during the swelling of the conidia as the intensity of the mobile contribution remains constant. This is coherent with the need for the cell wall to be softened in order to adapt quickly to an increased intracellular osmotic pressure and to allow the isotropic growth.

During the last step of the germination, the newly synthetized rigid β-glucan is highly

A B

produced, as the cell might need to reinforce its architecture in order to balance the polarized growth-effect.

Furthermore, the proportion of α-glucan continuously increases during the germination process and, apparently, a part of the mobile α-glucan is progressively converted into rigid counterpart. Once again, this polysaccharide would be required during the germ tube emergence to support the conidial cell wall integrity. However, the α-glucan was found to be more polymorphic in the swollen step as compared to the dormant and germinating state. This could be coherent with the export of α-glucan at the surface of swelling spores, creating a new electronic environment for the polysaccharides and leading to the aggregation of conidia 121 .

Finally, the unsaturated lipids observed using [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C- [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C DP-INADEQUATE experiments within the cell wall is present in a mobile state and with the same proportion in every conidial sample. Even though the role of this lipid, potentially TAG, remains unknown, its presence in the same electronic environment seems to be constant throughout the germination.

The conidial cell wall architecture

All the dynamics-based modifications of the carbohydrates occurring within the cell wall suggest a noticeable remodeling of the conidial architecture during the germination, as observed by SSNMR. To investigate this architecture, we chose to characterize the interaction of the water with the different rigid compounds of the cell wall. The aim was here to inquire about the proximity between one polysaccharide and the external medium. All the experiments listed below rely on a CP-based transfer between the water proton and the carbons of the cell wall components. Using this methodology, we only probed the rigid contribution of each compound. We first recorded 2D [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C-1 H HETCOR with or without a 1 H-1 H spin diffusion (SD) mixing time of 1.5 ms (Fig. 30A). In the NMR experiment, the protons from water molecules are first excited and the magnetization diffuses between protons via dipolar couplings, then it is transferred to rigid carbohydrates, and then detected on [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C using a 1 H-13 C CP transfer. Thus, the efficiency of this transfer is mediated by distance-and mobility-dependent 1 H-1 H dipolar couplings. Briefly, a molecule close to the water will get excited first and will exhibit a 1 H water- [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C polysaccharide cross peak more intense as compared to a molecule embedded into the cell wall and/or less water-accessible. We first questioned how water molecules are associated with the cell wall. For bulk water, we expected a sharp 1 H signal of dozens of hertz-width due to the fast tumbling of water molecules in solution [START_REF] White | Water-Polysaccharide Interactions in the Primary Cell Wall of Arabidopsis Thaliana from Polarization Transfer Solid-State NMR[END_REF] . The broad width of the 1 H water signal giving a cross peak with [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C carbohydrates (Fig. 30B) indicates that the water is immobilized, and could potentially lead to a higher chemical exchange rates with the polysaccharides [START_REF] White | Water-Polysaccharide Interactions in the Primary Cell Wall of Arabidopsis Thaliana from Polarization Transfer Solid-State NMR[END_REF] . Contrary to swollen and germinating conidia, the surface of the dormant conidia is highly hydrophobic due to the presence of both RodA rodlets and melanin. Nevertheless, this covering is not continuous and its displays holes through which carbohydrates can infiltrate and reach the surface. We hypothesize here that water molecules are immobilized at the surface or into these holes.

On the 2D [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C-1 H HETCOR spectra, we extracted 1D [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C traces reflecting the correlations of conidial compounds with water molecules (highlighted in light pink, Fig. 30A). Using the signal intensity of peaks corresponding to each conidial component, we were able to estimate the proportion of carbohydrates accessible to water (Fig. 30C). We observed that the relative intensities of α-glucan, β-glucan and chitin signals vary according to the

A B C

germination states indicating a polysaccharide-dependent regulation of the water accessibility of polysaccharides network. We demonstrated that germination prevents the water accessibility of both chitin and β-glucan. At the same time, the α-glucan exhibits the opposite behavior demonstrating its presence at the surface of the cell while the conidia starts to germinate; confirming its implication into conidial aggregation. Nevertheless, this experiment only gives preliminary results in term of cell wall architecture as the short 1 H-1 H spin diffusion only allows to transfer polarization to molecules close enough to water and prevents to get information about cell wall-embedded molecules.

Thus, to go further in this analysis -and still using experiments based on the waterpolysaccharide magnetization transfer -we performed water-edited experiments. In these experiments, the magnetization of rigid carbohydrates is first destroyed using a 1 H T 2 filter, then an initial magnetization is created on water 1 H and finally transferred to the [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C cell wall components as function of their water accessibility using a CP transfer. To obtain the wateredited fingerprint of the three conidial states, we recorded 2D water-edited [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C- [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C PDSD experiments with a short or a long 1 H-1 H spin diffusion for all the germination states (Fig. 32A). We expected the molecules closest (in space) to the water to be polarized even with a short mixing time. On the contrary, molecules embedded into the cell wall would require a longer time for the magnetization to be transfered to them. A) 2D water-edited 2D [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C- [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C PDSD experiments recorded with a short (blue) or a long (pink) [START_REF] O'meara | The Cryptococcus Neoformans Capsule: A Sword and a Shield[END_REF] H-1 H spin diffusion on [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C/ [START_REF] Fisher | Emerging Fungal Threats to Animal, Plant and Ecosystem Health[END_REF] N labeled dormant and germinating conidia. B) 1D [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C

A B

traces extracted from 2D 13 C- [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C PDSD (black), 2D [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C- [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C PDSD with 5 ms (blue) or with 20 ms (pink) for the dormant conidia and 1 H-1 H spin diffusion.

In these experiments, we observe that the three conidial states exhibit different 2D

water-edited fingerprints. Once the resting conidia starts to germinate, the two layers of RodA rodlets and DHN-melanin start to be degraded. This degradation reduces the thickness of the cell wall and subsequently increases the water-accessibility of the conidial carbohydrates network. In this context, we anticipated a global higher water-accessibility for cell wall carbohydrates resulting in intense NMR signal for the two last states of the germination.

Indeed, the Fig. 31A highlights two different patterns: (1) in the dormant conidia, the polysaccharides do not exhibit any signal when the experiment is performed using a short 1 H-1 H spin diffusion; (2) on the contrary, the carbohydrates network found in the germinating conidia seems to be highly water-accessible as it is polarized even using a short 1 H-1 H spin diffusion. Nevertheless, even if these two fingerprints clearly illustrate the global different organization of both dormant and germinating conidia, all the carbohydrates do not behave the same way. For instance, even at the end of the process, the chitin remains not as accessible to the water magnetization as compared with β-glucan (Fig. 31B).

To investigate these carbohydrates-dependent water-accessibility profiles, we recorded a series of 1D water-edited [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C CP spectra (Fig. 32). To get insights about the wateraccessibility of each compounds, we plotted the signal intensity of each compounds as function of the 1 H-1 H spin diffusion mixing time. Thus, the slope of the resulting build-up curves depends on the water-accessibility of the molecule. For instance, a fast build-up refers to a molecule highly accessible to water. While 2D 1 H- [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C HETCOR experiments provide surface-limited information, the build-up curves reported above refer to the whole architecture of the cell wall. By comparing the build-up curves of the same compound in the three conidia states, we expected to follow their changes in location within the cell wall throughout the germination. First, surprisingly, no significant differences in the water-accessibility profile of α-glucan, β-glucan or the unsaturated lipid were observed between the three conidial states. This suggests the three compounds to be equally water-accessible in the dormant, swollen or germinating cell wall.

This illustrates potentially the same location for the rigid part of the α-glucan and β-glucan within the conidia cell wall throughout the germination process.

On the contrary, chitin exhibits a remarkable change in water-accessibility during the polarized growth. At this step, the slope of the build-up curve significantly decreases indicating a much lower water-accessibility of the polysaccharide as compared with the dormant or swollen conidial cell wall. Thus, at the end of the process, the location of the chitin changes making the magnetization transfer slower to reach the polysaccharide. Our main hypothesis would be that the chitin is more embedded within the cell wall (i.e., closer to the plasma membrane) at the end of the germination process. This rigid polysaccharide could act as a skeleton, compensating the loss of melanin and RodA, and maintaining the integrity of the germinating conidia while the polarized growth starts and the germ tube.

Structural effects of the germination process

The conidial cell wall of A. fumigatus is a complex structure submitted to impressive remodeling throughout the germination process. In this section, we demonstrated the variations occurring as for the composition, the dynamics and the location of compounds within the cell wall.

Briefly, the whole conidial cell wall rigidify once the spores starts to germinate but the dynamics of each compounds is regulated in a precise manner. Indeed, the main dynamic contribution of α-glucan, β-glucan, chitin and the putative TAG are summarized in Fig. 33.

Then, on the same figure and based on our water-edited study, we tried to schematize the location of the different compounds within the cell wall according to the different states of development.

Fig. 33: Proposed cell wall remodeling upon the germination process.

THE CELL WALL OF α-GLUCAN-DEFICIENT ΔAGS MUTANT

The α-1,3-glucan was initially described in Schizosaccharomyces pombe 122 to play a role in the fission yeast morphogenesis. In pathogens such as A. fumigatus, the α-1,3-glucan is synthetized by three α-1,3-glucan synthases (AGS) consisting in transmembrane enzymes with very high molecular weight (<200 kDa). This molecule is described as an amorphous polysaccharide accounting for 20-25 % of the conidial cell wall 107 and mediating conidial and hyphae aggregation 121 . Moreover, the α-1,3-glucan would be involved in maintaining the cell rigidity as its presence prevents the conidial swelling [START_REF] Valsecchi | The Puzzling Construction of the Conidial Outer Layer of Aspergillus Fumigatus[END_REF] . In this section, we used the triple mutant Δags1 Δags2 Δags3 (named Δags mutant below) 123 to identify the effect of the depletion of α-glucan on the dormant conidial cell wall architecture. Indeed, this mutant does not express any α-1,3-glucan but, surprisingly, does not exhibit any reduction in viability or growth in vitro 123 .

Composition and dynamic-based study

In order to validate the non-production of α-1,3-glucan in the Δags mutant, we first recorded a 1D [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C CP fingerprint of the Δags mutant and compared it with the (WT) dormant conidia (Fig. 34A). The 13 C CP fingerprint recorded on the Δags dormant conidia proves the α-1,3-glucan to be totally absent from the cell wall as the signals corresponding to its spin system disappear in the mutant. Chitin from the Δags dormant conidial cell wall exhibits a more polymorphic [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C- [START_REF] Fisher | Emerging Fungal Threats to Animal, Plant and Ecosystem Health[END_REF] N pattern as compared with the WT. This was coherent with our hypothesis that the cell wall is remodeled in response to the mutation of the α-1,3-glucan. Moreover, preliminary evidence for the existence of this compensatory mechanism were already described using biological assays: the chitin was found to be overexpressed in the alkali-soluble fraction of the cell wall 123 . We could add to our hypothesis that, as seven chitin synthases were found in the genome of A. fumigatus 124 , they could be differentially activated to compensate the loss of one structural polysaccharide like the α-1,3-glucan. Furthermore, electron microscopy-based analysis proved the cell wall to be as thick for the mutant than for wild type. Thus, the overproduction and/or the modified location of chitin could be linked with the need for the cell to reinforce the rigidity of the cell wall affected by the loss of α-glucan 123 .

These results were confirmed by the relative quantification of both chitin and β-glucan in the cell wall of Δags dormant conidia using quantitative 1D [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C DP spectra (Fig. 36A).

We proved both polysaccharides -especially the chitin -to be overproduced in the mutant as compared with the WT. Moreover, for the Δags dormant conidia, the intensity of the mobile molecules detected in the DP spectrum appears lower than its equivalent for the WT dormant conidia (Fig. 36B). We assumed that the cell wall has the tendency to be more rigid for the mutant; which is coherent with a higher production of chitin. Indeed, as described in previous sections, chitin is known to be one of the polysaccharides conferring rigidity to the cell wall by folding on itself and being stabilized by intra-chains hydrogen bonds [START_REF] Fernandes | The Importance of Subclasses of Chitin Synthase Enzymes with Myosin-like Domains for the Fitness of Fungi[END_REF] .

Fig. 36:

The dynamic of the Δags dormant conidia cell wall.

A) Relative quantification of cell wall compounds obtained from quantitative 1D [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C DP spectra recorded on [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C/ 15 N labeled-WT or Δags dormant conidia.

Water-accessibility of conidial compounds

Then, we were wondering whether the architecture of the conidial cell wall evolves in response to the loss of α-1,3-glucan. We first recorded 2D 1 H- [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C HETCOR spectra with a 1 H-1 H spin diffusion of 1.5 ms to obtain correlations between the water protons and the carbons of the carbohydrates present at the water/cell wall interface. Then, we extracted the 1D [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C trace reflecting the cross peaks of carbohydrates carbons with water proton (Fig. 37A)

and we compared it with the data obtained for the WT (Fig. 37B).

Fig. 37: Water-accessibility of the cell wall carbohydrates.

A B A B

A) 1D [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C traces extracted from 2D 1 H- [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C HETCOR experiment at δ 1 H 4.9 ppm recorded on both [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C/ 15 N labeled-WT or Δags dormant conidia.

In this experiment, the peaks corresponding to the β-glucan and to the unsaturated lipids are more intense for the Δags dormant conidia as compared to the WT. This result illustrates a higher water accessibility of these two molecules in the mutant conidial cell wall.

On the other hand, even though chitin is overexpressed to compensate the loss of the αglucan, this location in the cell wall does not seem to vary as its not more exposed at the surface of the mutant spores.

Furthermore, we recorded water-edited experiments to decipher the effect of the depletion of α-glucan on the hydration of the cell wall. We chose to record the 2D 13 C-13 C water-edited PDSD fingerprints of the Δags dormant conidia with 5 and 20 ms 1 H-1 H spin diffusion. We compared the water-edited profile of the Δags dormant conidia with its WT counterpart (Fig. 38A). The global fingerprint of both WT dormant and Δags dormant conidia looks the same: the magnetization of water molecules is not transferred efficiently to the molecules of the fibrillar core of both conidia with a short 1 H-1 H spin diffusion mixing time indicating the carbohydrate network to be as far from the water in WT or mutant dormant conidia. This is coherent with the presence of both rodlets and melanin layers at the surface of both WT and Δags dormant conidia [START_REF] Valsecchi | The Puzzling Construction of the Conidial Outer Layer of Aspergillus Fumigatus[END_REF] that prevents the magnetization to reach the carbohydrates network.

A B

A) 2D water-edited 2D 13 C- [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C PDSD experiments recorded with a short (blue) or a long (pink) 1 H-1 H spin diffusion on [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C/ 15 N-labeled WT and Δags dormant conidia. B) Build-up curves with the relative intensity of peaks referring to one cell wall component plotted as function of the 1 H-1 H mixing time for the WT and Δags dormant conidia.

Then, as presented earlier, we recorded water-edited 1D [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C CP spectra with varying 1 H-1 H spin diffusion used to draw the build up curves presented Fig. 38B. Both β-glucan and chitin exhibit a slower build-up curve in the Δags dormant conidia as compared to the WT one. Thus, these two carbohydrates seem to be less accessible to water and potentially further from the surface. Moreover, dormant conidia -WT or Δags mutant -are covered by a layer of DHN-melanin and rodlets. We do not know whether the mutation of α-glucan impacts the thickness and/or the structure of these two layers. Following our first hypothesis that the surface of the spores is not homogenous but patchy, we could here assume that: (1) the suppression of α-glucan negatively impact the hydration of the cell wall by influencing the permeability of the external layers or (2) that the deletion of α-glucan impact the thickness of the melanin and/or rodlet layer preventing the magnetization the reach the fibrillar core as fast as for the WT dormant conidia.

THE DHN-MELANIN

Melanin is a pigment expressed ubiquitously from humans to plants. This pigment is involved in many biological processes, including the resistance against environmental stresses 125,126 . In airborne fungal pathogens -such as A. fumigatus dormant conidia -melanin also plays a role during infection. For instance, it is involved in the invasion of the host 127 or in the virulence of the pathogen 128,129 . Among the different types of fungal melanin (i.e., DHN-melanin, DOPA-melanin, or pyomelanin), A. fumigatus was described to produce mainly DHN-melanin responsible for the characteristic dark green color of the conidia. DHN-melanin is a negatively charged and hydrophobic pigment of high molecular weight. It mediates the interactions between pathogens and host components in the lung (e.g., fibronectin or laminin) 130 . In A. fumigatus, this pigment also plays a structural role in the assembly of conidial cell wall as it is important for the its structure and stiffness [START_REF] Bayry | Surface Structure Characterization of Aspergillus Fumigatus Conidia Mutated in the Melanin Synthesis Pathway and Their Human Cellular Immune Response[END_REF]131 . The synthetic pathway of DHN-melanin (Fig. 39) involves six enzymes encoded as a cluster on the chromosome 2. The first enzyme of this pathway is the polyketide synthase (PKSP), catalyzing the formation of heptaketide naphthopyrone from acetyl coenzyme A (acetyl-CoA) and malonyl-CoA 132 . The immunological role of this pigment in A. fumigatus has been studied using either melanin ghost or pigment-depleted mutant of the dormant conidia. Interestingly, the DHNmelanin efficiently protects the conidia against reactive oxygen species and masks the spores from their recognition by the host immune system 133 . Regarding all the elements cited above, the DHN-melanin seems to be a key component of the conidial cell wall of A. fumigatus. Nevertheless, despite its clear contribution to the infectious process making it an interesting potential therapeutic target, the structure of the pigment remains unknown. To date, SSNMR investigations proved eumelanins to exhibit aromatic content associated with cell wall-extracted polysaccharides and lipid-like aliphatic moieties 134,135 . Thus, in this section we aim at solving the structure of this enigmatic DHN-melanin using different samples ranging from whole conidia to in vitro synthesized pigment.

The conidial DHN-melanin

In the first part of this study, we were investigating the DHN-melanin structure in its conidial environment. To do so, we performed our experiments only on resting conidia. Indeed, in A. fumigatus, melanin is degraded immediately after the breaking of the dormancy.

The Δpksp dormant conidia are mutated for the pksp gene involved in the melanin synthesis pathway: the synthesis of the melanin is inhibited; the spores remain white.

We recorded a 1D [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C CP fingerprint of the Δpksp dormant conidia. We anticipated the disappearance of [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C signals corresponding to the melanin when compared with the 13 C CP fingerprint of the WT dormant conidia (Fig. 40). As described above, the DHN-melanin results from the condensation of 1,8-DHN which is an aromatic molecule. Thus, we expected the pigment to mainly exhibit aromatic signals. To our surprise, no such evidence was identified; spectra recorded on either WT or Δpksp dormant conidia do not contain signals encoding for aromatic groups. Moreover, the two fingerprints appear to be superimposable.

Based on this observation, we hypothesize that: (1) that the action of a compensatory mechanism occurring in response to the deletion of the pksp gene and leading to the synthesis of a new molecule with the same structural features as the melanin; thus with the same NMR pattern; or (2) that the DHN-melanin amount within the conidial cell wall is below the sensitivity of our SSNMR experiment.

To overcome this potential mechanism, our collaborator (Dr. Vishu Aimanianda, Pasteur Institute, Paris) provided us white dormant conidia. Briefly, the WT conidia were submitted to harsh chemical treatment to remove the superficial layers such as the DHNmelanin. The resulting conidia lose their characteristic dark-green color. We then recorded the white conidia 1D [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C CP fingerprint and compared it with WT and Δpksp dormant conidia spectra (Fig. 40). Still, no evidence for the absence of aromatic molecules was identified. Alignment of 1D [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C CP fingerprints recorded on [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C/ 15 N-labeled WT, Δpksp and white dormant conidia. The pink square highlights the δ 13 C range of expected aromatic signal.

The melanin ghost

Because the analysis of DHN-melanin polymer directly on conidia remains impossible, we decided to study melanin ghosts structures. The melanin ghosts are hollowed structure of DHN-melanin extracted from WT dormant conidia. These electron-dense structures result in black particles of the same size and shape as the conidia. We first recorded the 1D [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C CP fingerprint of this sample. To our surprise, this extracted-melanin sample exhibits only strong carboxyl and aliphatic signals (Fig. 41A). Furthermore, using 2D 13 C- [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C experiments, we observed one spin system in the carbohydrate area indicating the presence of a polysaccharide associated with the melanin ghost. By comparing the spin system resonances with the whole dormant conidia profile, we assigned this spin system to chitin (Fig. 41B). We concluded that the formation of melanin ghost using harsh chemical treatments damages the conidial cell wall and trapped the chitin within the ghost while extracting DHN-melanin. This polysaccharide seems to be strongly associated with the pigment as they are found in a rigid

A B

A) 1D [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C-CP spectrum recorded on [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C/ 15 N-labeled melanin ghost. B) Superimposition of 2D [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C- [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C spectra recorded on [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C/ 15 N-labeled WT dormant conidia (black) or melanin ghost (pink) with the [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C assignment of chitin.

The synthetic DHN-melanin

In order to establish whether 1,8-DHN could condense to form an aliphatic or aromatic pigment, we analyzed the commercially available 1,8-DHN. We polymerized it in vitro (without enzymatic catalysis) following the protocol presented in Fig. 42A. Briefly, the 1,8-DHN is incubated at 50°C in basic buffer for four hours under strong agitation. The solution, initially pink, becomes dark-brownish after four hours of incubation; visible particles start to precipitate. The pellet is recovered by centrifugation and analyzed by CP-based experiment (Fig. 42B). However, as the commercial 1,8-DNH is not [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C-labeled, it was not possible to record 2D spectra on this sample. After polymerization, DHN-melanin can also be recovered using acidic precipitation instead of ultracentrifugation. This sudden change in pH (from pH 11 to 1.7) induces aggregation of floating particles that immediately precipitate. Surprisingly, the [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C CP fingerprint of this sample displays a different pattern as compared with the synthetic DHNmelanin presented above (Fig. 43). First, no aliphatic pattern was identified. Then, an important set of aromatic signals was observed. This is coherent with the polymerization of aromatic cycles from 1,8-DHN. Nevertheless, it raises the question of the impact of the acidic precipitation onto the DHN-melanin structure; which has not been clarified yet.

New insights about the conidial DHN-melanin

In A. fumigatus, the DHN-melanin plays key roles either in the structural organization of the cell wall, by mediating host/pathogen interactions during fungal infections, or by protecting the spores against the host immune system. This pigment thus appears as a good potential target for the development of antifungal therapies and a better characterization of its structure would be crucial.

In this section, we tried to elucidate at least partially the structure of both in vivo (within the conidial cell wall) and in vitro (from commercial precursors) DHN-melanin using SSNMR. This DHN-melanin results from the condensation of 1,8-DHN which is an aromatic molecule; we would expect thus to observe a strong aromatic contribution in our analysis.

First, we tried to assign [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C melanin signal in cellular conditions. To do so, we used melanindepleted conidia (following Δpksp mutation or chemical extraction of the melanin on WT spores) to compare their [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C CP fingerprint with the WT dormant conidia. Unexpectedly, no significant changes were observed between melanin-depleted and melanized cells spectra. These results were not dependent upon the dynamics of the melanin (that could escape to CPbased detection) as we probed also the molecule using DP-and INEPT-based experimentreflecting mobile and highly flexible molecules respectively -with the same result.

Nevertheless, we noticed modification of the aliphatic content of the spectra for melanindepleted conidia. We had hypothesized that the overlapping effect occurring in whole cell [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C-detected spectra may prevent the observation of the melanin signals (hidden by other strong contributions).

Then, as we failed at identifying the melanin in the conidial context, and to reduce of the complexity of the analysis of whole cells structures, we extracted DHN-melanin from dormant conidia to obtain black melanin ghosts; theoretically containing only the DHNmelanin in hollow structures similar in size and shape to the original conidia. Moreover, based on the uniform black color of the melanin ghosts, we assumed the presence of a large amount of melanin within the sample. These melanin ghosts exhibit only [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] Next we investigated in vitro polymerized DHN-melanin. In basic medium, such large particles result from the condensation of 1,8-DHN precursors into hydrophobic polymer of high molecular weight. The biggest particles pellet down after 4h whereas the smallest ones remain partially soluble; the total amount of polymerized melanin is recovered after ultracentrifugation. The 13 C-detected SSNMR profile of this synthetic melanin was similar to the one of the melanin ghosts: the spectrum is dominated by aliphatic, alkene and carboxyl contributions. We only observed broad aromatic signals potentially associated with remaining 1,8-DHN monomers as their [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C chemical shift is consistent with the solution-NMR data obtained by our collaborator (Dr. Iñaki Guijarro, Pasteur Institute, Paris). We concluded here that the 1,8-DHN might condense into linear pigment in vitro when submitted to basic buffer. This mechanism remains to be documented. Moreover, the acid precipitation of the DHN-melanin particles led to a modified [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C fingerprint of the synthetic melanin; which is then dominated by aromatic signals. This result would imply that, in acidic conditions, the linear pigment obtained during the condensation of 1,8-DHN in basic buffer becomes cyclic. The mechanistic involved in this linearization/cyclization process is still under investigation.

THE RECOMBINANT HYDROPHOBIN RodA

Hydrophobins are secreted proteins found in filamentous fungi. These hydrophobic proteins are low molecular weight and display interesting physicochemical properties. Even if hydrophobin does not share a high sequence homology, they are characterized by an idiosyncratic pattern of eight conserved cysteine residues involved in four disulfides bridges.

Once secreted in a soluble form, they self-assemble at hydrophobic/hydrophilic interfaces (e.g., air/water interface) to form amphipathic layers essential for the development of fungal aerial structures such as hyphae. Based on their physicochemical properties, hydrophobins are divided into two classes: (1) Class I hydrophobins are functional amyloid proteins forming fibers organized in layers with rodlet morphology; (2) Class II hydrophobins do not form layer with well defined morphology. Recently Class III hydrophobins were identified.

In A. fumigatus, RodA self-assembles at the surface of the dormant conidia to form a patchy rodlets outer layer [START_REF] Valsecchi | The Puzzling Construction of the Conidial Outer Layer of Aspergillus Fumigatus[END_REF]136 . The high hydrophobicity of this layer that facilitates air dispersion helps the conidia to reach lung alveoli. The mechanical properties of RodA help to maintain the integrity of the cell wall and impact from external stresses like desiccation, physical damage, and drugs 137 . Moreover, this layer masks the recognition of conidial cell wall carbohydrates by the human innate system.

In addition to RodA, six hydrophobins were identified in A. fumigatus: RodB, C, D, E, F and G. Among them, only RodA, RodB and RodC are able to form rodlet layers and thus belong to Class I hydrophobins. To date, the biological role of RodB and C remains unclear.

Nevertheless, the structural features of these three proteins were recently investigated in the thesis of B. Rodríguez de Francisco 138 . Briefly, RodA, B and C self-assemble as amyloid fibers at interfaces. The nature of these interfaces determines the morphology of the hydrophobin assemblies. Moreover, the three hydrophobins share important physicochemical properties as well as structural modifications while forming fibers.

In the past decades, RodA was extensively studied as it confers immunological inertia to the conidia. Finally, the structure of the monomeric RodA in solution was elucidated by NMR in 2019 [START_REF] Valsecchi | Assembly and Disassembly of Aspergillus Fumigatus Conidial Rodlets[END_REF] (PDB access code: 6GCJ). No similar studies were carried on either RodB or C despite their strong structural similarities as compared with RodA. Moreover, getting more structural insights about RodB and C could highlight new potential biological role of these two proteins. Furthermore, the structure of RodA, B and C assembled as fibers has never been investigated despite their potential crucial importance as regard to the A. fumigatus pathogenicity understanding.

In this section, we focused of the structural characterization of RodA, B and C. We aimed at characterizing the structural features of the three hydrophobins assembled as fibers.

The SSNMR study of these proteins in the cellular context was not possible: the complexity of the cell wall and the weak amount of Rod proteins as compared to the rest of the cell wall would lead to overcrowded spectra preventing the in cellulo assignment of RodA, B and C.

Then, extracting fibers from the cell without affecting their supramolecular structure appears to be impossible, as it would require the use of harsh chemical treatments [START_REF] Aimanianda | Surface Hydrophobin Prevents Immune Recognition of Airborne Fungal Spores[END_REF] . Thus, we chose to perform our analysis (using Xray diffraction and SSNMR methods) on recombinant protein produced in vitro in E. coli bacteria.

Xray analysis of Rod proteins

RodA was described to be an amyloid protein. The less restrictive definition of an amyloid protein would be "a polypeptide, which upon polymerization exhibits the characteristical cross-β pattern" 139 . This cross-β pattern can be highlighted using Xray diffraction of amyloid fibers: this technique provides spatial information about the characteristic distances occurring within the amyloid fiber. Indeed, the cross-β quaternary structure in which β-sheets are stacked along the fibril axis with a 4.7 Å spacing distance as inter-strand interactions, and 10 Å as inter-sheet contacts 140 (Fig. 44). 45). This strongly suggests that the three hydrophobins share a common amyloid structure. However, the pattern of RodC seems slightly different as compared to the two others hydrophobins. Added to the characteristic cross-β pattern, we also observed peaks corresponding to 4.3 Å and 32 Å. These two peaks correspond to other repeated distances involved in the RodC packing into fibers.

Nevertheless, Xray diffraction alone does not give enough structural information to conclude on the structures of these three proteins.

SSNMR analysis of Rod proteins

To obtain complementary information at the atomic scale, we recorded SSNMR on the three proteins. As they all exhibit an amyloid fold, the core of each monomer composing the fibrils is structurally ordered. This ordering as well as all the compressed packing of monomers within the fiber make the whole structure rigid. Thus, we used CP-based experiments for the structural characterization of such assemblies.

Amyloid pattern of the three hydrophobins

We proved by WAXS technique that the three proteins share the same packing and cross-β pattern core structure. However, RodB and RodC sequences share only 42% and 59% homology with RodA (Fig. 46). This relatively low sequence homology percentage seems to be contradictory with structural evidence suggesting a conserved tertiary and quaternary structure as demonstrated by X-ray diffraction methods. Thus, we wanted to investigate the structure of these three proteins using SSNMR. We first recorded a [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C CP-detected fingerprint of each hydrophobin (Fig. 47). Even though the 1D fingerprints of RodA, B and C look similar (Fig. 47A), the superimposition of 2D fingerprints highlight differences between the three hydrophobins (Fig. 47B). The positions of the peaks remain superimposable; then we could confirm that the three hydrophobins share a common rigid and identical cross-β core. However, RodA exhibits the best NMR spectral quality illustrating its well ordered structure. Comparatively, RodB and C do not give resolved spectra: the SNR is low, peaks are broadened and the number of spin systems accounted is low as compared with RodA. Following our observations, RodB and C did not give 2D spectra with a sufficient resolution to attempt any assignment. We thus focused our structural analysis on RodA only.

Assignment of RodA fibers

First, we recorded a 2D 13 C- [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C PDSD with a 50 ms mixing time. In this experiment, [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C-13 C through-space correlations of two carbons within one amino acid are probed. Indeed the short mixing time allows the identification and enumeration of spin systems (Fig. 48A).

The recombinant RodA contains 143 amino acids. In our analysis, we identified 83 different spin systems and we unambiguously assigned 49 spin systems in the primary sequence. Thus, we first concluded that 60 amino acids were not rigid enough to be probed by CP-based experiments. They might be present in flexible moieties such as loops or mobile N-and Cterminal domains. Then, we derived the secondary structure based on [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] Cα and [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] Cβ chemical shifts in order to get some insights about the protein molecular conformation (Fig. 48B).

Indeed, residues in α-helices have positive secondary chemical shifts, while residues in βsheet conformation have negative secondary chemical shifts. Our results suggest that RodA mainly comprises β-sheet secondary structure elements reinforcing the hypothesis of its amyloid fold structured by a cross-β architecture.

A B

Moreover, we recorded a 2D 13 C- [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C PDSD with a 150 ms mixing time in order to access sequential connections between residues. The superimposition of 2D 13 C- [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C PDSD intra-and inter-residues exhibit new cross peaks referring to the through-space connection of two amino acids adjacent in the primary sequence (Fig. 48C). Our preliminary analysis allowed us to establish several amino acid sequential connections as this work is still in progress (Fig. 48D).

SUMMARY

In the first part of this study, we aimed at investigating the cell wall of the dormant conidia; especially its carbohydrates network. We used multidimensional SSNMR to characterize its composition as well as its dynamics (rigidity/mobility of the polysaccharides).

We assigned unambiguously the main rigid cell wall polysaccharides: β-1,3-glucan, α-1,3glucan and chitin (Table. S1). Our results suggest that the β-1,3-glucan is mainly synthetized as rigid and ordered fibrils organized as an homogenous layer within the cell wall. On the contrary, we noticed that both α-glucan and chitin exhibit polymorphism. α-glucan is an amorphous polysaccharide accounting for approximately 25% of the total cell wall. Thus, it might be present in different locations within the cell wall in order to achieve many biological functions. On the other hand, chitin was previously described to play an important role for maintaining the cell wall rigidity. Thus, it was not expected to exhibit structural polymorphism as seen in our SSNMR experiment. We speculate the origin of polymorphism to be linked to its molecular structure, more prone to undergo structural variations compared to α-glucan. Using SSNMR experiments dedicated to the detection of highly mobile molecules, we observed an unexpected amount of fatty acids trapped into the cell wall. We hypothesized these fatty acids to esterify a glycerol skeleton forming TAG, DAG or MAG.

Recently, these molecules were described as a suitable scaffold for the polymerization of melanin in the cell wall of C. neoformans. Our results might suggest the same phenomenon to happen in A. fumigatus.

Once we proved our method to be robust enough, we applied it to investigate the germination process in A. fumigatus. Indeed, the germination of dormant conidia appears crucial for infection and its prevention might be a new therapeutic target. In this context, a better understanding of this biologic phenomenon through the atomic remodeling of the conidial cell wall during the process could open up the way to the discovery of antigermination targets. We proved that the entire cell wall rigidifies throughout the germination process. We also demonstrated a component-dependent regulation of their dynamic states.

During the isotropic growth, important modifications of the cell envelope are required to adapt to the conidial size doubling. The cell wall has to softened. Thus, β-glucanases are secreted to digest β-glucans involved in the cell wall rigidity maintenance. These βglucanases only degrade the rigid pool of β-glucans during the swelling of the conidia as the intensity of the mobile contribution remains constant. During the last step of the germination, the newly synthetized rigid β-glucans are highly produced, as the cell might need to reinforce its architecture in order to balance the polarized growth-effect. Then, the relative amounts of α-glucans as well as rigid contribution continuously increase throughout the conidial germination. Once the germination starts, α-glucans might be produced to compensate the degradation of β-glucans. Its plasticity might be involved in maintaining the cell wall integrity without preventing the remodeling of the softened cell wall. The polymorphism of α-glucans suggests that thes carbohydrate might be present in different separated location within the cell wall. We assumed at least two possibilities: (1) one layer of α-glucan remains embedded into the cell wall, maintaining the cell wall integrity; (2) one part of the α-glucan is located at the surface of the spore and plays role in the swollen conidial agglutination.

Furthermore, chitin exhibits also a remarkable polymorphism even though its relative amount within the cell wall is not drastically modified throughout the germination process.

We hypothesized that this remodeling of the chitin network follows the modification of the βglucan core. First linked to the β-glucans in the dormant conidia, the chitin network would be remodeled after its released from the degraded β-glucan skeleton. The newly less-constrained chitin then would take place in different locations within the cell wall to achieve new biological functions. Finally, we notably assigned the mobile N-acetylgalactosamine composing the GAG; one of the main carbohydrates composing the cell wall of germinating conidia. This polysaccharide mediates the adhesion of germinating conidia to other materials, and it is thus supposed to be expressed at the surface of the conidia; explaining its apparent flexibility.

In the last part of the study of the germination process, we investigated the hydration of the three-conidial states. We discovered that the water is tightly bound to conidia, resulting in a broaden peak in 1 H-detected NMR. Indeed, contrary to swollen and germinating conidia, the surface of the dormant conidia is highly hydrophobic due to the presence of both RodA rodlets and melanin. Nevertheless, this patchy layer displays gaps through which carbohydrates can infiltrate and reach the surface. We hypothesize here that water molecules are trapped into these holes and/or between the hydrophobic layers and the fibrillar core. We demonstrated that germination continuously prevents the water accessibility of both chitin and β-glucans throughout this process. On the contrary, α-glucans are located at the surface of the spores as soon as the isotropic growth starts confirming its involvement in mediating interaction with the external medium. The water-accessibility profiles of α-glucan and βglucan were not modified throughout the isotropic or polarized growth, suggesting them to be equally water-accessible in the dormant, swollen or germinating cell wall. We could assume that the rigid part of these polysaccharides is globally found at the same location within the cell wall of the three conidial states. Chitin exhibits as well a remarkable change in wateraccessibility during the polarized growth. Our main hypothesis is that chitin is more embedded within the cell wall (i.e., closer to the plasma membrane) at the end of the germination process. This rigid polysaccharide could act as a skeleton, compensating the loss of melanin and RodA, and maintaining the integrity of the germinating conidia while the polarized growth starts and the germ tube emerges.

In a second part of this study, we were interested in deciphering structural insights about the DHN-melanin of A. fumigatus dormant conidia. The DHN-melanin plays key roles either in the structural organization of the cell wall, by mediating host/pathogen interaction during fungal infections or by protecting the spores against the host immune system. First, we tried to assign [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C conidial melanin signal in whether cellular conditions or in melanin ghosts structures. To do so, we used melanin-depleted conidia (following Δpksp mutation or chemical extraction of the melanin on WT spores). Unexpectedly, no significant changes were observed between melanin-depleted and melanized cells spectra. We hypothesized that the overlapping effect occurring in whole-cell [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C-detected spectra may prevent the observation of the melanin signals (hidden by other strong contributions). Moreover, the melanin ghost [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C spectra do no exhibit signals corresponding to aromatic molecules. Finally, we investigated the in vitro polymerized DHN-melanin. We observed broad aromatic signals potentially associated with remaining 1,8-DHN monomers as their [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C chemical shifts are consistent with the solution NMR data. We concluded here that the 1,8-DHN might condense into linear pigment in vitro when submitted to basic buffer. Moreover, the acidic precipitation of DHN-melanin particles gave a [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C fingerprint dominated by aromatic signals which suggests a cyclic pigment. The molecular mechanism for this linearization/cyclization process is still under investigation.

In the third, and last, step of this study, we wanted to investigate the structure of A.

fumigatus hydrophobins. We focused of the structural characterization of recombinant RodA, B and C. Both SSNMR and WAXS analyses confirm that these three hydrophobins share a common rigid amyloid cross-β structure. Because of the low resolution of RodB and C

SSNMR spectra, we attended to assign only the recombinant RodA protein. Using both [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] Cand 1 H-detection methods, we identified 83 rigid spin systems out of the 143 amino acids of the protein. Among these 83 spin systems, 49 were assigned and the analysis is still in progress. This work will pave the way to achieve a complete assignment of RodA in its amyloid form. Our SSNMR chemical shifts will be compared to solution NMR chemical shifts obtained on soluble RodA molecules to unravel the conformational changes occurring during RodA folding into amyloids.

CHAPTER 2: CRYPTOCOCCUS NEOFORMANS CHAPTER 2: CRYPTOCOCCUS NEOFORMANS

MATERIAL AND METHODS

All the Cryptococcus neoformans samples as well as the capsular extract were kindly provided by our collaborators Drs. Vishu Aimanianda, Alexandre Alanio and Guilhem Janbon from the Pasteur Institute, Paris. Moreover, all the SSNMR analyses of C. neoformans H99 capsule were performed by Dr. A. Lends from the IECB, Bordeaux.

1.1 Biological samples 1.1.1 Cell culture [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C/ [START_REF] Fisher | Emerging Fungal Threats to Animal, Plant and Ecosystem Health[END_REF] N-labeled yeast-Yeast-extract-peptone-dextrose (YPD) agar culture plate was streaked with frozen stocks (-80°C) of Cryptococcus neoformans H99 and incubated at 30°C until the growth of yeast-colonies. Following, a single colony was transferred to [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C/ 15 Nlabeled minimal-medium (containing 1% [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C-glucose and 0.1 M 15 [START_REF] Fisher | Emerging Fungal Threats to Animal, Plant and Ecosystem Health[END_REF] NH 4 Cl) and incubated at 30°C in a shaking incubator maintained at 150 rpm. Yeast-culture was arrested at its exponential growth phase, centrifuged to harvest the yeasts (1600 g, 10 min), washed them twice with sterile water, fixed with 2.5% para-formaldehyde in phosphate-buffered saline (pH 7.2) at ambient temperature for 1 h and then overnight at 4°C, quenched upon repeated addition of 0.1 M ammonium chloride and centrifugation (1600 g,10 min; three times), followed by washing twice with water and suspending fixed yeast cells in water; these fixed yeasts were stored at 4°C until further use.

The [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C/ 15 N-labeled Δcap59 141 (capsular glucuronoxylomannan deficient), Δuxs1 142 (xylose deficient), Δugd1 143 (glucuronic acid deficient), Δuge1 144 Δcap59 (galactose and capsule deficient) mutants were cultivated as described above.

13 C/ [START_REF] Fisher | Emerging Fungal Threats to Animal, Plant and Ecosystem Health[END_REF] N-labeled clinical isolates 145 -Cryptococcus neoformans from the frozen stock was streaked on yeast-extract-dextrose agar plates and incubated at 30°C until the colony growth. Following, a single colony was transferred to minimal medium (containing 1% [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] Cglucose and 20 mM 15 NH 4 Cl, supplemented with salt solution and trace elements) and incubated at 30 o C in a shaking incubator (150 rpm) for 24-48 h (depending on the clinical isolates). The yeast-cells were then collected upon centrifugation (5000 rmp, 10 min), washed with MilliQ water twice, PFA-fixed, quenched with ammonium chloride and washed thrice with water.

1.1.2 [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C/ [START_REF] Fisher | Emerging Fungal Threats to Animal, Plant and Ecosystem Health[END_REF] N-labeled capsular extract [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C/ 15 N-labeled C. neoformans cells were prepared as presented above and decapsulated using the protocol adapted from Goren et al., 1967 146 . Briefly, cells are incubatied into DMSO for 2h under gentle agitation at room temperature and then centrifuged (1600 g, 10 min; three times) to recover the supernatant containing the dissolved capsular substances.

Solid-state NMR analysis

Experimental data were processed using TopSpin and analyzed using CCPNMR. [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C/ [START_REF] Fisher | Emerging Fungal Threats to Animal, Plant and Ecosystem Health[END_REF] N-labeled C. neoformans H99, Δuxs1,

The cell wall of

Δugd1 and Δuge1Δcap59 mutants

All experiments were performed on a 600MHz 1 H Larmor frequency spectrometer (Bruker Biospin) using 3.2 mm MAS rotors filled with [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C/ 15 N-labeled C. neoformans H99, Δuxs1, Δugd1 143 and Δuge1Δcap59 mutants. The MAS frequency was set at 11kHz and the sample temperature was set to 276K with the internal reference DSS [START_REF] Carole | Characterization of Different Water Pools in Solid-State NMR Protein Samples[END_REF][START_REF] Markley | Recommendations for the Presentation of NMR Structures of Proteins and Nucleic Acids IUPAC-IUBMB-IUPAB Inter-Union Task Group on the Standardization of Data Bases of Protein and Nucleic Acid Structures Determined by NMR Spectroscopy[END_REF] .

One-dimension [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C CP experiments (512 scans) were recorded using a 1 ms contact time for the 1 H- [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C polarization transfer to detect the rigid molecules of the cell wall. Onedimension [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C DP spectra (512 scans) were recorded with a 35 s recycle delay to target all the cell wall components independently from their rigidity state (used as a quantitative spectra) or a 2 s recycle delay to detect only the mobile compounds. Two-dimension [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C- [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C DP INADEQUATE (64 scans) were performed with a recycle delay of 2 s and 13 C-13 C CP-INADEQUATE (64 scans) with a 0.5 ms contact time for the 1 H- [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C polarization transfer. Two-dimension [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C- [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C PDSD experiments (96 scans) with 50 ms mixing time and a 1 ms contact time for the 1 H- [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C polarization transfer were used to identify the polymorphism of the carbohydrates. Proton decoupling with a frequency of 90 kHz was applied during acquisition times, using the SPINAL-64 decoupling sequence.

To determine the water accessibility of cell wall components, one-dimension [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C CP spectra (256 scans) using a 1 ms contact time for the 1 H- [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C polarization transfer with a 1 H-T 2 filter of 1.5 ms and a variable 1 H-1 H mixing time (from 0.25 to 150 ms) were used to plot build-up curves using the intensities of the peaks assigned as the C2 of the chitin, the C6 of the β-glucan and the C4 of the α-glucan. Results were plotted as the square root of the 1 H-1 H mixing time for reflecting the relayed nature of the spin diffusion. Two-dimension 1 H- [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C HETCOR experiments (128 scans) were recorded with and without 5 ms 1 H-1 H spin diffusion and using a 1 ms contact time for the 1 H-13 C polarization transfer. A 1D [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C trace was extracted at the chemical shift of the bulk water (4.97 ppm) and peaks intensities were used to estimate the proportion of sugars highly accessible to water.

The highly flexible part of the cell wall was investigated using 2D 1 H- [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C INEPT experiments (96 scans) recorded using 90 kHz proton decoupling SPINAL-64.

The nitrogen content of the cell wall was partially investigated using [START_REF] Fisher | Emerging Fungal Threats to Animal, Plant and Ecosystem Health[END_REF] N- [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C NCO (576 scans), and NCA (576 scans) were recorded using a CP transfer from 1 H to [START_REF] Fisher | Emerging Fungal Threats to Animal, Plant and Ecosystem Health[END_REF] N of 1 ms and a double CP transfer 15 N- [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C and 1 H- [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C with a contact time of 3 ms.

13 C/ 15 N acapsular C. neoformans Δcap59 mutants

All experiments were performed on a 600MHz 1 H Larmor frequency spectrometer (Bruker Biospin) using a 3.2 mm MAS rotor filled with [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C/ 15 N acapsular C. neoformans Δcap59 mutants. The MAS frequency was set at 11kHz and the sample temperature was set to 276K with the internal reference DSS [START_REF] Carole | Characterization of Different Water Pools in Solid-State NMR Protein Samples[END_REF][START_REF] Markley | Recommendations for the Presentation of NMR Structures of Proteins and Nucleic Acids IUPAC-IUBMB-IUPAB Inter-Union Task Group on the Standardization of Data Bases of Protein and Nucleic Acid Structures Determined by NMR Spectroscopy[END_REF] .

One-dimension [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C CP experiments (512 scans) recorded using a 1 ms contact time for the 1 H- [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C polarization transfer to detect the rigid molecules of the cell wall. One-dimension [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C DP spectra (512 scans) were recorded with a 35 s recycle delay to target all the cell wall components independently from their rigidity state (used as a quantitative spectra) or a 2 s recycle delay to detect only the mobile compounds. Two-dimension [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C- [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C DP INADEQUATE (64 scans) were performed with a recycle delay of 2 s to and 13 C-13 C CP-INADEQUATE (64 scans) with a 0.5 ms contact time for the 1 H-13 C polarization transfer. Two-dimension [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C- [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C PDSD experiments (96 scans) with 50 ms mixing time and a 1 ms contact time for the 1 H- [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C polarization transfer were used to identify the polymorphism of the carbohydrates. Proton decoupling with a frequency of 90 kHz was applied during acquisition times, using the SPINAL-64 decoupling sequence.

13 C/ 15 N clinical isolates

All experiments were performed on a 800MHz 1 H Larmor frequency spectrometer (Bruker Biospin) using a 3.2 mm MAS rotor filled with [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C/ [START_REF] Fisher | Emerging Fungal Threats to Animal, Plant and Ecosystem Health[END_REF] N C. neoformans clinical isolates. The MAS frequency was set at 11kHz and the sample temperature was set to 276 K with the internal reference DSS [START_REF] Carole | Characterization of Different Water Pools in Solid-State NMR Protein Samples[END_REF][START_REF] Markley | Recommendations for the Presentation of NMR Structures of Proteins and Nucleic Acids IUPAC-IUBMB-IUPAB Inter-Union Task Group on the Standardization of Data Bases of Protein and Nucleic Acid Structures Determined by NMR Spectroscopy[END_REF] .

One-dimension [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C CP experiments (2048 scans) recorded using a 1 ms contact time for the 1 H- [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C polarization transfer to detect the rigid molecules of the cell wall. Onedimension [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C DP spectra (2048 scans) were recorded with a 2 s recycle delay. Two-dimension [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C- [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C PDSD experiments (160 scans) with 50 ms mixing time and a 0.75 ms contact time for the 1 H- [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C polarization. Proton decoupling with a frequency of 90 kHz was applied during acquisition times, using the SPINAL-64 decoupling sequence. Two-dimension [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C- [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C DP INADEQUATE (96 scans) were performed with a recycle delay of 2 s.

1 H-detected spectra of C.neoformans H99

All spectra were acquired on a 800 MHz spectrometer 1 H Larmor frequency spectrometer (Bruker Biospin) and MAS rate of 100 kHz Bruker 0.7 mm HCN probe. Sample temperature was maintained at about 20°C with the internal reference DSS. The temperature of this gas measured just before reaching the sample was 265 K at 100 kHz MAS respectively. For all 1 H-detected spectra, MISSISIPPI water suppression scheme was applied. Two-dimension [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C-CP hCH experiment were used to target the rigid cell wall and 2D [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C-hCH INEPT-HSQC (otimized delay used during two periods of 1 H proton transverse magnetization: 1.4 ms) were used to target the mobile capsule.

CRYPTOCOCCUS NEOFORMANS EXTERNAL STRUCTURES

Cryptococcus neoformans is the etiological agent of cryptococcosis which consists in mycosis affecting the central nervous system [START_REF] Kwon-Chung | Cryptococcus Neoformans and Cryptococcus Gattii, the Etiologic Agents of Cryptococcosis[END_REF] remaining a significant cause of mortality worldwide [START_REF] Rajasingham | Global Burden of Disease of HIV-Associated Cryptococcal Meningitis: An Updated Analysis[END_REF]147,[START_REF] O'meara | The Cryptococcus Neoformans Capsule: A Sword and a Shield[END_REF] . Moreover, the setting of new strategies is hampered by the lack of structural information at the atomic level for the main components of C. neoformans. Hence, new insights about the molecular organization of the yeast external structure could lead to a better understanding of the pathogenicity of C. neoformans. C. neoformans, is an encapsulated yeast with a size of 5-7 µm [START_REF] Reese | Loss of Cell Wall Alpha(1-3) Glucan Affects Cryptococcus Neoformans from Ultrastructure to Virulence[END_REF] . Its external structures consist in three main layers: the plasma membrane, the cell wall and the capsule (Fig. 50). First, the cell wall of C. neoformans is composed of polymers of α-1,3-glucan, β-1,6glucan, β-1,3-glucan, chitin (polymer of β-1,4-N-acetylglucosamine) and chitosan (deacetylated chitin) [START_REF] O'meara | The Cryptococcus Neoformans Capsule: A Sword and a Shield[END_REF][START_REF] Doering | How Sweet It Is ! Cell Wall Biogenesis and Polysaccharide Capsule Formation in Cryptococcus Neoformans[END_REF]148 . Additionally, glycoproteins, lipids and melanin molecules have also been described in C. neoformans outer parts [START_REF] Doering | How Sweet It Is ! Cell Wall Biogenesis and Polysaccharide Capsule Formation in Cryptococcus Neoformans[END_REF] . Then, the capsule of C. neoformans is anchored to the cell wall via α-1,3-glucan [START_REF] Reese | Cell Wall α-1,3-Glucan Is Required to Anchor the Cryptococcus Neoformans Capsule[END_REF] and formed of meshed long fibers. By interfering with the synthesis of the capsule, the whole cell cycle and infectivity of the pathogen can be abolished; thus, this layer seems to plays an essential role into the virulence of the yeast, making it a potential target for drug development [START_REF] Doering | How Sweet It Is ! Cell Wall Biogenesis and Polysaccharide Capsule Formation in Cryptococcus Neoformans[END_REF][START_REF] Gates | Molecular Architecture of the Cryptococcus Neoformans Capsule[END_REF] . The capsule of C. neoformans is mainly composed of carbohydrates among which the glucuronoxylomannan (GXM) is the most abundant one (up to 90%). This polymer is made of differently branched xylose, mannose and glucuronic acid residues [START_REF] Heiss | The Structure of Cryptococcus Neoformans Galactoxylomannan Contains β-d-Glucuronic Acid[END_REF] . Moreover, depending on the serotype of the yeast, at least 6 different types of GXM have been reported [START_REF] Doering | How Sweet It Is ! Cell Wall Biogenesis and Polysaccharide Capsule Formation in Cryptococcus Neoformans[END_REF]149 (Fig. 51). The other remaining components of the fungal capsule are glucuronoxylomannogalactan (GXMGal) [START_REF] Kwon-Chung | Cryptococcus Neoformans and Cryptococcus Gattii, the Etiologic Agents of Cryptococcosis[END_REF] , mannoproteins and lipids. Nevertheless, the presence and the complexity of this fungal capsule associated with the cell wall increase significantly the complexity of the analysis of such cells. Thus, in this section, we had two objectives: (1) to get more insights about C. neoformans both cell wall and capsular organization; (2) to develop a new methodology using ultra-fast 1 H-detection SNMR as powerful and emergent tool to study the external layer of fungal whole cells.

The fast MAS proton detection

Most SSNMR studies performed on fully-labeled parts of different species were based on a relatively large amount of sample (around 20 mg), employing [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C based detection methods. Very recently, Schanda et al [START_REF] Bougault | Studying Intact Bacterial Peptidoglycan by Proton-Detected NMR Spectroscopy at 100 kHz MAS Frequency[END_REF] Previously, correlations between 1 H, [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C and [START_REF] Fisher | Emerging Fungal Threats to Animal, Plant and Ecosystem Health[END_REF] N nuclei in multidimensional NMR experiments have been successfully applied to proteins at a fast MAS regime. The same experiments can also be transferred for assignment procedures in polysaccharides. The fast MAS frequencies in [START_REF] O'meara | The Cryptococcus Neoformans Capsule: A Sword and a Shield[END_REF] H detection experiments are crucial to improve the line-widths and sensitivity. Moreover, faster MAS frequencies would also require smaller rotors, needing smaller amount of samples (e.g., ~0.5 mg for 0.7 mm rotor). This feature is especially attractive for samples with limited production amounts.

In this study, we apply multidimensional [START_REF] O'meara | The Cryptococcus Neoformans Capsule: A Sword and a Shield[END_REF] H detection methods at 100 kHz MAS in combination with [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C detection methods at 11 kHz MAS to assess the molecular organization of the cell wall and capsule of C. neoformans. Despite the large number of different molecules and heterogeneity of such yeast cells, we expected to obtain well-resolved spectra and assign the majority of peaks. These findings could push the boundaries for the rapidly growing field of fast MAS NMR applied to whole-cell analysis.

The cell wall

Composition

We assessed the rigid contribution of whole C. neoformans cell wall and capsule (Fig. 52A) using CP-based 13 C-detected experiments. First, we recorded 2D 13 C-13 C PDSD to probe the carbohydrates expressed in a rigid form (Fig. 52B). Surprisingly, we observed only signals coming from α-1,3-glucan, β-1,3-glucan and chitin (Fig. 52C). This strongly suggests that only the cell wall components are expressed in a rigid state. Additionally, we recorded 1D [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C CP experiment. The resulting spectrum is dominated by the contributions of cell wall components and a low contribution of lipids which role will be discussed further (Fig. 52D).

Altogether, these data suggest that the capsular components escaped from CP-based detection; then only the cell wall carbohydrates of the yeast would form a rigid network. Then, we recorded a 2D 13 C-13 C CP-INADEQUATE spectrum in order to split carbohydrates signals over a double-quantum dimension and get more information about the cell wall composition (Fig. 53A). This experiment highlights a high number of chitin polymorphs as many CO and -CH 3 groups could be assigned to this carbohydrate. Moreover, at least two spin systems can be assigned to α-glucan, highlighting at least two polymorphs of this carbohydrate.

Moreover, there were still couples of peaks that cannot be unambiguously identified.

One of them might be β-1,6-glucan due to downshifter carbon at 65 ppm, which has been reported to be present in C. neoformans samples by other studies. Interestingly, a similar A B C D composition of polysaccharides with a high number of chitin polymorphs was reported for Aspergillus fungus [START_REF] Kang | Molecular Architecture of Fungal Cell Walls Revealed by Solid-State NMR[END_REF] .

In order to go further in the analysis of the rigid cell wall, we recorded 2D CP hCH spectrum at 100 kHz MAS (Fig. 53B). The spectrum exhibits well-dispersed but rather broad lines (FWHM ~190-400 Hz) for 1 H nucleus. These values were rather high compared to the ones measured for various protonated proteins at 100 kHz MAS frequencies (~91-200 Hz).

Nevertheless, we anticipated such a large heterogeneous contribution (~200 Hz) due to the high molecular diversity in the C.neoformans sample. This 2D CP hCH experiment lacked sufficient resolution to determine whether the rigid part of the yeast envelope contains other types of carbohydrates. Nevertheless, it indicates the presence of a significant amount of the proteins within the cell wall that might correspond to protein conjugates of polysaccharides 146 .

Since the resolution in a 2D was not sufficient due to the severe peak overlap, we applied 3D hCCH experiment with TOCSY magnetization transfer between [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C nuclei. The extracted 2D CC projection spectrum (Fig. 53C) shows similar CC correlations to the ones obtained using classical [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C-detected experiments (e.g., PDSD). Hence, the 1 H-detected 3D hCCH experiment could be use as the equivalent for 13 C-detected experiments. We analyzed the 3D hCCH spectrum as 2D 1 H -13 C correlation strip projections. We started our assignment with identification of anomeric carbons around 100 ppm and sequentially connected them to other carbons. Well-resolved peaks allowed us to intrinsically bond 1 H nuclei to corresponding 13 C resonances. However, no additional spin systems were identified using both 2D and 3D 1 H-detected experiments. Taken together, all these data confirmed that only the cell wall contains rigid carbohydrates contributions (Table 2). Indeed, this layer is designed to maintain the integrity of the whole cell and to protect the internal part of the cell from external stresses. Moreover, the polymorphism described for the chitin and the α-1,3-glucan are consistent with their functions within the cell wall. They are known to play a structural role within the cell wall and to anchor the capsule to the cell wall. Thus, we can assume that: (1) one form of the polysaccharide, highly rigid, would be embedded into the cell wall, associated with chitin and β-glucans to form a skeleton protecting the cell (most intense spin systems); (2) the other

Quantification and dynamic-based study

In order to estimate the relative amount of polysaccharides within the cell wall we extracted the intensities in the 1 H dimension from the 2D hCH spectrum (Fig. 53B). These data revealed that the cell wall consists of approximately same amounts of β-1,3 and α-1,3glucans, while chitin is found to be in ~3 times lower amounts (Table 3).

Table 3: Relative quantification of cell wall polysaccharides.

Intensities of 1 H trace extracted from CP hCH spectrum.

1,3-α-glucan

Intensities (x10 7 a.u)

11.24

1,3-β-glucan 10.19

Chitin

3.32

Then, we wanted to investigate these differences in dynamic profiles between the main components of the cell wall. Hence, we recorded a 1D [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C DP experiment with a short or a long recycle delay to probe either mobile or all the molecules respectively and we compared it with the 1D [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C CP experiment probing only rigid and highly rigid molecules (Fig. 54A).

Furthermore, we established the relative proportion of the mobile and rigid contributions of each main compound of the cell wall (Fig. 54B). First, the β-1,3-glucan is mainly produced as rigid polysaccharide. This polysaccharide can fold on itself to form fibrils rigidifying the cell wall and maintaining the whole cell integrity 150 . Then, the rigid part of either chitin (main part) and α-1,3-glucan (minor part) might be used to rigidify the cell wall. On the contrary, the flexible contribution of these two polysaccharides is located at the interface between the cell wall (rigid) and the capsule (mobile).

Hydration

In order to estimate the hydration of the cell wall, we recorded 1 H- [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C HETCOR with [START_REF] O'meara | The Cryptococcus Neoformans Capsule: A Sword and a Shield[END_REF] H-1 H spin diffusion (Fig. 55A). We identified characteristic [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C carbohydrates/ 1 H water cross peaks and extracted the corresponding 1D 1 H and [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C traces. First, the width of the water peak associated with carbohydrates is broader than for A. fumigatus. This broad peak suggests that the water is tightly associated with the cell wall (Fig. 55B). We assumed that this water pool is comprised between the cell wall and the capsule (described as porous);

preventing the fast tumbling of water molecules. Then, the [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C trace does not exhibit any evidence for the close proximity of water with one specific carbohydrate; this trace fits well when superimposed with the classical 1D 13 C CP spectrum. Hence, we suspect the complete cell wall to be hydrated.

A B

Fig. 55: The water-accessibility of cell wall carbohydrates.

A) 2D 1 H- [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C HETCOR recorded on [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C/ 15 N-labeled C. neoformans H99 with [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C carbohydrates/ 1 H water cross peaks highlighted in red. B) The 1 H and C) the [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C traces extracted from the carbohydrates/water cross peaks and assigned with α-1,3-glucan (pink), β-1,3-glucan (light purple), chitin (dark purple) and TAG (green).

To further investigate the water-accessibility profiles of the different carbohydrates within the cell wall, we recorded 2D 13 C-13 C water-edited PDSD with a T 2 filter to destroy the magnetization of rigid compounds and a 20 ms 1 H-1 H spin diffusion mixing time. The resulting spectrum superimposes perfectly with the conventional 13 C- [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C PDSD (Fig. 56A).

We were not able to evidence any differences in the water-accessibility for the three main carbohydrates (Fig. 56B). We then recorded a series of 1D water-edited [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C CP spectra. To get insights about the water accessibility of each compounds, we plotted this signal intensity as a function of the 1 H-1 H spin diffusion mixing time (Fig. 56C). The slope of the resulting build-up curves depends on the water accessibility of the molecule. We observed a similar behavior for all the cell wall components. Thus, the hydration of the cell wall seems to be homogenous.

The cell wall architecture

In this section, we investigated the composition and the architecture of the cell wall of C. neoformans (Fig. 57). We first observed that the glucans are three times more expressed than the chitin. Then, the β-1,3-glucan is mainly expressed in a rigid state: this polysaccharide could form long fibrillar structures responsible for the rigidity of the cell wall. Then, the α-

The capsule

In the second part of this section, we aimed at investigating the capsular content. As demonstrated earlier, the capsular polysaccharides seem to be too flexible to be revealed by CP-based experiment. Thus, we used DP-and INEPT-based pulse sequences to probe only flexible and highly flexible molecules.

Carbohydrates network

In order to identify the different anomers of carbohydrates, we recorded a 2D hCH HSQC spectrum. In this experiment, a 1 H decoupling occurs during the acquisition leading to a unique 1 H- [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C cross peak with the δ 1 H as the mean of the δ 1 H corresponding to the diastereotopic protons. Thus, we can recover the signal due to the two diastereotopic protons of anomeric carbons by suppressing the decoupling during the acquisition time (Fig. 58A).

These lead in a double cross peaks corresponding to the same [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C those coupling constant depend on whether the carbohydrate is in α or β conformation (Fig. 58B). That way, we determined the conformation of the different carbohydrates present in the capsule of C. neoformans (Fig. 58C). In addition to the literature-based data, these finding helped characterizing the different capsular carbohydrates. Then, to define the spin system corresponding to each carbohydrate, we recorded a 2D

hCH HSQC spectrum (Fig. 59A). The spectrum exhibited narrow 1 H line-widths (~30 Hz), similar to the ones obtained for the shaded capsule in solution NMR. The distinct downfield chemical shifts for anomeric carbons were good starting points for the assignments. From 85 ppm to 70 ppm we found peaks with typical chemical shifts corresponding to C2, C3, C4 and C5 carbons in saccharides. Around 65 ppm we observed C5/C6 carbons with diastereotopic protons appearing as separate 1 H shifts for the same carbon. Furthermore, we estimated the relative amount of polysaccharides within the capsule by measuring the intensities of peak corresponding the anomeric protons in the 1 H trace extracted from the 2D hCH spectrum.

These data revealed that the capsule consists in α-1,3-mannan chain highly ramified with xylose and potentially galactose residues (Table 4) which remains consistent with the previously described structure of GXM and GXMGal molecules. Based on the spectra presented above, we assigned mannose and xylose residues that might come from GXM of GXMGal. Moreover, we strongly assume that the component MY (β-Man/Gal) would be potentially a galactose as the mannose was never described to form βglycosidic linkage. Moreover, we partially assigned three more components, which could be gluxoronic acid, galactose and also differently branched mannose. Surprisingly, we were not able to find typical carbonyl group resonance for gluxoronic acid, but we cannot fully exclude the presence of it in our sample. Due to its low abundance as compared to mannose/galactose/xylose moieties, it could be below the detection limit of SSNMR methods.

Lipid

For both 2D hCH (Fig. 60A) and 2D hCCH TOCSY (Fig. 60B) spectra, we observed that the region around 15-45 ppm exhibits a correlation peaks pattern that might correspond to aliphatic structures. Based on our observations and on the analysis of 2D 13 C-13 C DP INADEQUATE spectrum (Fig. S1), we established the sequential assignment of aliphatic carbons. Moreover, we also established the presence of a spin system that could be related to glycerol skeleton (discussed further). Thus, we could determine that this lipid results in an unsaturated acyl chain of at least 10 carbons; those δ 13 C and δ 1 H are summarized Table 6. Based on the literature, we first hypothesized that this fatty acid could be part of glycosphingolipids 153 previously found in C.

neoformans. (Fig. 61). They would act as a suitable scaffold for the polymerization and folding of the DOPA-melanin. Moreover, these lipids were identified even in non-melanized cells like the cells we are using. Furthermore, the 13 C chemical shifts of both the identified unsaturated A B fatty acid and the glycerol skeleton are coherent with our data. Thus, in this section, we assumed that the unsaturated fatty acid and the potential glycerol spin system identified in our analysis would confirm the presence of such kind of lipids in the envelope of C. neoformans H99. Moreover, these TAGs are associated with the melanin pigment within the cell wall.

Thus, they share a common location at the interface between the cell wall and the capsule of C. neoformans. This is coherent with our observations where the TAG are probed by both rigidity-and flexibility-based experiments. Hence, we conclude that these lipids were located at the interface between the rigid cell wall and the flexible capsule and their dynamics are affected by these two compartments.

The architecture of C. neoformans outer layers

Using the data from our SSNMR experiments and from the literature, we built a representative model of the encapsulated C.neoformans cells (Fig. 62). The inner layer would consist in the plasma membrane protecting the cytoplasm. This lipid bilayer is covered by the cell wall maintaining the integrity and the rigidity of the cell. The cell wall is composed of rigid α-1,3-glucan, β-1,3-glucan, chitin and various associated proteins.

Then, we managed to investigate the highly dynamic and porous meshed network of the capsule. We assigned different kinds of carbohydrates (e.g., xylose, mannose and galactose) composing the two main constituents of the capsule: GXM and GXMGal from GXM polymer. However, we were not able to establish the exact arrangement of these carbohydrates residues as polysaccharides structures. Moreover, despite the well-documented presence of glucuronic acid within the capsule, this carbohydrate escaped from our detection;

it is thus absent from our model.

Finally, we observed a strong pattern of lipid-related signals. Following the latest publications about this topic, we strongly assumed these signal to be due to the presence of TAG within the external layers of C. neoformans. Moreover, these TAGs are associated with DOPA melanin, a pigment present in the outer layers of melanized cells (C. neoformans cells grown in the presence of DOPA; the precursor of DOPA melanin). The exact structure of the DOPA-melanin is still tedious 134,154 but this polymer described to be located at the interface between the cell wall and the capsule. Thus, we anticipated the lipid to be also located at the interface between the two layers. Furthermore, that hypothesis is reinforced by the presence of TAG in both rigid and highly flexible state; the dynamic different pool of the TAG could be influenced by either the cell wall rigidity or the high capsule flexibility.

Hence, the complete architecture of the yeast cannot be solved only using SSNMR: In this study, we showed that the overall resolution in CP based experiments was not sufficient to unambiguously identify all polysaccharide protons, especially C6 bonded diastereotopic protons. On the other hand, it has been shown that higher MAS frequencies can reduce the homogenous contributions for fully protonated proteins 155,156 and small molecules 157,158 to gain resolution and sensitivity. We would expect such "faster is better" 159 consensus in [START_REF] O'meara | The Cryptococcus Neoformans Capsule: A Sword and a Shield[END_REF] H detection experiments also for the fully labeled yeast samples. Hence, we successfully applied and implemented 1 H detected SSNMR experiments at the fast MAS regime in combination with classical [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C based experiments to unravel C.neoformans external structures.

CHARACTERIZATION OF CAPSULAR MUTANTS

In this section, we focused on the structural characterization of both the cell wall and capsule of C. neoformans upon different capsular carbohydrates biosynthesis pathways mutations. We chose to study the well-characterized acapsular Δcap59 mutant as well as two other single mutants (Δuxs1 and Δugd1) and one double mutant (Δuge1Δcap59). All these mutants do not exhibit any thick capsular structure and might be useful in order to simplify and improve our previous capsular analysis.

Mutant Δcap59

Several genes are essential for C. neoformans capsular carbohydrates synthesis or export into external structures. Among all these genes, cap59 would be involved in the process of GXM (polysaccharides) export during capsule synthesis. This 1.9-kb CAP59 gene encodes a 458-amino-acid protein of unknown function and is located on chromosome 1.

However, cap59 gene deletion mutant does not exhibit capsule (India ink staining or immunofluorescence assays). On the opposite, the cell diameter and volume for the Δcap59 strain are higher than for WT. This would suggest an accumulation of cellular material in the cytoplasm 141 .

We were first wondering whether the deletion of the cap59 gene affects the cell wall constitution even though this mutation was supposed to prevent only the capsule formation.

Thus, we recorded 2D 13 C-13 C CP-INADEQUATE (Fig. 63A) and 2D 13 C- [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C PDSD (Fig. We observed a perfect superimposition of both WT and Δcap59 mutant. Thus, we concluded that the mutation of cap59 did not affect the cell wall of C. neoformans. Then, we recorded 2D [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C- [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C DP-INADEQUATE spectra to probe the capsular components (Fig. 64). Δcap59 does not seem to be affected by the mutation. However, as reported in the literature, the mutation of cap59 does not prevent the synthesis of capsular carbohydrates but affects their transport to an external location. Thus, even though carbohydrates are not able to arrange in a thick and porous capsular layer, we assumed that they are still produced and exported at the surface. This last point will be further discussed.

Mutant Δuxs1

In capsular polysaccharides, xylose is incorporated within GXM or GXMGal polysaccharides via the processing of the sugar donor UDP-Xylose (UDP-Xyl). The biosynthesis of this UDP-Xyl occurs in two steps: (1) UDP-Glucose dehydrogenase (UGE1) catalyzes the dehydrogenation of its substrate to form UDP-glucuronic acid (UDP-GlcA);

UDP-GlcA is converted into UDP-Xyl upon the activity of a UDP-GlcA decarboxylase named UXS1 (a soluble 47-kDa protein) 160 .

Here, we focused our analysis on the Δuxs1 mutant. Hence, we tried to probe the potential effects of the deletion of xylose onto the composition/organization of both the capsule and cell wall layers. To do so, we recorded a set of spectra probing the different external compartments of the Δuxs1 mutant (Fig. 65).

First, we recorded CP-based experiments to probe the cell wall components (Fig. 65A andB). We did not anticipate any changes in the cell wall content as the mutation was supposed to only affect the biosynthesis of the xylose -a capsular component. Nevertheless, we observed superimposable spectra for all the cell wall carbohydrates except for the β-1,3glucan which escaped to the CP-based detection. On the same way, the intensity of the rigid chitin spin system also drastically decreased. We assumed that either (1) the rigid contribution of these two polysaccharides is converted into flexible one or (2) the amount of these polysaccharides decreases in response to the mutation of uxs1. Thus, we probed the flexible part of C. neoformans external structure in a 2D [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C- [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C DP-INADEQUATE experiment (Fig. 65C). Then, we observed that all the capsular components -including xylose -were still present in the capsule of the Δuxs1 mutant. We hypothesized that either the mutation was not effective or xylose could be produced and incorporated into GXM and GXMGal polysaccharides through side synthesis pathways, which do not depend on the activity of ux1.

A B C D E

NMR spectroscopy alone does not permit to answer this question: a biological approach would be required to investigate whether the synthesis of xylose is still effective within the cytoplasm of Δuxs1 mutants and how this carbohydrate is exported into the capsule and associated with GXM and GXMGal molecules.

Finally, on the same spectrum, we assigned the spin systems corresponding to chitin and β-1,3-glucan. The intensities of both spin systems are comparable with the one of the WT. Thus, only the flexible part of the two polysaccharides is still produced upon the uxs1 mutation. The rigid part of the chitin is strongly decreased while the rigid contribution of β-1,3-glucan is totally abolished. We here concluded that the total amount of chitin and β-1,3glucan is decreased in response to the uxs1 mutation. Despite UDP-Glc sugar donor, no metabolic connections were established between β-1,3-glucan, chitin and xylose synthesis pathways. Hence, the link between the mutation of uxs1 and the production of β-1,3-glucan or chitin remain to be elucidated.

Following these results, we investigated the dynamic state of the cell wall carbohydrates, as they seem strongly affected by the mutation. Thus, as described earlier, we recorded two types of 1D [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C DP and CP spectra (Fig. 65D) to obtain information about the rigidity/mobility states of the cell wall carbohydrates. Results were plotted Fig. 65E for α-1,3glucan and chitin; β-1,3-glucan being only present as a flexible polysaccharide. As demonstrated earlier, chitin is mainly produced as a flexible polysaccharide. Moreover, α-1,3glucan is mainly rigid maybe in order to balance the effect of the extinction of the rigid β-1,3glucan and to maintain the cell wall rigidity and cell integrity. These two behaviors are in opposition with the WT dynamic-state described earlier in this study highlighting an adaptive remodeling of the cell wall upon the uxs1 mutation. We could here assume that the decrease strongly rigid components such as chitin and β-1,3-glucan is required to soften the cell wall, thus conferring a higher plasticity to the Δuxs1 mutant as compared to the WT. Indeed, it was already proved that the cell diameter increases upon the uxs1 mutation potentially requiring a less rigid cell wall to adapt to this new phenotype.

Mutant Δugd1

In order to investigate further the role of the synthesis pathway described above, we then focused on the analysis of Δugd1 mutant. This gene encodes UDP-glucose dehydrogenase, which catalyzes the conversion of UDP-glucose into UDP-glucuronic acid.

The literature suggests that this UDP-dehydrogenase represents the major, and maybe only, biosynthetic pathway for UDP-glucuronic acid in C. neoformans. Consequently, the deletion of UGD1 blocked not only the synthesis of UDP-glucuronic acid but also that of UDPxylose 143 . As described previously, the deletion of this gene leads to an acapsular phenotype.

In order to investigate both the rigid cell wall and flexible capsular polysaccharides, we recorded the same set of experiments as described in the previous section (Fig. 66). As demonstrated earlier, both rigid β-1,3-glucan and chitin are strongly negatively impacted by the mutation of ugd1: the spin system of the β-1,3-glucan is absent whereas the intensity of chitin is closed to the noise level (Fig. 66A andB). Nevertheless, the flexible part of the two polysaccharides also strongly decreases as compare to the WT signals (Fig. 66C). Thus, the production of rigid polysaccharides is almost abolished whereas the one of the flexible contribution significantly decreases. We here conclude that the total synthesis of chitin and glucan is hampered by the mutation of ugd1.

Interestingly, no significant evidence for the presence of glucuronic acid within the capsule of WT cells was highlighted by comparing with the Δugd1 mutant (glucuronic-acid deficient). Hence, this polysaccharide might escape our detection either due to its low amount, as compared with other polysaccharides, or to its dynamic state which cannot be probed by our SSNMR experiments.

Then, following the same methodology as presented above, we investigated the dynamics of the cell wall carbohydrates despite the low amount of chitin within the cell wall (Fig. 66D andE). As presented for that Δuxs1 mutant, the α-1,3-glucan is mainly expressed as a rigid polysaccharide and the low amount chitin seems also to be rather rigid.

Hence, any mutation of the synthesis pathway converting UDP-Glc into UDP-GlcA and then converted into UDP-Xyl seems to lead to a decrease of the β-1,3-glucan content.

Even though no evidence shows interconnections between both xylose and β-1,3-glucan synthesis pathways, we could suppose that the mutation of ugd1 or uxs1 leads to the accumulation of UDP-Glc within the cytoplasm retro-negatively regulating the synthesis of β-1,3-glucan or chitin from UDP-Glc. This hypothesis needs to be confirmed by biological analysis.

Mutant Δuge1Δcap59

Unexpectedly, the three other mutants tested above did not exhibit any capsular modification when analyzed by SSNMR. We then chose to study a double mutant:

Δuge1Δcap59 in order to induce significant changes within the capsule of the yeast.

A B C D E

Alone, the mutation of Δcap59 leads to an acapsular phenotype; in addition, to more dramatically affect the capsule structure, we mutated Δuge1. Indeed, the genome of C.

neoformans carries two UDP-glucose epimerase genes: uge1 and uge2. UGE1 catalyzes the conversion of UDP-Glc into UDP-galactose (UDP-Gal) and has been identified as necessary for GXMGal biosynthesis. Thus, we anticipated strong modifications of the capsular content as well as a potential cell wall remodeling in response to the mutation of both cap59 and uge1.

We recorded the same set of spectra described above to investigate both cell wall and capsular polysaccharide content (Fig. 67). Surprisingly, our results were similar to the ones obtained for the two single mutants: (1) the rigid β-1,3-glucan and chitin were negatively affected;

(2) the α-1,3-glucan is mainly expressed in a rigid form (contrary to the WT) and ( 3) the flexible capsular polysaccharides were not affected by the double mutation. Moreover, the rigid β-1,3-glucan is absent from our results but this polysaccharide is still produced as a flexible form. None of these modifications were observed for the single Δcap59 mutant; thus we concluded that this cell wall remodeling occurs in response to the mutation of uge1 which prevents the formation of UDP-Gal.

These unexpected results led us to the hypothesis that the synthesis of galactose could involve other metabolic pathways, which were not influenced by the mutation of uge1.

Indeed, our data do not suggest that one carbohydrate disappears from the capsule as it was expected for the galactose. Then, this carbohydrate might be still produced in the Δuge1Δcap59 mutant and the capsule structure would still be present and organized as for the WT. In order to validate our hypothesis, our collaborator from Pasteur Institute performed immunolabeling assay onto Δuge1Δcap59 (Fig. 68). Surprisingly, both capsular and cell wall structures were present within the mutant outer layers. Fluorescent microscopy of Δuge1Δcap59 mutant. The cell wall (blue) is labeled using calcofluor white; the capsule (green) is labeled anti-capsular rabbit polyclonal antibody, revealed using anti-rabbit FITC-conjugated secondary antibody.

These results associated with our data strongly suggest that the mutation of cap59 do not prevent the total assembly of the capsule but only affect the thickness of this layer.

The unexpected effects of the capsular mutations

The study of these four mutants provided new insights about how they might affect the two external layers of C. neoformans. First, our results strongly suggest that the acapsular phenotype observed upon the mutation of cap59 does not consist in a total removal of the capsule. However, the cellular diameter of the Δcap59 cells increases and might underline the accumulation of polysaccharides within the cytoplasm of the yeast. Based on our data, we assume that a part of the capsule polysaccharides are still exported at the surface of the yeast to form a thin capsule layer. Moreover, this assumption is reinforced by fluorescent microscopy, which also probes some capsular components at the surface of the cells.

Furthermore, the mutations of uxs1, ugd1 or uge1 do not impact the capsular components visible by SSNMR. Indeed, we still probe the same polysaccharides as WT C.

neoformans. Assuming that the effective mutation of these three genes was already demonstrated, we proposed that their mutation does not prevent the production of xylose, glucuronic acid or galactose. Some secondary synthesis pathways could be activated in response to the deletion of the three enzymes. On the opposite, they all decrease or totally prevent the production of the cell wall β-1,3-glucan in rigid or both rigid and flexible states.

The role of such genes into the synthesis of this polysaccharide was never described to date.

Nevertheless, the three metabolic pathways involved in the production of xylose, glucuronic acid or galactose and theoretically blocked by these mutations involved the UDP-Glc as the initial sugar donor. Even though no connections were described between these synthesis pathways, the accumulation of UDP-Glc upon the mutation of one of these three genes could lead to a negative retro-control of the production of β-1,3-glucan favoring only the production of the flexible state of this polysaccharide.

Finally, as all the mutation of uxs1, ugd1 or uge1 unexpectedly impact only the cell wall of the mutants, we were interested in the hydration of this layer. Indeed, β-1,3-glucan and chitin were both described as a rigid scaffold maintaining the cell integrity via their rigidification effect. Thus, the loss of rigid β-1,3-glucan within the cell wall could lead to a global softening effect of the external structure modifying its hydration.

As described earlier, we recorded several 1D [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C CP spectra with a T 2 -filter and varying 1 H-1 H spin diffusion. We then plotted the intensities of reference peaks as function of the mixing time to obtain build-up curves. Those slopes indicate how hydrated a polysaccharide is (Fig. 69). As these experiment are based on a CP transfer, probing only the rigid contribution of each polysaccharide, such analysis was not conducted on the β-1,3glucan.

Our results do not probe significant differences concerning the hydration profile of chitin within the cell wall of the different mutants. On the contrary, the hydration of the α-1,3glucan is modified upon the mutation of ugd1. The mutation of this gene prevents the production UDP-GlcA from UDP-Glc and leads to an increased proportion of rigid α-1,3glucan as compared to the WT. According to these results, α-1,3-glucan would be less wateraccessible and thus more embedded into the cell wall maybe to balance the effect of the loss of β-1,3-glucan by self-organizing in a dense layer conferring rigidity to the cell wall. None of the three isolates exhibit a rigid cell wall β-1,3-glucan or chitin. The rigid core of the isolates cell wall seems to be exclusively made of α-1,3-glucan.

Then, we recorded DP-based spectra to evaluate the flexible content of the yeast envelope (Fig. 71). First, the clinical isolates 2.86 and 1.83 do not contain any signal corresponding to the chitin; signifying that the polysaccharide escape from our detection probing either rigid or flexible contributions. This might suggest that the chitin is produced in a low amount, below our detection threshold. Then, the α-1,3-glucan is expressed mainly as a rigid polysaccharide as its SNR remains low when probed by DP-based experiment. Thus, this polysaccharide might act as the scaffold of the cells: it could form a dense and rigid network all around the plasma membrane, maintaining the cell integrity. Despite the global [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C fingerprint shared by all the clinical isolates, the 2.86 exhibits one new specific spin system. This unknown spin system (named U in the Fig. 71) corresponds to a 5 carbons ring. Moreover, the high δ 13 C of the C1 and C3 indicates that the unit is linked in position 1 and 3 (Table 7). The intensity of the spin system is consistent with the structure of a polysaccharide composed of the same repeated unit. Moreover, δ 13 C of the C1 (>103 ppm) would preferentially correspond to a β anomer. Finally, the δ 13 C might correspond to a xylose unit; which is the most expressed five-carbons carbohydrates with C. neoformans envelope. Hence, we would have identified a β-1,3-xylan flexible structure. Such structure has never been described in C. neoformans but already reported in C. albidus. To conclude, the three clinical isolates displayed similar [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C fingerprint in both flexible-and rigid-probed experiments. They all do not contain rigid β-1,3-glucan and only a low amount of rigid chitin. Moreover, the cell wall rigidity seems to be based only on the presence of rigid α-1,3-glucan. Then, the capsular structure, mainly flexible, relies on the same set of polysaccharides (i.e., mannose, xylose). The only difference is the presence of a putative β-1,3-xylan; those role remains unknown.

Finally, our structural data have to be correlated with biological evidence in order to determine the impact of the yeast external structures remodeling in the context of the virulence of the different clinical isolates.

A NEW TOOL FOR STUDYING YEAST ENVELOPE

In this chapter, we successfully developed a new methodology based on both [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C and 1 H-detction using fast MAS respectively to study the complex external structures of yeast cells. We demonstrated the efficiency and the robustness of our methodology through the structural characterization of either the WT H99 strain, the different mutants or the clinical isolates of C. neoformans.

The analysis of such cells remains really challenging. First, the external envelope of the yeast is composed of two distinct layers (i.e., cell wall and capsule) displaying two different dynamic states. We overcome this difficulty by using the versatility of SSNMR pulse sequences in order to probe differentially the rigid or the flexible-states of the cell wall and capsule. Then, the molecular composition of these two layers relies mainly on a polysaccharide network; those δ 13 C remain similar. This leads to an important [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C spectral overlapping if we do not use experiment with a double-quantum dimension in order to split all the carbohydrates signals. Additionally, the 1 H-detection method might significantly help in reducing the ambiguities during the polysaccharides assignment.

Moreover, the emergent 1 H-detection experiments require spinning the sample at ultrafast MAS frequency (a hundred thousand hertz). This can be achieved only by using a small rotor; that diameter is 0.7 mm and only requires a low amount of sample (less than one mg).

On the other hand, such kind of rotors -and associated SSNMR probes-are not routinely used as they remain costly and not wide spread (only two 0.7 mm SSNMR probes used in France).

Nevertheless, this study lays the groundwork for the development of 1 H-detection methods at fast MAS frequencies in the context of the structural characterization of highly complex structures such as the external multilayers structure of whole yeast cells.

GENERAL CONCLUSIONS AND PERSPECTIVES GENERAL CONCLUSIONS AND PERSPECTIVES

Severe fungal infections are due to opportunistic pathogens that infect however the polysaccharide network remains composed of rigid β-1,3-glucan, α-1,3-glucan and chitin. Additionally, our detection method was sensitive enough to probe the presence of one specific lipid within the conidial cell wall of A. fumigatus. We assigned an unsaturated fatty acid esterifying a glycerol skeleton that might correspond to a TAG lipid. We observed a constant amount of this lipid produced mainly in a mobile state throughout the germination process.

Then, we investigated the dynamics and the hydration of the cell wall to determine whether these two parameters evolve throughout the germination. We observed a global tendency of all the cell wall to rigidify during the isotropic growth, maybe to maintain the conidial integrity during the polarized growth and the hyphae formation. Moreover, the production and the dynamics of each polysaccharide vary differently during the process. For instance, the production on β-1,3-glucan depends on the conidial state as it is strongly degraded during the swelling to soften the cell wall and allows the increase of the cell diameter.

Taken together, our results allow to schematize a molecular model of the evolution of the conidial cell wall through out the germination process taking into account the relative amount, the dynamic state and the location of the molecules within the cell wall of A.

fumigatus conidia. In the future, we will employ 1 H-detected experiments in order to exploit the sensitivity of the proton dimension. Indeed, some constituents of the conidial cell wall (e.g., chitosan) escaped from the 13 C-detection. Furthermore, the role of the TAG and its potential link with other cell wall components (e.g., DHN-melanin) has to be investigated.

Thus, we plan to apply our 1 H-detection methodology described in Chapter 2 onto the dormant conidia of A. fumigatus.

In the second part of this chapter, we performed SSNMR analysis on both the melanin ghosts (extracted from the dormant conidia) and the synthetic DHN-melanin produced via the polymerization of DHN precursor in a basic buffer. We observed, from chemical shift analysis, that the aromatic content of this pigment is only recovered when the DHN-melanin is submitted to acidic precipitation (pH 1.7), highlighting a pH sensitivity of this synthetic DHN-melanin. Hence, we analyzed different types of DHN-melanin either in conidial context or synthetized in vitro. The unexpected [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C-fingerprints of conidial DHN melanin as well as the potential pH-dependent cyclization/linearization process of the synthetic DHN-melanin highlighted the complex and polymorphic structure of this pigment. The elucidation of the DHN-melanin architecture is still under investigation. We plan to use [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C-labeled 1,8-DHN precursor to synthetize the DHN-melanin in order to record 2D SSNMR spectra and to get more insights about the pigment structure. This study could be the first step of the conidial DHN-melanin characterization.

In the last part of this chapter, the structure of the three recombinant hydrophobins RodA, B and C assembled as fibers was investigated by both X-ray diffraction and SSNMR.

Despite their low sequence homology, they all share a common cross-β core stabilized by disulfide bridges. The recombinant hydrophobin RodA fibers expressed in vitro and uniformly [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C/ 15 N or 2 H/ 13 C/ 15 N-labeled was studied using 13 C-detected or 1 H-detected experiments respectively. These experiments allowed assigning the rigid residues of the protein; potentially those involved in the cross-β structure characteristic of the amyloid fold.

We identified 83 out of the 143 residues of the protein, which assignments are still in progress. The secondary [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C chemical shift calculation highlights a strong content of residues present in β-sheet conformation. The comparison of the assignments of RodA assembled in amyloid fibers or in a soluble monomeric state will help us to highlight the structural changes occurring within the protein structure once it self-assembles into amyloid fibrils. Furthermore, we plan to record another data set on a new batch of recombinant RodA using higher magnetic field spectrometers in order to decipher its complete structure when self-assembled as fibers. In the future, this fibrillar structure could be compared with the one of recombinant RodA assembled at the surface of ΔrodA dormant conidia to determine whether the in vitro structure is biologically relevant.

Established characterization methods of cell wall organization, such as GC/MS analysis, require the extraction of cellular compounds, their modification (e.g., derivation into volatile compounds) and their analysis by various biochemical methods. This long process (up to many months) often involves harsh chemical treatments and damages supramolecular assemblies. In this first chapter, we developed a comparably fast (five days only) [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C-detected SSNMR methodology to probe the different component of cell wall structures according to their dynamics-, hydration state or their location within the cell wall at the same time.

Moreover, we successfully applied our robust and powerful method to probe slight modifications in composition or biophysical parameters (i.e., rigidity/mobility, hydration) variations throughout the different steps of a complex biologic process such as the filamentous fungal germination.

In the second chapter, we combined our [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C-detected SSNMR methods described above with the use of 1 H-detection experiments to study more complex yeast surface architecture, namely C. neoformans. These yeast cells are surrounded by a rigid cell wall and a unconventional flexible capsule. This double-layered architecture significantly increased the difficulty of our analysis as it increases both the number of molecules to identify and the difference in their dynamic states.

states. The role of such genes into the synthesis of this polysaccharide was never described to date. Nevertheless, the three metabolic pathways involved in the production of xylose, glucuronic acid or galactose and theoretically blocked by these mutations involved the UDP-Glc as the initial sugar donor. Even though no connections were described between these synthesis pathways, the accumulation of UDP-Glc upon the mutation of one of these three genes could lead to a negative retro-control of the production of β-1,3-glucan favoring only the production of the flexible state of this polysaccharide.

Then, we probed the impact of these mutations onto the cell wall hydration. We observed a similar hydration profile of the chitin within the cell wall for the different mutants.

Hydration of the α-1,3-glucan is modified upon the mutation of ugd1. The mutation of this gene prevents the production UDP-GlcA from UDP-Glc and leads to an increased proportion of rigid α-1,3-glucan as compared with the WT. According to these results, α-1,3-glucan would be less water-accessible and thus more embedded into the cell wall; maybe to balance the effect of the loss of β-1,3-glucan by self-organizing in a dense layer conferring rigidity to the cell wall. Because unexpected impacts of theses mutation onto the cell wall composition and architecture raise new biological questions, additional approaches such as fluxomic methods will be of great help to complement our SSNMR data to understand C. neoformans carbohydrate metabolism.

The last part of this chapter deals with the characterization of C. neoformans lipids.

We assigned lipid signals as TAG which presence was already described within the external layers of C. neoformans. Moreover, this TAG would be associated with DOPA melanin, a pigment present in the outer layers of melanized cells. Even though the structure of DOPAmelanin is still unknown, its location at the interface between the cell wall and the capsule is well described. Thus, we anticipate unsaturated lipids to be also located at the interface between the two layers. Furthermore, that hypothesis is reinforced by the presence of TAG in both rigid and highly mobile states as seen by SSNMR; the dynamic different pool of the TAG could be influenced by either the cell wall rigidity or the high capsule flexibility.

Once we demonstrated our method to be powerful enough to target composition and architectural remodeling within the cell wall and/or the capsule of C. neoformans, we applied it to three clinical isolates obtained from patients diagnosed with cryptococcosis to investigate whether they share common features or exhibit different external layer structures. We observed that the three clinical isolates displayed similar flexible and rigid [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C fingerprints.

Moreover, none of them contain rigid β-1,3-glucan and they expressed only a low amount of rigid chitin. Moreover, their cell wall rigidity seems to be based only on the presence of rigid α-1,3-glucan. Finally, the capsular structure, still mainly flexible, relies on the same set of polysaccharides (i.e., mannose, xylose) as compared with the H99 model strain. The only difference is the presence of a putative β-1,3-xylan in the isolates capsules; those role remains unknown. These structural data have to be correlated with biological evidence in order to determine the impact of the yeast external structures remodeling on the virulence profile of the different clinical isolates.

All the results obtained in this chapter underlined the power of the combination of both To conclude, during this thesis project, we developed and/or improve the elucidation of microbial external structures using emergent SSNMR technologies. Our methodology was applied to whole cells in order to study the fungal structure as close as possible to their native states. We used a combination of carbon-and proton-detection at conventional or fast MAS to study the complex organization of A. fumigatus and C. neoformans outer layers. Moreover, the conventional MAS does not damage the A. fumigatus dormant conidial cells integrity; the viability of these samples and their infectious abilities after SSNMR experiments will be investigated in the next steps of these study. Using a versatility of SSNMR pulse sequences, we successfully investigated the different mobile or rigid outer layers of yeast or filamentous fungi. We were able to decipher their composition (i.e., polysaccharides, lipids, proteins) and to probe specific biophysical parameters such as the dynamic-state, the hydration or the water accessibility of the molecules within a compartment.

2. 1

 1 Effect of the PFA-fixation on A. fumigatus conidia 2.1 The dormant conidial cell wall 2.1.1 The rigid part of the conidial cell wall 2.1.2 The mobile part of the conidial cell wall 2.1.3 Identification of cell wall lipids 2.1.4 Discussion on the dormant conidia cell wall 3. INVESTIGATION OF THE GERMINATION PROCESS 3.1 The germination of A. fumigatus 3.1.1 Dormant conidia 3.1.2 Swollen conidia 3.1.3 Germinating conidia 3.2 The conidial cell wall composition 3.2.1 Remodeling of the rigid conidial cell wall 3.2.2 Dynamic-based regulation of polysaccharides 3.2.3 The conidial cell wall architecture 3.2.4 Structural effects of the germination process 4. THE CELL WALL OF α-GLUCAN-DEFICIENT ΔAGS MUTANT 4.1 Composition and dynamic-based study 4.2 Water-accessibility of conidial compounds 5. THE DHN-MELANIN 5.1 The conidial DHN-melanin 5.2 The melanin ghost 5.3 The synthetic DHN-melanin 5.4 New insights about the conidial DHN-melanin 6. THE RECOMBINANT HYDROPHOBIN RodA 6.1 Xray analysis of Rod proteins 6.2 SSNMR analysis of Rod proteins 6.2.1 Amyloid pattern of the three hydrophobins 6.2.2 Assignment of RodA fibers

3 . 5 Fig. 1 :

 351 Fig. 1:The global burden of fungal diseases.

Fig. 2 :

 2 Fig. 2: Structural organization of fungal cell walls.

Fig. 3 :

 3 Fig. 3: Saprophytic and pathogenic cycles of A. fumigatus.

Fig. 4 :

 4 Fig. 4: The cell wall of Aspergillus fumigatus in conidia and hyphae.

Fig. 5 :

 5 Fig. 5: The cell wall of Cryptococcus neoformans in conidia and hyphae.

Fig. 6 :

 6 Fig. 6: Schematic 2D NMR spectra.

Fig. 7 :

 7 Fig. 7: Magic Angle Spinning in SSNMR.

Fig. 8 :

 8 Fig. 8: The 1D 13 C-detected CP experiment.

Fig. 9 :

 9 Fig. 9: The 2D 13 C-13 C PDSD experiment.

Fig. 10 :

 10 Fig. 10:The 2D 1 H-[START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C HETCOR with 1 H-1 H spin diffusion mixing time experiment.

Fig. 11 :

 11 Fig. 11: The 1D 13 C-detected DP experiment.

Fig. 12 :

 12 Fig. 12: The 2D 1 H-13 C INEPT experiment.

Fig. 13 :

 13 Fig. 13: Water-edited experiments.

Fig. 14 :

 14 Fig. 14: The 2D 13 C-13 C DP-INADEQUATE experiment.

Fig. 15 :

 15 Fig. 15: The 2D 13 C-13 C CP-INADEQUATE experiment.

Fig. 16 :

 16 Fig. 16:The proton-detected[START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C-[START_REF] Fisher | Emerging Fungal Threats to Animal, Plant and Ecosystem Health[END_REF] N-1 H 3D correlation experiments.

Fig. 17 :

 17 Fig. 17: Chemical reactions of formaldehyde and paraformaldehyde.

Fig. 18 :

 18 Fig. 18: Effect of the PFA-fixation of the conidia.

Fig. 19 :

 19 Fig. 19: Identification of the main carbohydrates of the conidial cell wall.

Fig. 20 :

 20 Fig. 20: Identification of the dormant conidial cell wall components.

Fig. 21 :

 21 Fig. 21: Assignment of mannose from the cell wall.

Fig. 22 :

 22 Fig. 22: Assignment of the flexible part of the conidial cell wall.

Fig. 23 :

 23 Fig. 23: Structure of a putative TAG identified with the conidial cell wall.

Fig. 24 :

 24 Fig. 24: The three different steps of the germination process of A. fumigatus.

Fig. 25 :

 25 Fig. 25: The rigid part of the conidial cell wall.

Fig. 26 :

 26 Fig. 26: Influence of the germination onto the cell wall carbohydrates.

Fig. 27 :

 27 Fig. 27: Identification of the GAGs within the cell wall of the germinating conidia.

Fig. 28 :

 28 Fig. 28: Evolution of the conidial cell wall dynamic during the germination process.

Fig. 29 :

 29 Fig. 29: Dynamics changes of the cell wall through the germination process.

Fig. 30 :

 30 Fig. 30: The conidial surface.

Fig. 31 :

 31 Fig. 31: Water-edited fingerprints of dormant and germinating conidia.

Fig. 32 :

 32 Fig. 32: Evolution of the architecture of the conidial cell wall.

Fig. 33 :

 33 Fig. 33: Proposed cell wall remodeling upon the germination process.

Fig. 34 :

 34 Fig. 34: The rigid part of the Δags dormant conidia cell wall.

Fig. 35 :

 35 Fig. 35: The polymorphism of the chitin.

Fig. 36 :

 36 Fig. 36: The dynamic of the Δags dormant conidia cell wall.

Fig. 37 :

 37 Fig. 37: Water-accessibility of the cell wall carbohydrates.

Fig. 38 :

 38 Fig. 38: Architecture of the Δags dormant conidia.

Fig. 39 :

 39 Fig. 39: The synthetic pathway of the DHN-melanin.

Fig. 40 :

 40 Fig. 40: The conidial DHN-melanin.

Fig. 41 :

 41 Fig. 41: The ghost DHN-melanin.

Fig. 42 :

 42 Fig. 42: The synthetic DHN-melanin.

Fig. 43 :

 43 Fig. 43: Impact of the acidic precipitation on the synthetic DHN-melanin.

Fig. 44 :

 44 Fig. 44: Theoretical X-ray diffraction pattern of amyloid fibers.

Fig. 45 :

 45 Fig. 45: WAXS diffraction patter of the three hydrophobins.

Fig. 46 :

 46 Fig. 46: Sequences alignment of RodA, B and C.

Fig. 47 :

 47 Fig. 47: The 13 C fingerprint of RodA, B and C.

Fig. 48 :Fig. 49 :

 4849 Fig. 48: The rigid core of the recombinant RodA Fig. 49: Proton-detection SSNMR of the amyloid RodA.

Fig. 50 :

 50 Fig. 50: The capsule and the cell wall of C. neoformans.

Fig. 51 :

 51 Fig. 51: Capsule polysaccharide structures.

Fig. 52 :

 52 Fig. 52: The rigid cell wall of C. neoformans.

Fig. 53 :

 53 Fig. 53: 1 H-detection of the cell wall carbohydrates.

Fig. 54 :

 54 Fig. 54: The different dynamic-based profiles within the cell wall.

Fig. 55 :

 55 Fig. 55: The water-accessibility of cell wall carbohydrates.

Fig. 56 :Fig. 57 :

 5657 Fig. 56: The homogenous hydration of the cell wall Fig. 57: Proposed architecture of C. neoformans cell wall.

Fig. 58 :Fig. 59 :

 5859 Fig. 58: Different anomers of capsular carbohydrates Fig. 59: The flexible capsule of C.neoformans.

Fig. 60 :

 60 Fig. 60: The TAG of C.neoformans.

Fig. 61 :

 61 Fig. 61: Structure of the potential TAG in the envelope of C. neoformans.

Fig. 62 :Fig. 63 :

 6263 Fig. 62: Proposed structure of the encapsulated yeast C. neoformans H99. Fig. 63: The cell wall carbohydrates of the Δcap59 mutant.

Fig. 64 :

 64 Fig. 64: The capsular carbohydrates of the Δcap59 mutant.

Fig. 65 :

 65 Fig. 65: The capsular and cell wall layers of the Δuxs1 mutant.

Fig. 66 :

 66 Fig. 66: The capsular and cell wall layers of the Δugd1 mutant.

Fig. 67 :

 67 Fig. 67: The capsular and cell wall layers of the Δuge1Δcap59 mutant.

Fig. 68 :

 68 Fig. 68: The capsule and the cell wall of the Δuge1Δcap59 mutant.

Fig. 69 :

 69 Fig. 69: The cell wall hydration of the mutant cells.

Fig. 70 :

 70 Fig. 70: The rigid part of C. neoformans clinical isolates envelope.

Fig. 71 :

 71 Fig. 71: The flexible part of C. neoformans clinical isolates envelope.

  ABBREVIATIONS

Fig. 2 :

 2 Fig. 2: Structural organization of fungal cell walls. (taken from Gow et al. 2017)

Fig. 3 :

 3 Fig. 3: Saprophytic and pathogenic cycles of A. fumigatus.

Fig. 8 :

 8 Fig. 8: The 1D 13 C-detected CP experiment. A) The 1D 13 C-detected CP pulse sequence where the magnetization transfer pathway is highlighted in light pink. B) An example of the use of 1D 13 C-detected CP experiment to highlight the global content of bacterial cell wall (extracted from Molina-Santiago et al., 2019) 83 .

Fig. 9 :

 9 Fig. 9: The 2D 13 C-13 C PDSD experiment.

  During a direct-polarization (DP)-based experiment, the nucleus of interest is directly exciting without using any type of polarization transfer making these experiments less sensitive as compared to CP-based ones. For the mobile molecules, due to fast molecular tumbling, the dipolar couplings are average out. Hence, CP based experiments cannot be used A B to detect them. By applying a short recycle delays the molecules with fast tumbling time can be unrevealed during a DP-based experiment.

  2019, Kang et al., illustrated dynamic-based differences occurring within the mycelial cell wall of A. fumigatus by recording a set 1D 13 C-DP experiments with both long or short recycle delays 84 (Fig. 11B).

Fig. 11 :

 11 Fig. 11: The 1D 13 C-detected DP experiment. A) The 1D 13 C-detected DP pulse sequence where the magnetization transfer pathway is highlighted in light pink. B) 1D 13 C DP spectra of intact A. fumigatus cells recorded with recycle delay of 35 s or 2 s and giving respectively a quantitative (black) or mobility-based (red) spectra. In these experiment, authors were able to assign all the cell wall components including polysaccharides, proteins, lipids and small molecules (adapted from Kang et al., 2018) 84 .

  these scalar couplings are unaffected by the presence or absence of dynamics, but the relatively long spin echo periods of the INEPT scheme are highly sensitive to magnetization losses due to T 2 relaxation. Then, only highly flexible (motion faster than the us) molecules with a long T 2 are targeted by INEPT-based pulse sequences. The INEPT transfer can be encoded into a one 13 C-detcted or two-dimension 1 H-13 Cdetected pulse sequences. The heteronuclear 2D spectra reflect the couples of 1 H-13 C covalently linked located in a mobile molecule or in a highly mobile moieties (e.g., loop of proteins) as illustrated by Kashefi et al., in 2017, to probe the functional complexes of the E.coli Asp receptor cytoplasmic fragment prepared from methylated cytoplasmic fragment CF4Q predicted to have a flexible tail 87 (Fig.12B).

Fig. 12 :

 12 Fig. 12: The 2D 1 H-13 C INEPT experiment. A) The 2D 1 H-13 C INEPT pulse sequence where the magnetization transfer pathway is highlighted in light pink and the coupling constant J CH used for the INEPT transfer in blue. B) 2D 1 H-13 C INEPT of CF4QΔ34 spectrum representing the most likely ∼50-residue mobile region of the protein (adapted from Kashefi et al., 2017) 87 .

Fig. 13 :

 13 Fig. 13: Water-edited experiments.

Fig. 14 :Fig. 15 :

 1415 Fig. 14: The 2D 13 C-13 C DP-INADEQUATE experiment. A) The 13 C-13 C DP-INADEQUATE pulse sequence the magnetization transfer pathway is highlighted in light pink. B) 13 C-13 C DP-INADEQUATE assigned with the different polysaccharides present in the mobile part of the cell wall of Arabidopsis thaliana (extracted from Dick-Pérez et al., 2011) 89 .

Fig. 16 Fig. 16 :

 1616 Fig. 16: The proton-detected 13 C-15 N-1 H 3D correlation experiments. The (H)CANH (A) and (H)CONH (B) pulse sequences used to establish intra-and interresidues connectivity; the cartoons refer to the connectivity involved in each experiment (extracted from Barbet-Massin et al., 2014) 90 .

  mM) -EDTA (50 mM) -SDS (2 %)beta-mercaptoethanol (40 mM) buffer (pH 7.4) for 15 min. The synthetic unlabeled DHN-melanin was polymerized by dispersing 40 mg of 1,8-Dihydroxynaphthalene (DHN) (Sigma) in bicarbonate (50 mM) buffer (pH 11) and incubated for 4 h at 50°C (12000g, 10 min). The pigment was recovered by centrifugation (16000g); the pellet was washed three times by resuspension into MilliQ water and ultracentrifugation (70000g, 10 min).

  C direct-polarization (DP) spectra (512 scans) were recorded with a 35 s recycle delay to target all the cell wall components independently from their rigidity state (used as a quantitative spectra) or a 2 s recycle delay to enhance mobile signals compared to 35 s recycle delay. Two-dimension 13 C-13 C DP INADEQUATE experiments (64 scans) were performed with a recycle delay of 2 s and 13 C-13 C CP-INADEQUATE (64 scans) with a 0.5 ms contact time for the 1 H-13 C polarization transfer.

Fig. 17 :

 17 Fig. 17: Chemical reactions of formaldehyde and paraformaldehyde. A) Structure of the formaldehyde. B) Example of the crosslinking between a lysine residue and another amino acid using formaldehyde. C) Mechanism of paraformaldehyde depolymerization.

  using the same experimental conditions (number of scans, CP contact time…).

Fig. 18 :

 18 Fig. 18: Effect of the PFA-fixation of the conidia. 1D 13 C CP spectra of 13 C/ 15 N-labeled PFA-fixed (black) and unfixed dormant conidia (purple) of A. fumigatus.

Fig. 19 :

 19 Fig. 19: Identification of the main carbohydrates of the conidial cell wall. A) 2D 13 C-13 C PDSD of 13 C/ 15 N-labeled dormant conidia assigned with α-glucan (pink), βglucan (red) and chitin (black). On the top left corn, an excerpt of the 13 C-13 C PDSD illustrates the α-glucan polymorphism. B) The structures of the three main carbohydrates of the cell wall with their 13 C chemical shift.

Fig. 20 :

 20 Fig. 20: Identification of the dormant conidial cell wall components.

  C-[START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C CP-INADEQUATE spectrum in which the second dimension is encoded as double-quantum to split more efficiently NMR signals (Fig.21). As this experiment is 1 H-13 C CP-based, it only probes rigid molecules.

Fig. 21 :

 21 Fig. 21: Assignment of mannose from the cell wall.2D 13 C-13 C CP-INADEQUATE spectrum recorded on 13 C/ 15 N-labeled dormant conidia assigned with α-glucan (pink), β-glucan (red), chitin (black), mannose (purple) and two other unassigned spin systems Y and X (blue).

  Furthermore, both α-glucan and β-glucan are detected using either CP or DP-based experiments. This could indicate that they are present in both mobile and rigid states indicating different regulation and location of these polysaccharides within the cell wall.Nevertheless, the contribution of their mobile signals is low as compared with other polysaccharides. Thus, we can here suggest that both α-glucan and β-glucan are secreted mainly as rigid polysaccharides probably maintaining the cell wall integrity and rigidity but a part of these polysaccharides remains in a mobile state. This could reflect: (1) the end of the polysaccharide chains or ramifications (especially for the β-1,3-glucan containing 4% of β-1,6-branched point or ramified with β-1,4-glucan) or (2) a complete chain in another more mobile environment (especially α-glucan exhibiting polymorphism).

Fig. 22 :

 22 Fig. 22: Assignment of the flexible part of the conidial cell wall. A) Zoom on the carbohydrates region and B) Full spectrum recorded using 2D 13 C-13 C DP-INADEQUATE on 13 C/ 15 N-labeled dormant conidia assigned with α-glucan (pink), β-glucan (red), chitin (black), mannose (purple), unknown carbohydrate Z (blue). Fatty acid tails and putative glycerol from glycerolipids are displayed in green.

Fig. 24 :

 24 Fig. 24: The three different steps of the germination process of A. fumigatus. Scanning electron microscopy images provided by Dr. V. Aimanianda (Pasteur Institute, Paris, France).

Fig. 25 :

 25 Fig. 25: The rigid part of the conidial cell wall. Alignment of the three 1D 13 C CP fingerprint of the dormant, swollen and germinating conidia with the assignment of the main components of the cell wall: α-glucan (pink), βglucan (red), chitin (black) and lipid (green).

Fig. 26 :

 26 Fig. 26: Influence of the germination onto the cell wall carbohydrates. A) 2D 13 C-13 C PDSD of the dormant, swollen and germinating conidia assigned with the three main carbohydrates: α-glucan (pink), β-glucan (red) and chitin (black). B) Superimposition of 13 C traces extracted at the β-glucan C 1 chemical shift (105.8 pmm) for both dormant and swollen conidia. C) Relative quantification of the contribution of each cell wall component during the three steps of the germination process.

Fig. 27 :

 27 Fig. 27: Identification of the GAGs within the cell wall of the germinating conidia. Superimposition of 2D 13 C-13 C DP-INADEQUATE recorded on both 13 C/ 15 N-labeled dormant and germinating conidia assigned with the three main carbohydrates: α-glucan (pink), β-glucan (red) and chitin (black).

Fig. 28 :

 28 Fig. 28: Evolution of the conidial cell wall dynamic during the germination process. Superimposition of 1D 13 C DP spectra recorded with a long (35 s) or short (2 s) recycle delay and giving quantitative (purple) or mobility-based (black) information respectively.

Fig. 29 :

 29 Fig. 29: Dynamics changes of the cell wall through the germination process. A) 1D 13 C DP spectra recorded with a short (2 s) recycle delay reflecting mobile compounds on 13 C/ 15 N labeled dormant, swollen and germinating conidia. Main components of the cell wall are assigned as: α-glucan (pink), β-glucan (red), chitin (black) and lipid (green). B) Relative quantification of both mobile and rigid parts of each cell wall components during the three steps of the germination process.

Fig. 30 :

 30 Fig. 30: The conidial surface. A) 2D 13 C-1 H HETCOR spectrum recorded with (black) or without (purple) a 1 H-1 H spin diffusion (SD) of 1.5 ms on 13 C/ 15 N labeled dormant, swollen and germinating conidia. The 1 H bulk water chemical shift is highlighted in light pink. B) 1D 1 H trace extracted at 4.9 ppm showing the width of the water peak correlated with polysaccharides. C) Relative quantification of water-accessible polysaccharides.

Fig. 31 :

 31 Fig. 31: Water-edited fingerprints of dormant and germinating conidia.

Fig. 32 :

 32 Fig. 32: Evolution of the architecture of the conidial cell wall. Build-up curves with the relative intensity of peaks referring to one cell wall component plotted as function of the 1 H-1 H mixing time for the three steps of the germination process.

Fig. 34 :Fig. 35 :

 3435 Fig. 34: The rigid part of the Δags dormant conidia cell wall.

Fig. 38 :

 38 Fig. 38: Architecture of the Δags dormant conidia.

Fig. 39 :

 39 Fig. 39: The synthetic pathway of the DHN-melanin. Enzymes are represented in purple, their function in pink.

  However, we observed that the chemical extraction of the external layers would have damaged the cell wall. Indeed, the intensities of the α-glucan signals are lower in the white dormant conidia as compared with WT one. This polysaccharide, mainly found at the surface of the spores, seem to have been partially extracted during the chemical release of both RodA and DHN-melanin layers.

Fig. 40 :

 40 Fig. 40: The conidial DHN-melanin.

  state (probed by CP-based experiments). The melanin ghosts do not contain any aromatic contribution expected from the 1,8-DHN polymerization. We could suggest that: either (1) the amorphous pigment escapes from our SSNMR detection using DP-, CP-or INEPT-based transfers or (2) the 1,8-DHN is converted into a linear pigment explaining the carboxyl and aliphatic signals. Nevertheless, this last hypothesis do not seem consistent with the absence of high content in alternated C=C bond required for the electron delocalization and the light absorbance of the pigment.

Fig. 41 :

 41 Fig. 41: The ghost DHN-melanin.

Fig. 42 :

 42 Fig. 42: The synthetic DHN-melanin. A) The DHN-melanin synthesis protocol starting from the commercial 1,8-DHN. Pictures of the solution at 1h, 2h and 4h illustrating its change in color. B) 1D 13 C CP spectrum of the synthetic DHN-melanin assigned with the different 13 C chemical shift regions and the spectral area of expected aromatic signals highlighted in pink.

Fig. 43 :

 43 Fig. 43: Impact of the acidic precipitation on the synthetic DHN-melanin. Alignment of 13 C CP spectra recorded on synthetic DHN-melanin recovered either by ultracentrifugation (black) or by acidic precipitation (purple) with lipid assignment. The pink square represents the aromatic 13 C chemical shift spectral area.

  C CP signals corresponding to an unsaturated fatty acid comparable to the one described earlier within the conidial cell wall. Based on these observations, we hypothesized that: (1) the low amount of DHN-melanin does not allow probing the pigment structure by SSNMR methods; (2) the DHN-melanin might escape to our detection by CP-, DP-or INEPT-based experiment, as it would be too amorphous and/or too heterogeneous; (3) the DHN-melanin is strongly associated with cell wall carbohydrates and unsaturated lipids assigned earlier in this study.

Fig. 44 :

 44 Fig. 44: Theoretical X-ray diffraction pattern of amyloid fibers. To investigate whether the structure of RodA, B and C exhibit a cross-β core indicating an amyloid fold, our collaborator (Dr. B. Kauffmann, IECB, Bordeaux) recorded WAXS experiments on the three samples. The resulting diffraction patterns indicate the presence of 10 Å and 4.7 Å peaks, characteristic of a cross-β structure (Fig. 45). This strongly

Fig. 45 :

 45 Fig. 45: WAXS diffraction patter of the three hydrophobins. The arrows indicate characteristic peaks of an amyloid fold.

Fig. 46 :

 46 Fig. 46: Sequences alignment of RodA, B and C. Homologous amino acids are highlighted in light blue; the idiosyncratic cysteine pattern in dark blue.

Fig. 47 :

 47 Fig. 47: The 13 C fingerprint of RodA, B and C. A) 1 D 13 C CP spectra and B) 2D 13 C-13 C PDSD spectra of RodA, B and C recorded on 13 C/ 15 N-labeled proteins.

Fig. 48 :Fig. 49 :

 4849 Fig. 48: The rigid core of the recombinant RodA A) 2D intra-residual 13 C-13 C PDSD spectrum recorded on 13 C/ 15 N labeled recombinant RodA showing the different regions of the spectrum B) Superimposition of 2D intra-residual (black) and inter-residual (blue) 13 C-13 C PDSD spectra recorded on 13 C/ 15 N labeled recombinant RodA. C) Secondary chemical shift of RodA amino acids. D) The amino acids sequence of recombinant RodA with the amino acids assigned sequentially underlined in blue.

Fig. 50 :

 50 Fig. 50: The capsule and the cell wall of C. neoformans. Immunolabeling of the capsule and the cell wall of C. neoformans. The cell wall was specifically labeled with Calcofluor white and the capsule with rabbit anti-capsular polyclonal antibody revealed with goat anti-rabbit IgG-FITC-conjugated secondary antibody (provided by Dr. V. Aimanianda, Pasteur Institute, Paris)

Fig. 51 :

 51 Fig. 51: Capsule polysaccharide structures. A) The six different patterns of glucuronoxylomannan (GXM). B) The structure of glucuronoxylomannogalactan (GXMGal). Linkages are indicated on the diagram. In both panels all sugars are in pyranose form (except the galactofuranose of the GXMGal), and single polymer repeat units are shown. Shapes and colors follow the recommendations of the editors of Essentials of Glycobiology: mannose, green spheres; xylose, red stars; glucuronic acid, half-filled diamonds, galactose as yellow spheres.

  added[START_REF] O'meara | The Cryptococcus Neoformans Capsule: A Sword and a Shield[END_REF] H detection methods at 100 kHz MAS to A B detect intact bacterial peptidoglycans. Phyo and Hong 77 also applied 1 H-detected methods at 50-60 kHz MAS frequencies to elucidate the structure of the matrix of polysaccharides found in plant cell walls. These studies showed good resolution and sensitivity in 1 H-detected experiments for polysaccharide molecules. Thus, the introduction of 1 H detection to multidimensional SSNMR experiments can simplify the identification of polysaccharides and other molecules. At fast MAS frequencies, the resolution in SSNMR spectra becomes closer to the one of liquid-state NMR spectra. With the addition of J-couplings-based[START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C-[START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C magnetization transfers (like TOCSY), different connections for assignment purposes can be tailored together 150 .

Fig. 52 :

 52 Fig. 52: The rigid cell wall of C. neoformans. A) The schematic envelope of C. neoformans with the plasma membrane highlighted in yellow, the cell wall in purple, the capsule in blue. This color code will be used throughout this study. B) 2D 13 C-13 C PDSD recorded on 13 C/ 15 N labeled C. neoformans H99 assigned with α-glucan (pink), β-glucan (light purple) and chitin (dark purple). C) Structure of α-1,3glucan, β-1,3-glucan and chitin. D) 1D 13 C CP spectrum recorded on 13 C/ 15 N labeled C. neoformans assigned with α-1,3-glucan (pink), β-1,3-glucan (light purple), chitin (dark purple) and TAG (green).

Fig. 53 :

 53 Fig. 53: 1 H-detection of the cell wall carbohydrates. A) 2D 13 C-13 C CP-INADEQUATE, B) 2D hCH, C) Slices extracted from 3D hCCH recorded on 13 C/ 15 N-labeled C. neoformans H99 assigned with α-1,3-glucan (pink), β-1,3-glucan (light purple) and chitin (dark purple).

  polysaccharide, more flexible, would be present at the interface between the capsule and the cell wall (secondary spin systems) 151,152 .

Fig. 54 :

 54 Fig. 54: The different dynamic-based profiles within the cell wall. A) Superimposition of 1D 13 C CP and DP recorded with a long mixing time on 13 C/ 15 Nlabeled C. neoformans H99. B) Relative quantification of the mobile and rigid part of the cell wall polysaccharides.

Fig. 56 :

 56 Fig. 56: The homogenous hydration of the cell wall A) Superimposition of 13 C-13 C PDSD (black) and 13 C-13 C water-edited PDSD (red) with highlights of the α-1,3-glucan (pink), β-1,3-glucan (light purple), chitin (dark purple). B) Excerpt of the spin systems corresponding to α-1,3-glucan, β-1,3-glucan and chitin. C) Buildup curve of α-1,3-glucan, β-1,3-glucan and chitin.

1, 3 -Fig. 57 :

 357 Fig. 57: Proposed architecture of C. neoformans cell wall.

Fig. 58 :

 58 Fig. 58: Different anomers of capsular carbohydrates A) Superimposition of 2D hCH HSQC acquired with (black) or without 1 H decoupling (yellow). B) The two possible anomers of pyranose. C) The different anomers of capsular carbohydrates.

Table 4 :

 4 Relative quantification of capsular polysaccharides.Intensities of 1 H trace extracted from the hCH HSQC spectrum. order to remove the ambiguous assignment of anomeric[START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C exhibited in the 13 C-13 C DP INADEQUATE spectrum, we employed the 14 ms TOCSY long transfer in 2D hCCH experiment (Fig.59A,B). Indeed, in this experiment, the magnetization is transferred through up to 4-5 chemical bonds, allowing identifying all the carbons within the same molecule.

Fig. 59 :

 59 Fig. 59: The flexible capsule of C.neoformans. A) 2D hCH HSQC B) 2D hCCH 14 ms TOCSY C) Excerpt of 2D 13 C-13 C DP INADEQUATE spectra recorded on 13 C/ 15 N C. neoformans H99.

Fig. 60 :

 60 Fig. 60: The TAG of C.neoformans. A) 2D hCH HSQC B) 2D hCCH 14 ms TOCSY spectra recorded on 13 C/ 15 N C. neoformans H99.

Fig. 61 :

 61 Fig. 61: Structure of the potential TAG in the envelope of C. neoformans.

  some molecules escape from our detection and the structure of capsular polysaccharides remains unclear. For all these reasons, and based on our preliminaries results, we established only an initial model of the molecular architecture of the encapsulated C.neoformans outer layers.

Fig. 62 :

 62 Fig. 62: Proposed structure of the encapsulated yeast C. neoformans H99.

63B)

  spectra on Δcap59 mutant and compared it with spectra recorded on WT cells. Using CP-based experiments, we investigated the rigid part of the C. neoformans envelope. Indeed, we demonstrated earlier that the rigid part of the yeast external structures only contains cell wall carbohydrates.

Fig. 63 :

 63 Fig. 63: The cell wall carbohydrates of the Δcap59 mutant. A) 2D 13 C-13 C CP-INADEQUATE and B) 2D 13 C-13 C PDSD recorded on 13 C/ 15 N Δcap59 mutant (red) or WT (black) assigned with α-1,3-glucan (pink), β-1,3-glucan (light purple) and chitin (dark purple).

Fig. 64 :

 64 Fig. 64: The capsular carbohydrates of the Δcap59 mutant.

Fig. 65 :

 65 Fig. 65: The capsular and cell wall layers of the Δuxs1 mutant. A) 2D 13 C-13 C CP-INADEQUATE and B) 2D 13 C-13 C PDSD C) 2D 13 C-13 C DP-INADEQUATE D) 1D 13 C CP recorded on 13 C/ 15 N-labled Δuxs1 mutant (red) or WT (black) assigned with α-1,3-glucan (pink), β-1,3-glucan (light purple), chitin (dark purple), mannose (light blue) and xylose (dark blue). E) Relative quantification of both rigid or mobile α-1,3glucan and chitin within the cell wall of C. neoformans Δuxs1.

Fig. 66 :

 66 Fig. 66: The capsular and cell wall layers of the Δugd1 mutant. A) 2D 13 C-13 C CP-INADEQUATE and B) 2D 13 C-13 C PDSD C) 2D 13 C-13 C DP-INADEQUATE D) 1D 13 C CP recorded on 13 C/ 15 N-labled Δugd1 mutant (red) or WT (black) assigned with α-1,3-glucan (pink), β-1,3-glucan (light purple), chitin (dark purple), mannose (light blue) and xylose (dark blue). E) Relative quantification of both rigid or mobile α-1,3glucan and chitin within the cell wall of C. neoformans Δugd1.

Fig. 67 :

 67 Fig. 67: The capsular and cell wall layers of the Δuge1Δcap59 mutant. A) 2D 13 C-13 C CP-INADEQUATE and B) 2D 13 C-13 C PDSD C) 2D 13 C-13 C DP-INADEQUATE D) 1D 13 C CP recorded on 13 C/ 15 N-labled Δuge1Δcap59 mutant (red) or WT (black) assigned with α-1,3-glucan (pink), β-1,3-glucan (light purple), chitin (dark purple), mannose (light blue) and xylose (dark blue). E) Relative quantification of both rigid or mobile α-1,3-glucan and chitin within the cell wall of C. neoformans Δuge1Δcap59.

Fig. 68 :

 68 Fig. 68: The capsule and the cell wall of the Δuge1Δcap59 mutant.

Fig. 69 :

 69 Fig. 69: The cell wall hydration of the mutant cells.

Fig. 70 :

 70 Fig. 70: The rigid part of C. neoformans clinical isolates envelope. 2D 13 C-13 C PDSD spectra recorded on 13 C/ 15 N-label clinical isolates (green, yellow or red) or WT (black) assigned with α-1,3-glucan (pink), β-1,3-glucan (light purple) and chitin (dark purple).

Fig. 71 :

 71 Fig. 71: The flexible part of C. neoformans clinical isolates envelope. 2D 13 C-13 C DP-INADEQUATE spectra recorded on 13 C/ 15 N-label clinical isolates (green, yellow or red) or WT (black) assigned with α-1,3-glucan (pink), β-1,3-glucan (light purple), chitin (dark purple), xylose (dark blue), mannose (light blue) and the unknown new polysaccharide (red).

  immunosuppressed individuals and are, most of the time, associated with other diseases or medical conditions such as asthma, leukemia, organ transplants, AIDS or immunosuppressive corticosteroid therapies. Despite the number of deaths and the increase in severe mycosis, fungal infections remain neglected by public health authorities. The virulence of fungal pathogens often relies on their external structures which could be a new potential target for the development of new therapeutic molecules as well as prophylactic strategies. Fungal external structures provide strength and rigidity, protecting cells from environmental stresses while maintaining cell shape and regulating permeability. Remarkably, these powerful structures are also very plastic, allowing significant morphologic changes during germination or differentiation. Moreover, they enable fungal cells to transition between environments.In the first chapter, we aimed at investigating the composition and the architecture of the cell wall of A. fumigatus using 13 C-detected SSNMR experiments. This way, we probed at atomic resolution the molecular changes and remodeling occurring within the cell wall throughout the germination of the filamentous fungus. Using a set of multidimensional experiments, we provided an atomistic description of the cell wall composition and mobility based on polysaccharide signals. Polysaccharides such as glucans and chitin have been identified, quantified and probed in their different mobility states. By comparing SSNMR experiments of A. fumigatus cells at various germinating states, we establish the molecular basis of structural and compositional changes occurring throughout the process. Our analysis highlighted that the composition of the cell wall varies through the germination process;

  [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C-detected and 1 H-detected SSNMR methods to characterize yeast surface architecture. Our approach is applied on intact cells and thus does not require additional extraction of cell wall (or capsular) components. Additional experiments will be developed to further analyze capsular carbohydrates in order to build a relevant model of this structure. Indeed, the surface of the yeast is in direct contact with host cellular structures and immune cells: getting more structural insights about the outer layer of C. neoformans might bring a better comprehension of host/pathogen interactions occurring during the host invasions and mediate the development of new therapeutic strategies.

Fig. S1 .

 S1 Fig. S1. 2D 13 C-13 C DP-INADEQUATE spectrum recorded on 13 C/ 15 N-labeled C. neoformans H99.

  Institut de Chimie et Biologie des Membranes et Nano-objets, UMR 5248,

	1.1.1 Conidial samples
	1.1.2 Melanin samples
	1.1.3 Production and purification of hydrophobins
	Allée Geoffroy Saint Hilaire, B14 1.2 Solid-state NMR experiments
	1.2.1	33600 Pessac

Table 1 :

 1 Examples of human mycosis.

Table 2 :

 2 Main δ 13 C and δ1 H assignments of the rigid cell wall carbohydrates

Table 3 :

 3 Relative quantification of cell wall polysaccharides.

Table 4 :

 4 Relative quantification of capsular polysaccharides.

Table 5 :

 5 δ[START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C and δ 1 H assignements of the flexible capsular carbohydrates

Table 6 :

 6 δ[START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C and δ 1 H assignements of the putative capsular TAG

Table 7 :

 7 The δ[START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C of the putative β-1,3-xylan present in the 2.86 clinical isolate.

Table 1 :

 1 Examples of human mycosis. (adapted from Fausto et al, 2019). Aspergillus spp. mainly in immunocompromised individuals. There are more than 250 species of Aspergillus, of them fewer than 40 are reported to cause infections in humans. Aspergillus fumigatus and Aspergillus flavus are the most common human pathogens.

	Fungal disease	Distribution and etiological agents
	Aspergillosis Invasive infection caused by Blastomycosis Endemic mycosis located to the south central and north central United States, caused by Blastomyces dermatitidis and Blastomyces gilchristii that can affect
		immunocompetent and immunocompromised individuals.

1 H-13 C HETCOR pulse sequence where the magnetization transfer pathway is highlighted in light pink and 1 H spin diffusion mixing time t SD in green. B) The water- accessibility of carbohydrates close to the surface of B. subtilis is investigated by

	superimposing 2D 1 H-13 C HETCOR spectra recorded on the extracellular matrix-deficient
	mutant without (in red) and with (in blue) a 1H spin diffusion mixing time. The bulk water
	frequency is indicated in yellow. (Extracted from Molina-Santiago et al., 2019) 83 .

Table 2 :

 2 Main δ 13 C and δ1 H assignments of the rigid cell wall carbohydrates

		C1	C2	C3	C4	C5	C6	CH 3	CO
	1,3-α-glucan	103.1	73.7	86.9	69.80	74.2	63.3	-	-
	1,3-β-glucan	105.8	76.4	88.7	70.3	79.8	63.2	-	-
	Chitin	106.2	57.6	75.1	84.6	76.5	61.6	25.0 177.4
		H1	H2	H3	H4	H5	H6/H6' CH 3
	1,3-α-glucan	5.54	4.11	4.12	3.16	3.97	3.8	-
	1,3-β-glucan	4.84	3.66	3.67	3.55	3.75	3.94	-
	Chitin	3.60	3.87	3.86	3.42		4.01	2.15

Table 5 :

 5 δ[START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C and δ 1 H assignements of the flexible capsular carbohydrates

		C1/H1	C2/H2	C3/H3	C4/H4	C5/H5/H5'	C5/H6/H6'
	Xylose (β-1-?-)	105.8/4.56	76.2/3.62	79.6/3.79	72.4	63.9	-
	MX (Man/Gal-α-1-?-)	102.6/5.43	74.5	76.25/3.37	71.9	72.9	63.8
	MY (β-Man/Gal)	98.99/4.67	77.1/3.28	79.37	72.0/3.95
	MZ (α-Man/Gal)	94.93/5.26	74.2/3.61	76.4		71.62	64.97/4.3/4.09
	MD (α-1,3-Man/Gal)	105.8/4.7	76.1	87.1	70.9	78.6	63.7

Table 6 :

 6 δ[START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C and δ 1 H assignements of the putative capsular TAG

	C1	C2	C3	C4	C5	C6	C7	C8	C9 C10 C11	C12
	Fatty acid 130.7 36.1 27.4 31.6 28.0 130.5 132.2 29.8 31.9 34.1 23.60 14.96
		H2	H3	H4	H5	H6	H7	H8	H0 H10 H11	H12
		5.33 2.77 2.05 1.32 5.33	5.33 0.90 1.59 2.26 0.97	0.56
	However, recently, a new study based on the analysis of both melanized and non-
	melanized cells demonstrated the presence of lipids trapped within the capsule and cell wall
	of C. neoformans H99 110 . These lipids would be glycerolipids such as triacylglycerides (TAG)

Table 7 :

 7 The δ 13 C of the putative β-1,3-xylan present in the 2.86 clinical isolate.

		C1	C2	C3	C4	C5
	β-1,3-xylan	105,4	75,9	80,7	74,1	63,4

Remerciements

A) 1D [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C CP spectra recorded for both [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C/ [START_REF] Fisher | Emerging Fungal Threats to Animal, Plant and Ecosystem Health[END_REF] N-labeled dormant and Δags dormant conidia assigned with α-glucan (pink), β-glucan (red), chitin (black) and lipid (green). B) Superimposition of 2D [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C- [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C PDSD recorded for [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C/ [START_REF] Fisher | Emerging Fungal Threats to Animal, Plant and Ecosystem Health[END_REF] N-labeled dormant and Δags dormant conidia.

Here, we were particularly interested in the compensation of the mutation by the production of other sugar and/or by an overproduction of preexisting polysaccharides. Indeed, the cell wall thickness of the Δags dormant conidia remains identical as compared with the WT indicating the occurrence of a compensatory mechanism in response to the α-glucan depletion [START_REF] Valsecchi | The Puzzling Construction of the Conidial Outer Layer of Aspergillus Fumigatus[END_REF] . As no significant evidence for this kind of conidial cell wall remodeling were deciphered only using the 1D [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C CP fingerprint, we recorded a 2D [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C- [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C PDSD on the Δags dormant conidia to overcome the 1D spectral overlapping of carbohydrates signals area (Fig. 34B). The resulting spectrum suggests that no new carbohydrate has been secreted to compensate the absence of the α-glucan as no new spin system has been identified. On the other hand, even though the β-glucan does not seem to be affected by the mutation, the spin systems corresponding to the chitin are more intense and display a higher polymorphism as compared the WT conidia. To investigate this polymorphism, we recorded 2D [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C- [START_REF] Fisher | Emerging Fungal Threats to Animal, Plant and Ecosystem Health[END_REF] N NCO and NCA spectra (Fig. 35A). In these experiments, the magnetization is transferred from the nitrogen of the amide bond to either the C2 of the glucose ring or to the carboxyl of the amide bond as schematized Fig. 35B. In these conditions, as electronic environment of each chitin polymorph is supposed to be slightly different, and as the chemical shift of an atom is dependent upon its electronic environment, we would expect to obtain a higher number of peaks for a polysaccharide displaying high polymorphism.

A B

Build-up curves of the chitin and α-1,3-glucan for the WT and the three mutants of C. neoformans.

To conclude, even though, unexpectedly, the mutations studied in this section did not affect the capsular structure, they considerably modified the cell wall composition and/or organization. In this context, other types of analysis would be required to precisely define the connections between the three pathways mutated here and the production of the β-1,3-glucan.

CHARACTERIZATION OF CLINICAL ISOLATES

In the last section of the C. neoformans analysis, we were interesting in characterizing the differences between different clinical isolates. Indeed, we aimed at applying our methodology to nearly in vivo samples. As our method required isotope labeling, and thus cannot be applied directly on primary clinical isolate, we used clinical isolates extracted from patients diagnosed with cryptococcosis and then grown in vitro in a [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C/ 15 N-labeled medium.

This kind of study would be significantly relevant for a better understanding of C. We focused our analysis on three clinical isolates (2.86, 1.07 and 1.83) of the strain C.

neoformans kindly provided by our collaborators Drs. Guilhem Janbon, Alexandre Alanio and Vishu Aimanianda (Pasteur Institute, Paris). First, we recorded CP-based experiments to investigate the rigid content of the yeast envelope (Fig. 70).

First, we investigated the composition of the rigid cell wall using [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C-detected methods. We showed the presence of highly rigid α-1,3-glucan, β-1,3-glucan, chitin and various associated proteins as observed by SSNMR. We revealed that glucans are three times more expressed than chitin, while β-1,3-glucan is mainly expressed in a rigid state and could form long fibrillar structures responsible for the rigidity of the cell wall. α-1,3-glucan and chitin are present in two different locations within the cell wall: (1) one form (rigid) might be associated with β-1,3-glucan to form a rigid network supporting of the cell integrity; (2) one form (mobile) at the interface with the capsule; anchoring the capsular components to the cell wall. We probed the hydration of polysaccharides and chitin and observed an homogenous behavior, contrary to the case of A. fumigatus. We worked on the methodological development of 1 H-detected SSNMR experiments to decipher both the composition and structure of the highly dynamic and porous meshed polysaccharide network of the capsule.

Using the additional 1 H dimension, we assigned various additional carbohydrates (e.g., xylose, mannose and galactose) composing the two main complex polysaccharides of the capsule: GXM and GXMGal.

Then, we analyzed various capsular mutants of C. neoformans. We chose to study a fully acapsular mutant (Δcap59) as well as single mutant xylose-deficient (Δuxs1), glucuronic acid-deficient (Δugd1) or the double mutant acapsular and galactose-deficient (Δcap59Δuge1). We used both [START_REF] Gaynes | The Discovery of Penicillin-New Insights After More Than 75 Years of Clinical Use[END_REF] C-detected and 1 H-detected NMR experiments to investigate the cell wall and the capsule of these mutants and decipher how these mutations impact their architecture.

First, we still observed SSNMR signals of capsular components in the Δcap59 and Δcap59Δuge1 mutants. These data were confirmed by immunolabeling assays. They strongly suggest that the acapsular phenotype observed upon the mutation of cap59 does not consist in a total removal of the capsule. Indeed, a part of the capsule polysaccharides seem to be still exported at the surface of the yeast to form a thin capsule layer. Moreover, the mutations of uxs1, ugd1 or uge1 do not impact the capsular components visible by SSNMR as it was expected for such mutations. Indeed, we still probe the same polysaccharides content in the mutant capsules as for the WT one. We proposed that the mutation of these three genes do not totally prevent the production of xylose, glucuronic acid or galactose. Some secondary synthesis pathway could be activated in response to the deletion of the three enzymes.

Unexpectedly, the mutation of uxs1, ugd1 or uge1 leads to a significant decrease or a total prevention of the production of the cell wall β-1,3-glucan in rigid or both rigid and flexible

In future perspective, this relatively fast characterization method could be applied to the routine elucidation of mutations impacts onto the organization of external structures or to study different clinical isolates in order to identify new structural features related to their virulence rates. In conclusion, we anticipate that the improvement of MAS technologies and the new multidimensional pulse sequences appearing at the horizon will pave the way for a much precise structure determination of such complex systems.

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