Gal Galactose

Fluorescein Isothiocyanate FKBPs FK506-binding proteins Fuc Fucose GAGs Glycosaminoglycans

AKGNOWLEGMENTS

This project was a great challenge full of obstacles that would not have been possible to overcome without the help and collaboration of a huge list of people, to whom I would like to dedicate a few words.

First of all, I would like to thank the members of my thesis committee: Dr. Thibaut Crépin, Dr. Ute Krengel, Dr. Angelina de Palma, and Prof. Geert-Jan Boons for taking the time to examine this thesis and join me in the final defense of this work.

I also would like to thank the members of my CSI committee: Dr. Robert de Vries, and Dr. Olivier Lerouxel for their advice and commentaries during our meetings. Without a doubt, your critical thinking and questions were key pieces in the search for answers beyond the obvious.

To our collaborators, Prof. Muriel Cornet, Dr. Julia Novion and Dr. Liu Yan, for their great disposition and their faith in this project. Without your contributions, none of this would have been possible.

To Dr. Serge Perez and Dr. Anne Imberty, thank you for sharing your great knowledge and for always bringing interesting discussions to our meetings.

A very special thank you also to Valérie Chazalet and Emilie Gillon, for sharing all their experience with me and for always being there when I had a question.

Thanks to both of you, because working in such an organized environment made my days at CERMAV much more relaxed and productive. But above all, thank you because your enthusiasm and great personality make the lab a wonderful place to work. It's great to always start the day with your smiles.

To my sisters Diana and Nadia, for their support and patience throughout the development of this project and for never losing faith in me. For being my source of inspiration and the engine that drives me to keep going every day. I want to thank my husband Rodrigo Escobar, for being my greatest support in the adverse moments and letting me share with him my joys and achievements in the good moments. For his immense patience and love, for being always present (even at distance), for being willing to cross the world just to make me smile. Rodrigo, it is difficult to resume all the reasons that I am grateful to you.

So, I will mention only the most important one: thank you for being in my life! To the Priem De la Rosa family, for having welcomed me with open arms since I arrived at France. Aida, Bernard, Emma and Rafa, thank you for making me feel part of the family, for all those afternoons full of games, laughter, and delicious food. But more than anything, thank you because I never felt alone in difficult times, you were always there to lend a hand when I needed it most.

To my colleagues and friends who were present in the most representative stages of this project, always ready to support me and encourage me to move forward.

Thank you, Simona, Dylan, Jalaa, Nathan, Isaty, Olga, Aurore, Oriane, Roosmarijn, Torben, Vito, Gael and Luca, for the shared experiences, your support, and your contributions, for making lunchtime a very nice experience to wait for during the morning. Thank you for all the laughs, and for all the nice moments we shared.

To the board members of the PhD4GlycoDrug consortium, for making this great project possible, and for being a constant source of inspiration to me. I also want to thank you for your technical support and for your interest in our professional and personal development. A very special thanks to Dr. Marko Anderluh who has accompanied me throughout this journey in a unique manner, and who is always there to support me in undertaking new challenges. Marko, I sincerely appreciate that you care about every ESR and you are always looking new opportunities for us. Now that I mentioned the PhD4GlycoDrug, I want to especially thank the other ESRs who have shared this great adventure with me and who, without a doubt, have become part of my family: Rafael, Kanhaya, Margherita, Nives, Mujtaba, Sjors, Benedetta, Cyril, Elena, Tiago, and Gabriele. Thank you very much because this adventure would not have been the same without your company and support. Our adventures will be forever in my memory, and you all have a very special place in my heart. I consider myself a very lucky person because, well, what are the possibilities to bring together such a particular group of people? jajaja. I cannot imagine what our advisers would have thought during the recruitment, but I am very happy for having met you. I know this is getting too long, but I really need to thank Elena Loi and Gabriele Conti for their technical advice in chemistry, for staying up late in the lab to discuss and help me interpret results. Also, thanks for your patience while teaching me the basics of chemistry. From all the people I have thanked, Margherita Duca has a very very very special place on the list. Somehow our projects are interconnected and together we live the joys and also the failures of the other. Maybe for this reason we created a very strong bond (not hydrogen, jaja terrible joke). Marghe was my teacher and the person who guided me through the part of the project that involved organic synthesis, in which I had no experience. When I arrived at the laboratory, I didn't know how to do a liquid extraction, I didn't know how to interpret an NMR, I didn't know how to clamp the material in the fumehood, I didn't know anything at all. Nevertheless, Marghe was always there to teach me and to avoid I died. But farther than an incredibly patient teacher, she is also an exceptional friend and ABSOLUTELY NOTHING would have been the same without her company in this journey. Thank you forever my Margarita, for supporting me in difficult times, for your unconditional love. I am only sure of one thing, wherever I will go from now on, you will always be an important part of my life.

To Dr. Rafael Bermeo Malo... well, maybe you will never realize the watershed that your presence in this world has meant for my life. But it doesn't matter, the only think you need to know that I will be eternally grateful to you because there is a Dania before and after Rafael. I really hope that your lessons will accompany me for the rest of my life, because they'll be essential to achieve happiness. Thank you for having crossed this path by my side. We started this together, and we finished it together. Until forever Mr. Malo! Finally, I would like to express my most sincere gratitude to my advisors Dr.

Annabelle Varrot and Prof. Roland Pieters for giving me the opportunity to be a part of their teams, and for all their guidance during these years. Words are not enough for me to express the joy of have been your student. I hope one day to be lucky enough to work together again.

Honorable Professor Pieters, XD! Thank you very much for welcoming me to Utrecht. Without a doubt, the time I spent in your laboratory was a great opportunity to learn and develop new skills both in the work and personal aspects. I appreciate the opportunity to work together and your willingness to contribute to the maturation of my scientific skills, but above all, I want to thank you for trusting me.

Although transitory, my stay in Utrecht was magnificent and I definitively learned a lot more than I would have ever thought possible in a year. I also enjoyed our personal talks and your always active sense of humor. So, I am very grateful to you for giving to me this opportunity. If there is something that I will always remember about my stay with you, it is that "Sometimes the answer to the most complicated question is found in the simplest details".

Annabelle, I didn't tell you this before, but when I arrived to your lab I was terrified. I had the impression that the project was too ambitious and that it would hardly be possible for me to acquire the necessary skills to be up to the task. Then, the first challenges arrived almost at the beginning of the project, with SapL1. I spent entire nights awake wondering if I was the right person to overcome the situation? If you made the right decision by giving me this opportunity? But you seemed pretty confident about it. You may not have realized it at the time, but your support made me feel deeply grateful and I was determined not to disappoint you. So, I kept trying, harder and harder until we were finally able to overcome that obstacle. Later, as other challenges came (because things did not go very often as were expected), you always stayed by my side. Your technical advice and support helped me to get through all the difficulties of this project, and, one by one, we overcome all of them. It's funny because during the "good times", when I thought I had done a magnificent job, you always had something new to teach me. I must admit that on many occasions I felt frustrated because I was totally sure I had done my best.

But precisely there, when I thought it was impossible to improve, you always pushed me beyond what I thought was my limit. I personally believe that you have a brilliant mind an enormous talent to enhance the skills of your students.

But, beyond the scientific field, I admire you because you are a great woman, full of strength but with a great heart.

There are so many other things that I would like to tell you, but words hardly could express how grateful I am, and will always be, to you. So, I'll just say: Thank you Annabelle, for sharing your research experience with me and for being there when I needed you. For never leaving me alone, for pushing me through both, difficult and good times. But above all, I want to thank you for trusting me and my ideas.

Once upon a time there was a bunny, sitting in the sun and munching on some brownies.

A fox comes along and asks the bunny what she's up to. The bunny says: "I'm writing my dissertation on how bunnies eat foxes". "What?! Are you serious?" says the fox. "Everyone knows foxes eat bunnies!". "Come and see", says the bunny, and the fox follows her into her rabbit hole. A few minutes later, out comes the bunny, with a few small stains of blood on her fur, which she promptly cleans up and sits down to munch on brownies.

A wolf comes along, and asks the bunny what she's up to. The bunny says: "I'm writing my dissertation on how bunnies eat wolves". "What?! Are you serious?" says the wolf. "Everyone knows wolves eat bunnies!". "Come and see", says the bunny, and the wolf follows her into her rabbit hole. A few minutes later, out comes the bunny, using a toothpick to clean between her teeth. She sits down to munch on brownies.

A tiger comes along, and asks the bunny what she's up to. The bunny says:

"I'm writing my dissertation on how bunnies eat tigers". "What?! Are you serious?" says the tiger. "Everyone knows tigers eat bunnies!". "Come and see", says the bunny, and the tiger follows her into her rabbit hole.

The tiger follows the bunny into the burrow, where a lion sits up as soon as they arrive, kills the tiger with one stroke, and waits patiently for the bunny to collect herself and go back outside.

The moral of the story is that it does not matter what your thesis topic is; it only matters who your advisor is. [START_REF] Garnica | Infecciones fúngicas emergentes[END_REF]

LIST OF FIGURES

LIST OF TABLES

GalNAc

N-acetylgalactosamine

CGFs

Glycosides coated filters

GlcNAc

N-acetylglucosamine

Glu

Glucose

GMO

Genetically modified organisms

GNA

Galanthus nivalis lectin

GPS-SAM

GPS-Self Assembled Monolayer

HMOs

PBS

Phosphate buffer saline

PCR

Polymerase chain reaction

PPIases

Peptidyl-prolyl isomerases

PRMs Peptidorhamnomannans

PVL

Psathyrella velutina lectin

PT Palladium

Rh

Rhamnose

RFU

Relative fluorescent units

RT

Room temperature

SAM

Self assembled monolayer

SapL1

Scedosporium apiospermum lectin 1

SapL6

Scedosporium apiospermum lectin 6

SapCP

Scedosporium apiospermum cerato platanin

SapFKBP12

Scedosporium apiospermum lectin FK506 binding lectin

SEC

Size exclusion chromatography

SDS

SUMMARY

Scedosporium apiospermum is an emerging opportunistic fungal pathogen responsible for life-threatening infections in immunocompromised patients. This fungus exhibits limited susceptibility to all current antifungals and displays high mortality rates. S. apiospermum represents a therapeutic challenge since; on one hand, the options for its treatment are limited when the infection has progressed and, on the other hand, its prevention by antifungal prophylaxis carries significant risks for health. In this context, therapies aimed at precluding its binding to human tissues seem to represent a better alternative for high-risk populations. Unfortunately, due to its emerging character, S. apiospermum pathogenicity and virulence factors remain largely unknown.

Some lectins (carbohydrate-binding proteins) are important mediators of hostpathogen interactions during the early stages of infections. Therefore, they are considered valuable therapeutic targets and several approaches have suggested their inhibition as a strategy to prevent infections. The most popular one, proposes the use of sugar-based inhibitors and glycomimetics specifically designed to block the binding site of those proteins as a strategy to avoid microbial adhesion to the host cells. Nevertheless, in the case of S. apiospermum, the development of these therapies is hindered because its lectins remain unidentified.

The main aim of this work was the identification and characterization of lectins from this emerging opportunistic fungal pathogen. This has been addressed by two different approaches; the first strategy comprises the in-silico prediction of putative lectins codified on its genome by data mining, while the second strategy lies in the identification of native lectins directly from fungal protein extracts.

Through the in-silico approach, we have identified two novel lectins from S.

apiospermum genome, SapL1 and SapL6. Both proteins were produced in Escherichia coli and their full biochemical/structural characterization was achieved. For the second strategy, we have analysed the intracellular proteins and secretomes of S. apiospermum in conjunction with another opportunistic fungal pathogen of medical relevance, Aspergillus fumigatus. The analysis of peptides obtained by mass spectrometry led to the identification of several proteins, from which 4 corresponded to new hypothetical proteins with carbohydrate-binding activity from S. apiospermum (PVL-like lectin, SapCP, CcMBL-like lectin and SapFKBP12). The preliminary analysis of those proteins revealed that some might be attractive pharmacological targets, and others could find application as biotechnological tools.

The second objective of this thesis was the development of a new tool that allows the identification and purification of unknown lectins directly from crude protein extracts. This tool assembles the fusion of the three classic steps of lectin purification train: clarification, concentration, and separation, in a single operating unit. It also allows the simultaneous purification of lectins with different specificities in a quick and simple process. In addition, it can be used as a "mini glycan array" for the study of carbohydrate lectin interactions, without the need for fluorescent labeling. Hence, we consider that this device might greatly contribute to the study of the lectinome of species.

Overall, this work represents a general strategy to address the study S.

apiospermum lectinome. Our findings contribute to the understanding of glycosylated surface recognition by this emerging opportunistic pathogen and, we expect, will contribute to design targeted therapies against it. Finally, we hope that the information provided here encourages the study and characterization of the new proteins discovered, in the search for biomedical applications and new drug targets.

RÉSUMÉ

GENERAL INTRODUCTION

Fungal infections

Fungal infections are an important worldwide cause of human morbidity and mortality that is often overlooked due to their underreporting. This is largely due to limited access to accurate and inexpensive tests for the detection of fungal species during co-infections by other pathogens, such as bacteria [1]. Therefore, it is considered that the actual number of deaths attributable to those microorganisms could be greatly underestimated. Despite this, their epidemiological overview points to a dramatic increase in the occurrence of lifethreatening infections since 1980, primarily in immunosuppressed patients [START_REF] Ellis | Epidemiology: Surveillance of fungal infections[END_REF].

Although it is true that these incidences can be correlated to the growing size of risk populations, strongly reinforced by the use of immunosuppressive agents and corticosteroids, these are not the major triggers. This phenomenon has been mainly attributed to the appearance of new species of yeasts and molds, previously harmless to humans, which have developed the capacity to generate invasive infections [1][START_REF] Ellis | Epidemiology: Surveillance of fungal infections[END_REF][START_REF] Garnica | Infecciones fúngicas emergentes[END_REF]. These species collectively cause enormous damage to human health and affect billions of people each year [1].

Scedosporium apiospermum an emerging fungal pathogen

Emerging opportunistic fungal pathogens

During the last decades, an increased incidence of invasive infections, especially in immunosuppressed patients, has been caused by previously rare fungal species [START_REF] Tuite | Overview of invasive fungal infections[END_REF]. These organisms are called "emerging opportunistic fungal pathogens" and represent a major health problem due to the scarce information about their virulence factors, host-binding mechanisms and treatment. Among those emerging organisms, species from the Scedosporium complex are particularly dangerous since their mortality rate may be over 80% [START_REF] Ramirez-Garcia | Scedosporium and Lomentospora: An updated overview of underrated opportunists[END_REF].

Scedosporium complex

The genus Scedosporium comprises more than ten species (Scedosporium angustum, S. apiospermum, S. aurantiacum, S. boydii, S. cereisporum, S. dehoogii, S. desertorum, S. ellipsoideum, S. fusoideum, S. minutisporum, S. rarisporum and S. sanyaense) whose taxonomy has been previously described based on molecular phylogenetic by using the sequences of four genetic loci [START_REF] Ramirez-Garcia | Scedosporium and Lomentospora: An updated overview of underrated opportunists[END_REF][START_REF] Gilgado | Molecular phylogeny of the Pseudallescheria boydii species complex: proposal of two new species[END_REF] (Figure 1.1A). However, only the genome of S. boydii, S. aurantiacum, S. dehoogii and S. apiospermum have been completely sequenced to date [START_REF] Duvaux | Draft genome sequence of the human-pathogenic fungus Scedosporium boydii[END_REF][START_REF] Pérez-Bercoff | Draft genome of Australian environmental strain WM 09.24 of the opportunistic human pathogen Scedosporium aurantiacum[END_REF][START_REF] Vandeputte | Draft genome sequence of the pathogenic fungus Scedosporium apiospermum[END_REF].

Scedosporium infections mainly affect immunocompromised patients such as solid organ transplant (SOT) or hematopoietic stem cell transplant (HSCT) recipients or patients with chronic granulomatous disease (CGD). However, these pathogens also represent a high risk for patients with other immune deficiencies such as cancer and cystic fibrosis (CF). Finally, although with low frequency, Scedosporiosis infections have also been reported in healthy persons [START_REF] Luplertlop | Pseudallescheria/Scedosporium complex species: From saprobic to pathogenic fungus[END_REF][START_REF] Cortez | Infections Caused by Scedosporium spp[END_REF].

Within the genus, the main responsible for human's infections is S. apiospermum, formerly considered as the asexual form of Pseudallescheria boydii (Figure 1.1B-E).

This cosmopolitan microfungus is responsible for localized to severe or fatal disseminated infections and is one of the most common Scedosporium species (with S. boydii and S. aurantiacum) capable to chronically colonize the lungs of CF patients [START_REF] Pereira De Mello | Conidial germination in Scedosporium apiospermum, S . aurantiacum, S . minutisporum and Lomentospora prolificans: influence of growth conditions and antifungal susceptibility profiles[END_REF][START_REF] Goldman | Scedosporium apiospermum infections and the role of combination antifungal therapy and GM-CSF: a case report and review of the literature[END_REF].

Scedosporium spp pathogeny

Infections caused by any member of the genus Scedosporium are commonly known as scedosporiosis and originate predominantly in the lungs and soft tissues, with occasional spread to bones. The range of diseases caused by these infections is wide, ranging from localized cutaneous infections to full dissemination in both, immunocompromised and immunocompetent hosts [START_REF] Luplertlop | Pseudallescheria/Scedosporium complex species: From saprobic to pathogenic fungus[END_REF][START_REF] Cortez | Infections Caused by Scedosporium spp[END_REF][START_REF] Goldman | Scedosporium apiospermum infections and the role of combination antifungal therapy and GM-CSF: a case report and review of the literature[END_REF]. In humans scedosporiosis can be divided into 4 main categories according to the clinical conditions associated with the pathologies: (i) mycetoma, (ii) colonization of the respiratory tract, (iii) nonmycetoma deep infections and (v) disseminated infections (Figure 1.2A).

Mycetomas are subcutaneous infections characterized by the formation of compact aggregates of mycelium. These structures are known as granules or sclerotia and their main function is to allow these opportunistic pathogens to evade the defenses of the host's immune system [START_REF] Santiago Reis | Mycetomas: an epidemiological, etiological, clinical, laboratory and therapeutic review[END_REF]. The color and morphology of the sclera can vary according to the species, but they are usually granules of 2 to 20 µm in diameter. These type of infections are relatively uncommon in immunocompromised hosts [START_REF] Branscomb | Mycetoma: An Overview[END_REF].

Colonization of the respiratory tract is very common in the presence of pathologies that affect bronchial secretions, such as tuberculosis, chronic obstructive pulmonary disease (COPD), CF, Job's syndrome, etc. This is because the abnormally viscous mucus favors the anchorage of pathogens and interfere with the clearance mechanisms of the lungs. Scedosporium colonization can be transient or permanent. Although, some patients with chronical colonization may have minimal or no symptoms, others develop pulmonary infiltrates or allergic reactions [START_REF] Cortez | Infections Caused by Scedosporium spp[END_REF][START_REF] Borghi | Chronic airway colonization by Scedosporium apiospermum with a fatal outcome in a patient with cystic fibrosis[END_REF].

Nonmycetoma deep infections comprises all those local infections that do not

present clinical manifestations characteristic of mycetomas (such as sclerotia formation). These diseases, in turn, can be subclassified into sinopulmonary and extrapulmonary infections [START_REF] Cortez | Infections Caused by Scedosporium spp[END_REF]. [START_REF] Badiee | Opportunistic invasive fungal infections: Diagnosis & clinical management[END_REF]. The respiratory tract and skin lesions are the most common routes of entry for pathogens that can lead towards disseminated infections.

Those affectations are usually seen among immunocompromised patients; however, they have also been reported in immunocompetent patients [START_REF] Ellis | Disseminated infections: A clinical overview[END_REF]. In both cases, dissemination carries a bad prognosis. et al., 2008 [11].

Distribution and epidemiological overview

Scedosporium apiospermum is a globally distributed soil fungus that is capable of assimilating numerous aromatic or polycyclic hydrocarbons. This is possibly why it is found mainly in areas affected by men, such as industrial areas, urban parks, roadsides and agricultural lands [START_REF] Luplertlop | Pseudallescheria/Scedosporium complex species: From saprobic to pathogenic fungus[END_REF]. Its role in the environment is still limited and there is no real knowledge of its geographical distribution, however, the main affected countries have been identified based on the incidence of infections caused by this pathogen (Figure 1.3) [START_REF] Bouchara | Scedosporium apiospermum[END_REF]. Sinopulmonary infections mainly affect immunosuppressed patients and often result in systemic dissemination. Together with Fusarium species and some dematiaceous fungi, the Scedosporium complex is responsible for approximately 10% of the mycosis caused by filamentous fungi in hematopoietic cell transplant recipients and up to 19% in solid organ transplant patients [START_REF] Goldman | Scedosporium apiospermum infections and the role of combination antifungal therapy and GM-CSF: a case report and review of the literature[END_REF]. In addition, after

Aspergillus fumigatus, Scedosporium spp are the most common among the filamentous fungi that colonize the respiratory tract of people with cystic fibrosis (CF). Furthermore, because of their propensity to disseminate in case of immune deficiency, the airway colonization by those fungi is considered as a contraindication for lung transplantation, which, in many CF patients, remains as the ultimate line of treatment [START_REF] Rammaert | Perspectives on Scedosporium species and Lomentospora prolificans in lung transplantation: results of an international practice survey from ESCMID fungal infection study group and study group for infections in compromised hosts, and European confederation of medical mycology[END_REF]. Hence, CF patients are considered as one of the main risk populations for scedosporiosis and there is growing concern about them [START_REF] Ramirez-Garcia | Scedosporium and Lomentospora: An updated overview of underrated opportunists[END_REF].

Cystic Fibrosis; a high-risk population for scedosporiosis

Cystic Fibrosis (CF) is a serious condition caused by an autosomal recessive disorder of the CF Transmembrane Regulator (CFTR). CFTR is an integral membrane glycoprotein that acts as a chloride channel in epithelial cells, and whose dysfunction leads to the production of abnormally viscous bronchial secretions [START_REF] Borghi | Chronic airway colonization by Scedosporium apiospermum with a fatal outcome in a patient with cystic fibrosis[END_REF][START_REF] Turcios | Cystic fibrosis lung disease: An overview[END_REF]. This pathology not only hinders the natural process of mucociliary clearance but also affects the glycosylation pattern of pulmonary mucins [START_REF] Scanlin | Terminal glycosylation in cystic fibrosis[END_REF][START_REF] Lamblin | Human airway mucin glycosylation: a combinatory of carbohydrate determinants which vary in cystic fibrosis[END_REF]. In conjunction, those effects favor the binding of pathogens and trigger a colonization process that usually starts in the adolescence and becomes chronic in up to 19% of the patients. This colonization may also lead to a chronic inflammation and sometimes to an allergic bronchopulmonary mycosis [START_REF] Ramirez-Garcia | Scedosporium and Lomentospora: An updated overview of underrated opportunists[END_REF].

Clinical treatment

The treatment of Scedosporium infections is challenging because its efficacy depends on timely diagnosis, which is based on morphological detection by microscopy, histological analysis and culture on selective media. This process is time consuming and may lead to false negative results, especially from respiratory secretions of patients with CF because of the common co-colonization by other fungi or bacteria [START_REF] Ramirez-Garcia | Scedosporium and Lomentospora: An updated overview of underrated opportunists[END_REF]. Furthermore, Scedosporium spp display a primary resistance to classical antifungals such as 5-flucytosine, amphotericin B and the first-generation triazole drugs, fluconazole or itraconazole and exhibit a limited susceptibility to the newest generations of antifungal drugs, e.g., echinocandins, voriconazole and isavuconazole [START_REF] Ramirez-Garcia | Scedosporium and Lomentospora: An updated overview of underrated opportunists[END_REF][START_REF] Tortorano | ESCMID and ECMM joint guidelines on diagnosis and management of hyalohyphomycosis: Fusarium spp., Scedosporium spp. and others[END_REF]. Nowadays, the first-line treatment for Scedosporium infections involves a combination therapy that includes the use of voriconazole in conjunction with other antifungals [START_REF] Tortorano | ESCMID and ECMM joint guidelines on diagnosis and management of hyalohyphomycosis: Fusarium spp., Scedosporium spp. and others[END_REF][START_REF] Troke | Treatment of scedosporiosis with voriconazole: clinical experience with 107 patients[END_REF]. However, due to common recurrences, even without interruption of treatment, the recovery rates are poor and mortality remains over 65% while it is almost 100% when dissemination occurs, a reason why it has aroused special attention despite its emerging character [START_REF] Troke | Treatment of scedosporiosis with voriconazole: clinical experience with 107 patients[END_REF][START_REF] Rodriguez-Tudela | Scedosporium apiospermum: changing clinical spectrum of a therapy-refractory opportunist[END_REF].

Due to its high incidence and recurrence, the constitutive administration (prophylaxis) of antifungals in immunocompromised people has been proposed.

However, some of the side effects of these drugs are dangerous during long-term administration. In this context, therapies aimed to prevent the development scedosporiosis represent a better alternative for these patients.

Host-binding; the first step towards infection

In its adult stage, the S. apiospermum mycelium is composed of a network of elongated and cylindrical cells that are surrounded by a cell wall. These fungal filaments are called hyphae and originate from the germination of oval spores (2 to 5 μm) known as conidia when they originate from asexual reproduction [START_REF] Pereira De Mello | Conidial germination in Scedosporium apiospermum, S . aurantiacum, S . minutisporum and Lomentospora prolificans: influence of growth conditions and antifungal susceptibility profiles[END_REF].

Scedosporium infections begin with conidia adherence to tissues, followed by germination and hyphal elongation [START_REF] Cortez | Infections Caused by Scedosporium spp[END_REF]. This adherence process allows it to avoid cleansing mechanisms aimed to eradicate the invading pathogens and represents the initial step towards infection [START_REF] Cortez | Infections Caused by Scedosporium spp[END_REF][START_REF] Theuretzbacher | Non-traditional antibacterial therapeutic options and challenges[END_REF][START_REF] Krachler | Targeting the bacteria-host interface strategies in anti-adhesion therapy[END_REF][START_REF] Ofek | Anti-adhesion therapy of bacterial diseases: prospects and problems[END_REF].

Conidial adhesion is mediated by cell surface molecules (CSM), including different types of carbohydrates such as polysaccharides and glycoconjugates.

The presence and/or abundance of these molecules on the cell surface varies according to the stage of development and is of great relevance to understand fungal pathobiology [START_REF] Pinto | Involvement of peptidorhamnomannan in the interaction of Pseudallescheria boydii and HEp2 cells[END_REF]. Some of the most important carbohydrate CSM described to date for Scedosporium species include peptidorhamnomannans (PRMs) [START_REF] Pinto | Involvement of peptidorhamnomannan in the interaction of Pseudallescheria boydii and HEp2 cells[END_REF] α-glucan [START_REF] Bittencourt | An a-glucan of Pseudallescheria boydii is involved in fungal phagocytosis and Toll-like receptor activation[END_REF], melanin [START_REF] Ghamrawi | Cell wall modifications during conidial maturation of the human pathogenic fungus Pseudallescheria boydii[END_REF], ceramide monohexosides [START_REF] Pinto | Involvement of peptidorhamnomannan in the interaction of Pseudallescheria boydii and HEp2 cells[END_REF][START_REF] Rollin-Pinheiro | Characterization of Scedosporium apiospermum glucosylceramides and their involvement in fungal development and macrophage functions[END_REF], Nacetyl-D-glucosamine-containing molecules [START_REF] Mello | Scedosporium apiospermum, Scedosporium aurantiacum, Scedosporium minutisporum and Lomentospora prolificans: a comparative study of surface molecules produced by conidial and germinated conidial cells[END_REF] and mannose/glucose-rich glycoconjugates [START_REF] Pinto | Involvement of peptidorhamnomannan in the interaction of Pseudallescheria boydii and HEp2 cells[END_REF]. Some carbohydrate binding proteins (lectins) display an essential role during fungal pathogenesis and can be considered CSMs.

Lectins

Definition and generalities

Lectins are ubiquitous proteins that include at least one domain with the ability of binding, in a reversible and specific way, to glycans without modifying them [START_REF] Sharon | Lectins as cell recognition molecules[END_REF][START_REF] Kocourek | A note on the regent discussion on definition of the term "LECTIN[END_REF]. The spectrum of carbohydrates that can be recognized by lectins is considerably broad and ranges from simple monosaccharides to complex structures containing polysaccharides and glycoconjugates, such as glycoproteins, glycolipids, proteoglycans or glycosaminoglycans (GAGs) (Figure 1.5) [START_REF] Sharon | How proteins bind carbohydrates: Lessons from legume lectins[END_REF]. Carbohydrates represent the third alphabet of life and cover the surface of every cell. The information encoded in carbohydrate called glycocode can be deciphered by the lectins.

Since lectins are generally capable of agglutinating erythrocytes, they are often referred to as hemagglutinins. However, it is important to clarify that this is an oversimplification, since not all lectins are hemagglutinins nor all hemagglutinins are lectins. This is, some lectins do not display hemagglutination activity because their ligands are not present on the cell surface of red blood cells and not all hemagglutinins agglutinate erythrocytes through the reversible binding of sugars found on their cell surface.

Carbohydrate-binding properties of lectins

Most of the lectins described to date have complex structures and some of them may even include prosthetic groups, e.g., ions and glycosylations. In some cases, these prosthetic groups are essential to exert their biological activity [START_REF] Weis | Structural basis of lectin-carbohydrate recognition[END_REF],

however, it is important to highlight that the carbohydrate-binding properties of lectins are essentially attributed to the Carbohydrate Recognition Domain (CRD),

where the binding pocket (BP) is embedded [START_REF] Dodd | Lectin-like proteins in model organisms: implications for evolution of carbohydrate-binding activity[END_REF]. The selectivity of this interactions is given by i) a hydrogen bond network established with the hydroxyl groups of the sugar and ii) π-CH stacking interactions and iii) a Van der Waals packing, which often includes the coverage of the hydrophobic portion of the sugar by apolar residues of the protein (Figure 1.6) [START_REF] Weis | Structural basis of lectin-carbohydrate recognition[END_REF][START_REF] Dodd | Lectin-like proteins in model organisms: implications for evolution of carbohydrate-binding activity[END_REF]. Lectins tend to present a shallow binding site resulting, usually, in low affinity in the millimolar order in particular for monosaccharides [START_REF] Weis | Structural basis of lectin-carbohydrate recognition[END_REF]. This low affinity can be compensated by multivalency, meaning the lectins present multiple binding sites. Upon multivalent interactions, there is indeed an extensive enhancement of binding, i.e., avidity, that is a well-known phenomenon for

Specificity of lectins

The set of glycans that lectins can recognize is very wide. Some of them have affinity for single monosaccharides such as the case of GNA from Galanthus nivalis, which recognize mannose [START_REF] Raemaekers | Functional phytohemagglutinin (PHA) and Galanthus nivalis agglutinin (GNA) expressed in Pichia pastoris. Correct N-terminal processing and secretion of heterologous proteins expressed using the PHA-E signal peptide[END_REF], while in other cases, a specific arrangement of carbohydrate linkages is necessary to achieve lectin binding.

PHA:L from the common bean (Phaseolus vulgaris) is a clear example of this condition since its minimal structural unit for high-affinity binding is the pentasaccharide Gal(β1-4)GlcNAc(β1-2)[Gal(β1-4)GlcNAc(β1-6)]Man [START_REF] Nagae | Phytohemagglutinin from Phaseolus vulgaris ( PHA-E ) displays a novel glycan recognition mode using a common legume lectin fold[END_REF]. There are also some lectins with affinity for more than one monosaccharide, such as the members of the "Glucose/mannose" or "Galactose/N-acetyl-D-galactosamine"

subfamilies [START_REF] Martínez-Alarcón | Expression of lectins in heterologous systems[END_REF]. In those examples, lectins specificity can be considered degenerated since the interaction with different sugars is established through the same binding site. On the other hand, the recently discovered subfamily of "superlectins" also displays affinity for more than one type of sugar, however, their dual recognition is due to the presence of multiple CRDs with specificity for different sugars [START_REF] Bermeo | BC2L-C N-Terminal lectin domain complexed with histo blood group oligosaccharides provides new structural information[END_REF]. BC2L-C from Burkholderia cenocepacia was the first superlectin to be characterized [START_REF] Šulák | Burkholderia cenocepacia BC2L-C is a super lectin with dual specificity and proinflammatory activity[END_REF]. et al., 2009 [62].

Classification of lectins

Lectins are distributed among all kingdoms including viruses, protozoa, bacteria, animals, plants and fungi [START_REF] Branco | Expression and purification of the recombinant SALT lectin from rice (Oryza sativa L.)[END_REF][START_REF] Lannoo | Expression of the nucleocytoplasmic tobacco lectin in the yeast Pichia pastoris[END_REF]. They are a very heterogeneous group of proteins, that vary greatly from each other, both in structure and in function [START_REF] Dodd | Lectin-like proteins in model organisms: implications for evolution of carbohydrate-binding activity[END_REF].

Therefore, their classification is complex to address and there is not a global consensus in the scientific community. Originally, lectins classification was based on taxonomic origin (e.g., plants lectins, animal lectins, viral lectins, bacterial lectins and fungal lectins). However, incompatible functions of this classification have aroused the need for more functional classifications. Over the last years, different proposals for classification emerged based on common characteristics such as cell location, carbohydrate recognition specificity, functions, structure, among others [START_REF] Lakhtin | Lectins of living organisms. The overview[END_REF]. For some of those, there are very well defined parameters that allow consider lectins from all living organisms, however, some others, are usually established for a defined set of organisms [START_REF] Peumans | Classification of plant lectins in families of structurally and evolutionary related proteins[END_REF].

With the dramatic increase in the number of lectin structures released over the last decade, the recently created UniLectin3D platform has proposed a classification based on their fold. This is particularly interesting since there is a wide variety of fold and arrangements very well defined among organisms that, in addition to a classification criterion, allow the prediction of specificity and biological functions based on homology. UniLectin3D comprises a curated database of 3D lectin structures that provides a regularly updated classification and predicts new hypothetical lectins from genome sequence databases [START_REF] Bonnardel | Unilectin3D, a database of carbohydrate binding proteins with curated information on 3D structures and interacting ligands[END_REF].

Fungal lectins: roles and biotechnological applications

Fungi are an invaluable source for novel lectins with unique specificities that, are essential for their survival and development. Although, they are poorly studied, in comparison those from plants and animals, a broad spectrum of biotechnological and biomedical applications is emerging for those proteins.

Fungal lectins participate in diverse biological roles (Figure 1.9). For example some are involved on defense mechanisms due their toxicity against insects, virus and worms [START_REF] Varrot | Fungal lectins : structure , function and potential applications[END_REF], while others display functions related to metabolism or are key elements during the stages of dormancy, growth and morphogenesis [START_REF] Cioci | β-Propeller crystal structure of Psathyrella velutina lectin: an integrin like fungal protein interacting with monosaccharides and calcium[END_REF].

They might also serve as storage proteins or participate in the process of anchoring, necessary for both, ectomycorrhizal symbiosis and infection by pathogenic fungi [START_REF] Cioci | β-Propeller crystal structure of Psathyrella velutina lectin: an integrin like fungal protein interacting with monosaccharides and calcium[END_REF][START_REF] Houser | A soluble fucosespecific lectin from Aspergillus fumigatus conidia -structure, specificity and possible role in fungal pathogenicity[END_REF]. They play an important role in fungal innate immunity, the sole line of defense to recognize foreign epitopes from invaders.

On the other hand, fungal lectins are also valuable tools in glycobiology, since they can be coupled to resins for the isolation of industrially-relevant glycans [START_REF] Monzo | Lectin-immobilization strategies for affinity purification and separation of glycoconjugates[END_REF][START_REF] Khan | Fungal Lectins: Current molecular and biochemical perspectives[END_REF]. Due to their high specificity, some of those proteins are being used as biomarkers to detect/monitor the changes that occur in the glycoconjugates of the surface of cell membranes during physiological and pathological processes [START_REF] Varrot | Fungal lectins : structure , function and potential applications[END_REF] and, some others, have shown immunological and antitumor activity [START_REF] Kobayashi | Fungal lectins: a growing family[END_REF][START_REF] Singh | Mushroom lectins as promising anticancer substances[END_REF].

Furthermore, since some fungal lectins are important mediators of host-pathogen interactions, they represent a valuable tool for biomedical research on antifungals development. [START_REF] Krachler | Targeting the bacteria-host interface strategies in anti-adhesion therapy[END_REF][START_REF] Koharudin | The evolutionarily conserved family of cyanovirin-N homologs : structures and carbohydrate specificity[END_REF][START_REF] Mohamed | Mushroom lectins : Specificity , structure and bioactivity relevant to human disease[END_REF][START_REF] Shoaf-Sweeney | Adherence, anti-adherence, and oligosaccharides: preventing pathogens from sticking to the host[END_REF][START_REF] Varki | Microbial lectins: hemagglutinins, adhesins, and toxins[END_REF][START_REF] Lehot | Multivalent fucosides with nanomolar affinity for the Aspergillus fumigatus lectin FleA prevent spore adhesion to pneumocytes[END_REF][START_REF] Goyard | Multivalent glycomimetics with affinity and selectivity toward fucose-binding receptors from emerging pathogens[END_REF][START_REF] Akkouh | Lectins with anti-HIV activity: A review[END_REF][START_REF] Fang | A lectin with anti-HIV-1 reverse transcriptase, antitumor, and nitric oxide inducing activities from seeds of Phaseolus vulgaris cv. Extralong Autumn Purple Bean[END_REF].

Figure 1.9: Roles and potential applications of fungal lectins. Reprinted with permission from Varrot, et al., 2013 [52].

The anti-adhesive approach

Host-binding; a prerequisite for infection

Host recognition and anchoring are the first steps in infection processes. They are carried out through the recognition of determinants exposed on the surface of human tissues by microbial surface molecules and is critical for colonization [START_REF] Sharon | Safe as mother's milk: carbohydrates as future antiadhesion drugs for bacterial diseases[END_REF].

The main function of adhesion is to help pathogens to avoid the human clearance mechanisms that aim at their eradication, such as their elimination through urine, sweat and mucous membranes. In addition, it also provides them with better access to sources of nutrition and creates the right conditions for their growth [START_REF] Lannoo | Expression of the nucleocytoplasmic tobacco lectin in the yeast Pichia pastoris[END_REF]. In fungi, this process is fundamental for differentiation of the conidia towards the hypha and, in some cases, it is also a universal prerequisite for starting the transcription of their virulence factors of second line, including delivery of toxins and biofilm formation [START_REF] Krachler | Targeting the bacteria-host interface strategies in anti-adhesion therapy[END_REF][START_REF] Sattin | Glycoconjugates and glycomimetics as microbial anti-adhesives[END_REF].

Because host-binding is so crucial for infection, pathogens have devised a vast repertoire of attachment mechanisms. In general, the host-anchoring process can be visualized in three very well-defined stages adsorption, initial adhesion and high-affinity interactions. During the first stage, the microorganisms bind to the host cell surface through weak nonspecific interactions. These interactions are transient and are determined by general physicochemical properties of cell surfaces, such as charge and hydrophobicity. Then, the process is reinforced by the initial adhesion and high affinity interactions, which are mediated by specific interactions involving components exposed on the cellular surfaces of both, host and pathogen cells [START_REF] Krachler | Targeting the bacteria-host interface strategies in anti-adhesion therapy[END_REF].

Since carbohydrates are abundantly distributed in human cell membranes, microbial lectins are often involved in the initial mechanism of host-adhesion. In fact, their role is so fundamental that it has been observed that mutants of pathogenic bacterial strains with lectin deficiency are often unable to initiate their infectious process [START_REF] Sharon | Carbohydrates as future anti-adhesion drugs for infectious diseases[END_REF]. Therefore, lectins can be considered as virulence factors indispensable for host-pathogen interactions during the early stages of infection and represent promising therapeutical targets for the development of antiadhesives molecules.

Anti-adhesive therapy

The alarming increase in the incidence of multidrug-resistant pathogens is an urgent public health problem. Infections are becoming one of the leading causes of death worldwide and it is increasingly difficult to develop new generations of drugs at the speed that the situation demands [START_REF] Asadi | A review on anti-adhesion therapies of bacterial diseases[END_REF]. Thus, there is an urgent need for therapeutic strategies capable of resisting the ever-increasing repertoire of microbial resistance mechanisms [START_REF] Krachler | Targeting the bacteria-host interface strategies in anti-adhesion therapy[END_REF].

Since attachment is strict condition for virulence, it is logical to assume that the blocking of host-pathogen interactions will prevent the development of infections. Hence, a new approach that proposes the use of antagonists of such interactions has emerged as a valuable alternative for infectious diseases. This has been called the anti-adhesive therapy.

Anti-adhesive therapy represents an efficient substitute and/or complement to conventional antimicrobials because it explores different pharmacological routes and the development of resistance is rare and slow [START_REF] Sharon | Carbohydrates as future anti-adhesion drugs for infectious diseases[END_REF]. This is probably one of its biggest advantages and is due to the fact that its mechanism of action does not intended to kill the pathogen or stop its cell cycle. Consequently, there is no selective pressure for resistant pathogens to survive and multiply, inheriting their resistance to the progeny [START_REF] Krachler | Targeting the bacteria-host interface strategies in anti-adhesion therapy[END_REF]. Furthermore, this approach is particularly promising as a treatment for fungal infections because the administration can be local and directed, therefore the expected side effects on non-target tissues are less than those caused by conventional antifungals. This may allow its implementation as prophylactic therapy for immunosuppressed patients [START_REF] Ofek | Anti-adhesion therapy of bacterial diseases: prospects and problems[END_REF][START_REF] Sharon | Safe as mother's milk: carbohydrates as future antiadhesion drugs for bacterial diseases[END_REF].

Carbohydrates and glycomimetics as microbial anti-adhesives

Since it is well known that some microbial lectins participate in the initial adhesion to human epithelial surfaces, it has been proposed that carbohydrates could be used to inhibit pathogen anchoring infection during the early stages of infection. The first indications of the protective effect of sugars against pathogens, dates back to the late 19 th century, when it was observed the mortality rate of breast-fed infants was 7 times lower than infants fed with cow's milk [START_REF] Kunz | Historical aspects of human milk oligosaccharides[END_REF].

Later, it was discovered that this survival phenomenon was due to the fact that the oligosaccharides present in human breast milk (HMOs) hinder the anchoring of pathogens and protect infants during lactation [START_REF] Ayechu-Muruzabal | Diversity of human milk oligosaccharides and effects on early life immune development[END_REF]. Nowadays, the protective role of HMOs in newborn babies is well established and they are widely used in the formulation of nutritional products.

The use of sugars as protective therapy against infections in animals was first reported in 1979 by Aronson and coworkers [START_REF] Aronson | Prevention of colonization of the urinary tract of mice with Escherichia coli by blocking of bacterial adherence with methyl a-d-mannopyranoside[END_REF]. In their published work, they report that the administration of α-D-mannopyranoside to mice, prevented the colonization of their urinary tract by Escherichia coli. Subsequently, many studies have confirmed the feasibility of using saccharides to prevent infections caused by different pathogenic microorganisms. Modified from Ofek et al., 2003 [29] Although sugars have been shown to be effective in hindering microbial adhesion, lectin-carbohydrate interactions are generally very weak and some of these molecules are susceptible to enzymatic degradation [START_REF] Sattin | Glycoconjugates and glycomimetics as microbial anti-adhesives[END_REF]. Therefore, an evolved approach to lectin-targeted antiadhesive therapy propose the use of carbohydrate mimics (glycomimetics) as a replacement for natural sugars. These compounds mimic the bioactive function of carbohydrates while displaying better drug-like properties, which leads to higher metabolic stability and higher selectivity [83]. However, the development of efficient and specific glycomimetics requires prior knowledge about the lectin that is being targeted. This is particularly problematic in the case of emerging pathogens whose anchor lectins remain anonymous and evidences the imperative need for their identification.

Aspergillus fumigatus lectins

A. fumigatus is also an ubiquitous opportunistic saprophytic mold that is the most prevalent airborne fungal pathogen causing infections in humans. It is responsible for 16 million bronchopulmonary infections a year in immunocompromised patients with high mortality rate and resistance to actual antifungals [84]. Its lectinome has been under study in our research group for a decade. Two lectins named FleA and AFL6 have been especially under study.

FleA is a lectin found on the surface of the A. fumigatus conidia with a strong proinflammatory effect on human bronchial cells. Its structure is a six-bladed βpropeller homodimer with 6 binding pockets (BP) per protomer (Figure 1.11A) [START_REF] Houser | A soluble fucosespecific lectin from Aspergillus fumigatus conidia -structure, specificity and possible role in fungal pathogenicity[END_REF]. FleA is specific for fucosylated carbohydrates, notably, human blood group antigens (BGA). Those can be found on airway mucins and macrophage

AIM OF THE THESIS AND OBJECTIVES

The overall goal of this work was to contribute to the general understanding of the host-pathogen interactions by the identification and characterization of new lectins from the emerging opportunistic fungal pathogen Scedosporium apisospermum. This has been addressed by two different strategies; the first strategy comprises the in-silico prediction of putative lectins encoded in the S.

apisospermum genome by data mining, followed by their recombinant production and subsequent characterization. The second strategy lies in the identification of native lectins directly from fungal protein extracts of S. apiospermum. In this latter approach, we have also included the analysis of protein extracts from another medically relevant pathogenic fungi, Aspergillus fumigatus.

A second aim of this project was to develop a new tool that allows the identification and purification of unknown lectins directly from crude extracts of proteins without the need for fluorescent labelling. This tool assembles the fusion of the tree classic steps of lectin purification train: clarification, concentration and purification, in a single operating unit. It also allows the simultaneous purification of lectins with different specificities in a simple and fast process. It can also be used as a "mini glycan array" for the study of carbohydrate lectin interactions.

To achieve those main goals, 4 objectives were defined:

1-To identify putative lectins from the genome of S. apiospermun by data mining.

2-To carry out the heterologous production of the lectins identified and proceed with their biochemical and structural characterization.

3-To identify native lectins from protein extracts of S. apiospermum.

4-To develop a new tool for purification and identification of lectins from crude extracts.

57

RESULTS

Chapters III-VII

The following five chapters describe the main results of the project, which are schematically summarized in the following diagram.

BIOCHEMICAL AND STRUCTURAL STUDIES OF

TARGET LECTIN SapL1 FROM THE EMERGING OPPORTUNISTIC MICROFUNGUS S. apiospermum

Summary

In this chapter, we present the first report of the identification and characterization of a lectin from S. apiospermum. This lectin has been named

SapL1 and it is homologous to the conidial surface lectin from Aspergillus fumigatus (FleA), which is known to be involved in the adhesion to host glycoconjugates present in human lung epithelium. Therefore, the discovery and characterization of SapL1 contribute to the understanding of glycosylated surface recognition by this fungal pathogen and might guide the development of antiadhesive glycodrugs by targeting its inhibition.

The main findings of this part of the project are described in a paper that has been accepted for publication in the Scientific Reports Journal. This work includes a detailed strategy to achieve the soluble expression of SapL1 in E. coli, its biochemical characterization, an analysis of its specificity and affinity by Glycan In this chapter, we describe the identification and characterization of a Cyanovirin-like lectin from S. apiospermum (SapL6). SapL6 codifying sequence was identified by data mining using the sequence of a homologous protein from A. fumigatus, which has been previously studied by our research group (AFL6).

Both, AFL6 and SapL6 belong to the recently discovered Cyanovirin-N homolog (CVNH) family.

Although the biological function of this family of proteins is unclear, the relevance of SapL6 fungal life is evidenced by its constitutive expression during the whole life cycle of S. spiospermum [87]. Hence, it represents interesting target for the development of antifungal therapies. Besides, its evolutive relationship with the powerful antiviral protein Cyanovirin-N (CN-V), brightens up the interest to evaluate its possible applications as biotechnological tool. Together, these factors evidence the relevance of approaching the study of this protein.

This work includes a detailed strategy to achieve the recombinant expression of SapL6 in E. coli, its biochemical characterization and its 3D-structure solved by X-ray crystallography. The analysis of its specificity was also addressed by different approaches, such as glycan array and ITC and co-crystallization with glycans. However, to date, it has not been possible to identify its ligand. A graphical abstract of this part of the project is shown in figure 4.1.

Introduction

Cyanovirin-N

Cyanovirin-N (CV-N) is a small protein from the cyanobacterium Nostoc ellipsosporum that binds mannosides and possesses strong virucidal properties against a wide range of viruses (e.g., HIV, ebola, influenza, hepatitis C, herpes virus, etc.) [86,88]. Although the natural ligand and the biological role of CV-N in N. ellipsosporum is unknown, its potential pharmacological use has motivated a large number of studies to elucidate its antiviral mechanism of action, which has turned out to be specific to each case. In HIV, for example, inhibition is mediated by high affinity interactions with the viral envelope glycoproteins gp120 and gp41, which are highly mannosylated [START_REF] Akkouh | Lectins with anti-HIV activity: A review[END_REF]. This interaction disables the recognition of the host cell CD4 and CCR5 receptors and prevents conformational changes that are necessary for membrane fusion and viral entry into the target cells [88,89].

The CV-N fold

Cyanovirin-N is composed by two homologous repeats of ~50 amino acids that fold into a pair of two interconnected pseudosymmetric domains formed by strand exchange called domains A and B (Figure 4.2) [START_REF] Koharudin | The evolutionarily conserved family of cyanovirin-N homologs : structures and carbohydrate specificity[END_REF][START_REF] Bewley | Solution Structure of a Cyanovirin-N : Mana 1-2Mana Complex : Structural Basis for High-Affinity Carbohydrate-Mediated Binding to gp120[END_REF]. Each domain comprised one helical turn a three-stranded β-sheet, a β-hairpin and an intramolecular disulfide bond. This provides the protein with great stability and forms two symmetry-related carbohydrate binding pockets located at opposite ends of the protein's long axis (BP1 and BP2) [85]. These binding sites exhibit differing affinities for the disaccharide Manα1-2Man. BP2 shows the highest affinity (Ka of ~ 7.2 X10 -6 M -1 ), whilst the affinity forBP1 is 10 times lower (Ka ~ 6.8X10 -5 M -1 ) [START_REF] Bewley | Solution Structure of a Cyanovirin-N : Mana 1-2Mana Complex : Structural Basis for High-Affinity Carbohydrate-Mediated Binding to gp120[END_REF].

Cyanovirin-N homologs (CVNHs)

Given the efficacy of CV-N as a virucide, there was great interest in the identification of similar proteins after its discovery. In 2005, an in-silico screening conducted by Percudani and coworkers leaded towards the identification of a group of CVN-related proteins that were named CyanoVirin-N Homologous (CVNHs) [85]. Originally, all members of this protein family were identified from eukaryotic organisms such as filamentous ascomycetes and seedless plants, however, in further studies CVNHs have also been identified from prokaryotic cells [88,[START_REF] Kehr | A mannan binding lectin is involved in cell-cell attachment in a toxic strain of Microcystis aeruginosa[END_REF]. The phylogenetics of this family has been divided into three major subgroups of CVNHs:

• CVNH I is represented by the fern Ceratopteris richardii lectin (CrCVNH).

This group includes CV-N, the prokaryotic CV-like proteins MVN and CaVN, five fungal CVNHs and two fern CVNHs.

• CVNH II is represented by the truffle Tuber borchii lectin (TbCVNH). This group comprise proteins from filamentous Ascomycetes whose sequence is closely related with TbCVNH.

• CVNH III is represented by the mold Neurospora crassa lectin (NcCVNH).

This group is entirely composed of CVNH polypeptides from filamentous

Ascomycetes whose sequences closely related with NcCVNH.

• Others: CVNHs, that did not cluster with any of the three major groups. On the other hand, despite their high structural similarity, noteworthy divergences are identified for some CVNH members, such as the absence of S-S bonds in the fungal members and the poor conservation of the putative sugarbinding pockets, which is common in protein families that interact with multiple ligands [START_REF] Koharudin | The evolutionarily conserved family of cyanovirin-N homologs : structures and carbohydrate specificity[END_REF]85]. Additionally, extended loops are present in the fungal members of the family and this may stabilize the conformation of local regions.

CVNHs carbohydrate binding properties

Chemical shift perturbations monitored by 1 H-15 N HSQC spectroscopy, showed that the three representative members of each CVNH subfamily bind to Manα1-2Man as Cyanovirin-N. Only the fern homolog has two functional binding sites, while for the fungal proteins, only one binding site is active [START_REF] Koharudin | The evolutionarily conserved family of cyanovirin-N homologs : structures and carbohydrate specificity[END_REF]. Functional NcCVN. Adapted with permission from Koharudin et al., 2008 [59].

Additional NMR titrations performed with sugars no represented in the glycan array, resulted in the identification of sucrose binding. According to the pattern of the affected resonances, it was concluded that the sucrose binding pocket was the same than the mannose one [START_REF] Koharudin | The evolutionarily conserved family of cyanovirin-N homologs : structures and carbohydrate specificity[END_REF]. Sucrose binding appears to be exclusive of TbCVN and it is speculated that this disaccharide could mimic the shape of an unidentified natural ligand.

CVNHs biological roles

It has been suggested that fungal CVNHs could be nutrient state-regulated lectins. This theory arose from the observation, by northern blot and microarray hybridization, of a severe down-regulation of TcCVN mRNA (M5G11) upon carbon or nitrogen starvation [85]. This was later supported by the reduced immunodetection of TbCVNH in mycelia grown under glucose-limiting conditions [START_REF] Koharudin | The evolutionarily conserved family of cyanovirin-N homologs : structures and carbohydrate specificity[END_REF]. On the other hand, a recently identified N. crassa mRNA encoding a protein identical in sequence to NcCVNH could be related to the metabolic/nutritional changes that accompany the circadian rhythm in this organism [START_REF] Lakin-Thomas | Circadian rhythms in microorganisms: New complexities[END_REF]. Taken together, these data could implicate potential physiological functions of CVNH, e.g., cell-cell interaction through the recognition of specific sugars (and/or glycoproteins) bound to the surface or the detection/transduction of associated specific metabolic signals. Furthermore, nitrogen deficiencies are believed to be important environmental signals that trigger plant infection, as well as other adaptive morphogenetic transitions in fungi [START_REF] Soragni | A nutrient-regulated, dual localization phospholipase A2 in the symbiotic fungus Tuber borchii[END_REF]. Hence a putative role during pathogenesis should not be ruled out.

CVNHs subcellular localization

Although the subcellular localization of CV-N in N. ellipsosporum is unknown, it is predicted to be a secreted protein since it lacks its N-terminal methionine. The secretory theory is reinforced by the presence of disulfide bonds, another signature characteristic of extracellular proteins [85]. CVNHs and in particular the fungal CNVHs are also predicted to be secreted despite the absence of signal peptide. Both TbCVNH and NcCVNH are found primarily in sedimentary subcellular fractions (membrane and organelles) of macerated fungal cells and associate with the surface of hyphae [START_REF] Koharudin | The evolutionarily conserved family of cyanovirin-N homologs : structures and carbohydrate specificity[END_REF]. The secretion mechanism of these proteins is unknown as for many fungal lectin which are also found extracellularly without secretion signal.

AFL6

Previously, our research group has undertaken the study of AFL6, a hypothetical CVNH from A. fumigatus [START_REF] Hündling | Biochemical and structural studies of lectines from opportunistic filamentous fungi[END_REF]. This protein was originally predicted by [START_REF] Koharudin | The evolutionarily conserved family of cyanovirin-N homologs : structures and carbohydrate specificity[END_REF] and it was one of the 39 sequences used to establish the phylogeny of CVNH proteins. AFL6 was produced recombinantly in E. coli and its structure was solved by X-ray crystallography. As expected, AFL6 shares the common CV-N fold and, in agreement with the other fungal members of the CVNH family, it does not present hemagglutinating activity.

According to the original classification of CVNHs, AFL6 belongs to the subfamily represented by the N. crassa lectin (NcCVNH), however a glycan array assay showed that, instead of highly mannosylated structures, AFL6 preferentially binds to glycans containing biantenary polylactosamine, such as TbCVNH from T. borchii. Nevertheless, no interaction with lactosamine could be confirmed by ITC and its affinity for mannose was very low.

Material and methods

Genetic construction

The coding sequence for SapL6 was optimized for expression in E. coli and ordered at Eurofins Genomics (Ebersberg, Germany). SapL6 was excised from the commercial vector where it was inserted, through standard digestion (2 h at 37° C) with the restriction enzymes NdeI and XhoI. pProNde and pET-TEV [START_REF] Houben | Interaction of the C-terminal domains of sendai virus N and P proteins: comparison of polymerase-nucleocapsid interactions within the paramyxovirus family[END_REF] vectors were digested with identical restriction patterns. pProNde is a homemade vector which results from the mutation of the NcoI to NdeI restriction site in pProEx HTb vector (EMBL, Heidelberg). All products of digestion were verified by electrophoresis in 0.8% agarose gels, from where the linearized vectors and SapL6 codifying sequence were recovered and purified by using the commercial kit of BioLabs "Monarch DNA Gel Extraction". Then, SapL6 insert and digested plasmids were ligated using the Fermentas T4 ligase, in a ratio of 3:

1. Reactions were incubated at 25°C for 1 h and were used to transform chemocompetent cells of E. coli TOP10 by thermic shock at 42 °C. Transformed cells were recovered for growth in LB medium plates with the corresponding antibiotic (Ampicillin 100 μg/mL for pET-TEV and Kanamycin 30 μg/mL for pProNde) and incubated at 37 °C. After 16 h, a PCR colony screening was performed using a set of primers designed to hybridize sequences flanking the gene insertion in each vector and positive hits were grown in LB media to perform plasmid extraction. Finally, gene insertion was confirmed on the purified plasmids by digestion and the correct reading frame was validated by sequencing.

Production of SapL6

For heterologous production of SapL6, the recombinant plasmids (previously validated) were introduced into E. coli BL21(DE3) strain by thermal shock at 42°C. Transformed cells were recovered for growth in LB plates with the corresponding antibiotic (Ab) and colony PCR screening was performed to confirm the plasmid insertion. A positive hit from each vector was grown in LB+Ab media and incubated at 37°C with orbital agitation (160 rpm) until it reaches an optical density (OD600) of 0.6. The temperature was then lowered to 16°C and when OD600 reached 0.8, the induction was carried out overnight by the addition of isopropyl-β-D-1-thiogalactoside (IPTG) to a final concentration of 0.1 mM.

Purification of SapL6

Cultures were harvested by centrifugation at 5000 g for 10 min and resuspended on buffer A (50 mM Tris-HCl, 500 mM NaCl, pH 7.5) with moderate agitation for 30 minutes. Then, cells were disrupted at 1.9 kbar using one shot cell disrupter

Size exclusion chromatography

Size exclusion chromatography (SEC) was performed using a High-Resolution

ENrich™ SEC 70 column (Bio-Rad) on NGC™ chromatography system (Bio-Rad). Prior assay, protein sample was prepared at 10 mg•mL -1 and centrifuged 30 min at 12000 rpm. Column was equilibrated with 50 ml of buffer D (20 mM MES, 100mM NaCl, pH 6) and 200 µl of sample were injected followed by 40 ml isocratic elution on buffer D. Fractions were monitored by absorbance at 280 nm and collected every 0.5 mL.

Dynamic light scattering (DLS)

DLS analyses were performed using a Zetasizer™ NanoS (Malvern Panalytical) using a 40 μl quartz cuvette. Prior to measurements, a solution of pure protein at 1 mg•mL -1 on buffer C (50 mM TRIS-HCl, 100 mM NaCl, pH 7.5) was prepared and centrifuged during 30 minutes at 12,000 rpm in microcentrifugue tubes.

Corresponding quartz cells were filled with sample solution and tree successive DLS measurements were performed per sample.

Thermal Shift Assay (TSA)

The thermal stability of SapL6 was analyzed by TSA with the MiniOpticon realtime PCR system (Bio-Rad, Hercules, CA, USA). Prior assay, buffer stocks at 100 mM and a mixture containing 70 μl of SapL6 at 1 mg mL

Hemagglutination assay

Agglutination test was performed with fresh rabbit erythrocytes (BioMerieux, Lyon) in 96-well plates. For the test, 150 mM NaCl was used as a negative control and rPVL from Psathyrella velutina, as a positive control. Aliquots of each sample were prepared at 0.1 mg•mL -1 and serial double dilutions were made from the first to the 12 lines. Once the dilutions were made, 50 μL of rabbit erythrocytes 3% were placed into each well and the plate was incubated at room temperature (RT). After 2 hours the result of the experiment was evaluated and agglutination activity was calculated according to the dilution of the protein.

Mammalian Glycan array assay (CFG)

SapL6 was labeled with Fluorescein Isothiocyanate (FITC) (Sigma-Aldrich, St Louis, MO) according to the supplier's instructions with slight modifications.

Briefly: Two milligrams of protein were dissolved in 1 mL of buffer E (100 mM Na2CO3, 100 mM NaCl, pH 9), then, 40 microliters of FITC at 1 mg•mL -1 , previously dissolved in DMSO, were gradually added to the protein solution and the mixture was gently stirred at room temperature overnight. Next day, the solution was supplemented with NH4Cl to a final concentration of 50 mM and free FITC was removed using PD10 desalting column with PBS as mobile phase.

Protein concentration was determined at Abs280 and FITC at Abs490 using a NanoDrop TM 200 (Thermo Scientific, Hercules, CA, USA), and Fluorescein/Protein molar ratio (F/P) was estimated by the following formula:

Where MW is the molecular weight of the protein, 389 is the molecular weight of FITC, 195 is the absorption E0.1% of bound FITC at 490 nm at pH 13.0, (0.35 X A495) is the correction factor due to the absorbance of FITC at 280 nm, and E 0.1% is the absorption at 280 nm of a protein at 1.0 mg mL -1 , Being an ideal F/P should be 0.3 > 1.

Labeled lectin was sent to the Consortium for Functional Glycomics (CFG), (Boston, MA, USA) and binding properties were assayed at 5 and 50 μg mL -1 on a "Mammalian Glycan Array version 5.4" which contain 585 glycans in replicates of 6. The highest and lowest signal of each set of replicates were eliminated and the average of the remaining data were normalized to the percentages of the highest RFU value for each analysis, finally, the percentages for each glycan were averaged at different lectin concentrations.

Crystallization and data collection

Crystal screening was performed using the hanging-drop vapour diffusion technique by mixing equal volumes of pure protein at 5-10 mg•mL -1 and precipitant solutions from commercial screenings of Molecular Dimensions (Newmarket, UK). 2 µL drops were incubated at 19°C until crystals appeared. A subsequent optimization of positive conditions for SapL6 crystallization was carried out and crystals suitable for X-ray diffraction analysis were obtained under solution containing 3 M ammonium sulphate in buffer HEPES 0.1 M pH 7.

Data collection was performed on PX1 and PX2 beamlines at SOLEIL Synchrotron (Saint Aubin, FR) using a Eiger detector.

Structure determination

The diffraction patterns collected were processed using XDS 14 software [96] and were converted to structure factors using the CCP4 [START_REF] Winn | Overview of the CCP4 suite and current developments[END_REF] program package v.6.115, with 5% of the data reserved for Rfree calculation. The structure was determined using the molecular-replacement method with Phaser MR v.2.5 16 [START_REF] Mccoy | Phaser crystallographic software[END_REF], with the structure of AFL6 (unpublished data) as starting models. Models' refinement were performed using REFMAC v.5.17 [START_REF] Murshudov | REFMAC 5 for the refinement of macromolecular crystal structures[END_REF] alternated with manual model building in Coot v.0.718 Sugar residues and other compounds that were present were placed manually using Coot.

Results and discussion

Identification of SapL6

The codifying sequence for a hypothetical Cyanovirin-N homolog protein (EMBL accession number XM_016790607.1) was identified through data mining by using AFL6 lectin as input in the genome of Scedosporium apiospermum. This sequence is produced by the transcription of the SAPIO_CDS9133 gene and it corresponds to a hypothetical lectin of ~11 kDa hereinafter referred to as SapL6.

As it is characteristic in CVNHs, the sequence of SapL6 is composed by two homologous tandem repeats (TR) that are predicted to have the same fold and display high identity between them (> 30%). The first tandem (TR1) is comprised from residues 1-49, while TR2 covers from 50-106. Surprisingly, during a multiple alignment of CVNHs sequences and phylogeny, we identified that SapL6-TR1 seems to have more similarities with TbCVNH than with NcCVNH (Figure 4.5).

This was unexpected since AFL6 belong to the subfamily of CVNH-III and this family is represented by NcCVNH. Hence, we perform a new phylogenetic analysis with 25 of the original 39 sequences used by Koharudin et al in 2008 (Figures 4.6 and 4.18) [START_REF] Koharudin | The evolutionarily conserved family of cyanovirin-N homologs : structures and carbohydrate specificity[END_REF]. The analysis suggests that, although SapL6 also belongs to the CVNH-III subfamily, its evolutionary distance with TbCVNH is shorter than in the case of AFL6 and NcCVNH. Therefore, their biological role and specificity could also be closely related.

Production and purification of SapL6

The coding sequence for SapL6 was fused to an N-terminal His-tag cleavable by

Molecular size and oligomeric stage of SapL6

In crystal structures, CV-N can be observed as a domain swapped dimer, where domain A of one monomer interacts with domain B from the other monomer.

Originally, this was considered a crystallization artifact, however, nowadays it has been demonstrated that CV-N can exist both in monomeric and in domainswapped dimeric form also in solution [START_REF] Hündling | Biochemical and structural studies of lectines from opportunistic filamentous fungi[END_REF]. Conversely, no exchange or dimerization has been observed in the CVNH characterized so far.

The molecular size and oligomerization state of SapL6 were evaluated through

Thermal stability of SapL6

The stability of the protein was assayed in 26 different buffers in a pH range from 5 to 10 by thermal shift assay (TSA). No significant differences were found between the tested conditions, which indicates that the pH or buffer type don't have a big influence on the stability of SapL6 (at least not on the gradient studied).

Addressing the small differences in detail, it can be generalized that the most stable condition ranged between pH 6.5 and 9 with a Tm between 45-48 °C where the denaturation occurred in a single event. On the other hand, multiple Tm were predominantly observed at low pH, but it is pertinent to highlight that the additional signals were originated from small peaks that could have been generated from automatic baseline adjustment (Figure 4.9). When stored in MES buffer pH=6 or PBS pH=7.4, SapL6 is stable up to 12 months at 4°C and no evidence of degradation of misfolding were identified even after 24 months storage at -20 °C. The protein is therefore quite stable despite a low Tm.

Hemagglutination activity of SapL6

Since it is well known that many lectins have the capacity to agglutinate blood cells, we have performed an hemagglutination assay on rabbit erythrocytes.

However, no hemagglutination activity was detected even at high protein concentrations (Figure 4.10). This result is consistent with what has been previously reported for AFL6 and other homologous proteins from the CNVhomologs in fungi. The absence of hemagglutination can be explained by the fact that SapL6 as other fungal CVNHs would be monovalent with only one the two potential binding sites functional. However, the possibility that the determinants recognized by this lectin are absent in the analyzed cells, cannot be discarded.

SapL6 carbohydrate binding properties ITC

The affinity of SapL6 for Manα1-2Manα was addressed by ITC, however, it was not possible to identify a clear pattern of interaction. Since it is known that TbCVNH also can bind glycans composed of glucose, galactose and Nacetylgalactosamine, we also titrated SapL6 using those sugars and a large set of other glycans available in our laboratory, but no binding was detected. In some cases, titrations with high concentrations of ligands (i.e., mannose, Nacetylgalactosamine, sucrose, etc.) display patterns that resemble a very weak interaction, however, this was not confirmed when the molar ratio protein: ligand was increased. Therefore, this phenomenon was attributed to the energy realized by the sugar dilution in the sample cell. A table summarizing ITC experiments is presented in Table 1. where it was possible to detect only some low interaction with the polylactosamine containing glycans. It also agrees with the data reported by [START_REF] Koharudin | The evolutionarily conserved family of cyanovirin-N homologs : structures and carbohydrate specificity[END_REF] where TbCVN was bound to mannose-lacking glycans composed exclusively by glucose or a combination of glucose, galactose, and Nacetylglucosamine [START_REF] Koharudin | The evolutionarily conserved family of cyanovirin-N homologs : structures and carbohydrate specificity[END_REF]. This leads us to raise three possible scenarios:

i) SapL6 and AFL6 are involved in host recognition process but their target was not present in the glycan array.

ii) SapL6 and AFL6 are not involved in host recognition and therefore no interaction with mammal's glycans should be expected.

iii) SapL6 and AFL6 are host binding lectins that should display affinity for the carbohydrates of the glycan arrays respectively tested, but they are not active.

Assuming the first and/or second scenarios are correct, it was determined to expand the search with other chips that contain glycans from different sources, not just mammals. However, due to the previously observed inconveniences during the labeling of SapL6, on this occasion, it was decided to proceed with its detection by using anti-his antibodies (anti-His Ab) fluorescent-labeled. For this, a new batch of SapL6 was produced; on this occasion, the histidine tail was not cleaved the elution parameters were modified during the first affinity chromatography to improve the sample purity (Figure 4.12). Then, the sample was additionally purified by size exclusion chromatography and were tested on two new glycan matrix platforms; 1) the microbe-based glycan array matrix [104],

developed by the groups of Professor Peter H. Seeberger, at Max Planck Institute (Germany and 2) the broad-spectrum microbial glycan array of the Imperial College of London (UK). According to our contacts in these platforms, it was not possible to identify any interaction during the screening, although the formal reports are still pending.

SapL6 crystallization

After thousands of tested conditions, small SapL6 crystals were obtained in a solution containing 3 M (NH₄)₂SO₄ and 100 mM HEPES buffer pH = 7. Crystals diffracted at 2 Å resolution and the protein structure was solved by molecular replacement. However, we encountered problems during refinement leading to poor statistics and we decided to optimize nucleation. For this, we studied the influence of small variations of each of the components of the, already simple, mother liquor. Through this screening, we identify that SapL6 crystallizes in a very narrow window of conditions, mainly influenced by the concentration of ammonium sulfate.

From all conditions tested, large and well-defined symmetric crystals were obtained only with 2.8 M ammonium sulfate. Interestingly, even when the rest of the components remained identical, the protein precipitated at slightly higher concentrations (≥ 2.9 M) and the droplets remained clear at ≤ 2.7 M (Figure 4.13).

A great influence was also observed for buffers systems (both, in the protein sample and mother liquor). On the other hand, protein concentration does not

Chapter IV: Production and characterization of SapL6 103 seem to be as strict a parameter such as the other components, since it was possible to obtain crystals in protein concentrations ranging from 7 to 10 mg mL - 1 with high reproducibility. The new crystals were obtained for the apo protein or during cocystallization assay with different sugars. They diffracted up to 1.35 Å, several high-quality dataset were obtained, the best one was chosen but its refinement is still ongoing.

Hence, the original structure solved at 2 Å is reported in this draft of the thesis.

SapL6 structure

SapL6 structure was solved by molecular replacement at 2 Å with AFL6 structure as searching model. The crystal space group was found to be P41212 with one molecule per asymmetric unit and 30.69% solvent (Table 1). The overall structure of the model closely resembles the CV-N fold; where the two tandem repeats

Comparison of SapL6 with other CVNHs

The structural comparison of SapL6 and the most representative members of the CNVH family shows that although the sequences of these proteins display <50% of identity, their 3D-fold is extremely conserved (Figure 4.15 A). SapL6 is an elongated protein presenting largely beta-sheets and displays internal two-fold pseudosymmetry.

Consistently with our previous data, the topological distribution of SapL6_TR1 reassembles TbCVNH_TR1 better than NcCVNH_TR1, mainly due to the absence of the first helical turn of NcCVNH_TR1 (Figure 4.15 C and E, rmsd 1.55 and 1.43 respectively). This is particularly relevant since the inactivity of the BP1 of NcCVNH_TR1 has been attributed to the substitution of this α1 by a flexible tail, resulting in the loss of the concave pocket required for sugar binding [START_REF] Koharudin | The evolutionarily conserved family of cyanovirin-N homologs : structures and carbohydrate specificity[END_REF].

Hence, the funtionality of this binding site in AFL6 and SapL6 seems plausible.

Surprisingly, in addition to AFL6 (rmsd 0.522), the CV-N structure is the one with the most similarities to SapL6 in both domains (rmsd 1.26). Except for disulfide bridges, the most important difference is the presence of 5 additional residues in SapL6_TR2 . This small sequence leads to the elongation of the loop connecting the two strands β6 and β7 as well as the stands themselves in SapL6 compared to all other CNVHs and CV-N (Figure 4.15 B-F).

Structural insights of putative carbohydrate-binding pockets in SapL6

SapL6 has two shallow grooves at both ends of the ellipse that perfectly overlap the carbohydrate-binding pockets of CV-N. Although the topology of these regions is very similar, the residues directly involved in the binding of Man1-2Man in CV-N greatly differ from those found in SapL6. In the cyanobacteria members, the binding pocket of the domain A (BP1), the disaccharide interaction is mediated by hydrogen bonds with two aspartic acids that are replaced by

Conclusions

In this study, we have identified and recombinantly produced a new cyanovirinlike lectin from the human opportunistic microfungi S. apiospermum. Although SapL6 was successfully produced and characterized, it has not been possible to identify any carbohydrate-binding activity. Due to its low affinity for mannosylated glycans and monovalency, SapL6 is not expected to present CV-N activity against viruses such as HIV. However, its applications as a pharmacological target are still valid.

Cyanovirin-like lectins are a great enigma of nature. Although it is clear that its biological role must be very important for living, the functions of the already identified members of this family remain unclear and their characterization has only aroused questions such as:

❖ Which evolutive force leads to the preservation of such structure among so divergent organisms even when the sequences are not conserved?

❖ What is their natural ligand?

❖ Do all these proteins share the same function in their organisms of origin?

❖ Which function is this?

❖ Why are they only present in cyanobacteria, fungi and ferns?

❖ What is the process of evolutionary divergence that led to the transmission of CV-N genes only in these species and the differences between bacterial and fungal members?

We believe that the identification and characterization of new CVNHs could provide important information to contribute to the general understanding of such proteins and answer these questions. For this reason, despite we were aware of the enormous challenge that this objective entails, we have approached the study of SapL6, not entirely unsuccessfully. We believe that the structural information gathered here will be useful to address possible interactions with glycans through in silico analysis and might guide towards the discovery of its natural ligand. Which, we consider, is the ultimate clue to finally understanding the biological role of this family of proteins and to evaluate their potential as drug targets.

For our part, we continue to search for strategies that allow us to overcome the current obstacle in which we are stopped, and we are testing the cocrystallization of the protein with diverse glycans, but so far, only the apo structure was obtained.

Contributions

Dania

IDENTIFICATION OF NEW NATIVE LECTINS FROM PROTEIN EXTRACTS OF MEDICAL RELEVANT

FUNGAL STRAINS

Summary

Fungi are an invaluable source of novel lectins that are key elements for the development of glycobiology and biomedical research, either as tools or as drug targets. So, there is great interest in their identification and characterization.

Nevertheless, fungal lectins have been poorly studied and their reports are scarce compared to those from other organisms. This represents a problem in the case life-threatening species for humans, such as Aspergillus fumigatus and Scedosporium apiospermum. Therefore, the main aim of this part of the project was to identify new wild-type lectins from those pathogens. For this, we have analyzed their intracellular proteins and secretomes trough sugar-affinity chromatography, SDS-page and mass spectrometry. A total of 54 bands (presumably corresponding to carbohydrate-binding proteins) were recovered from gels and 22 of them have been sequenced to date. The analysis of the peptides obtained by mass spectrometry, allowed us to identify 7 hypothetical lectins from those samples, four of them from S. apiospermum (PVL-like lectin, SapCP, CcMBL-like lectin and SapFKBP12). The preliminary analysis of those proteins revealed that some might be attractive pharmacological targets and others could find application as biotechnological tools. In this chapter, we present a detailed analysis of their sequences, structure and putative role in the fungal life, as well as their phylogenetic distribution and potential applications.

Introduction

Due to their biological relevance and possible applications, fungal lectins have arisen the attention of the scientific community, either to explore their implementation as tools or to propose them as drug targets. This interest, has led to a growing rate of new fungal lectins discovery in the recent years, however, there is still a considerable gap compared to lectins from other organisms, such as plants, animals or bacteria. The lack of information is particularly alarming in the case of microfungi, whose represent only 15% of the total lectins identified from the complete kingdom, while the remaining 85% come from mushrooms and yeasts [START_REF] Varrot | Fungal lectins : structure , function and potential applications[END_REF][START_REF] Khan | Fungal Lectins: Current molecular and biochemical perspectives[END_REF][START_REF] Kobayashi | Fungal lectins: a growing family[END_REF].

S. apiospermum and A. fumigatus are opportunistic microfungal pathogens that affect mainly immunocompromised patients. The rate of infections caused by those microorganisms has increased in the recent decades [START_REF] Tuite | Overview of invasive fungal infections[END_REF] and nowadays they are most common filamentous fungus to colonize the lungs of patients with cystic fibrosis [START_REF] Pereira De Mello | Conidial germination in Scedosporium apiospermum, S . aurantiacum, S . minutisporum and Lomentospora prolificans: influence of growth conditions and antifungal susceptibility profiles[END_REF][START_REF] Goldman | Scedosporium apiospermum infections and the role of combination antifungal therapy and GM-CSF: a case report and review of the literature[END_REF][START_REF] Sakai | Fucose-specific lectin of Aspergillus fumigatus: binding properties and effects on immune response stimulation[END_REF]. Furthermore, they have a high rate of recidivism and display resistance towards several antifungals [START_REF] Ramirez-Garcia | Scedosporium and Lomentospora: An updated overview of underrated opportunists[END_REF]106]. Since lectins are involved in several mechanisms elemental for the fungal development and host-adhesion process, there is an emerging interest in the identification and characterization of lectins from those two microfungal pathogens. Nevertheless, their identification reports are limited to date, particularly in the case of S. apiospermum.

There are two main strategies for new lectins identification; i) the in silico approach, which use data mining to predict putative proteins encoded on the DNA of species whose genome has been sequenced and, ii) the traditional approach, which takes advantage of the carbohydrate-binding properties of those proteins and uses affinity chromatography for their purification. Each one of those strategies have their advantages/disadvantages.

On one hand, the in silico prediction requires a priori knowledge of similar lectins from other species and the identification must be followed by the recombinant expression of the predicted proteins and their subsequent validation. This implies a slow process that limits the study of a large number of proteins simultaneously. In addition, this strategy is only available for organisms whose genomes have been sequenced and incomplete or partially assembled genomes could drive to false negatives during the identification. Finally, it is important to consider that genomic sequences can vary according to the database or even between strains of the same species.

On the other hand, the major disadvantages of the traditional approach for the identification of new lectins are that some of these proteins are expressed differentially during different stages of fungal development or are produced only under certain conditions, such as abiotic stress, colonization, etc. Furthermore, some of those lectins are present in very low quantities, hindering their detection.

Even if the experimental approach manages to evade those inconveniences, it is important to consider that some lectins need a specific arrangement of carbohydrate linkages to achieve binding and they would not be retained by conventional carbohydrate-based matrixes usually constituted of monosaccharides. Additionally, when using peptide-based mass spectrometry to sequence the identified proteins, sequence databases are also needed. This brings back the problems associated with incomplete or partially assembled genomes and sequence variations between databases or strains.

The in silico approach has been used during this work to predict SapL1 and SapL6 lectins from S. apiospermum. Whose production and characterization are described in chapters III and IV. Now, the main aim at this stage of the project was to identify native lectins of medical relevance from mycelium cultures of A.

fumigatus and S. apiospermum. For this, we have performed an extraction of total proteins from mycelia of both microfungi and we also have analyzed the fraction of proteins secreted to the culture media. All fractions were purified by carbohydrate-affinity chromatography and proteins recovered from SDS-PAGE gels were proteolyzed and the peptide identified by mass spectrometry. A general overview of the project is shown in figure 5.1.

Material and methods

Fungal strains

The identification of native lectins from medically relevant fungal strains was carried out from protein extracts of S. apiospermum IHEM 14462 and A. fumigatus IHEM 6951, kindly provided by Prof. Muriel Cornet, TIMC, CHU Grenoble.

Sample preparation

Three weeks after inoculation, culture media was removed by filtering with 0.45 µm membranes. After filtration, 50 mL of each culture media were collected for further analysis and the biomass was frozen in liquid nitrogen, grinded and suspended in Phosphate buffer saline (PBS) buffer pH 7.4. Finally, samples were centrifuged at 24000 g for 30 minutes to remove cellular debris and soluble fraction was filtered again with 0.45 µm filters.

Lectins purification

Six different matrixes for carbohydrate affinity chromatography were assembled in a consecutive purification train. Then, columns were equilibrated with 10 column-volumes (CV) of PBS buffer pH 7.4 and each sample was sequentially eluted through all of them as described in the figure 5.1.2. Immediately after the sample was eluted, resins were washed with 10 CV of PBS buffer pH 7.4

supplemented with 1 M of NaCl to disrupt unspecific interactions. Finally, bounded proteins were recovered from each column by eluting 4 CV of PBS buffer pH 7.4 supplemented with 250 mM of the corresponding sugar.

SDS-PAGE

Fractions eluted from each column were concentrated to 200 µl via ultrafiltration with Vivaspin® filters of 10 kDa cutoff (Sartorius). Then, samples were boiled 10 minutes in presence of LDS sample buffer 4X (Expedeom Ltd), centrifuged and ran in a 4-20% Mini-PROTEAN® TGX™ Precast Protein Gels (Bio-Rad Ltd) at 200 mV during 40 minutes. Gels were stained with InstantBlue Coomassie staining solution (Abcam) overnight. In the following day, the visible bands were manually sectioned and stored in 10% EtOH at 4°C for further analysis.

Proteins sequencing

Samples were sent to EDyP-service platform, CEA Grenoble, where mass spectrometry analysis were performed. For this, samples were digested using trypsin, which is an endopeptidase cutting after arginines and lysines. Then, the peptides generated from the digestion of each sample were separated and their MS spectrum experimentally obtained were compared to a database of proteins digested in silico. Three different databases were used for the analysis of the peptides: N. Fumigata database, which contains 9600 proteins (96.9% annotation):

S. apiospermum database, which contains 8300 proteins (81,1% annotation) and an extended database containing all proteins of the Fungi taxonomy (11,000,000 proteins).

Results and discussion

Purification of native lectins

3-week-old fungal mycelia of A. fumigatus and S. apiospermum were used for the analysis. It is important to highlight that, even when both cultures were grown under identical conditions, they presented considerable morphological differences at harvesting time. Scedosporium mycelia showed highly organized All the bands were recovered from the gels and stored in EtOH 10% and the 22 stronger bands were selected for mass spectrometry analysis while the rest remained at 4°C until further analysis.

Sequencing of native lectins

Several peptides were successfully identified from each one of the bands that were analysed. However, when they were compared with the in silico digested databases of Neosartorya Fumigata (9600 proteins, 96.9% annotated) and S.

apiospermum (8300 proteins, 81.1% annotated), some of the bands with a lot of material (e.g., bands 1, 2 and 4) appeared to have a low signal (few identified peptides). This result was unexpected since the concentration of a protein sample can be roughly estimated using the "spectral count" corresponding to the number of mass spectra that allow the identification of a peptide in a sample. Therefore, abundant proteins will generally be identified with high spectral count and these results might imply that those proteins were not identified because their sequence was absent from the databases used. For this reason, we performed again the analysis using an extended database that contains all the proteins from the Fungi taxonomy (11,000,000 proteins) and, through this strategy, the problematic samples were successfully identified with high spectral counts.

These results can mean either that the identified proteins are a contamination of another species of fungi or that the N. Fumigata and S. apiospermum databases are not sufficiently annotated leading to the absence of those proteins in them whilst they display homology with proteins from another fungal species. This latest explanation seems to be more plausible since all the proteins identified in another species of fungi are lectins and their carbohydrate binding specificity is congruent with the one of the lectins identified as homologous. The possibility of cross contamination can, however, not be ruled out. The individual cases will be discussed in the following sections, where we present a detailed analysis the 22 protein bands analyzed, their evolutive relationship across the fungal kingdom and their putative role in the life cycle of S. apiospermum and A. fumigatus.

All details regarding the samples and their analysis are listed in Table 1. The list of peptides identified for each protein can be found in the supplementary information of the chapter.

123

Identification of SapCP from S. apiospermum

Four small proteins of ~13 kDa were recovered from the extracellular fraction of S. apiospermum (B40, B41, B43 and B53). Initially, we consider that those bands corresponded to different proteins since they were recovered using distinct affinity resin (Fetuin, Gal, Fuc and GlcNAc). Yet, according to the peptides identified by mass spectrometry, they all correspond to a hypothetical protein (accession number A0A084FZZ4) from the genome of S. apiospermum. This uncharacterized protein contains a secretory signal peptide of 18 residues and seems to belong to the Cerato-platanin proteins family (CPPs). This is a group of small secreted proteins with oligosaccharide-binding activity that are involved

Cerato-platanin proteins (CPPs) -generalities and biological roles

The family of CPPs is a group of small fungal proteins rich in cysteines that are relatively new to science [108]. This family was created in 2004 and owes its name to the phytotoxin ceratoplatanin (CP) produced by the ascomycete Ceratocystis platani, which is a 120 amino acids protein secreted extracellularly. In general, all the member of the CPPs family are highly secreted to the medium, so they are abundantly found in culture filtrates [109,110]. However, some studies have also reported their localization on fungal cell walls [111,112].

CP is considered as microbe-associated a molecular pattern (MAMP) due its

CPPs distribution

CPPs appear to exist only in fungi, bacteria, oomycota, plants but animals do not possess CP homologs. Genes encoding for CPPs have been found, so far, in more than 100 fungal genomes, including species from the ascomycota and basidiomycota branches of dikarya [109]. It is remarkable that, while basidiomycota possess multiple CPP encoding genes (>12), the ascomycota only possesses one homolog with few exceptions that might have up to 2 or 4 [108].

CPPs genes seem to be lost in early branches of jelly fungi as well as in some groups of yeast or yeast-like forms in their life cycle [108].

CPPs biochemical properties

Almost all CP homologues contain a secretory signal peptide, which explain their extracellular prevalence. The length of mature CPPs ranges from 105-134 amino acids per monomer and they can exist as dimers, trimers, tetramers or a mixture of those forms [112,128]. An N-glycosylation site is also present in most CPPs sequences, although no evidence of glycosylation has been found in CP in vivo [110]. Interestingly, during our analysis, the complete sequence of mature SapCP was identified in three out of four samples with exception of the peptide LTYQGKS (residues 82 -88), which was consistently missing in all of them. The absence of this peptide could be explained by the presence of glycosylations, which has been previously reported for some others CPPs, such as CS-Ag.

However, given that this peptide does not contain the signal sequence for Nglycosylation (Asn-X-Ser/Thr), we consider that the lack of its spectra must be due to an O-glycosylation at the residue of Threonine-83, Serine-88 or both.

CPPs structure

Although only five CPPs structures have been solved to date, the overall topology is highly conserved (Figure 5.6). CP presents a globular fold that contains two α-helices and six β-strands that form a double ψβ-barrel fold [113].

In CP and MpCP3-5, the external face of barrel reveals a shallow crevice rich in polar and aromatic residues, which is responsible for the interaction with N- SapCP contains all the conserved elements of the CPPs proteins family for its fold and for the recognition of polysaccharides. Its recovery from distinct affinity columns during our study can be explained by the fact that CPPs have been reported to bind different carbohydrate structures.

Potential applications of SapCP

Given the relevance of CPP in fungal development and its possible role during host recognition and colonization processes, there is an immeasurable number of possible uses for SapCP. Therefore, we consider that would be very interesting to achieve its full characterization.

SapCP could be used:

• As specific antigen for the detection of Scedosporium as in the case of CS-Ag from C. immitis.

• As a sensitization agent of the immune system for immunosuppressed patients.

• To elucidate its role during host-recognition in order to evaluate its potential as target for the development of anti-adhesive molecules.

• To evaluate if it is involved in remodelling and growth of the fungal cell wall. In such a case, it could become a target for the development of novel antifungal therapies.

• To elucidate the first crystallographic structure of a CP from human pathogens that could be used for drug design.

Identification of PVL-like lectins from S. apiospermum and A. fumigatus

A GlcNAc binding protein was recovered from the intracellular fraction of proteins from S. apiospermum and A. fumigatus (

Psathyrella velutina Lectin (PVL)

PVL is a 7 beta-propeller lectin of ~42 kDa with affinity for terminal GlcNac that has been purified from the fruiting bodies of the mushroom recently renamed Lacrymaria lacrymabunda (Figure 5.9) [START_REF] Cioci | β-Propeller crystal structure of Psathyrella velutina lectin: an integrin like fungal protein interacting with monosaccharides and calcium[END_REF].

PVL distribution in fungi kingdom and phylogeny

The presence of PVL-like sequences has been found in the genomes of other mushrooms and of several bacteria [START_REF] Cioci | β-Propeller crystal structure of Psathyrella velutina lectin: an integrin like fungal protein interacting with monosaccharides and calcium[END_REF] but its presence has not been reported in any microfungi to date. To better understand the implications of this distribution, first it is necessary to briefly review the fungi evolution.

The fungal kingdom is divided in "Early diverging fungi" and the "Dykaria subkingdom" (Figure 5.10). In turn, dikarya is composed by ascomycota and basidiomycota phylums, to which belong the vast majority of fungal species known to date. Ascomycetes are fungi with septate mycelium that produce endogenous ascospores. It is the largest taxonomic group and includes a great diversity of types of fungi, such as false mushrooms, truffles, terrestrial languages, most lichens, some molds and yeasts. On the other hand, basidiomycetes are fungi that contain a microscopic structure called basidia, which produces haploid spores by meiosis. This division includes macroscopic fungi such as the classic mushrooms, represented in edible fungi, toxic fungi, hallucinogenic fungi and phytopathogenic fungi. Due to the early divergence of these two phylums, there are considerable differences between their life cycle, reproduction and morphology. To date, the main fungal specimens where PVL-homologous lectins have been identified belong to the basidiomycota phylum, while S. apiospermum and A.

fumigatus belong to the ascomycota one. Therefore, excited by the possibility of having discovered for the first time a PVL-like lectin in ascomycetes, we look all the proteins registered in the Swiss database UniProt that display percentages of identity superior to > 50 % with PVL and as expected, all these proteins were found only in basidiomycetes. Surprisingly, the phylogenetic analysis of those sequences placed B1 and B7 as the proteins closer related to PVL (Figure 5.11), that is possible, but not plausible due to the evolutionary distance between these fungal species. Therefore, and considering the fact that we have previously worked in our laboratory with a recombinant version of PVL, the suspicion of a possible sample contamination aroused. To evaluate this, we compared the peptides identified for B1 and B7 with PVL-like proteins and we found that most them correspond to highly conserved regions, which, in conjunction with the size discrepancy between B1 and B7, seems to detract from the possibility of crosscontamination. However, further studies are required to confirm the presence of those PVL-like lectins in S. apiospermum and A. fumigatus.

Identification of the fucose-binding lectins FleA and SapL1

This section describes the identification of the fucose-binding lectins from A. fumigatus (FleA) and S. apiospermum (SapL1), which have been widely described 134 in other sections of the manuscript. Hence, generalities of these lectins are omitted here.

Identification of FleA from A. fumigatus

The fucose binding lectin FleA (accession numbers Q4WW81 and Q8NJT4) from Even thought, some SapL1 peptides were identified in B45, their spectral account was too low to be considered as evidence the identification of this lectin. This might be explained by the stage of development of the cultures, since it is known that this lectin is mainly expressed during the conidial stage. A different approach is being carried out to confirm the presence of SapL1 transcripts in mRNA from S. apiospermum conidia as they could not be isolated in mRNAs from mycelium.

Identification of AfFG-GAP repeat protein from A. fumigatus

A protein of approximately 33 kDa from the extracellular fraction of proteins of Some peptides matching with the same sequence were also found from a band of ~10 kDa with galactose binding properties (sample B25), however, the coverage of sequences and the spectral count were considerably lower than those found for B23 and B28. Therefore, we consider that the peptides identified in this sample

AfFG-GAPs putative role

In 2011, four protein samples covering 9-43% of the sequence of AfFG-GAP were also identified in a broad proteomic approach aimed to identify A. fumigatus antigens. The authors proposed that, due to its possible interaction with the βchain of human integrins, microbial secreted FG-GAP repeat proteins may be responsible for integrin-mediated cell adhesion and signal transduction during pathogenesis [136,137]. However, additional studies need to be conducted to validate AfFG-GAP biological roles and evaluate its potential for biotechnological applications.

Chapter V: Identification of native lectins from medical relevant fungal strains 140

Identification of CcMBL-homologs in A. fumigatus and S. apiospermum

Six of the samples sequenced display high identity with a hypothetical protein of ~13,7 kDa from the mushroom Coprinopsis cinerea (Figure 5.17

CcMBL-like proteins from S. apiospermum

In the case of Scedosporium, two fucose-binding lectins were identified from both intracellular and extracellular fractions. They have a molecular weight of ~13 kDa (B2 and B47) and ~20 kDa (B3 and B48). According to peptides recovered from mass spectrometry and the analysis performed with the three in silico digested databases, those four proteins are similar to CcMBL with B1 covering 100% of the sequence, B3 and B48 covering both 95.28% and B47 covering 78.74% (Table 5.1 and Figure 5.20). Initially, we considered that the size discrepancies between the pairs of proteins B2/B3 and B47/B48 could be due the presence of glycosylation in the biggest proteins (B3 and B47), since glycosidic moieties increase the size of peptides hindering their identification in the expected region for their mass spectra, which result in the lack of the corresponding sequence. So, following this idea, we proposed the residue of serine-80 as the most plausible candidate to be glycosylated since the only difference between the reconstructed sequences for B3 and B2 is the SPVGR peptide, which is missing only in B3. However, when bands of B47 and B48 were latter analyzed, SPVGR peptide could not be identified in any of them. So, there are two possible explanations for it: a) serine-80 is glycosylated in B3/B47 but not in B2/B48 and SPVGR peptide could not be identified in B48 due the low quantity of material in this sample.

b) this peptide was only identified in B2 because small peptides are difficult to detect in samples with low quantity of material. Hence, none of the proteins is glycosylated and that B3 and B47 correspond to thermostable protein dimers, that were not disrupted during the denaturation.

CcMBL-like proteins from A. fumigatus

During our second purification attempt, four fucose-binding lectins were recovered from the extract of A. fumigatus intracellular proteins (Figure 5.4). Only two were selected for identification: FleA, previously discussed and a small protein of ~13 kDa (B8) which corresponded to the most abundant protein whose peptides covered 83% of the CcMBL sequence. On the other hand, a CcMBL-like protein (B29) was also identified from the extracellular fraction of Aspergillus proteins but its peptides only gave a 78.74% sequence coverage. The only difference between those two samples was, again, the absence of SPVGR peptide in B29 (Figure 5.21).

As in Scedosporium samples, fucose-binding proteins of ~20 kDa were identified in both intracellular and extracellular fractions but those bands have not been selected for identification in that round (B9 and B30, Figure 5.4). The possibility of finding similar proteins to CcMBL on those samples is not ruled out.

CcMBL-like galactose-binding proteins

CcMBL similar proteins were also detected in the intracellular fraction of both fungi, S. spiospermum (B4) and A. fumigatus (B15) after galactose chromatography.

The analysis of mass spectrometry reveals that B4 actually contains a mixture of two different proteins. From one of them, 13 different peptides covering 99.21%

of the sequence of CcMBL were identified, while the second one corresponds to a peptidylprolyl isomerase from S. spiospermum that will be latter discussed. The presence of this second protein explains the drastic difference on the amount of material between B4 and B15.

B15 was originally listed as CcMBL related protein with a high confidence score (level 1), however in a second analysis this score was degraded to level 3 since the spectral count of this sample was very low [START_REF] Santiago Reis | Mycetomas: an epidemiological, etiological, clinical, laboratory and therapeutic review[END_REF]. This is common in samples with low concentration. Despite this fact, the 6 different peptides identified from this sample actually cover 55.12% of the CcMBL sequence and its presence is

Discussion

CcMBL-like proteins were identified from six of the selected bands from both fucose and galactose affinity chromatography. Their sequence identity with CcMBL seems to suggest an evolutionary relationship between them.

Nevertheless, it is not clear why these proteins were not annotated as hypothetical protein in both genomes. So, in order to inquire about the function of this protein, we performed a blast search for similar sequences in other organisms. Surprisingly, no hits were found in UniProt and no protein family was related to this sequence. This could be explained by the fact that C. cinerea paralog multi-copy genes are found in highly recombinant regions but this contrasts with our findings of highly similar sequences in both microfungi.

The affinity of those proteins is particularly interesting since it has been shown that mucins of cystic fibrosis lungs (CF) have an increased content of both fucose and galactose. The increment of those sugars, derives from the abundance of the blood antigen group Lewis-X at the non-reducing end of the mucins glycosylations [START_REF] Scanlin | Terminal glycosylation in cystic fibrosis[END_REF]. Therefore, the sugar-binding specificity of these proteins, would support the mucin-binding activity predicted for CcMBL. In conjunction, this information and the well-known role of mucins during fungal pathogenesis leads to the conclusion that the CcMBL-like proteins identified in this study might represent interesting drug targets. So, further studies need to be performed to confirm the presence of those proteins in the genomes of those microfungi and on their characterization.

Identification of SapFKBP12 from S. apiospermum

A strong band of ~12 kDa was recovered from galactose purification in the intracellular fraction of S. apiospermum (B4). This band contains CcMBL as previously discussed and a hypothetical peptidyl-prolyl isomerase (accession number A0A084G113), further referred as SapFKBP12. We don't know why this protein display affinity by galactose, but it is plausible that the overall fold of this monosaccharide resembles the structure of the SapFKBP12 substrate (peptidylproline). Surprisingly, SapFKBP12 could not be found when the peptide spectra were compared to the Scedosporium in silico digested database, but it was possible to identify it when the files were re-interrogated against an expanded database containing all the proteins of the fungal taxonomy. Paradoxically, it is listed as a S. apiospermum protein in the fungal database, which seems to be a good example of how incomplete annotation of databases and strains sequences variations might greatly influence the identification of proteins by mass spectrometry. The peptides identified cover 92.79% of the sequence of SapFKBP12, whose prevalence in B4 seems to correspond only approximately ~25% of the total amount of protein in the band according to the spectral counts (Figure 5.23).

SapFKBP12and immunophilins

SapFKBP12 belongs to the family of immunophilins that is a highly conserved cluster of intracellular peptidyl-prolyl isomerases (PPIases) catalyzing the interconversion between the cis and trans isomers of peptide bonds containing proline [138]. These proteins are important pharmacological targets against fungal infections and act as intracellular receptors for immunosuppressive drugs such as rapamycin, cyclosporine and tacrolimus (originally called FK506).

Nevertheless, their isomerase activity does not appear to be related to their binding to these compounds [139].

Immunophilins-functions and classification

Immunophilins act as proteins chaperons and are fundamental for the proper folding of diverse proteins. Some of them are also involved in specific regulatory process including cellular signaling, apoptosis and transcription mechanisms [140,141]. Immunophilins are traditionally classified into two subfamilies:

"cyclosporin-binding cyclophilins (CyPs)" and "FK506-binding proteins (FKBPs)". Besides, they can be grouped into a subclassification according to their cellular distribution [142]. According to our analysis, the ~12 kDa protein that we have identified (A0A084G113) belongs to the FKBPs and more specifically to the subgroup of cytoplasmic immunophilins FKBP12 so, we named it SapFKBP12.

FKBP12 subfamily owes its name to the first member discovered, which is a small protein constitutively expressed in all human tissues (hFKBP12). hFKBP12

homologs have been identified in a very wide range of eukaryotes [143]. The sequences of all the member of this subfamily are highly conserved and, in all cases, they correspond to small cytosolic proteins of ~12 kDa with at least one peptidyl-prolyl isomerases (PPIase) domain. As all the other subfamilies of FKBPs proteins, FKBP12 members display affinity by the immunosuppressive drug FK506 (tacrolimus).

Calcineurin inhibition as antifungal therapy

In the presence of tacrolimus (FK506), FKBP12 proteins form a stable intracellular complex (FKBP12-FK506) that inhibits the phosphatase calcineurin (CaN) and Consequently, calcineurin is considered as one of the most promising targets for antifungal drug development.

• In human cells, the function of CaN is to dephosphorylate the nuclear factor of activated T cells (NFAT). This phosphorylation allows it to be translocated to the nucleus where it cooperates with other nuclear transcription factors to induce the synthesis of interleukin-2 (IL-2) and initiate the transcription program for the activation of T cells. Therefore, CaN inhibition leads towards suppression of the immune system in humans.

FKBP12 and the panfungal strategy

Due to its central role in fungal pathogenesis, calcineurin is an attractive pharmacological target. Nevertheless, the development of specific antifungals targeting CaN inhibition is complex to address due the great similarity between human and fungal calcineurins whose catalytic (CnA) and a regulatory (CnB) subunits are nearly identical [143,147,148].

In this context, FKBPs play an essential role since it has been shown that fungal-FKBPs share only 40-50% of identity with their human counterpart [143].

Consequently, the development of novel drugs targeting FKBPs is emerging as an alternative for the upstream inhibition of CaN in a highly specific manner. An additional advantage of this approach, lies in the fact that FKBPs are very well distributed among the fungal kingdom and their sequences are highly conserved between human pathogens. Hence, fungal FKBP12s have emerged a prime candidate for the development of broad-spectrum antifungals and their discovery from emerging fungal pathogens will significantly aid in the development of a panfungal strategy (Figure 5.25). . In all the fungal complexes, the overall fold of calcineurin was very similar to its human homologous (PDB ID: 4OR9) previously published by Griffith et al. 1995 [149]. While, the conformation of fungal and human FKBP12 (hFKBP12) revealed relevant differences, especially in the loops 40s and 80s, where most of the residues involved in FK506 are located (Figure 5.26). Most of those residues are also conserved in SapFKBP12 (Figure 5.26B, black triangles). They have also identified a phenylalanine (F88) residue that is critical for binding and is conserved among the fungal FKBP12 whilst it is replaced by a histidine in hFKBP12 (Figure 5.26B, yellow star). Hence, this residue has been proposed as in important guide in the development of FK506 analogs specific for the inhibition of fungal CaN.

SapFKBP12 potential applications

We have shown that the residues critical for FK506 binding are well conserved among SapFKBP12 and the FKBP12s from other fungal pathogens that display susceptibility towards tracolimus (FK506) treatment, such as the case of A.

fumigatus, C. albicans, C. neoformans and C. immitis. This information is of utmost importance because it suggests the clinical use of tracolimus as a potential treatment in non-systemic scedosporiosis such as topical infections. This is the most common affection caused by this pathogen in immunocompetent patients and whose treatment is very challenging. Furthermore, the identification of SapFKBP12 will also contribute to the development of new treatments not only against Scedosporium infections but also in the development of a broad-spectrum antifungal therapy.

Conclusions

The analysis of peptides obtained by mass spectrometry led to the identification 8 different proteins from the 22 bands that were analysed by mass spectrometry.

Five of them were found in the protein extracts of S. apiospermum and 4 were identified from A. fumigatus extracts. The preliminary analysis of those proteins revealed that some might be attractive pharmacological targets, and others could find application as biotechnological tools. We consider that the proteins that deserve special attention are SapFKBP12 and CcMBL as pharmacological targets and SapCP as allergen or as antigens for the specific detection of Scedosporium in humans. The two PVL-like lectins could also be of medical relevance; however, their identification needs to be confirmed. On the other hand, we don't discard the possibility that in the 32 samples no sequenced there are also lectins that remain unidentified. Hence, the study of these samples should be addressed in the future.

The information provided here represents a general strategy for approaching the study of the lectinome of those two species of these fungal pathogens and, we hope that our findings will encourage the study and characterization of those new proteins, in the search for biomedical applications and new drug targets.

Contributions

Supplementary information

GCFs; A NEW MINI GLYCANARRAY FOR IDENTIFICATION AND PURIFICATION OF LECTINS

Summary

Due to their biological relevance and possible applications, lectins have arisen the attention of the scientific community, either to explore their implementation as tools or to propose them as drug targets. Among the strategies for lectin purification, sugar-based affinity chromatography is by far the most widely used.

However, this strategy has several disadvantages that slow down considerably the purification process, preventing the discovery of new lectins. For this reason, there is a growing interest in the development of non-column-based methodologies for lectin purification.

In this chapter, we report the development of a new tool that allows the identification and purification of unknown lectins directly from crude protein extracts. This tool assembles the fusion of the three classical steps of lectin purification : clarification, concentration and separation in a single operating unit. It also allows the simultaneous purification of lectins with different specificities in a quick and simple process. In addition, it can be used as a "mini glycan array" for the study of carbohydrate lectin interactions without the need for any labeling. Hence, we consider that this device might greatly contribute to the study of the lectinome.

Introduction

Lectins are powerful tools in the study of complex oligosaccharides and can be used for purification of industry-relevant glycans [150]. They also provide valuable insights for diagnostics and drug discovery. Therefore, it is expected that those proteins will occupy an important place in the biopharmaceutical industry in the coming decades [151]. Some of the most relevant biotechnology [159,160], development of antiadhesive therapies against fungi, bacteria and virus [START_REF] Krachler | Targeting the bacteria-host interface strategies in anti-adhesion therapy[END_REF][START_REF] Koharudin | The evolutionarily conserved family of cyanovirin-N homologs : structures and carbohydrate specificity[END_REF][START_REF] Mohamed | Mushroom lectins : Specificity , structure and bioactivity relevant to human disease[END_REF][START_REF] Shoaf-Sweeney | Adherence, anti-adherence, and oligosaccharides: preventing pathogens from sticking to the host[END_REF][START_REF] Varki | Microbial lectins: hemagglutinins, adhesins, and toxins[END_REF][START_REF] Lehot | Multivalent fucosides with nanomolar affinity for the Aspergillus fumigatus lectin FleA prevent spore adhesion to pneumocytes[END_REF][START_REF] Goyard | Multivalent glycomimetics with affinity and selectivity toward fucose-binding receptors from emerging pathogens[END_REF][START_REF] Akkouh | Lectins with anti-HIV activity: A review[END_REF][START_REF] Fang | A lectin with anti-HIV-1 reverse transcriptase, antitumor, and nitric oxide inducing activities from seeds of Phaseolus vulgaris cv. Extralong Autumn Purple Bean[END_REF], among others. For all these reasons, there is an emerging interest in the identification of new lectins and in the development of novel approaches to generate efficient alternatives for their purification.

Common strategies for lectins purification

Native lectins are purified from macerated tissues through a purification train that typically includes from three to five steps. Depending on the type of sample, it may be necessary to pre-treat the crude extracts, either by using ammonium sulphate or ethanol to precipitate proteins. Then, the process continues employing one or several chromatography steps based on gel-filtration, ionexchange, hydrophobic interaction, etc. but affinity chromatography is, by far, the most commonly used. This latter one takes advantage of the carbohydratebinding properties of lectins and use sugar-based matrices to purify them. Sugars are usually used for elution but EDTA can be also applied in the case of cation dependent binding [161,162]. The most popular matrices nowadays are glucosebased Sephadex TM and the galactose-based Sepharose TM , however, the number of requirements for the purification and/or identification of lectins that bind exclusively to complex glycans, such as in the case of PHA:L, for example. This generates a gap in the study and identification of an immeasurable number of unknown lectins that could have important applications or could be a key piece to elucidate cellular processes that remain unclear. Furthermore, this system requires hard maintenance when crude extracts are loaded into the columns because such samples contain pigments, oily components, proteolytic enzymes, and other complex substances that could damage the column and impair the purification [237].

One of the strategies that have been most widely used to avoid the drawbacks associated with the purification of native lectins is their heterologous production in genetically modified organisms (GMO) such as bacteria and yeast, among others. These recombinant proteins generally include fusion tag to facilitate their expression in soluble form (MBP, GST tags) and their purification (6-His tag).

However, since it is necessary to know the sequence of the protein a priori, this technology is limited to lectins that have been previously identified. In exceptional cases, their sequences can also be tracked by data mining when the genome of the source organism is available. For all these reasons, there is a growing interest in the development of non-column-based methodologies for lectin purification, with a particular focus on those with the plasticity to adapt to specific requirements of particular cases.

Here, we report the development of a new tool that assembles the fusion of the tree classic steps of lectin purification train: clarification, concentration and separation in a single operating unit. Furthermore, this tool allows the identification of unknown lectins from crude extracts and can also be used as a mini array to identify their carbohydrate specificity.

Brief description of the device

The device comprises the modification of borosilicate microfiber filters by coating their surfaces with a homogeneous layer of epoxides that can be covalently attached to any glycan, protein, antibody or compound with a nucleophilic nature. In this particular work, we have use glycosides attached to spacers with primary amines to guaranty their proper exposure, however, any sugar is susceptible to coupled. In the case of non-modified sugars, it is important to consider that, even when the alpha hydroxyl is the most susceptible to react, all the hydroxyls are susceptible to coupling.

The novelty of the device lies in the fact that, due to its porous nature, once the filter is modified it can be coupled to a vacuum filtration system to treat large volumes of samples. This allows lectins, even those found in very small concentrations, to be retained by interactions with the sugar-coated surface. Once the sample is eluted, the filter is rinsed and sectioned (in the case of multi-glycans filters). Then, bounded lectins can be eluted by two different ways:

1) Competitive inhibition where the filter is immersed in a solution containing the same sugar that is on the surface.

2) Denaturation where the filter is immersed in PBS and heated to later proceed to SDS-PAGE gel electrophoresis.

Elution by competitive inhibition allows recovering of native lectins without affecting their ability to bind to glycans, making it very useful in the cases where future activity studies are required. The elution by denaturation is exclusively used for the identification of lectins (Figure 6.1). In this case, the lectin profile obtained from each organism will contribute to lay the foundations for the study of its lectinome. Finally and most important of all, we have discovered that a simple protein staining can be applied to identify the area of the surface where lectins are bound. This allows to identify carbohydrate specificity in a similar way to other glycan arrays but without the need for labeling nor specialized equipment. This makes it an accessible strategy for any laboratory.

Material and methods

Synthesis of 2´-Chloroethyl 2,3,4-Tri-O-acetyl-α-L-fucopyranoside

A mixture of L-fucose (1 g), 2-chloroethoxyethanol (7.5 mL), and Amberlite IR-120 Resin (H +) (0.6 g) was stirred under reflux at 80 °C for 5 h. Then, the mixture was cooled to room temperature (RT) and the resin was removed by filtration.

The excess of solvent was evaporated at low pressure and residues were dissolved in pyridine (12 mL) and placed in a cool bath with stirring, while acetic anhydride was periodically added (6 mL). The ice bath was removed and the reaction proceeded at RT overnight. After the reaction was complete, a liquid extraction was performed with water and ethyl acetate. The organic layer was dried by adding sodium sulfate, filtered and evaporated. Then, compounds were purified by flash column chromatography on silica gel using EtOAc-Petroleum ether (8:2, v/v) as mobile phase to obtain separately the α and β conformations of the per-O-acetylated fucoside, which was obtained in a α:β ratio of approximately 7:3.

Synthesis of 2-(2-cloroethoxy)ethylper-O-acetylated glucosides

Mixtures containing 2-(2-cloroethoxy)ethanol (7.5 mL), Amberlite IR-120 Resin (H +) (0.6 g) and 1 gr of the respective glycoside (D-mannose, D-glucose and Dgalactose) were stirred at 80 °C for 5 h. The mixtures were cooled to RT and filtrated. Then, solvent was evaporated and glycosides were purified by flash column chromatography on silica gel using DCM:MeOH (8:2, v/v). Acetylation was carried out as described previously for the fucoside, followed by liquid extraction (also described previously). Per-O-acetylated glycosides were purified by flash column chromatography on silica gel using EtOAc-Petroleum ether (8:2, v/v).

General procedures for azidation of glycosides

A suspension of the respective glycoside (0.5 mM, 1 eq), TBAI (0.2 eq), and sodium azide (5 eq) in N,N-dimethylformamide was stirred at 90 °C for 18 h. The N,Ndimethylformamide was evaporated at reduced pressure and the residue was partitioned between dichloromethane (150 mL) and water (30 mL). The organic layer was separated, washed with brine (3X 30 mL), dried over sodium sulfate, filtered and evaporated.

General procedures for deprotection of glycosides

The per-O-acetylated glycosides were treated with a catalytic amount of MeONa in MeOH (10 mL) for 30 min and then the reaction was neutralized with Amberlite IR-120 Resin (H +). The resin was removed by filtration and the solvent was evaporated.

General procedures for the reduction of azido glycosides

Solutions of the respective 2-(2-aminoethoxy)ethyl galactoside, 2-(2aminoethoxy)ethyl mannoside, 2-(2-aminoethoxy)ethyl glucoside and 2´-azidoethyl fucoside were prepared in methanol at 1 mM. Then, palladium (Pd was added in catalytic amount and solutions were stirred at room temperature under hydrogen atmosphere for 12-18 h, until the disappearance of the starting material. Mixtures were filtered through a pad of celite, solids were washed with methanol and the filtrates were evaporated at a reduced pressure.

Hydroxylation of borosilicate fibers

The borosilicate microfiber filters were rinsed with acetone for 5 min in orbital agitation to remove any adsorbed chemical species from the surface. Then, filters were allowed to dry at RT until complete evaporation of acetone, since traces of organic solvent react explosively during the next step. Once acetone was completely evaporated, filters were immersed into sulphuric acid and hydrogen peroxide (3:1 ratio) for 10 s. After, filters were immediately submerged in toluene (99.8%, v/v) for 15 seconds and rinsed twice with sterile PBS buffer pH 7.4.

Finally, the filters were dry at 110° C for 45 minutes.

Silane coupling (Self-assembled monolayer)

Filters were covered with 4% GPS ((3-glycidyloxypropyl)trimethoxysilane) dissolved in dry toluene and placed in a nitrogen environment in absence of light.

This allows a homogeneous coupling of the silane with the hydroxides previously generated on the borosilicate microfibers and prevents selfpolymerization. After 16 h, the GPS-coated filters were removed from this solution, rinsed with ethanol and were allowed to dry at RT.

Monodeposition of glycosides on GPS-coated filters

Activated filters were placed in individual containers and covered by a 5 mM solution of the respective glycoside (1, 2, 3 and 4) dissolved in sterile PBS pH 7.4.

Containers were closed and incubated at slow orbital agitation at room temperature for 2 h. Afterwards, the substrate was rinsed several times with PBS buffer to remove any unbound compound from the surface. Finally, the glycoside-coated filters (GCFs) were incubated 2 h in a solution containing 50 mM of urea and 50 mM of glycine to open remaining epoxides.

Multi-spot deposition of glycosides on GPS-coated filters

Compounds 1, 2, 3 and 4 were dissolved at 50 mM in MeOH and a 2 µl of each solution were pipetted onto different areas of the GPS-coated filters surface.

Spots were allowed to dry at RT and the unbound glycosides were removed by eluting 100 mL of PBS buffer trough the filters by applying vacuum into a Buchner funnel. Finally, filters were incubated 2 h in a solution containing 50 mM of urea and 50 mM of glycine to open remaining epoxides.

Lectin binding to glycoside-coated filters

Solutions containing 200 µg/mL of purified lectins that have affinity for the prepared glycosides. Each sample also contained 200 µg/mL BSA as a negative control for testing protein adsorption. The GCFs were placed in a Buchner funnel, incubated and 10 mL of the respective proteins cocktail was eluted applying vacuum. To visualize proteins attached to the surface InstantBlue R protein stain was used.

Protein denaturing electrophoresis (SDS-PAGE)

All SDS-PAGE electrophoresis were performed on 15% polyacrylamide gels with a current of 180 mV in a Mini-Protean Tetra Electrophoresis Cell kit from Bio-Rad (Hercules, CA, EU.), using a molecular weight marker wide range. To visualize the result of the runs, the gels were stained with InstantBlue R 10 min.

Results and discussion

Glycosides synthesis

The derivatized monosaccharides 1, 2, 3 and 4 were synthesized through a sequence of reactions that involves attaching a chloro-alcohol to the anomeric xposition of the sugars, followed by nucleophilic substitution of chlorine for an azido group (N3) and its subsequent reduction to a primary amine. Intermediate steps of protection and deprotection were performed to facilitate purification of the intermediate products by flash column chromatography and to avoid unspecific reactions with the remaining OHs of the ring (Figure 6.2).

Borosilicate surface modification

Borosilicate (ZnO) microfiber filters were hydroxylated by immersion in piranha solution. Then, a GPS-Self Assembled Monolayer (GPS-SAM) was achieved by liquid deposition under N2 atmosphere. This reaction leaves available epoxide residue to interacts with the amino group of the previously prepared glycosides to covalently attach them to the surface (Figure 6.3). Glycosides-coated filters (GCFs) with compounds 1, 2, 3 and 4 were prepared to generate mannose-coated Filters (Man-CF), glucose-coated filters (Glu-CF), galactose-coated filters (Gal-CF) and fucose coated filters (Fuc-CF), respectively.

Specific interaction between lectins and glycosides-coated filters

To determine if lectins can bind to the carbohydrates on borosilicate surfaces, the previously coated filters (Man-CF, Glc-CF, Gal-CF and Fuc-CF) were incubated in a PBS solution containing a lectin with known affinity for the respective glycoside. Thus, FleA from A. fumigatus [START_REF] Sakai | Fucose-specific lectin of Aspergillus fumigatus: binding properties and effects on immune response stimulation[END_REF] was used for Fuc-CF, LecA from

Pseudomonas aeruginosa [238] for Gal-CF and due to its dual recognition for glucose and mannose, ConA from Canavalia ensiformis [239] was used for Man-CF and Glc-CF. Each solution also contained Bovine Serum Albumin (BSA) as a negative control to rule out protein adsorption. Then, filters were rinsed, immersed in 1 mLl of PBS and heated at 100 °C for 10 minutes. The presence of proteins in all fractions was verified by SDS-Page, trough it was found that only the respective lectin was attached to the filters after the washing steps (Figure 6.5A-D). This result indicates that the interaction lectin-sugar is taking place and confirms that non-interacting proteins can be removed through simple washing steps. It also evidences that the rest of the matrix does not display unspecific interactions with common proteins. Once the interaction between lectins and our glycosides coated filters (GCFs) was established, the next question to address was if the proteins could be recovered from the surface. Therefore, a preliminary study was conducted with Glc-CF and ConA where, the filter was split into two equivalent portions after washing and one of them was incubated in PBS containing 50 mM glucose, instead of heating.

ConA was recovered when glucose was added to the buffer as expected (Figure 6.4E). In a later study, a Fuc-CF was incubated with a solution of PBS containing FleA at 10 µg/mL and BSA at 200 mg/mL. After elution with fucose, the electrophoretic profile shows that FleA was retained on the filter and that its concentration in the elution fraction was considerable higher that in the original solution. This confirms that the filters can bind lectins, even when they are in a very low concentration with respect to other proteins in the sample and establishes the filter utility as concentration tools (Figure 6.4 F).

Mini chip for detection of protein-glycan interactions on unlabeled samples

One of the main characteristics of glass microfiber filters is that they are inert, do not retain small molecules and do not establish electrostatic interactions. So, theoretically, they should not interact with the anionic dye Coomassie R-250, present in the InstantBlue solution. If this was correct and we manage to find the right conditions to deposit several glycosides on different areas of the same filter, it would mean that the lectins attached to them could be in situ stained. Thus, generating a system for detection of lectin-carbohydrate interactions, equivalent to the glycan arrays but without the need for any labelling. From a practical point of view, this strategy has many advantages compared to conventional arrays, not only because it can be performed in unlabeled samples, but also, it is simple and can be carried out in any laboratory with minimal requirement of equipment.

To confirm the absence of interactions between filter and colorant, we dipped untreated filters in the InstantBlue solution during 1 h and rinsed them in water.

They were still completely white indicating that no dye particles remained on the surface after treatment (data not shown). Then, we proceeded to find the ideal conditions to achieve the localized deposition of glycosides on the surface of the filters (data not shown). In the final experiment, we use a pencil to draw a circle in both, a GPS-coated filter and an untreated filter. Then 2 µl of mannoside (1) (50 mM in MeOH) were pipetted inside of each circle. Solutions were allowed to dry and the excess of glycoside was removed with PBS. The filters were incubated 2 h in a solution containing urea and glycine to deactivate unreactive GPS (from the surface that was no in contact with the mannoside solution). Each filter was latterly incubated with ConA for 1 hour, followed by washing steps and staining with InstantBlue solution. After 15 minutes, the Man-CF displayed an intense blue coloration on the surface where mannoside had been spotted. The back also showed an intense coloration in this area, indicating that when the glycoside was deposited on the filter surface, it penetrated to deeper layers and was homogeneously attached throughout the thickness of the filter. In the case of the untreated filter, no deposition of the dye was observed, either in the front nor the backside (Figure 6.5A). The slight coloration on the rest of the Man-CF may be attributed to the interaction of the colorant with the free carboxyl group of glycine used to deactivate unreactive epoxides. Now, we are working on new protocols to deactivate the remaining epoxides through the use of other bases such as ethanolamine. This experiment evidences that the glycosides deposition can be guided and confirms that GCFs could be used as mini arrays for the study of carbohydrate-protein interactions. With the evidence provided by the previous experiments, we decided to make our first mini array prototype. For this, we attach the glycosides 1, 2, 3 and 4 to different quadrants of GPS-coated filters. This was done by duplicate and after the glycosides were attached, one of the filters was immersed in 4% sulphuric acid (H2SO4) and dry with a heat gun to reveal the presence of sugar spots on the surface. The other filter was incubated in a PBS solution containing 200 µg/mL of ConA.

Sulphuric acid staining confirmed that all glycosides were properly attached to the surface and the protein staining revealed that, as expected, ConA was mostly linked to the glucose/mannose-coated spots (Figure 6.5B). A slight change in coloration was also detected in the fucose-coated spot suggesting a possible interaction with this sugar. Further experiments will address the study of this interaction by ITC. On the other hand, no signal was detected at all on the galactose-spotted quadrant, despite the fact that its successful deposition had been previously confirmed. This result evidences that the differential binding of ConA to the surface is ruled by the sugar in the respective area and sets the GCFs as useful mini arrays for unlabeled samples.

Recovery of lectins from complex protein mixtures

The next step in our research was to validate if the GCFs would keep their functionality when complex mixtures of proteins are applied. For this, we expressed recombinant FleA in E. coli and treated the extract containing soluble intracellular proteins with a Fuc-CF. On the resulting electrophoretic profile, most of the protein bound to the filter corresponds to FleA, however, it is still necessary to optimize the washing steps to get rid of the contaminant proteins observed on the gel (Figure 6.6). The possibility that those other proteins correspond to endogenous fucose binding lectins from E. coli is not ruled out and it will be addressed in the near future.

Conclusions

The GCFs represent an interesting multipurpose tool that can be used both, as a lectin purification system for identification purposes and as a device to study carbohydrate-protein interactions.

Some of the advantages of GCFs as a purification tool are that: i) they avoid the inconveniences associated with prepackaging, maintenance and cleaning of columns; ii) the recovery of surface-bound lectins is easily accessible due to the flexible nature and easy handling of the filters; iii) the strategy allows the process of lectin purification quickly and efficiently since it merges several steps of the classical train of lectin purification and iv) the use of this tool is easy and costeffective. The most significant expenses are given by the cost of the sugars attached to the surface.

On the other hand, concerning its potential use as a glycan matrix, the device has the advantages that i) no prior purification and labeling of proteins is required;

ii) identification of interactions in situ is simple and iii) no specialized equipment is required for data acquisition nor manipulation.

Finally, since any nucleophilic molecule can bind to the surface (proteins, nucleic acids, natural glycans, antibodies, etc.), the adaptation of this methodology could

GENERAL CONSLUSIONS AND PERSPECTIVES

The emergence of new pathogenic species represents a global health problem that claims millions of lives each year. Furthermore, the limited susceptibility of these This project arises from the urgent need to generate information to guide the development of antifungals against the emerging pathogenic fungus Scedosporium apisospermum. The main objective of the thesis was the identification and characterization of its lectins, as a strategy to contribute to the general understanding of its host-pathogen interactions during the early stages of infection. For this, we have developed 4 independent but related projects:

Project 1-Identification and characterization of SapL1; a new target for the development of antiadhesive glycodrugs from S. apiospermum.

In this study, we present the identification, production, and characterization of a new carbohydrate-binding protein from S. apiospermum. To the best of our knowledge, this is the first studied lectin for this opportunistic pathogen. SapL1 is homologous to the conidial surface lectin FleA from A. fumigatus, known to be involved in adhesion to the host glycoconjugates present in mucins and human lung epithelium.

Here, we demonstrated that SapL1 is strictly specific for fucosylated carbohydrates and recognized all blood group types. These results are particularly interesting since it has been shown that there is six times more content of fucose α1,3/4 linked to glycoproteins in the CF airways, where Scedosporium colonization is more frequent. SapL1 adopts the 6-bladed βpropeller fold and forms a dimer with 6 carbohydrate-binding pockets in each face. Within our analysis, we have found that these binding pockets are nonequivalent, but they all share the features necessary for fucose recognition. The specificity and affinity of each one of the six binding pockets of SapL1 have been deeply analyzed to highlight the features that must be explored for the design of efficient inhibitors.

The detailed information exposed here places SapL1 as a promising target for the treatment of Scedosporium infections and will be of great value to guide the development of antiadhesive glycodrugs against this pathogen. SapL6 was successfully produced and characterized, both biochemically and structurally, however, despite our efforts it was not possible to identify any carbohydrate-binding activity. Nevertheless, we believe that the identification of new CVNH such as SapL6 will contribute to the general understanding of this family of proteins. Furthermore, the structural information gathered here will be useful to address possible interactions with glycans through in silico analysis and might guide towards the discovery of its natural ligand. Which, we consider, is the ultimate clue to finally understanding the biological role of CVNHs in fungi and to validate their potential uses as drug targets.

Project 3-Identification of new native lectins from protein extracts of medical relevant fungal strains

The main aim of this project was to identify new native lectins from the human opportunistic fungi S. apiospermum and A. fumigatus. The analysis of their intracellular proteins and secretomes leaded us to the identification of 8 different proteins. Five of them were found in the protein extracts of S. apiospermum and 4

were identified from A. fumigatus extracts. The preliminary analysis of those proteins revealed that some might be attractive pharmacological targets and others could find application as biotechnological tools.

SapCP: This protein was the most abundant protein secreted to the culture media of S. apiospermum. SapCP belongs to the family of cerato-platanin proteins (CPPs)

whose members bind to multiple polysaccharides and are essential for the fungal life cycle. Some of them also participate in the colonization and host recognition process, while others have found biotechnological applications as allergens or as antigens for detection of specific fungal pathogens.

PVL-like lectins: two GlcNAc-binding lectins were identified from the intracellular fraction of proteins of S. apiospermum and A. fumigatus. Both proteins display high similarity with the protein PVL from the saprophytic fungi Psathyrella velutina, which is a GlcNAc-binding lectin involved in the colonization of soils and host recognition. PVL-homologs are widely distributed in some branches of the fungal kingdom but it has never been identified in human pathogens. Therefore, in case of being confirmed, this finding might have important implications for the subsequent study of infections caused by those two microfungi.

FleA:

The fucose binding lectin FleA from A. fumigatus was unambiguously identified in both, the intracellular and extracellular fraction of proteins of Aspergillus. FleA is a lectin found on the surface of the Aspergillus conidia and shows a strong pro-inflammatory effect on human bronchial cells. This protein displays affinity by fucosylated carbohydrates and recognizes human blood group antigens (BGA). Therefore, it has been suggested that FleA mediates the binding of A. fumigatus to airways in a fucose-dependent manner.

AfFG-GAP:

The mannose/galactose binding protein AfFG-GAP was highly secreted to the extracellular media by A. fumigatus. The overall fold this protein is a β-propeller composed of two FG-GAP repeats. This domain is commonly found in the N-terminal region of the α-chains of integrins, a region shown to be important for ligand binding and protein-protein interactions with some components of the extracellular matrix. Hence, it has been proposed that AfFG-GAP could be involved in cell adhesion and/or the transduction of signals for immune recognition of A.

fumigatus.

CcMBL-like proteins: we identify six samples that display high similarity to a hypothetical mucin-binding lectin from the mushroom Coprinopsis cinerea (CcMBL). CcMBL-like proteins were recovered from both, fucose and galactose affinity chromatographies. This fact supports the mucin-binding activity predicted for CcMBL since it has been shown that mucins of cystic fibrosis lungs (CF) have an increased content of those monosaccharides. Therefore, considering the well-known role that aberrant glycosylation of mucins displays during fungal pathogenesis in CF patients, these proteins might represent interesting drug targets.

SapFKBP12: SapFKBP12 belongs to the family of cytoplasmic immunophilins FKBP12. These proteins are highly conserved among fungal pathogens and can interfere with the transcription of virulence factors and genes involved in hyphal growth. FKBP12 proteins own their name to their capacity to bind the immunosuppressant FK506. This drug has shown to be very effective against deadly fungal pathogens but is still contraindicated in systemic infections due to its immunosuppressant side effects. Hence, the synthesis of FK506 analogous with greater specificity is needed and the discovery of FKB12s from emerging fungal pathogens would significantly aid their development. On the other hand, we have shown that residues critical for FK506 binding are well conserved between SapFKBP12 and FKBP12 from other FK506-susceptible fungal pathogens, such as A. fumigatus and Candida albicans. This information suggests that Scedosporium might also be susceptible to this drug and future clinical approaches must explore its use as a treatment in non-systemic infections, such as the increasingly common topical scedosporiosis. The identification of SapFKBP12 and its future characterization will greatly contribute to explore new treatments not only against Scedosporium infections but also contribute to the development of a broad-spectrum antifungal therapy.

Project 4-GCFs; a new mini glycan array for identification and purification of lectins

The challenges we faced during the development of the first 3 projects of this thesis led us to raise the need to generate an efficient tool for the identification of new lectins. The original idea was to generate a device that would allow the simultaneous purification of lectins with different specificities, exclusively for identification purposes. However, as the project progressed, we realized that the tool we had created (GCFs) would not only allow the purification of multiple lectins but also facilitate the process and, additionally, could be used as a "miniglycan matrix". Hence, the GCFs represent an interesting multipurpose tool that can be used both, as a lectin purification system and to study carbohydrate lectin interactions.

Concerning its potential use as a glycan matrix, the device has the advantage that no prior protein labeling is required. Furthermore, the identification of binding interactions is simple and does not require specialized equipment for data acquisition nor manipulation. On the other hand, as a purification tool, the most outstanding advantage of GCFs is that they allow the process to be carried out in a fast and efficient way since it merges several steps of the classic lectin purification train. This might impulse the characterization of lectinomes in the coming decades. But farther than that, this methodology can be adapted to study any other interactions e.g., enzyme activity/inhibition assays, antibody screening, interactions of whole cells with surfaces, enzyme activities, DNA / RNA, etc.

Hence, we consider our strategy could be used beyond the glycobiology approach and to be applied in many other areas of science.

6. 1

 1 Summary .............................................................................................................. 6.2 Introducton .......................................................................................................... 6.2.2 Common strategies for lectins purification ...................................................... 6.2.3 Drawbacks of sugar-based affinity chromatography ....................................... 6.2.4 Brief description of the device ..........................................................................

6. 4

 4 Results and discussion ...................................................................................... 6.4.1 Glycosides synthesis ........................................................................................ 6.4.2 Borosilicate surface modification ..................................................................... 6.4.3 Specific interaction between lectins and glycosides-coated filters ................... 6.4.4 Mini chip for detection of protein-glycan interactions on unlabeled samples . 6.4.5 Recovery of lectins from complex protein mixtures ......................................... 6.5 Conclusions .................................................................................................................................................................................................................

Figure 1 . 1 :

 11 Figure 1.1: Scedosporium genus and S. apiospermum ................................................

Figure 1 . 2 :

 12 Figure 1.2: Scedosporium spp pathogeny ...................................................................

Figure 1 . 3 .

 13 Figure 1.3. Global distribution and relative burden of scedosporiosis ................

Figure 1 . 4 :

 14 Figure 1.4: Scedosporium apiospermum in the Cystic Fibrosis context....................

Figure 1 . 5 :

 15 Figure 1.5: Carbohydrates-lectin interactions. .........................................................

Figure 1 . 6 :

 16 Figure 1.6: Carbohydrate recognition domain of lectins .......................................

Figure 1 . 7 :

 17 Figure 1.7: Schematic representation of lectins symmetry ...................................

Figure 1 . 8 :

 18 Figure 1.8: Examples of glycan determinants recognized by different lectins. ..

Figure 1 . 9 :

 19 Figure 1.9: Roles and potential applications of fungal lectins. ..............................

Figure 1 . 10 :

 110 Figure 1.10: Fungal adhesion to epithelial cells.......................................................

Figure 1 . 11 :

 111 Figure 1.11: Overall structure of FleA and AFL6. ...................................................

Figure 2 :

 2 Figure 2: Thesis results overview ..............................................................................

Figure 3 . 1 :

 31 Figure 3.1: Graphical abstract of project 1. ...............................................................

Figure 4 . 1 :

 41 Figure 4.1: Graphical abstract of project 2 ................................................................

Figure 4 . 2 .

 42 Figure 4.2. CV-N Structure. ........................................................................................

Figure 4 . 3 .

 43 Figure 4.3. Phylogenetic tree of the CVNH Family. ...............................................

Figure 4 . 4 .

 44 Figure 4.4. CVNHs sugar binding specificity ..........................................................

Figure 4 . 5 :

 45 Figure 4.5: Comparison of CVNH tandem repeats ................................................

Figure 4 . 6 :

 46 Figure 4.6: Phylogenetic tree of CVNHs ..................................................................

Figure 4 . 7 .

 47 Figure 4.7. SapL6 expression and purification ........................................................

Figure 4 . 8 .

 48 Figure 4.8. SapL6 oligomerization stage ..................................................................

Figure 4 . 9 .

 49 Figure 4.9. SapL6 thermal Stability ...........................................................................

Figure 4 . 10 .

 410 Figure 4.10. Hemagglutination activity of SapL6 ...................................................

Figure 4 . 11 .

 411 Figure 4.11. SapL6 interaction with the mammalian glycan array of CFG .......

Figure 4 .

 4 Figure 4.12. optimization of SapL6 purification ....................................................

Figure 4 . 13 .

 413 Figure 4.13. SapL6 crystallization ............................................................................

Figure 4 . 14 .

 414 Figure 4.14. SapL6 Structure ....................................................................................

Figure 4 . 15 .

 415 Figure 4.15. Comparison of SapL6 with other CVNHs ........................................

Figure 4 . 16 .

 416 Figure 4.16. Structural comparison of CV-N and SapL6 carbohydrate BP .......

Figure 4 . 17 .

 417 Figure 4.17. Structural comparison of the chimera-LKAMG and SapL6 carbohydrate BP .........................................................................................................

Figure 4 . 18 :

 418 Figure 4.18: Multiple alignment of sequences of the CVNH for philogeny .....

Figure 5 . 1 :

 51 Figure 5.1: Graphical abstract of project 3 ..............................................................

Figure 5 . 2 :

 52 Figure 5.2: Native lectins identified from S. apiospermum mycelium cultures..

Figure 5 . 3 :

 53 Figure 5.3: Native lectins identified from A. fumigatus mycelium cultures ......

Figure 5 . 4 :

 54 Figure 5.4: Native lectins identified from fungal cultures 20X concentrated ...

Figure 5 . 5 :

 55 Figure 5.5: SapCP .......................................................................................................

Figure 5 . 6 :

 56 Figure 5.6: CPPs structural analysis ........................................................................

Figure 5 . 7 :

 57 Figure 5.7: Alignment of the peptides identified from B1 and the PVL ............

Figure 5 . 8 .

 58 Figure 5.8. Alignment of the peptides identified from B7 and the PVL ............

Figure 5 . 9 :

 59 Figure 5.9: Psathyrella velutina Lectin (PVL) ...........................................................

Figure 5 . 10 :

 510 Figure 5.10: Evolutionary tree of the major groups of fungi ...............................

Figure 5 . 11 :

 511 Figure 5.11: Phylogenetic analysis of PVL-like lectins .........................................

Figure 5 . 12 .

 512 Figure 5.12. Alignment of the peptides identified from B10, B31, B32 and the FleA sequence .............................................................................................................

Figure 5 . 13 :

 513 Figure 5.13: Alignment of the peptides identified from B45 and B46 and FleA sequences ....................................................................................................................

Figure 5 . 14 :

 514 Figure 5.14: Alignment of the peptides identified from B23/B28 and AfFG-GAP sequence. .....................................................................................................................

Figure 5 . 15 :

 515 Figure 5.15: Alignment of the peptides identified from B25 and AfFG-GAP sequence ......................................................................................................................

Figure 5 . 16 :

 516 Figure 5.16: Structural model of AfFG-GAP ..........................................................

Figure 5 .

 5 Figure 5.17: CcMBL ...................................................................................................

Figure 5 .

 5 Figure 5.18 Alignment of the peptides identified from B2, B3, B47 and B48 with the sequence of CcMBL .............................................................................................

Figure 5 . 19 :

 519 Figure 5.19: Alignment of the peptides identified from B8 and B29 with the sequence of CcMBL ...................................................................................................

Figure 5 . 20 :

 520 Figure 5.20: Alignment of the peptides identified from B15 and B4 with the sequence of CcMBL ...................................................................................................

Figure 5 . 21 :

 521 Figure 5.21: Alignment of the peptides identified from B4 and the SapFKBP12 sequence ......................................................................................................................

Figure 5 . 22 :

 522 Figure 5.22: Ambivalent effect of CaN inhibition in fungi and human cells ....

Figure 5 . 23 :

 523 Figure 5.23: The role of FKBPs in the panfungal strategy ...................................

Figure 5 . 24 :

 524 Figure 5.24: FKBP12 from pathogenic fungi: alignment and structure of complexes....................................................................................................................

Figure 5 . 25 :

 525 Figure 5.25: Alignment of sequences of highly conserved fungal FKBP12 .......

Figure 6 . 1 :

 61 Figure 6.1: Schematic representation of the device ...............................................

Figure 6 . 2 :

 62 Figure 6.2: Glycosides synthesis ..............................................................................

Figure 6 . 3 :

 63 Figure 6.3: Borosilicate surface functionalization and glycosides coating ........

Figure 6 . 4 :Figure 6 . 6 .

 6466 Figure 6.4: Electrophoretic profile of fractions recovered from the GCFs after protein incubation ..................................................................................................... 176

Figure 1 . 1 :

 11 Figure 1.1: Scedosporium genus and S. apiospermum. A) Molecular phylogenetic maximum likelihood analysis of the BT2 gene of Scedosporium complex species. B, C) Macroscopic appearance of S. apiospermum (top view and reverse, respectively).D, E) Microscopic appearance conidiogenous cells and conidia (magnification, 40 X and 100 X, respectively). Adapted with permission fromLuplertlop, 2018 [7].

Figure 1 . 2 :

 12 Figure 1.2: Scedosporium spp pathogeny. A) Diagram of diseases caused by Scedosporium complex species in humans. B) Anatomical origins (sites of infection) of 370 isolates submitted to the Fungus Testing Laboratory at the University of Texas Health Science System from January 2000 to May 2007. Panel B was adapted with permission fromCortez et al., 2008 [11].

Figure 1 . 3 .

 13 Figure 1.3. Global distribution and relative burden of scedosporiosis. Reprinted with permission fromLuplertlop, 2018 [10]

Figure 1 . 4 :

 14 Figure 1.4: Scedosporium apiospermum in the Cystic Fibrosis context. Reprinted with permission fromBouchara and Papon, 2019 [19]

Figure 1 . 5 :

 15 Figure 1.5: Carbohydrates-lectin interactions. A) Structure of the CCL2 lectin from Coprinopsis cinerea in complex with sialyl lewis X (PDB 4USO). B-D) glycosyl ligands of lectins, monosaccharide, glycogen and membrane glycoconjugates (from right to left, respectively).

Figure 1 . 6 :

 16 Figure 1.6: Carbohydrate recognition domain of lectins. The main forces that influence CRD selectivity and the hydrophobic pocket are indicated in the diagram. Figure created using LigandScaut 4.3.

  Figure 1.7) [40].

Figure 1 . 7 :

 17 Figure 1.7: Schematic representation of different types of symmetry observed in lectins. The symmetry family and symmetry axis are noted for each schematic representation. Reprinted with permission fromNotova et al., 2020 [41]

  Figure 1.8 shows some additional examples of glycans recognized by different lectins.

Figure 1 . 8 :

 18 Figure 1.8: Examples of glycan determinants recognized by different lectins. The determinants required for binding are indicated in the boxed areas. Yellow circles represent galactose, blue squares represent N-acetylglucosamine, red triangles represent fucose, green circles represent mannose. Reprinted with permission fromVarki et al., 2009 [62].

Figure 1 . 10 :

 110 Figure 1.10: Fungal adhesion to epithelial cells. Figure generated using Biorender.com.

  glycoproteins and it has been suggested that their recognition by FleA would mediate the binding of A. fumigatus conidia to host cells. The BPs all present a triad necessary for fucose recognition and have diverged in FleA to have low and high affinity BPs[START_REF] Mello | Scedosporium apiospermum, Scedosporium aurantiacum, Scedosporium minutisporum and Lomentospora prolificans: a comparative study of surface molecules produced by conidial and germinated conidial cells[END_REF] AFL6 belongs to the Cyanivirin-N homologue lectin subfamily (CNVH) in fungi(Figure 1.11B, unpublished work). The biological roles of this subfamily remain unclear, but it has been suggested that some of them play an important role in pathogenesis[85]. It seems to have a biological relevance as it is constitutively expressed during the whole fungal life cycle. CNVHs have caught the attention of the scientific community for being homologous to Cyanovirin-N, a powerful antiviral protein isolated from the cyanobacteria Nostoc ellipsosporum[86]. CN-V presents a tandem repeat leading to the formation of two pseudo-symmetrical domains with one BPs per domain recognising mannosides but in fungal CNVH, usually only on BP seems functional and the specificity is still under debate[START_REF] Koharudin | The evolutionarily conserved family of cyanovirin-N homologs : structures and carbohydrate specificity[END_REF].

Figure 1 . 11 :

 111 Figure 1.11: Overall structure of FleA and AFL6. A) crystal structure recombinant FleA from A. fumigatus in complex with alpha-L-fucopyranoside (PDB 4U4D). B) crystal structure recombinant AFL6 from A. fumigatus (unpublished data). Figure created using PyMOL Version 1.8.4 (Schrödinger).

Figure 2 :

 2 Figure 2: Thesis results overview. Figure created with Microsoft Office 365.

  array and Isothermal Titration Calorimetry (ITC), as well as the structural characterization of its binding mode by X-ray crystallography. The publication of this work was used for this chapter. A graphical abstract is shown in figure3.1.

Figure 3 . 1 :

 31 Figure 3.1: Graphical abstract of project 1. Production and characterization of Sapl1. Figure created with Microsoft Office 365.

Figure 4 . 1 :

 41 Figure 4.1: Graphical abstract of project 2. Production and characterization of SapL6. Figure created with Microsoft Office 365.

Figure 4 . 2 .

 42 Figure 4.2. CV-N Structure. Protein model was visualized and edited with PyMol Version 1.8.4 with the PDB 1IIY. The two sugar binding pockets are indicated as BP1 and BP2.

Figure 4 . 3 .

 43 Figure 4.3. Phylogenetic tree of the CVNH Family. Cladogram of 39 predicted CVNH polypeptide sequences (labeled in color along with the three CVNH representative membersand CV-N). Adapted with permission fromKoharudin et al. 2008 [59].

  domains were A in TcCVN and B in NcCVN (Figure 4.4 A-C). The specificity of these two proteins was further addressed by a glycan array screening, through which it was shown that NcCVNH interacts with high mannose glycocompounds, similarly to CV-N. On the other hand, TbCVNH only recognized two of the oligomannosides present in the array but also bound mannose-lacking glycans composed exclusively by glucose or a combination of glucose, galactose and N-acetylglucosamine (Figure 4.5 D) [59].

Figure 4 . 4 .

 44 Figure 4.4. CVNHs sugar binding specificity. A-C) Structural mapping of sugar-binding sites onto space-filling representations of the 3D structures of TbCVNH, CrCVNH, and NcCVNH. Residues corresponding to strongly, moderately, and weakly affected resonances are red, orange, and yellow, respectively. D) Glycan array screening of CV-N fungal homologous TbCVN and

(

  Constant system Ltd) and cell debris was pelleted by centrifugation at 22000 g for 30 minutes. The supernatant was filtered with 0.45 µm membranes and the protein purification was carried out by immobilized metal affinity chromatography (IMAC) with Nickel-NTA His-Trap FF columns (GE Healthcare Life Sciences) on a NGC™ chromatography system (Bio-Rad) with a flow rate of 1 mL•min -1 . Once the supernatant was loaded, column was rinse thoroughly with buffer A until stabilization of the baseline. Bound protein was subsequently eluted trough a gradient of 5-500 mM imidazole. SapL6 containing fractions were reconcentrated by ultrafiltration and sample buffer was replaced by buffer C (50mM Tris-HCl, 100 mM NaCl, pH 7.5) using PD10 desalting column (GE healthcare Life Sciences). Then, the protein sample was supplemented with 0.5 mM EDTA, 0.25 mM TCEP and TEV protease (1:50 ratio) for overnight His-tag cleavage at 19°C. TEV protease and uncleaved His-SapL6 were separated by IMAC and cleaved protein was collected into the flow through.

Figure 4 . 5 :

 45 Figure 4.5: Comparison of CVNH tandem repeats. A) Alignment of the sequences of the first tandem repeat of the most representative members of the CVNHs and their phylogenetic tree. B) Alignment of the TR2 sequences of the first tandem repeat of the most representative members and their phylogenetic tree. Sequence's alignments were performed using Clustal W [100] and visualized with ESPript3 [101]. Evolutionary histories were inferred using the maximum likelihood method based on the Jones et al. algorithm, 1992 [102]. The trees are drawn to scale, with branch lengths measured in the number of substitutions per site (next to the branches). Evolutionary analyzes were performed in MEGA7 [103].

Figure 4 . 6 :

 46 Figure 4.6: Phylogenetic tree of CVNHs. Phylogeny was inferred using 29 of the original 39 sequences used to define the original tree of CVNHs in 2008. Sequence's alignments was performed using Clustal W [100] and the evolutionary history was inferred using the maximum likelihood method based on the Jones et al. algorithm, 1992 [102]. Evolutionary analyzes were performed in MEGA7 [103].

  Tobacco Etch Virus protease (TEV) by cloning it into two different expression vectors (pET-TEV and pProNde), which were chosen to evaluate the best production performance (Figure 4.7 A and D). Then, expression was induced and purification was carried by Nickel affinity chromatography in two steps before and after TEV cleavage of the His-tag. The cleavage of this flexible fusion allow us to obtain pure protein in the flow-through of the second IMAC chromatography, would prevent possible problems during crystallization and avoids possible interferences with SapL6 folding and activity. The final yield was 15 mg•L -1 and 9.31 mg•L -1 for pProNde and pET-TEV vectors, respectively.

Figure 4 . 7 .

 47 Figure 4.7. SapL6 expression and purification. A and D) Schematic representation of the genetic constructs used for the expression of SapL6 in pProNde and pET-TEV vectors. B and E) Representative chromatograms of SapL6 purification before TEV cleavage with their respective profiles on 15% SDS-polyacrylamide gels (insoluble fraction, soluble fraction, flow through, washing, elution and molecular weight marker, from left to right respectively). Green lines represent Absorbance and dark lines represent the elution gradient. C and F) Representative chromatograms of SapL6 purifications after TEV cleavage, with their respective profiles on 15% SDS-polyacrylamide gels.

  SEC and DLS analyses. According with the data obtained from those experiments, SapL6 is a monomeric protein of ~10.5-12.8 kDa. No evidence of dimerization was found in any case(Figure 4.8).

Figure 4 . 8 .

 48 Figure 4.8. SapL6 oligomerization stage. A) Size exclusion chromatogram of SapL6. B) Size Distribution by Mass of SapL6 obtained by dynamic light scattering.

Figure 4 . 9 .

 49 Figure 4.9. SapL6 thermal Stability. Melt temperatures of SapL6 obtained through the Thermal Shift Assay (TSA). A temperature gradient from 20 to 100°C in was applied under 26 different buffer conditions with a pH range from 5 to 10.

Figure 4 . 10 .

 410 Figure 4.10. Hemagglutination activity of SapL6. assay performed on fresh rabbit erythrocytes. Negative and positive controls consist of 150 mM NaCl and the rPVL lectin from P. velutina, respectively.

  To expand our search, we used the mammalian glycan array version 5.4 of the Consortium for Functional Glycomics (USA) consisting of 585 mammalian glycans. For this, SapL6 was labeled with the FITC fluorophore. However, the low proportion of primary amines in its sequence (1%, 1 residue) entailed low labeling (molar ratio ~0.04) and weak fluorescence signals with elevated standard deviations were obtained from the glycan array screening(Figure 4.11).

Figure 4 . 11 .

 411 Figure 4.11. SapL6 interaction with the mammalian glycan array of CFG. Relative Fluorescent Units (RFU) plot of the glycan array matrix with SapL6 at 50 µg•mL -1 . The experiment was carried out in duplicate at 50 and 5 µg mL -1 of protein. Only the plot obtained at 50 µg mL -1 is included in the figure. The structures of the 5 best binders averaging both experiments are shown linked to their respective signals. Raw data can be consulted at https://docs.google.com/spreadsheets/d/1U8oGfSXKJ90RqfzHz5gISeIS4zvQO2dL/edit?usp=shar

Figure 4 .

 4 Figure 4.12. optimization of SapL6 purification. The figure shows a representative chromatograms of affinity chromatography purification of SapL6 after the optimization of elution parameters. Green lines represent Absorbance and dark lines represent the elution gradient. The electrophoretic profile of the purification 15% SDS-polyacrylamide gels is also shown (insoluble fraction, soluble fraction, flow through, washing, elution and molecular weight marker, from left to right respectively).

Figure 4 . 13 .

 413 Figure 4.13. SapL6 crystallization. Three pictures of representative conditions tested for SapL6 crystallization are shown in the figure. Drops were made by mixing equal volumes of SapL6 (9.5 mg mL -1 in PBS buffer, pH=7.4) and mother liquor (100 mM HEPES buffer pH=7, with the respective concentration of ammonium sulphate).

(

  TR1 and TR2) have an identical topological organization and form two domains interconnected by chain exchange(Figure 4.14). In SapL6, domain A is composed of residues 1-37 and 95-106, while domain B is positioned between amino acids 38-94. Each domain is composed of two helical turns and 5 β-strands divided by a 2-fold symmetry axis.

Figure 4 . 14 .

 414 Figure 4.14. SapL6 Structure. Alignment of the two tandem repeats of SapL6 (TR1 and TR2), accompanied by 3D-fold description. Sequence's alignment was performed using Clustal W [100] and visualized with ESPript3 [101]. Protein model was visualized and edited with PyMol Version 1.8.4.

Figure 4 . 15 .

 415 Figure 4.15. Comparison of SapL6 with other CVNHs. A) Structural alignment of SapL6, AFL6, CV-N and the most representative members of the CVNH family (TbCVNH, CrCVNH and NcCVNH). B) Structural comparison of SapL6 and CV-N (PDB: 1IIY). Green numbers indicate bondable cysteine pairs C) Structural comparison of SapL6 and TbCVNH (PDB: 2JZK). D) Structural comparison of SapL6 and CrCVNH (PDB: 2JZJ). Green numbers indicate bondable cysteine pairs E) Structural comparison of SapL6 and NcCVNH (PDB: 2JZL). F) Structural comparison of SapL6 and AFL6 [94].Structural alignments and protein models were visualized and edited with PyMol Version 1.8.4. Sequence alignments were performed using Clustal W [100] and visualized with ESPript3 [101].

  arginine and asparagine residues, respectively (D95 / N99 and D23 / R22) in SapL6(Figure 4.16 A). While in the binding pocket located in domain B, residues N53 and Q78 are replaced by glycines, only asparagine (N42) remains conserved.In fact, this residue is conserved in all members of the CV-N family (Figure2.16 B). Interestingly, while the composition of the binding pockets of SapL6 differs greatly from that of CV-N, these residues are identical among all the fungal members of this family showing divergence of the fungal members from the bacterial one.In 2009, a chimeric protein was synthetically engineered by fusing the A domain of TbCVNH with the B domain of NcCVNH. This chimera maintained its mannose-binding properties but also exhibited sucrose-binding activity and its structure was solved with this sugar attached to both pockets[105]. We have evaluated the similarities between the residues directly involved in such interactions and we did not find significant differences compared to previous comparison with CV-N. In domain B, the two asparagine residues remain conserved while Arg81 and Asp45 are substituted by very similar residues leading to the conservation of the hydrogen binding network (Figure4.17 A). In domain A, the only observed change consists in the substitution of glutamine Q98 by histidine H96. Surprisingly, a greater variability is observed among the residues involved in the interaction with sucrose among the fungal members of the family (Figure4.17 B).

Figure 4 . 16 .

 416 Figure 4.16. Structural comparison of CV-N and SapL6 carbohydrate binding pockets. A) Alignment of CV-N (purple) and SapL6 (cyan) structures. B) Sequence alignment of SapL6 and the most representative members of the CVNH family. Black triangles indicate the residues responsible for hydrogen bonding with Man1-2Man in CV-N (PDB: 1IIY). Sequences alignment was performed using Clustal W [100] and visualized with ESPript3 [101]. Protein models were visualized and edited with PyMol Version 1.8.4.

Figure 4 . 17 .

 417 Figure 4.17. Structural comparison of the chimera-LKAMG and SapL6 carbohydrate binding pockets. A) Alignment of the sructures of SapL6 (cyan) and the chimera-LKAMG (doman A in blue, domain B in green, PDB: 3HP8). B) Sequence alignment of SapL6 and the most representative members of the CVNH family. Black triangles indicate the residues responsible for hydrogen bonding with Man1-2Mana in CV-N (PDB: 1IIY). Sequences alignment was performed using Clustal W [100] and visualized with ESPript3 [101]. Protein models were visualized and edited with PyMol Version 1.8.4.

  figures and discussed all results. A.V. and Roland J. Pieters (RJP) administered

Figure 4 . 18 :

 418 Figure 4.18: Multiple alignment of sequences of the CVNH used to build the family philogeny. Sequence's alignment was performed using Clustal W [100] and visualized with ESPript3 [101].

Figure 5 . 1 :

 51 Figure 5.1: Graphical abstract of project 3. 1) protein extraction and sample preparation. 2) schematic representation of the purification train assembled for native lectins purification. Nacetilglucosamina, N-acetylgalactosamina, fucose, mannose, glucose and fetuin columns are shown from right to left respectively. 3) Schematic representation of data collection, analysis and identification of new lectins. 4) Graphical summary of identified proteins. Figure generated using Biorender.com and Microsoft Office 365.

  growth and did form robust spherical granules (hyphal pellets). The coloration of this culture was sparkly-deep brown and the weight of wet biomass recovered by filtration was 25 gr per liter of culture. While, Aspergillus culture was a turbid homogeneous solution with an opaque-light brown color and the mass of the wet pellet was only 10 gr per liter of culture. The carbohydrate binding proteins of each sample were purified and concentrated as described in materials and methods. Their electrophoretic profile is shown in figures 5.2 and 5.3, where it is possible to observe at least four clear bands in the intracellular fractions and multiple bands of lower concentrations in the extracellular fractions of both fungi.The molecular size of proteins identified from the intracellular fractions are slightly different between S. apiospermum and A. fumigatus, however they all follow the same pattern. On the other hand, it is clear that the size patterns of proteins secreted to the media are pretty different between the two fungal strains(Figures 5.2 and 5.3).In a second attempt to identify very diluted proteins, samples were concentrated a 2 nd time trough ultrafiltration until achieve the half of their original volume(Figure 5.4). This strategy allowed us to identify 23 additional proteins but five of the original bands disappeared possibly due to degradation. The missing bands in the second experiment are indicated in the figures 5.2 and 5.3 as green labels with the range numeration from 56-59. A total of 54 proteins bands with presumptive carbohydrate-binding activity were obtained from this second attempt of identification.

Figure 5 . 2 :

 52 Figure 5.2: Native lectins identified from S. apiospermum mycelium cultures. Left) SDS-PAGE purification of intracellular proteins. Lane 1: ladder; lane 3: 20 μL of culture media (concentrated 5X); Lane 4: 5 μL of total protein extract (soluble fraction); Lane 5: flow-through; Lines 7-12, 20 μL of samples obtained after elution from GlcNAc, GalNac, fucose, mannose, galactose and fetuin resins, from left to right respectively. Right) SDS-PAGE of purification of proteins secreted to the media. Samples as disposed as previously described for left panel.

Figure 5 . 3 :

 53 Figure 5.3: Native lectins identified from A. fumigatus mycelium cultures. Left) SDS-PAGE purification of intracellular proteins. Lane 1: ladder; lane 3: 20 μL of culture media (concentrated 5X); Lane 4: 5 μL of total protein extract (soluble fraction); Lane 5: flow-through; Lines 7-12, 20 μL of samples obtained after elution from GlcNAc, GalNac, fucose, mannose, galactose and fetuin resins, from left to right respectively. Right) SDS-PAGE of purification of proteins secreted to the media. Samples as disposed as previously described for left panel.

Figure 5 . 4 :

 54 Figure 5.4: Native lectins identified from fungal cultures 20X concentrated. Left) SDS-PAGE purification of proteins from the intracellular fraction of both mycelia. Lines 1-6: 20 μL of samples obtained from the purification of total protein extract from Aspergillus on GlcNAc, GalNac, Fucose, Mannose, Galactose and fetuin resins, from left to right respectively; Lines -12: 20 μL of samples obtained from purification total protein extract from Scedosporium. Right) SDS-PAGE of purification of proteins secreted to the media by Aspergillus and Scedosporium. Samples as disposed as previously described for left panel.

  in fungus-host interactions. We called it SapCP for Scedosporium apiospermum cerato-platanin. The alignment of the peptides identified for each one of those samples and the SapCP, as well as the in silico modeling of SapCP structure, are shown in figure 5.5.

Figure 5 . 5 :

 55 Figure 5.5: SapCP. A) Alignment of the sequences of SapCP (accession number A0A084FZZ4) and the peptides identified for samples B40, B41, B43 and B53. Sequence's alignment was performed using Clustal W [100] and visualized with ESPript3 [101]. B) Modeling the structure of SapCP. Model was generated by the RaptorX webserver [107]. The order of the polypeptide sequence is indicated by a gradual change of colour from blue (N-terminal end) to red (C-terminal end).

  125

  ability to induces host defense responses[109,113]. CPPs have been shown to play a dual role in fungi where, on one hand, they are elementary for the life cycle of those organisms and display multifunctional roles during the different stages of their development, such as the growth of hyphae, remodeling of fungal cell wall and the formation of chlamydospores[114,115]. On the other hand, they are also related to fungal colonization through various processes that can be beneficial or lethal to the host[116]. Some of those proteins, for example, are elicitors of defense responses in plants thought diverse mechanisms that include activation of reactive oxygen species, production of nitric oxide, MAPK activation, ethylene and jasmonic acid signaling, etc.[109]. In contrast to their role in plant resistance, some CPPs can act as phytotoxins and are key elements during various stages of pathogenicity[110,111,115,117]. For example, it has been shown that CPPs gene expression increases when Trichoderma virens and Trichoderma harzianum are grown in the presence of tomato or cotton roots[118,119], while the CPP knockout mutants of B. cinerea and M. grisea display reduced virulence on their host plants[120,121]. It is speculated that their carbohydrate binding activity might play an essential role during fungus-host interactions through the recognition of plant polysaccharides during early stages of infections[113,122]. Glycans recognized by CPPs that have been reported to date includeGlcNAc [123], chitin[122,123] cellulose and β3-glucan[124,125].To date, the study of CPPs has been exclusively devoted to fungi interacting with plants and there is extensive literature on them, whereas only two CPPs have been identified from human pathogenic fungi: CS-Ag from Coccidioides immitis which is a specific antigen ofCoccidioides [126] and rAsp f13 of Aspergillus fumigatus is an allergen for humans[127].

  acetylglucosamine[123]. This is also widely distributed among other families of proteins, e.g. expansins, fungal endoglucanases, and barwins plant defense proteins, where are involved in the recognition/modification of polysaccharides[113].

Figure 5 . 6 :

 56 Figure 5.6: CPPs structural analysis. 3D structures of CP from Ceratocystis platani (PDB: 2KQA) [113]; MpCP1 (PDB: 3SUM), MpCP2 (PDB: 3SUK), MpCP3 (PDB: 3SUJ), and MpCP5 (PDB: 3SUL) from Moniliophthora perniciosa [123]. The bottom right corner of the panel presents structural alignment of those five CPPs and the structure modelled for SapCP.

Figure 5 . 4 ,

 54 B1 and B7). As observed in electrophoretic profiles, the protein B1 corresponds to one of the most abundant proteins in Scedosporium sample while the apparent abundance of B7 in Aspergillus is considerably lower(Figures 5.2 and 5.3). It has been possible to identify 34 different peptides for sample B1 and 25 for B7. The overall analysis of those peptides did not display any match with proteins reported in the databases of Scedosporium nor Aspergillus but, interestingly, they both displayed high identity scores with the PVL lectin from the mushroom Psathyrella velutina (accession number Q309D1_9AGAR). The coverage percent were69.11% and 50.63 %, for B1 and B7, respectively (Figures 5.7 and 5.8).

Figure 5 . 7 :

 57 Figure 5.7: Alignment of the peptides identified from B1 and the PVL sequence. Sequence's alignment was performed using Clustal W [100] and visualized with ESPript3 [101].

Figure 5 . 8 .

 58 Figure 5.8. Alignment of the peptides identified from B7 and the PVL sequence. Sequence's alignment was performed using Clustal W [100] and visualized with ESPript3 [101].

Figure 5 . 9 :

 59 Figure 5.9: Psathyrella velutina Lectin (PVL). A) crystal structure recombinant PVL in complex with GlcNAc-D-1,3Galactoside (Reprinted with permission from Audfray et al., 2014, PDB:4UP4). B) Photo of P. velutina mushroom (taken from the French mycology database, mycodb.fr).

Figure 5 . 10 :

 510 Figure 5.10: Evolutionary tree of the major groups of fungi. Modified from Biology: Life on Earth. 2008, Pearson Prentice Hall, Inc.

Figure 5 . 11 :

 511 Figure 5.11: Phylogenetic analysis of PVL-like lectins. The evolutionary history was inferred using the maximum likelihood method based on Jones et al. algorithm 1992 [102]. The tree with the highest probability is displayed and drawn to scale. Evolutionary analyzes were performed in MEGA7 [103].

A

  . fumigatus was unambiguously identified in both, the intracellular and extracellular fraction of proteins of Aspergillus. In the intracellular fraction it corresponded to a protein of ~32 kDa(Figures 5.3 and 5.4, B10) that was recovered from the elution of the fucose-affinity chromatography and display 82.54% of coverage. A total of 33 different peptides were identified from this sample with high spectral count (231). In the extracellular fraction FleA was found in two different bands (B31 and B32) with a respective spectral count of 318 and 159. This is consistent with the amount of material observable in each band and serves as an indirect indication that most of the protein was secreted to the media. A total of 30 and 31 different peptides were identified from those samples, however, in both cases the percentage of coverage was the identical (76.51%). An alignment of identified peptides for B10, B31, B32 and FleA sequence is shown in figure5.12.

Figure 5 . 12 .

 512 Figure 5.12. Alignment of the peptides identified from B10, B31, B32 and the FleA sequence. Sequence's alignment was performed using Clustal W [100] and visualized with ESPript3 [101].

Figure 5 . 13 :

 513 Figure 5.13: Alignment of the peptides identified from B45 and B46 and FleA sequences. Sequence's alignment was performed using Clustal W [100] and visualized with ESPript3 [101].

A

  . fumigatus was recovered from mannose and galactose affinity chromatography(B23 and B28, respectively). The sequence analysis of those samples leaded towards the identification of >30 different peptides that display 89.9% of identity with a sequence of an hypothetical FG-GAP repeat protein deduced from the genome of A. fumigatus (accession number Q4WK08_ASPFU), further referred as AfFG-GAP. It presents a 25 residues long signal peptide inferring secretion that is consistent with the extracellular identification of B23/B28. The peptide signal is excised since we observed 100% of coverage with the sequence of the mature protein and 89.9% with the immature one. AfFG-GAP contains two repeats of the FG-GAP domain corresponding to residues 108-174 and 220-286 for the first and second repeat, respectively. They are indicated with dash lines in figure 5.14.

Figure 5 . 14 :

 514 Figure 5.14: Alignment of the peptides identified from B23/B28 and AfFG-GAP sequence. The dotted line indicates FG-GAP repeats. Sequence's alignment was performed using Clustal W [100] and visualized with ESPript3 [101].

  138 could be the product of degradation of B23. The comparison of the AfFG-GAP sequence and the peptides obtained for sample 25 is shown in figure 5.15.

Figure 5 . 15 :

 515 Figure 5.15: Alignment of the peptides identified from B25 and AfFG-GAP sequence. The dotted line indicates FG-GAP repeats. Sequence's alignment was performed using Clustal W [100] and visualized with ESPript3 [101].

Figure 5 . 16 :

 516 Figure 5.16: Structural model of AfFG-GAP. Model was generated using the RaptorX webserver [107] with the mature sequence of the protein identified from B23 and B28. The order of the polypeptide sequence is indicated by a gradual change of colour from blue to red (N-terminal to C-terminal ends).

  ). Although, this sequence corresponds to a hypothetical protein, it has been annotated in the Swiss database UniProt as a mucin binding lectin (accession numbers A8NDX2 and B3VS76). Therefore, we have called this protein Coprinopsis cinerea mucinbinding lectin (CcMBL). CcMBL-homologs were identified from both microfungi. Most of those samples were recovered from fucose-affinity chromatography but two were identified from galactose-affinity chromatography.

Figure 5 . 17 :

 517 Figure 5.17: CcMBL. A) Modeling the structure of CcMBL sequence. Model was generated using the RaptorX webserver [107]. B) Photo of Coprinopsis cinerea mushroom (taken from the French mycology database, mycodb.fr).

Figure 5 .

 5 Figure 5.18 Alignment of the peptides identified from B2, B3, B47 and B48 with the sequence of CcMBL. Sequence's alignment was performed using Clustal W [100] and visualized with ESPript3 [101].

Figure 5 . 19 :

 519 Figure 5.19: Alignment of the peptides identified from B8 and B29 with the sequence of CcMBL.

  consistent with the finding of similar peptides in B4 from Scedosporium. The comparison of the CcMBL sequence and the peptides obtained for samples B4 and B15 is shown in figures 5.22.

Figure 5 . 20 :

 520 Figure 5.20: Alignment of the peptides identified from B15 and B4 with the sequence of CcMBL. Sequence's alignment was performed using Clustal W [100] and visualized with ESPript3 [101].

Figure 5 . 21 :

 521 Figure 5.21: Alignment of the peptides identified from B4 and the SapFKBP12 sequence (accession number A0A084G113). Sequence's alignment was performed using Clustal W [100] and visualized with ESPript3 [101].

  prevent the dephosphorylation of its downstream targets [144]. This inhibition plays an ambivalent role in the treatment of fungal infections because fungal-CaN is fundamental for pathogenesis but, on the other hand, the inhibition of human-CaN leads towards immunosuppression (Figure 5.24).

Figure 5 . 22 :

 522 Figure 5.22: Ambivalent effect of calcineurin inhibition in fungi and human cells. A) Mechanism of the immunosuppressive action of FK506. B) Antifungal action of FK506 against fungal pathogens. Crz1: Calcineurin-responsive zinc finger 1. Adapted with permission from Jung et al., 2020 [146].

Figure 5 . 23 :

 523 Figure 5.23: The role of FKBPs in the panfungal strategy. Schematic representation of the FKBPs conservation among humans and fungal kingdom. Figure created with Microsoft Office 365.

Figure 5 . 24 :

 524 Figure 5.24: FKBP12 from pathogenic fungi: alignment and structure of complexes. A) Overlay of fungal CNs with the bovine complex (bovine: PDB 1TCO gray; fungal: CnA red, CnB blue, FKBP12 green and FK506 in orange). Adapted with permission from Juvvadi et al., 2019 [148]. UniProt accession number are given in parenthesis. B) Sequences alignment of fungal FKBP12s. A. fumigatus FKBP12 (6TZ7), C. albicans FKBP12 (P28870), C. neoformans FKBP12 (O94746), C. immitis (J3K5Z5), S. apiospermum FKBP12 (A0A084G113). Sequence's alignment was performed using Clustal W [100] and visualized with ESPript3 [101]. Residues within 5 Å of FK506 are indicated by black triangles. From those, conserved residues in fungi are highlighted red. The yellow star indicates the residue of phenylalanine 88 critical for FK506 binding.

Figure 5 . 25 :

 525 Figure 5.25: Alignment of sequences of highly conserved fungal FKBP12. The figure shows the alignment of all fungal FKBP12 that display >90% identity with SapFKBP12 from the UniProt database. Black triangles indicate the residues within 5 Å of FK506 in the crystal structures of the complexes with FKBP12 from A. fumigatus FKBP12 (6TZ7), C. albicans FKBP12 (P28870), C. neoformans FKBP12 (O94746), C. immitis (J3K5Z5). Sequence's alignment was performed using Clustal W [100] and visualized with ESPript3 [101].

Fungal

  cultures were provided by Prof. Muriel Cornet (MC). Valerie Chazalet (VC) performed extraction of total proteins. Dania Martínez Alarcón (DMA) performed protein purification. Mass spectrometry analysis was requested to the EDyP-service platform and were performed by Julia Novion (JN). The analysis of data was performed by DMA. DMA prepared all the figures and discussed results. Annabelle Varrot (AV) and Roland J. Pieters (RJP) administered the project, conceived the design of the study, obtained funding, contributed to data analysis, and supervised DMA.

Figure 6 . 1 :

 61 Figure 6.1: Schematic representation of the device. The red triangles represent fucose; green circles mannose; blue circles glucose and yellow circles galactose. Figure created using BioRender.com.

Figure 6 . 2 :

 62 Figure 6.2: Glycosides synthesis. Schematic representation of the synthetic pathway of 2-(2aminoethoxy)ethyl mannoside (1), 2-(2-aminoethoxy)ethyl glucoside (2), 2-(2-aminoethoxy)ethyl galactoside (3) and 2´-azidoethyl fucoside (4). Figure created using ChemDraw Version 15.

Figure 6 . 3 :

 63 Figure 6.3: Borosilicate surface functionalization and glycosides coating. Schematic representation of the synthetic pathway to covalently attach glycosides to the borosilicate microfiber filters. Figure created using ChemDraw Version 15.

Figure 6 . 4 :

 64 Figure 6.4: Electrophoretic profile of fractions recovered from the GCFs after protein incubation.Well 1, freshly prepared proteins mix containing 200 µg/mL of lectin and BSA; well 2, unbound proteins after 1 h incubation; wells 3 and 4 washing steps with PBS; well 4, proteins recovered from the filter after heat treatment at 100 °C. Fractions recovered from the fucosecoated filter (A), from the mannose-coated filter (B), from the galactose-coated filter (C) and from the glucose-coated filter (D). E) Fractions recovered from the glucose-coated filter with two elution methods. Well 1, freshly prepared proteins mix at 200 mg/mL; well 2, proteins mix recovered from the filter after 1 h incubation; wells 3 to 5 washing steps with PBS; well 5, proteins recovered after heat treatment at 100 °C; well 6 proteins eluted from the filter with 50 mM glucose. F) Lectins concentration. Well 1, freshly prepared proteins mix containing (10 µg/ml of FleA and 200 µg/ml of BSA); wells 2 to 5 washing steps with PBS; well 6, proteins recovered by heat treatment at 100 °C.

Figure 6 . 5 :

 65 Figure 6.5: GCFs mini chips for the detection of carbohydrate-lectin interactions. A) In situ labeling of ConA attached to Man-CF. Untreated filter spotted with mannoside (1) was used as a negative control. B) GCFs Miniarrays printing scheme and stainings. In the upper part is shown the filter stained with H2SO4 after glycosides deposition and in the lower part the filter stained with InstantBlue after incubation with ConA.

Figure 6 . 6 .

 66 Figure 6.6. Electrophoretic profile of fractions recovered from the GCFs after incubation with total proteins extracts. Well 1, soluble extract; well 2, unbound proteins after 1 h incubation; wells 3 and 5 washing steps with PBS; well 4, proteins eluted by heat treatment at 100 °C.

  emerging pathogens to conventional drugs and the rapid generation of resistance are urgent health problems. According to the Antimicrobial Resistance Review "By 2050, 10 million deaths per year are expected to be attributable to microbial resistance." Therefore, the World Health Organization (WHO) has declared antimicrobial resistance to be one of the top 10 public health threats facing humanity and has established the fight against antimicrobial resistance (AMR) as a priority worldwide.In this context, the European collaborative network PhD4GlycoDrug has emerged. The main objective of this program is the discovery and development of new Glyco-Drugs as a therapeutic alternative to evade the selective pressure that leads to AMR. For this, 12 independent projects have been established; each project seeks to contribute to the development of antiadhesive therapies against different human pathogens from varied perspectives; from exploration to evaluation. For pathogens whose virulence factors are known, the program aims to develop glycomimetics to antagonize microbial lectins involved in host binding. However, in the case of emerging pathogens, such as S. apiospermum, there is very limited information about its anchoring mechanism. Hence, it is first necessary to identify the virulence mechanisms involved in carbohydrate-lectin interactions during host recognition.

Project 2 -

 2 Biochemical and structural characterization of a Cyanovirin-like lectin from the human opportunistic fungi S. apiospermum In this study, we report the identification of a Cyanovirin-like lectin from Scedosporium apiospermum (SapL6). SapL6 belongs to the fungal Cyanovirin-N homolog (CVNH) subfamily III. Although the specificity and biological function of this group of proteins are not clear, it is believed that they play a fundamental role in fungal life. Consequently, they are considered potential targets for the development of new antifungals.

  fucopyranoside .............. 6.3.2 Synthesis of 2-(2-cloroethoxy)ethylper-O-acetylated glucosides ..................... 6.3.3 General procedures for azidation of glycosides ................................................ 6.3.4 General procedures for deprotection of glycosides ........................................... 6.3.5 General procedures for the reduction of azido glycosides ................................

6.3.6 Hydroxylation of borosilicate fibers 1721726.3.7 Silane coupling (Self-assembled monolayer) ................................................................................................................ 6.3.8 Monodeposition of glycosides on GPS-coated filters ....................................... 6.3.9 Multi-spot deposition of glycosides on GPS-coated filters .............................. 6.3.10 Lectin binding to glycoside-coated filters ...................................................... 6.3.11 Protein denaturing electrophoresis (SDS-PAGE) .........................................

Table 1 .

 1

1. Carbohydrates that prevent infection in vivo .........................................

Table 4 . 2

 42

. Data-collection statistics. ......................................................................... Table 5.1. Summary of mass spectrometry hits. ....................................................

Table 5 . 2

 52

. List of peptides identified by MS for sample B1 .................................

Table 5 . 3

 53

. List of peptides identified by MS for sample B2. ................................

Table 5 . 4

 54

. List of peptides identified by MS for sample B3. ................................ Table 5.5. List of peptides identified by MS for sample B4. ................................ Table 5.6. List of peptides identified by MS for sample B7. ................................ Table 5.7. List of peptides identified by MSy for sample B8. .............................. Table 5.8. List of peptides identified by MS for sample B10. .............................. Table 5.9. List of peptides identified by MS for sample B15 ............................... Table 5.10. List of peptides identified by MS for sample B23. ............................ Table 5.11. List of peptides identified by MS for sample B25 ............................. Table 5.12. List of peptides identified by MS for sample B28 ............................. Table 5.13. List of peptides identified by MS for sample B29 ............................. Table 5.14. List of peptides identified by MS for sample B31. ............................

Table 5 .

 5

15. List of peptides identified by MS for sample B32 .............................

Table 5 .

 5

16. List of peptides identified by MS for sample B40. ............................

Table 5 .

 5

17. List of peptides identified by MS for sample B41 .............................

Table 5 .

 5

18. List of peptides identified by MS for sample B43. ............................

Table 5 .

 5

19. List of peptides identified by MS for sample B45 ............................. Table 5.20. List of peptides identified by MS for sample B46 ............................. Table 5.21. List of peptides identified by MS for sample B47 .............................

Table 5 . 22

 522

. List of peptides identified by MS for sample B48. ............................

Table 5 . 23

 523

. List of peptides identified by MS for sample B53 .............................

Table 6 .

 6 1. Carbohydrate-based matrix for lectins purification……………….…

  Em: 490/530 nm and the data processing was performed with the CFX Manager software.

	4.3.7 Isothermal Titration Calorimetry (ITC)
	Experiments were performed using a Microcal ITC200 calorimeter (Malvern
	Panalytical, Malvern, UK) with 40 μL of ligand and 200 μM of protein according
	to the conditions described in Table 4.1. Both, protein, and sugars were gradually
	added to the sample cell by 2 µL injections in a range of 120 seconds while stirring
	at 1000 rpm. Experimental data were adjusted to a theoretical titration curve by
	the Origin ITC Analysis software.
	-1 , 7 μl of 500x Sypro
	Orange (Sigma-Aldrich) and 63 μl of H2O were prepared. Then, 7.5 μl of H2O,
	12.5 μl of the corresponding buffer and 5 μl of the protein/Sypro mixture were

mixed in 96-well PCR microplates. The heat exchange test was then carried out from 20°C to 100°C with a heating rate of 1°C•min -1 . Fluorescence intensity was measured with Ex/

Table 4 .

 4

	Protein concentration (mM)	Ligand	Ligand concentration (mM)	Molar ratio
	0.1 0.05	Manα1-2Man	50 100
	0.5		250
	0.2 0.06	D-Mannose	100 150
	0.06		30
		Methyl α-D-	30
		mannopyranoside	150
		Glucose	30 150
		GlcNac	30 150
		Galactose	30 150
	0.06	Lactose	30 150
		L-Rhamnose	30 150
		Sucralose	30 150
		Lactulose	30 150
		L-Xylose	30
		D-Xylose

1.

ITC experimental parameters (SapL6)

Table 4 . 2 .

 42 Data-collection statistics.

Table 5 .

 5 1. Summary of mass spectrometry hits.

		Sample			Major protein identified
	Organis m	ID	Sugar	Gene	Description	Cov. (%)	MW	# of pept	Spect ral count	Data base	Conf. score
					Q309D1_9AGAR Lectin PVL
		B1	GlcNAc	pvl	(Fragment) OS=Lacrymaria velutina	74.18	42420	39	393
					OX=71681 GN=pvl PE=1 SV=1
		B2			A8NDX2_COPC7 Uncharacterized	100		17	517
			Fuc		protein OS=Coprinopsis cinerea
	Sa	B3		CC1G_10558	MYA-4618 / FGSC 9003) OX=240176 (strain Okayama-7 / 130 / ATCC	95.28	13733	11	208
		B4			GN=CC1G_10558 PE=4 SV=2	99.21		13	90
	INTRACELLULAR	B7 B7 B8	Gal GlcNAc	SAPIO_CDS7060 pvl CC1G_10558	A0A084G113_PSEDA Peptidylprolyl isomerase OS=Pseudallescheria apiosperma OX=563466 GN=SAPIO_CDS7060 PE=4 SV=1 Q309D1_9AGAR Lectin PVL (Fragment) OS=Lacrymaria velutina OX=71681 GN=pvl PE=1 SV=1 A8NDX2_COPC7 Uncharacterized protein OS=Coprinopsis cinerea (strain Okayama-7 / 130 / ATCC	92.79 57.22 82.68	11835 42420 13733	14 30 14	28 175 50	Fungi
					MYA-4618 / FGSC 9003) OX=240176
			Fuc		GN=CC1G_10558 PE=4 SV=2 Q4WW81|LECF_ASPFU Fucose-
					specific lectin OS=Neosartorya
		B10		fleA	fumigata (strain ATCC MYA-4609 /	82.54	34661	33	231	Af
					Af293 / CBS 101355 / FGSC A1100)
					OX=330879 GN=fleA PE=1 SV=1
					A8NDX2_COPC7 Uncharacterized
	Af	B15	Gal	CC1G_10558	GN=CC1G_10558 PE=4 SV=2 protein OS=Coprinopsis cinerea (strain Okayama-7 / 130 / ATCC MYA-4618 / FGSC 9003) OX=240176	55.12	55.12	1373 3	6	Fungi
		B23			Q4WK08_ASPFU FG-GAP repeat protein, putative OS=Neosartorya	89.9		31	127
		B25		AFUA_1G04130	fumigata (strain ATCC MYA-4609 / Af293 / CBS 101355 / FGSC A1100)	40.39	33760	13	25	Af
		B28	Man		OX=330879 GN=AFUA_1G04130 PE=4 SV=2	89.9		30	139
					A8NDX2_COPC7 Uncharacterized
					protein OS=Coprinopsis cinerea
		B29		CC1G_10558	(strain Okayama-7 / 130 / ATCC	78.74	13733	10	58	Fungi
					MYA-4618 / FGSC 9003) OX=240176
		B_31	Fuc		GN=CC1G_10558 PE=4 SV=2 Q4WW81|LECF_ASPFU Fucose-specific lectin OS=Neosartorya	76.51		30	317
				fleA	fumigata (strain ATCC MYA-4609 /		34661			Af
	EXTRACELLULLAR	B_32 B_40 B_41 B_43	Neu5Ac Gal Man	SAPIO_CDS8583	Af293 / CBS 101355 / FGSC A1100) OX=330879 GN=fleA PE=1 SV=1 A0A084FZZ4_PSEDA Uncharacterized protein OS=Pseudallescheria apiosperma OX=563466 GN=SAPIO_CDS8583 PE=3 SV=1	73.97 40.74 82.22 82.19	13912	29 7 9 8	159 44 25 42	Sa
	Sa	B_45 B_46 B_47	Fuc	fleA	Q8NJT4|LECF_ASPFM Fucose-specific lectin OS=Neosartorya fumigata OX=746128 GN=fleA PE=3 SV=1 A8NDX2_COPC7 Uncharacterized protein OS=Coprinopsis cinerea	83.76 81.53 78.74	34520	30 30 9	92 189 62	Fungi
				CC1G_10558	(strain Okayama-7 / 130 / ATCC		13733
		B_48			MYA-4618 / FGSC 9003) OX=240176	95.28		11	104
					GN=CC1G_10558 PE=4 SV=2
					A0A084FZZ4_PSEDA
					Uncharacterized protein
		B_53	GlcNAc	SAPIO_CDS8583	OS=Pseudallescheria apiosperma	82.22	13912	8	31	Sa
					OX=563466 GN=SAPIO_CDS8583
					PE=3 SV=1
	Af: Aspergillus fumigatus, Sa: Scedosporium apiospermum, cov.: coverage
	1	Very confident
	2	Quite confident
	3	Low confidence

Table 5 . 2 .

 52 List of peptides identified by mass spectrometry for sample B1

	sequence	modifications	psm score	mass	accession number and description
	QALPVPTR		45.1	880.513046

Table 5 . 3 .

 53 List of peptides identified by mass spectrometry for sample B2.

	sequence	modifications	psm score	mass	accession number and description
	WAVSFSHNYTR		84.35	1366.64183
	DKVTTAYALPDNL		64.29	1419.724548
	LTLLTWGGLNNLR		100.96	1469.835434
	SVIGAIPTENTGVTK		72.15	1485.803879
	AIFHTGSELFIITR		86.79	1603.872223
	AIFHTGSELFIITR MAIFHTGSELFIITR	Acetyl (Protein N-term)	88.41 79.17	1645.882782 1734.912704	A8NDX2_COPC7 Uncharacterized protein
	MAIFHTGSELFIITR	Oxidation (M1)	103.79	1750.907623	OS=Coprinopsis cinerea
	GPGKLTLLTWGGLNNLR		94.31	1809.026093	(strain Okayama-7 / 130
	AIFHTGSELFIITRGPGK FSFIWEGQGEACYQIGNGLTR SVIGAIPTENTGVTKWAVSFSHNYTR	101.33 Carbamidomethyl (C12) 158.21 100.98	1943.062881 2432.121933 2834.43515	/ ATCC MYA-4618 / FGSC 9003) OX=240176 GN=CC1G_10558 PE=4 SV=2
	SWSSSSTIHWGSSTVITEDVTSVVPGAVNR		110.29	3145.531662
	SWSSSSTIHWGSSTVITEDVTSVVPGAVNRDK		88.37	3388.653564
	AIFHTGSELFIITRGPGKLTLLTWGGLNNLR		54.23	3394.887756
	SPVGRSWSSSSTIHWGSSTVITEDVTSVVPGAVNR		125.09	3641.807449
	SWSSSSTIHWGSSTVITEDVTSVVPGAVNRDKVTT AYALPDNL		32.37	4547.245651

Table 5 . 4 .

 54 List of peptides identified by mass spectrometry for sample B3.

	sequence	modifications	psm score	mass	accession number and description
	WAVSFSHNYTR		84.67	1366.64183
	DKVTTAYALPDNL		64.2 1419.724548
	LTLLTWGGLNNLR		81.3 1469.835434
	SVIGAIPTENTGVTK		75.67 1485.803879	A8NDX2_COPC7
	AIFHTGSELFIITR		82.9 1603.872223	Uncharacterized protein
	AIFHTGSELFIITR	Acetyl (Protein N-term)	78.06 1645.882782	OS=Coprinopsis cinerea (strain Okayama-7 / 130 /
	GPGKLTLLTWGGLNNLR		92.54 1809.026093	ATCC MYA-4618 / FGSC 9003)
	AIFHTGSELFIITRGPGK FSFIWEGQGEACYQIGNGLTR	Carbamidomethyl (C12)	77.94 1943.062881 51.68 2432.121933	OX=240176 GN=CC1G_10558 PE=4 SV=2
	SVIGAIPTENTGVTKWAVSFSHNYTR		116.74	2834.43515
	SWSSSSTIHWGSSTVITEDVTSVVPGAVNR		93.2 3145.531662

Table 5 . 5 .

 55 List of peptides identified by mass spectrometry for sample B4.

	sequence	modifications	pms score	mass	accession number and description
	WAVSFSHNYTR		68.58	1366.64183
	DKVTTAYALPDNL		32.49	1419.724548
	LTLLTWGGLNNLR SVIGAIPTENTGVTK AIFHTGSELFIITR AIFHTGSELFIITR GPGKLTLLTWGGLNNLR FSFIWEGQGEACYQIGNGLTR SVIGAIPTENTGVTKWAVSFSHNYTR SWSSSSTIHWGSSTVITEDVTSVVPGAVNR SWSSSSTIHWGSSTVITEDVTSVVPGAVNRDK	Acetyl (N-term) CarbaMet(C12)	83.49 43.71 64.95 67.77 50.07 121.35 75.99 98.04 40.52	1469.835434 1485.803879 1603.872223 1645.882782 1809.026093 2432.121933 2834.43515 3145.531662 3388.653564	A8NDX2_COPC7 Uncharacterized protein OS=Coprinopsis cinerea (strain Okayama-7 / 130 / ATCC MYA-4618 / FGSC 9003) OX=240176 GN=CC1G_10558 PE=4 SV=2
	AIFHTGSELFIITRGPGKLTLLTWGGLNNLR		60.34	3394.887756
	SPVGRSWSSSSTIHWGSSTVITEDVTSVVPGAVNR		69.13	3641.807449
	IGVGQVIR		29.82	840.518143
	GWEEGVTQMK		44.68	1163.528107
	GWEEGVTQMK	Oxidation (M9)	42.57	1179.523026
	TVITEGSGPIPK FDSSVGRGPFVTR ALLDISSDFAYGER PGQTVTIEYTGWLK VGEKALLDISSDFAYGER IGVGQVIRGWEEGVTQMK GFTGHIPPNADLLFEVELK PGQTVTIEYTGWLKGPNGEK		56.04 41.86 83.18 99.48 47.69 26.12 69.14 74.56	1197.660507 1423.72084 1555.751816 1591.824615 1968.979248 1986.03569 2096.094238 2174.100784	A0A084G113_PSEDA SEDA Peptidylprolyl isomerase OS=Pseudallescheria apiosperma OX=563466 GN=SAPIO_CDS7060 PE=4 SV=1
	GFTGHIPPNADLLFEVELKK		75.08	2224.189194
	GFTGHIPPNADLLFEVELKKVA		34.52	2394.294724
	TVITEGSGPIPKPGQTVTIEYTGWLK		111.24	2771.474564

Table 5 . 6 .

 56 List of peptides identified by mass spectrometry for sample B7.

	sequence	modifications	psm score	mass	accession number and description
	QALPVPTR		51.28	880.513046
	CVSSLTNK	CarbaMet (C1)	46.64	907.443314
	VVADLTGDR		62.16	944.492722
	NSLLIQVVK		53.52 1012.628067
	LIDDMTVSK		56.9 1020.516144
	GDGTFGPIMK		58.52	1021.49028
	DFGVNSGWR		39.71 1036.472656
	LIDDMTVSK	Oxidation (M5)	61.32 1036.511063
	GDGTFGPIMK	Oxidation (M9)	44.11 1037.485199
	VVADLTGDRK		30.46 1072.587677
	TVRYAANLYL		39.67 1182.639679
	DFGVNSGWRVEK		76.03 1392.678619
	VIDNFGYNQGWR		92.25 1467.689514	Q309D1_9AGAR
	VINNFGYDAGGWR		84.93 1467.689514	Lectin PVL (Fragment)
	MVLANFAYAAGGWR		82.81 1525.749985	OS=Lacrymaria velutina OX=71681
	ADIVGFGDGGIYISR		121.89 1538.772919	GN=pvl PE=1 SV=1
	MVLANFAYAAGGWR	Oxidation (M1)	97.35 1541.744904
	RVIDNFGYNQGWR		86.09 1623.790619
	LIDDMTVSKGWTLQK		104.13 1733.902206
	LIDDMTVSKGWTLQK	Oxidation (M5)	107.91 1749.897125
	DFGVNSGWRVEKHVR		39.66 1784.907059
	VIDNFGYNQGWRVDK		88.16 1809.879837
	VINNFGYDAGGWRVEK		77.02 1823.895477
	TGRADIVGFGDGGIYISR		76.45 1852.943176
	MVLANFAYAAGGWRVEK	Oxidation (M1)	67.72 1897.950867
	IPGVADLVGFGNGGVYIIR		108.44 1916.052002
	VIDNFGYNQGWRVDKHPR		67.54 2200.092621
	NNGGGQFAPAQLALNNFGYAQGWR		134.48	2550.21524

Table 5 . 7 .

 57 List of peptides identified by mass spectrometry for sample B8.

	sequence	modifications	psm score	mass	accession number and description
	WAVSFSHNYTR		74.46	1366.64183
	DKVTTAYALPDNL		64.16 1419.724548
	LTLLTWGGLNNLR		88.63 1469.835434
	SVIGAIPTENTGVTK AIFHTGSELFIITR AIFHTGSELFIITR GPGKLTLLTWGGLNNLR AIFHTGSELFIITRGPGK SVIGAIPTENTGVTKWAVSFSHNYTR SWSSSSTIHWGSSTVITEDVTSVVPGAVNR GPGKLTLLTWGGLNNLRSVIGAIPTENTGVTK AIFHTGSELFIITRGPGKLTLLTWGGLNNLR	Acetyl (N-term)	70.06 1485.803879 79.43 1603.872223 61.96 1645.882782 85.27 1809.026093 60.87 1943.062881 109.56 2834.43515 100.3 3145.531662 46.83 3276.819412 75.86 3394.887756	A8NDX2_COPC7 Uncharacterized protein OS=Coprinopsis cinerea (strain Okayama-7 / 130 / ATCC MYA-4618 / FGSC 9003) OX=240176 GN=CC1G_10558 PE=4 SV=2
	SPVGRSWSSSSTIHWGSSTVITEDVTSVVPGAVNR		115.62 3641.807449
	SWSSSSTIHWGSSTVITEDVTSVVPGAVNRDKV TTAYALPDNL		35.5 4547.245651

Table 5 . 8 .

 58 List of peptides identified by mass spectrometry for sample B10.

	sequence	modifications	psm score	mass	accession number and description
	AHGSQLGR		27.18	824.425293
	LGSPVAATSK		69.77	929.518188
	FQVAPYSR		51.6	966.49231
	IWFQTDDLK		48.44 1164.581528
	YTDYNGPSIR		70.75 1184.546188
	STPGAQQVLFR		72.31 1202.640778
	STPGAQQVLFR	Acetyl (N-term)	83.39 1244.651337
	LGSPVAATSKELK		83.61 1299.739792
	MSTPGAQQVLFR		77.47 1333.681259
	VYFQDVYGSIR		81.25 1345.666656
	GWYPDLVTIFDR IAAVFLAGTDALQLR ESLYEGSWANGTEK IWFQTDDLKLVQR TAIAATSFGAGNSSIYMR TAIAATSFGAGNSSIYMR EGTNLGGALPGTGIGATSFR GWYPDLVTIFDRAPPR AYDPHKGWYPDLVTIFDR APPRTAIAATSFGAGNSSIYMR ESLYEGSWANGTEKNVIGNAK YTDYNGPSIRIWFQTDDLK LYFQNGTYISAVSEWVWNR	Oxidation (M17) Oxidation (M21)	80.31 1480.735077 93.57 1557.887863 71.35 1569.694687 104.96 1660.893692 147.55 1816.877762 112.63 1832.872681 100.55 1874.948654 49.55 1901.978821 92.93 2192.069092 52.26 2254.116425 147.98 2266.086548 53.41 2331.117157 57.47 2332.127655	LECF_ASPFU Fucose-specific lectin OS=Neosartorya fumigata (strain ATCC MYA-4609 / Af293 / CBS 101355 / FGSC A1100) OX=330879 GN=fleA PE=1 SV=1
	IYFVNSDNTIWQVCWDHGK	Carbamidomethyl	72.26	2381.08992
	IYAQKPDNTIQEYMWNGDGWK		59.47 2556.174332
	IYAQKPDNTIQEYMWNGDGWK	Oxidation (M14)	65.13	2572.16925
	AYDPHKGWYPDLVTIFDRAPPR		61.19 2613.312836
	YTDYNGPSIRIWFQTDDLKLVQR		64.77 2827.429321
	VYFQDVYGSIRESLYEGSWANGTEK		95.13 2897.350784
	EGTNLGGALPGTGIGATSFRYTDYNGPSIR		83.16 3041.484283
	VYTLTEGNTLQEFAYDSGTGWYNGGLGGAK		123.78 3168.467636
	IYAQKPDNTIQEYMWNGDGWKEGTNLGGALPGTGIGATSFR		97.91 4413.112427
	IYAQKPDNTIQEYMWNGDGWKEGTNLGGALPGTGIGATSFR Oxidation (M14)	79 4429.107346

Table 5 . 9 .

 59 List of peptides identified by mass spectrometry for sample B15

	sequence	modifications	psm score	mass	accession number and description
	WAVSFSHNYTR		46.93	1366.64183
	DKVTTAYALPDNL		34.53 1419.724548	A8NDX2_COPC7
	LTLLTWGGLNNLR		77.45 1469.835434	Uncharacterized protein OS=Coprinopsis cinerea
	SVIGAIPTENTGVTK AIFHTGSELFIITR		68.59 1485.803879 76.72 1603.872223	(strain Okayama-7 / 130 / ATCC MYA-4618 / FGSC 9003) OX=240176 GN=CC1G_10558 PE=4 SV=2
	GPGKLTLLTWGGLNNLR		50.68 1809.026093

Table 5 .

 5 10. List of peptides identified by mass spectrometry for sample B23.

	sequence	modifications	psm score	mass	accession number and description
	ASNYQTR		48.84	838.393311
	LDLVYIK		49.9	862.516388
	DDLVYIK		49.02	864.459274
	PGPDLAYIK		78.64	972.528015
	DGVLDLAYVK		63.76 1091.586273
	LDLVYIKTR		77.36 1119.665176
	DGKLDLVYIK		63.32	1162.65976
	VEVHVASGASTYK		81.23 1346.683029
	DGKLDLVYIKTR		66.78 1419.808548
	VEVHVASGASTYKTR		86.54 1603.831818
	VEVHVASGASNYQTR		93.71	1616.79068
	VEVHIATAASNFQTR		99.86 1642.842712
	TSNTGTGKVEVHLASR		72.8	1655.8591
	DGVLDLAYVKTQNTGTGR		73.37 1906.974869
	TQNTGTGRIEVHVANGRN		48.15 1922.967087
	NTGTGRVEVHVASGASTYK		53.17 1932.965363	Q4WK08_ASPFU
	TRNTGTGRVEVHVASGASTYK NTGTGRVEVHVASGASNYQTR NTGTGRVEVHIATAASNFQTR		43.02 2190.114151 124.13 2203.073013 68.41 2229.125046	FG-GAP repeat protein, putative OS=Neosartorya fumigata (strain ATCC MYA-4609 / Af293 / CBS 101355 / FGSC A1100) OX=330879
	TRNTGTGRVEVHVASGASNYQTR		64.86 2460.221802	GN=AFUA_1G04130 PE=4 SV=2
	TRNTGTGRVEVHIATAASNFQTR		51.26 2486.273834
	VQEVGTTFYPEDNGFWQLIDFNK		46.6 2746.291519
	IQEVGTTFYPEDNGVWQMIDFNR		85.4	2758.26973
	IQEVGTTFYPEDNGVWQMIDFNR	Oxidation (M18)	80.66 2774.264648
	TLEVGTTFFPEDNGVWQLIDADGDGR		61.1 2851.330109
	DVGTTFLPEDNGTWQMADFDGDGILD LIYIK	Oxidation (M16)	85.9 3474.617752
	ILATGTTFNAPDPYGPWSLTPSANKPGPD LAYIK		73.44 3572.819122
	TLEVGTTFFPEDNGVWQLIDADGDGRDD LVYIK		86.7 3697.778824
	IKDVGTTFLPEDNGTWQMADFDGDGILD LIYIK	Oxidation (M18)	94.82 3715.796768
	VQEVGTTFYPEDNGFWQLIDFNKDGVLD LAYVK		53.79 3819.867233
	TLEVGTTFFPEDNGVWQLIDADGDGRDD LVYIKTR		83.53 3954.927612

Table 5 .

 5 11. List of peptides identified by mass spectrometry for sample B25

	sequence	modifications	psm score	mass	accession number and description
	LDLVYIK		44.8	862.516388
	DDLVYIK		43.17	864.459274
	PGPDLAYIK		78.64	972.528015
	DGVLDLAYVK		47.15 1091.586273
	DGKLDLVYIK		51.72	1162.65976	Q4WK08_ASPFU
	VEVHVASGASNYQTR VEVHIATAASNFQTR NTGTGRVEVHVASGASTYK		95.99 83.15 1642.842712 1616.79068 45.51 1932.965363	FG-GAP repeat protein, putative OS=Neosartorya fumigata (strain ATCC MYA-4609 / Af293 / CBS 101355 / FGSC A1100) OX=330879 GN=AFUA_1G04130
	NTGTGRVEVHVASGASTYKTR		52.6 2190.114151	PE=4 SV=2
	NTGTGRVEVHVASGASNYQTR		70.35 2203.073013
	NTGTGRVEVHIATAASNFQTR		65.74 2229.125046
	TRNTGTGRVEVHVASGASNYQTR		44.74 2460.221802
	IQEVGTTFYPEDNGVWQMIDFNR Oxidation (M18)	27.1 2774.264648

Table 5 .

 5 12. List of peptides identified by mass spectrometry for sample B28

	sequence	modifications	psm score	mass	Accession number and description
	ASNYQTR		48.76	838.393311
	LDLVYIK		52.77	862.516388
	DDLVYIK		43.72	864.459274
	PGPDLAYIK		78.65	972.528015
	DGVLDLAYVK		64.57 1091.586273
	DGKLDLVYIK		70.01	1162.65976
	VEVHVASGASTYK		71.38 1346.683029
	DGKLDLVYIKTR		66.79 1419.808548
	VEVHVASGASNYQTR		94.8	1616.79068
	VEVHIATAASNFQTR		99.74 1642.842712
	TSNTGTGKVEVHLASR		74.9	1655.8591
	DGVLDLAYVKTQNTGTGR		65.55 1906.974869
	TQNTGTGRIEVHVANGRN		47.79 1922.967087
	NTGTGRVEVHVASGASTYK		87.16 1932.965363	Q4WK08_ASPFU
	NTGTGRVEVHVASGASTYKTR NTGTGRVEVHVASGASNYQTR NTGTGRVEVHIATAASNFQTR		81.21 2190.114151 122.09 2203.073013 76.84 2229.125046	FG-GAP repeat protein, putative OS=Neosartorya fumigata (strain ATCC MYA-4609 / Af293 / CBS 101355
	TRNTGTGRVEVHVASGASNYQTR		69.42 2460.221802	/ FGSC A1100) OX=330879
	ILATGTTFNAPDPYGPWSLTPSANK		68.75 2618.301666	GN=AFUA_1G04130 PE=4
	VQEVGTTFYPEDNGFWQLIDFNK		40.89 2746.291519	SV=2
	IQEVGTTFYPEDNGVWQMIDFNR		97.1	2758.26973
	IQEVGTTFYPEDNGVWQMIDFNR	Oxidation (M18)	67.97 2774.264648
	TLEVGTTFFPEDNGVWQLIDADGDGR		72.54 2851.330109
	IQEVGTTFYPEDNGVWQMIDFNRDGK	Oxidation (M18)	43.41	3074.40802
	DVGTTFLPEDNGTWQMADFDGDGILDLIYIK	Oxidation (M16)	75.59 3474.617752
	ILATGTTFNAPDPYGPWSLTPSANKPGPDLAYIK		86.01 3572.819122
	TLEVGTTFFPEDNGVWQLIDADGDGRDDLVYIK		73.21 3697.778824
	IKDVGTTFLPEDNGTWQMADFDGDGILDLIYIK	Oxidation (M18)	110.86 3715.796768
	VQEVGTTFYPEDNGFWQLIDFNKDGVLDLAYVK		46.81 3819.867233
	TLEVGTTFFPEDNGVWQLIDADGDGRDDLVYIKTR		86.53 3954.927612

Table 5 .

 5 13. List of peptides identified by mass spectrometry for sample B29

	sequence	modifications	psm score	mass	accession number and description
	WAVSFSHNYTR		80.95	1366.64183
	DKVTTAYALPDNL		74.36 1419.724548
	LTLLTWGGLNNLR		101.05 1469.835434	A8NDX2_COPC7
	SVIGAIPTENTGVTK		74.03 1485.803879	Uncharacterized protein
	AIFHTGSELFIITR AIFHTGSELFIITR GPGKLTLLTWGGLNNLR	Acetyl (N-term)	79.99 1603.872223 61.83 1645.882782 85.85 1809.026093	OS=Coprinopsis cinerea (strain Okayama-7 / 130 / ATCC MYA-4618 / FGSC 9003) OX=240176
	AIFHTGSELFIITRGPGK		72.09 1943.062881	GN=CC1G_10558 PE=4 SV=2
	SVIGAIPTENTGVTKWAVSFSHNYTR		118.52	2834.43515
	SWSSSSTIHWGSSTVITEDVTSVVPGAVNR		102.51 3145.531662

Table 5 .

 5 14. List of peptides identified by mass spectrometry for sample B31.

	sequence	modifications	psm score	mass	accession number and description
	AHGSQLGR		32.83	824.425293
	LGSPVAATSK		67.44	929.518188
	FQVAPYSR		53.67	966.49231
	IWFQTDDLK		52.17 1164.581528
	YTDYNGPSIR		68.96 1184.546188
	STPGAQQVLFR		79.45 1202.640778
	STPGAQQVLFR	Acetyl (N-term)	86.71 1244.651337
	LGSPVAATSKELK		85.24 1299.739792
	MSTPGAQQVLFR		74.76 1333.681259
	VYFQDVYGSIR		74.2 1345.666656
	GWYPDLVTIFDR		80.36 1480.735077
	IAAVFLAGTDALQLR		100.63 1557.887863
	ESLYEGSWANGTEK		72.86 1569.694687
	NVIGNAKLGSPVAATSK		73.16 1625.910049	LECF_ASPFU
	IWFQTDDLKLVQR		105.28 1660.893692	Fucose-specific lectin
	TAIAATSFGAGNSSIYMR TAIAATSFGAGNSSIYMR EGTNLGGALPGTGIGATSFR	Oxidation (M17)	147.63 1816.877762 112.39 1832.872681 108.19 1874.948654	OS=Neosartorya fumigata (strain ATCC MYA-4609 / Af293 / CBS 101355 / FGSC A1100) OX=330879 GN=fleA PE=1 SV=1
	GWYPDLVTIFDRAPPR		48.97 1901.978821
	AYDPHKGWYPDLVTIFDR		90.75 2192.069092
	APPRTAIAATSFGAGNSSIYMR	Oxidation (M21)	96.96 2254.116425
	ESLYEGSWANGTEKNVIGNAK		134.29 2266.086548
	YTDYNGPSIRIWFQTDDLK		59.8 2331.117157
	IYFVNSDNTIWQVCWDHGK	Carbamidomethyl (C14)	72.2	2381.08992
	IYAQKPDNTIQEYMWNGDGWK	Oxidation (M14)	85.18	2572.16925
	AYDPHKGWYPDLVTIFDRAPPR		76.65 2613.312836
	YTDYNGPSIRIWFQTDDLKLVQR		71.43 2827.429321
	VYFQDVYGSIRESLYEGSWANGTEK		77.52 2897.350784
	VYTLTEGNTLQEFAYDSGTGWYNGGLGGAK		109.41 3168.467636
	IYAQKPDNTIQEYMWNGDGWKEGTNLGGA LPGTGIGATSFR		81.13 4413.112427

Table 5 .

 5 15. List of peptides identified by mass spectrometry for sample B32.

			psm		accession number and
	sequence	modifications	score	mass	description
	LGSPVAATSK		69.99	929.518188
	FQVAPYSR		54.1	966.49231
	IWFQTDDLK		50.17 1164.581528
	YTDYNGPSIR		67.25 1184.546188
	STPGAQQVLFR		89.62 1202.640778
	STPGAQQVLFR	Acetyl (N-term)	86.62 1244.651337
	LGSPVAATSKELK		88.77 1299.739792
	MSTPGAQQVLFR		75.84 1333.681259
	VYFQDVYGSIR		74.98 1345.666656
	GWYPDLVTIFDR		80.38 1480.735077
	IAAVFLAGTDALQLR		101.46 1557.887863
	ESLYEGSWANGTEK		71.82 1569.694687
	NVIGNAKLGSPVAATSK IWFQTDDLKLVQR TAIAATSFGAGNSSIYMR		73.53 1625.910049 115.02 1660.893692 143.2 1816.877762	LECF_ASPFU Fucose-specific lectin OS=Neosartorya fumigata
	TAIAATSFGAGNSSIYMR	Oxidation (M17)	112.51 1832.872681	(strain ATCC MYA-4609 /
	EGTNLGGALPGTGIGATSFR GWYPDLVTIFDRAPPR AYDPHKGWYPDLVTIFDR		129.01 1874.948654 45.63 1901.978821 90.7 2192.069092	Af293 / CBS 101355 / FGSC A1100) OX=330879 GN=fleA PE=1 SV=1
	ESLYEGSWANGTEKNVIGNAK		96.98 2266.086548
	YTDYNGPSIRIWFQTDDLK		53.74 2331.117157
	IYFVNSDNTIWQVCWDHGK	Carbamidomethyl (C14)	28.23	2381.08992
	IYAQKPDNTIQEYMWNGDGWK		58.46 2556.174332
	AYDPHKGWYPDLVTIFDRAPPR		63.17 2613.312836
	YTDYNGPSIRIWFQTDDLKLVQR		85.94 2827.429321
	VYFQDVYGSIRESLYEGSWANGTEK		89.8 2897.350784
	VYTLTEGNTLQEFAYDSGTGWYNGGLGGAK		108.68 3168.467636
	IYAQKPDNTIQEYMWNGDGWKEGTNLGGA LPGTGIGATSFR		57.96 4413.112427
	IYAQKPDNTIQEYMWNGDGWKEGTNLGGALP GTGIGATSFR	Oxidation (M14)	79.95 4429.107346

Table 5 .16.

 5 List of peptides identified by mass spectrometry for sample B40.

	sequence	modifications	psm score	mass	accession number and description
	FPTQGNLPK		58.59	1000.53418
	VEASAEQVDK		48.75 1074.519302
	VEASAEQVDKSNCGL SMLAVSCSDGSNGLASR SMLAVSCSDGSNGLASR	Carbamidomethyl (C13) Carbamidomethyl (C7) Oxidation (M2); Carbamidomethyl (C7)	90.09 1605.730438 109.89 1710.76651 127.55 1726.761429	A0A084FZZ4_PSEDA Uncharacterized protein OS=Pseudallescheria apiosperma OX=563466 GN=SAPIO_CDS8583 PE=3 SV=1
	SMLAVSCSDGSNGLASRFPTQGNLPK Carbamidomethyl (C7)	73.2 2693.290131
	SMLAVSCSDGSNGLASRFPTQGNLPK	Oxidation (M2); Carbamidomethyl (C7)	91.94 2709.285049

Table 5 .

 5 17. List of peptides identified by mass spectrometry for sample B41.

	sequence	modifications	psm score	mass	accession number and description
	FPTQGNLPK		44.32	1000.53418
	VEASAEQVDKSNCGL	Carbamidomethyl (C13)	75.72 1605.730438
	SMLAVSCSDGSNGLASR	Carbamidomethyl (C7)	129.7	1710.76651
		Oxidation (M2);
	SMLAVSCSDGSNGLASR	Carbamidomethyl	127.13 1726.761429	A0A084FZZ4_PSEDA
		(C7)			Uncharacterized protein
	FPYIGGVPAIGGWNSPNCGSCWK	Carbamidomethyl (C18, C21)	58.63 2523.146393	OS=Pseudallescheria apiosperma OX=563466 GN=SAPIO_CDS8583 PE=3
	SMLAVSCSDGSNGLASRFPTQGNLPK	Carbamidomethyl (C7)	92.32 2693.290131	SV=1
		Oxidation (M2);
	SMLAVSCSDGSNGLASRFPTQGNLPK	Carbamidomethyl	108.87 2709.285049
		(C7)
	SIYILGIDHSTSFNIALHAMNDLTNGNA VGLGR	Oxidation (M20)	62.23 3499.751785
	IGVGQVIR		29.82	840.518143
	GWEEGVTQMK		44.68 1163.528107
	GWEEGVTQMK	Oxidation (M9)	42.57 1179.523026
	TVITEGSGPIPK		56.04 1197.660507
	FDSSVGRGPFVTR		41.86	1423.72084
	ALLDISSDFAYGER PGQTVTIEYTGWLK VGEKALLDISSDFAYGER IGVGQVIRGWEEGVTQMK		83.18 1555.751816 99.48 1591.824615 47.69 1968.979248 26.12 1986.03569	A0A084G113_PSEDA Peptidylprolyl isomerase OS=Pseudallescheria apiosperma OX=563466 GN=SAPIO_CDS7060 PE=4 SV=1
	GFTGHIPPNADLLFEVELK		69.14 2096.094238
	PGQTVTIEYTGWLKGPNGEK		74.56 2174.100784
	GFTGHIPPNADLLFEVELKK		75.08 2224.189194
	GFTGHIPPNADLLFEVELKKVA		34.52 2394.294724
	TVITEGSGPIPKPGQTVTIEYTGWLK		111.24 2771.474564

Table 5 .

 5 18.List of peptides identified by mass spectrometry for sample B43.

			psm		accession number
	sequence	modifications	score	mass	and description
	FPTQGNLPK		58.36	1000.53418
	VEASAEQVDKSNCGL	Carbamidomethyl (C13)	75.57 1605.730438
	SMLAVSCSDGSNGLASR SMLAVSCSDGSNGLASR FPYIGGVPAIGGWNSPNCGSCWK SMLAVSCSDGSNGLASRFPTQGNLPK	Carbamidomethyl (C7) Oxidation (M2); Carbamidomethyl (C7) Carbamidomethyl (C18); Carbamidomethyl (C21) Carbamidomethyl (C7)	104.01 126.97 1726.761429 1710.76651 65.96 2523.146393 98.89 2693.290131	A0A084FZZ4_PSEDA Uncharacterized protein OS=Pseudallescheria apiosperma OX=563466 GN=SAPIO_CDS8583 PE=3 SV=1
	SMLAVSCSDGSNGLASRFPTQGNLPK	Oxidation (M2); Carbamidomethyl (C7)	101.73 2709.285049
	SIYILGIDHSTSFNIALHAMNDLTNGNAVGLGR Oxidation (M20)	44.98 3499.751785

Table 5 .

 5 19. List of peptides identified by mass spectrometry for sample B45.

	sequence	modifications	psm score	mass	accession number and description
	LGSPVAATSK		74.38	929.518188
	FQVAPYSR		51.55	966.49231
	IWFQTDDLK		49.39 1164.581528
	YTDYNGPSIR		69.3 1184.546188
	STPGAQQVLFR		89.59 1202.640778
	LGSPVAATSKELK		94.47 1299.739792
	MSTPGAQQVLFR		74.81 1333.681259
	VYFQDVYGSIR		70.59 1345.666656
	GWYPDLVTIFDR		80.28 1480.735077
	IAAVFLAGTDALQLR		95.64 1557.887863
	ESLYEGSWANGTEK		70.69 1569.694687
	IWFQTDDLKLVQR		105.08 1660.893692
	TAIAATSFGAGNSSIYMR EGTNLGGALPGTGIGATSFR GWYPDLVTIFDRAPPR		147.6 1816.877762 122.63 1874.948654 50.76 1901.978821	LECF_ASPFU Fucose-specific lectin OS=Neosartorya fumigata (strain ATCC
	AYDPHKGWYPDLVTIFDR		83.79 2192.069092	MYA-4609 / Af293 / CBS
	ESLYEGSWANGTEKNVIGNAK		75.97 2266.086548	101355 / FGSC A1100)
	YTDYNGPSIRIWFQTDDLK IYFVNSDNTIWQVCWDHGK	Carbamidomethyl (C14)	53.86 2331.117157 57.76 2381.08992	OX=330879 GN=fleA PE=1 SV=1
	FQVAPYSRIAAVFLAGTDALQLR		53.53 2506.369614
	IYAQKPDNTIQEYMWNGDGWK	Oxidation (M14)	64.18	2572.16925
	AYDPHKGWYPDLVTIFDRAPPR		85.95 2613.312836
	YTDYNGPSIRIWFQTDDLKLVQR		125.66 2827.429321
	EGTNLGGALPGTGIGATSFRYTDYNGPSIR		76.68 3041.484283
	LYFQNGTYISAVSEWVWNRAHGSQLGR		90.34 3138.542389
	VYTLTEGNTLQEFAYDSGTGWYNGGLGGAK		101.72 3168.467636
	HIRVYTLTEGNTLQEFAYDSGTGWYNGGLGGAK		71.83 3574.711716
	VYFQDVYGSIRESLYEGSWANGTEKNVIGNAK		43.72 3593.742645
	IYAQKPDNTIQEYMWNGDGWKEGTNLGGALP GTGIGATSFR		72.45 4413.112427
	TGIAAVNSTNHLR		88.01 1352.716049	LECF_ASPFM
	STPGAQQVLFRTGIAAVNSTNHLR	Acetyl (N-term)	93.45 2579.356827	Fucose-specific lectin OS=Neosartorya
	TGIAAVNSTNHLRVYFQDVYGSIR		105.76 2680.372147	fumigata OX=746128 GN=fleA PE=3 SV=1

Table 5 .

 5 20. List of peptides identified by mass spectrometry for sample B46.

	sequence	modifications	psm score	mass	accession number and description
	LGSPVAATSK		72.16	929.518188
	FQVAPYSR		51.59	966.49231
	IWFQTDDLK		45.25 1164.581528
	YTDYNGPSIR		67.27 1184.546188
	STPGAQQVLFR		85.97 1202.640778
	LGSPVAATSKELK		89.55 1299.739792
	MSTPGAQQVLFR		75.37 1333.681259
	VYFQDVYGSIR		74.96 1345.666656
	GWYPDLVTIFDR		80.21 1480.735077
	IAAVFLAGTDALQLR		93.53 1557.887863
	ESLYEGSWANGTEK		80.91 1569.694687
	NVIGNAKLGSPVAATSK		72.22 1625.910049
	IWFQTDDLKLVQR TAIAATSFGAGNSSIYMR EGTNLGGALPGTGIGATSFR		105.01 1660.893692 143.36 1816.877762 111.87 1874.948654	LECF_ASPFU Fucose-specific lectin OS=Neosartorya fumigata (strain ATCC MYA-4609 /
	GWYPDLVTIFDRAPPR		44.89 1901.978821	Af293 / CBS 101355 / FGSC
	AYDPHKGWYPDLVTIFDR APPRTAIAATSFGAGNSSIYMR	Oxidation (M21)	78.38 2192.069092 86.46 2254.116425	A1100) OX=330879 GN=fleA PE=1 SV=1
	ESLYEGSWANGTEKNVIGNAK		153.11 2266.086548
	YTDYNGPSIRIWFQTDDLK		45.91 2331.117157
	IYFVNSDNTIWQVCWDHGK	Carbamidomethyl (C14)	46.47	2381.08992
	IYAQKPDNTIQEYMWNGDGWK		53.45 2556.174332
	AYDPHKGWYPDLVTIFDRAPPR		74.68 2613.312836
	YTDYNGPSIRIWFQTDDLKLVQR		123.51 2827.429321
	GTITPVIQGSEVAIISWGSFANNGPDLR		98.78 2898.487579
	VYTLTEGNTLQEFAYDSGTGWYNGGLGGAK		131.26 3168.467636
	HIRVYTLTEGNTLQEFAYDSGTGWYNGGLGGAK		98.97 3574.711716
	IYAQKPDNTIQEYMWNGDGWKEGTNLGGALPG TGIGATSFR		87.22 4413.112427
	TGIAAVNSTNHLR		92.1 1352.716049	LECF_ASPFM
	STPGAQQVLFRTGIAAVNSTNHLR	Acetyl (N-term)	106.9 2579.356827	Fucose-specific lectin OS=Neosartorya fumigata
	TGIAAVNSTNHLRVYFQDVYGSIR		98.04 2680.372147	OX=746128 GN=fleA PE=3 SV=1

Table 5 .21.

 5 List of peptides identified by mass spectrometry for sample B47.

			psm		accession number and
	sequence	modifications	score	mass	description
	WAVSFSHNYTR		74.84	1366.64183
	DKVTTAYALPDNL		74.34 1419.724548
	LTLLTWGGLNNLR SVIGAIPTENTGVTK		106.48 1469.835434 77.33 1485.803879	A8NDX2_COPC7 Uncharacterized protein
	AIFHTGSELFIITR		80.3 1603.872223	OS=Coprinopsis cinerea (strain
	AIFHTGSELFIITR	Acetyl (N-term)	48.45 1645.882782	Okayama-7 / 130 / ATCC MYA-4618 /
	GPGKLTLLTWGGLNNLR AIFHTGSELFIITRGPGK		77.42 1809.026093 53.69 1943.062881	FGSC 9003) OX=240176 GN=CC1G_10558 PE=4 SV=2
	SVIGAIPTENTGVTKWAVSFSHNYTR		89.63	2834.43515
	SWSSSSTIHWGSSTVITEDVTSVVPGAVNR		90.53 3145.531662

Table 5 .

 5 22.List of peptides identified by mass spectrometry for sample B48.

			psm		accession number and
	sequence	modifications	score	mass	description
	WAVSFSHNYTR		87.98	1366.64183
	DKVTTAYALPDNL		74.47 1419.724548
	LTLLTWGGLNNLR		94.78 1469.835434
	SVIGAIPTENTGVTK		69.31 1485.803879
	AIFHTGSELFIITR AIFHTGSELFIITR GPGKLTLLTWGGLNNLR	Acetyl N-term)	79.95 1603.872223 59.69 1645.882782 83.02 1809.026093	A8NDX2_COPC7 Uncharacterized protein OS=Coprinopsis cinerea (strain Okayama-7 / 130 / ATCC MYA-
	AIFHTGSELFIITRGPGK		62.73 1943.062881	4618 / FGSC 9003) OX=240176
	AIFHTGSELFIITRGPGK	Acetyl (N-term)	25.41 1985.073441	GN=CC1G_10558 PE=4 SV=2
	FSFIWEGQGEACYQIGNGLTR	Carbamidomethyl (C12)	55.16 2432.121933
	SVIGAIPTENTGVTKWAVSFSHNYTR		111.37	2834.43515
	SWSSSSTIHWGSSTVITEDVTSVVPGAVNR		100.75 3145.531662

Table 5 .

 5 23.List of peptides identified by mass spectrometry for sample B53.

			psm		accession number
	sequence	modifications	score	mass	and description
	FPTQGNLPK		44.32	1000.53418
	VEASAEQVDKSNCGL	Carbamidomethyl (C13)	75.72 1605.730438
	SMLAVSCSDGSNGLASR	Carbamidomethyl (C7)	129.7	1710.76651	A0A084FZZ4_PSEDA
	SMLAVSCSDGSNGLASR	Oxidation (M2); Carbamidomethyl (C7)	127.13 1726.761429	_PSEDA Uncharacterized protein
	FPYIGGVPAIGGWNSPNCGSCWK	Carbamidomethyl (C18, C21)	58.63 2523.146393	OS=Pseudallescheria apiosperma OX=563466
	SMLAVSCSDGSNGLASRFPTQGNLPK SMLAVSCSDGSNGLASRFPTQGNLPK	Carbamidomethyl (C7) Oxidation (M2); Carbamidomethyl (C7)	92.32 2693.290131 108.87 2709.285049	GN=SAPIO_CDS8583 PE=3 SV=1
	SIYILGIDHSTSFNIALHAMNDLTNGNAVGLGR Oxidation (M20)	62.23 3499.751785

  Tuomanen, E. Oligosaccharides interfere with the establishment and progression of experimental pneumococcal pneumonia. J.Infect. Dis. 1997, 176, 704-712. 81. Wang, Q.; Singh, S.; Taylor, K.G.; Doyle', R.J. Ati-adhesins of Streptococcus sobrinus. Adv Exp Med Biol 1996, 408, 249-250. 82. Cywes, C.; Stamenkovic, I.; Wessels, M.R. CD44 as a receptor for colonization of the pharynx by group A Streptococcus. J. Clin. Invest. 2000, 106, 995-1002. 83. Ernst, B.; Magnani, J.L. From carbohydrate leads to glycomimetic drugs. Nat. Publ. Gr. 2009, 8, 661-677. 84. Arastehfar, A.; Carvalho, A.; Houbraken, J.; Lombardi, L.; Garcia-Rubio, R. Virol. Sin. 2010, 25, 432-439. 87. Vandeputte, P.; Dugé de Bernonville, T.; Le Govic, Y.; Le Gal, S.; Nevez, G.; Papon, N.; Bouchara, J.P. Comparative transcriptome analysis unveils the adaptative mechanisms of Scedosporium apiospermum to the microenvironment encountered in the lungs of patients with cystic fibrosis. Comput. Struct. Biotechnol. J. 2020, 18, 3468-3483. 88. Matei, E.; Basu, R.; Furey, W.; Shi, J.; Calnan, C.; Aiken, C.; Gronenborn, A.M. Structure and glycan binding of a new cyanovirin-N homolog. J. Biol.

	Fitzgibbon, M.J.; Fleming, M.A.; Caron, P.R.; Hsiao, K.; Navia, M.A. X-ray
	structure	of	calcineurin	inhibited	by	the	immunophilin-
	Chem. 2016, 291, 18967-18976. immunosuppressant FKBP12-FK506 complex. Cell 1995, 82, 507-522.

Aspergillus fumigatus and aspergillosis: From basics to clinics. Studies in Mycology 2021, 10, 100115. 85. Percudani, R.; Montanini, B.; Ottonello, S. The anti-HIV Cyanovirin-N domain is evolutionarily conserved and occurs as a protein module in eukaryotes. Proteins 2005, 60, 670-678. 86. Yu, H.; Liu, Z.T.; Lv, R.; Zhang, W.Q. Antiviral activity of recombinant cyanovirin-N against HSV-1. 89. Ql, X.; Yang, Y.; Su, Y.; Wang, T. Molecular cloning and sequence analysis of cyanovirin-N homology gene in Ceratopteris thalictroides. Am. Fern J. 150. O'Connor, B.; Monaghan, D.; Cawley, J. Lectin affinity chromatography (LAC). In Methods in Molecular Biology; Humana Press Inc., 2017; Vol. 1485,

Arabinose

.

Raw data of the glycan array assay can be consulted at https://docs.google.com/spreadsheets/d/1lyGh8bEw3kC_pgTcUaLepVxbAY7Xa

RRm/edit?usp=sharing&ouid=111416367677195406970&rtpof=true&sd=true

commercial and homemade sugar-based resins is increasing every day. Table one collects some examples of matrices that have been used for purification of lectins.

Drawbacks of sugar-based affinity chromatography

Although it is true that sugar-based columns are very useful and their variety is increasing every day, their current diversity does not meet the actual be used for enzyme activity/inhibition assays, antibody screening, interactions of whole cells with surfaces, enzyme activities, DNA / RNA interactions and noncarbohydrate molecule binding assays. Therefore, we consider that this device could contribute greatly not only to the study of the lectinome of species, but also that our strategy could be used beyond the glycobiology approach and applied in many other areas of science.

Contributions

Dania Martínez Alarcón (DMA) performed the synthesis of glycosidic derivatives, the borosilicate surface modification, prepared the Glycoside Coated Filters (GCFs) and performed protein binding experiments. DMA prepared all the figures, discussed results and conceived the design of the study. Roland J.

Pieters (RJP) and Annabelle Varrot (AV) administered the project, obtained funding, contributed to data analysis, and supervised DMA.

Overall, this thesis represents a general strategy to address the study S. apiospermum lectinome. Our findings have revealed the first insights about the recognition of human glycoconjugates by lectins from this microfungus and contribute to the general understanding of the host-binding process during the early stages of infection. In addition, we have developed a new tool that could mean a milestone in glycobiology and exponentially boost the discovery of new lectins in the coming years.

Finally, we expect that the information gathered here will contribute to design targeted therapies against this pathogen and will encourage the study and characterization of the new proteins discovered, in the search for new biomedical applications and drug targets.

APPENDIX

During the Ph.D., I also worked on three additional projects that are not in direct line with the topic of the thesis. One of these projects has already been already published and it is included in this annex.

SUMMARY

Scedosporium apiospermum is an emerging opportunistic fungal pathogen responsible for lifethreatening infections in humans. Due to its high mortality rate and low susceptibility to antifungals, there is an urgent need to develop new therapeutic strategies against it. Since microbial lectins are often involved in host recognition and adhesion, they constitute valuable drug targets and have aimed at for the development of antiadhesive therapy against other deadly pathogens. However, in the case of S. apiospermum, the development of such therapies is hindered because its lectins are unknown. Therefore, the main aim of this work was the identification and characterization of S. apiospermum lectins. This has been addressed by two different approaches: the first strategy comprises the in-silico prediction of putative lectins codified on its genome by data mining and their recombinant production, while the second strategy lies in the identification of native lectins from fungal protein extracts. These approaches have led to the identification of several proteins with carbohydrate-binding activity from this human pathogen. The preliminary analysis of those proteins reveals that some of them are potential pharmacological targets and others could find application as biotechnological tools.

A second objective of this thesis was the development of a new tool that allows the identification and purification of unknown lectins directly from crude protein extracts. This device also enables the study of lectin carbohydrate interactions without the need for fluorescent labeling. Therefore, it could greatly contribute to the future study of lectinome from other organisms.

Overall, this work represents a broad strategy to approach the study of S. apiospermum lectinome and our findings contribute to the understanding of the glycosylated surfaces recognition by this pathogen. We hope that the information gathered here will encourage the study of the proteins discovered to gain fundamental knowledge and for potential biomedical or biotechnological applications.

RESUMÉ

Scedosporium apiospermum est un pathogène fongique opportuniste émergent responsable d'infections potentiellement mortelles chez l'homme. En raison de son taux de mortalité élevé et de sa faible sensibilité aux antifongiques, il est urgent de développer de nouvelles stratégies thérapeutiques pour le combattre. Puisque les lectines microbiennes sont souvent impliquées dans la reconnaissance et l'adhésion à l'hôte, elles constituent des cibles pharmacologiques intéressantes et ont été ciblées pour le développement d'une thérapie antiadhésive contre d'autres pathogènes mortels. Cependant, dans le cas de S. apiospermum, le développement d'une telle thérapie est entravé car ses lectines sont inconnues. Par conséquent, l'objectif principal de ce travail a été l'identification et la caractérisation des lectines de S. apiospermum. Cette question a été abordée selon deux approches différentes; la première stratégie comprend la prédiction in-silico de lectines putatives encodées dans son génome par data mining tandis que la seconde réside dans l'identification de lectines natives à partir d'extraits de protéines fongiques. Ces approches ont conduit à l'identification de plusieurs lectines et protéines chez ce pathogène humain. L'analyse préliminaire de ces protéines révèle que certaines d'entre elles pourraient être des cibles pharmacologiques intéressantes tandis que d'autres pourraient trouver une application en tant qu'outil biotechnologique.

Un deuxième objectif de cette thèse a été le développement d'un nouvel outil permettant l'identification et la purification de lectines inconnues directement à partir d'extraits bruts de protéines. Ce dispositif permet également l'étude des interactions lectines-glucides sans avoir recours à un marquage fluorescent. Par conséquent, il pourrait grandement contribuer à l'étude future des lectinomes pour d'autres organismes. Dans l'ensemble, ce travail représente une stratégie pour aborder l'étude du lectinome de S. apiospermum et nos résultats contribuent à la compréhension de la reconnaissance des surfaces glycosylées par ce pathogène. Nous espérons que les informations recueillies ici encourageront l'étude des protéines découvertes pour acquérir des nouvelles connaissances fondamentales et en vue de potentielles applications biomédicales ou biotechnologiques.