Insights on Lulworthiales Inhabiting the Mediterranean Sea and Description of Three Novel Species of the Genus Paralulworthia

The order Lulworthiales, with its sole family Lulworthiaceae, consists of strictly marine genera found on a wide range of substrates such as seagrasses, seaweeds, and seafoam. Twenty-one unidentified Lulworthiales were isolated in previous surveys aimed at broadening our understanding of the biodiversity hosted in the Mediterranean Sea. Here, these organisms, mostly found in association with Posidonia oceanica and with submerged woods, were examined using thorough multi-locus phylogenetic analyses and morphological observations. Maximum-likelihood and Bayesian phylogeny based on nrITS, nrSSU, nrLSU, and four protein-coding genes led to the introduction of three novel species of the genus Paralulworthia: P. candida, P. elbensis, and P. mediterranea. Once again, the marine environment is a confirmed huge reservoir of novel fungal lineages with an under-investigated biotechnological potential waiting to be explored

Standardized nanomechanical atomic force microscopy procedure (SNAP) for measuring soft and biological samples

We present a procedure that allows a reliable determination of the elastic (Young's) modulus of soft samples, including living cells, by atomic force microscopy (AFM). The standardized nanomechanical AFM procedure (SNAP) ensures the precise adjustment of the AFM optical lever system, a prerequisite for all kinds of force spectroscopy methods, to obtain reliable values independent of the instrument, laboratory and operator. Measurements of soft hydrogel samples with a well-defined elastic modulus using different AFMs revealed that the uncertainties in the determination of the deflection sensitivity and subsequently cantilever's spring constant were the main sources of error. SNAP eliminates those errors by calculating the correct deflection sensitivity based on spring constants determined with a vibrometer. The procedure was validated within a large network of European laboratories by measuring the elastic properties of gels and living cells, showing that its application reduces the variability in elastic moduli of hydrogels down to 1%, and increased the consistency of living cells elasticity measurements by a factor of two. The high reproducibility of elasticity measurements provided by SNAP could improve significantly the applicability of cell mechanics as a quantitative marker to discriminate between cell types and conditions

Champignons marins d'éponges marines : biodiversité, chimiodiversité et applications biotechnologiques

Marine environment represents an untapped source of fungal diversity, where it has been estimated that about 10% of fungi have been explored until now. Due to the lack of knowledge on marine fungi and their incredible biotechnological potential, this Ph.D. thesis focuses on a highly promising group of fungi: those associated with marine sponges. These fungi are both characterized by high biodiversity and chemodiversity, being the most successful producers of new bioactive molecules. On these premises, the main goal of the research was to cover the firsts and fundamentals aspects of the natural products discovery pipeline: from the isolation and identification of fungi from sponges to the isolation of molecules and the evaluation of their biological activity. This resulted in a multidisciplinary Ph.D. project that enclosed mycology, chemistry, biochemistry and biotechnology. In a “funnel-like” perspective, using multidisciplinary experimental approaches three main parts were developed: - The first aim was to isolate the fungal communities associated with sponges using several isolation techniques to increase the number of cultivable fungi. Four and three sponges were respectively collected in the Atlantic Ocean and in the Mediterranean Sea. Overall, 129 taxa were obtained; thanks to a polyphasic approach based on morphological, molecular and phylogenetic techniques, 84.5% of them were identified at the species level. Two fungal species Thelebolus balaustiformis and Thelebolus spongiae were here first described, updating the knowledge on marine fungal diversity. This work underlined the specificity of the fungal community for each sponge, leading to think that these animals are able to recruit their own mycobiota. - The second part was based on the investigation of the chemical diversity of marine fungi associated with the sponge Grantia compressa, using the OSMAC approach (One Strain – Many Compounds). Not surprisingly, it has been difficult to define a condition that promotes both the development of the mycelium and the secondary metabolites production for all fungi; generally, rich nutrients media are the best candidates to achieve the above-mentioned results. Among the tested fungi, Eurotium chevalieri MUT 2316 produce more metabolites than any other fungus and ten pure compounds were isolated. - The third part of this Ph.D. project aimed to test the biological activity of the ten fungal molecules. Two main research fields, pharmaceutical and environmental, were chosen as potential targets. Six compounds showed antibacterial activity, with isodihydroauroglaucin active against most of the Grampositive bacteria tested also with bactericidal activity. Dihydroauroglaucin and physcion were able to completely inhibit the replication of Influenza A virus, while neoechinulin completely inhibited Herpes Simplex Virus 1. Finally, the last series of bioassays aimed to face the urgent need of environmentally friendly antifouling and highlighted several molecules already active at extremely low concentrations, inhibiting the adhesion and growth of both bacteria and microalgae. As result, a mix of few compounds produced by E. chevalieri MUT 2316 would inhibit all the bacteria and microalgae tested. In conclusion, this Ph.D. project highlighted the outstanding biodiversity and chemodiveristy of marine fungi inhabiting sponges. The molecules isolated from E. chevalieri MUT 2316 found applications in different research fields and represent promising candidates for the development of new drugs and antifouling paints.L'environnement marin est doté d’une diversité fongique encore trop faiblement explorée puisqu’on estime qu’environ 10% des champignons marins ont fait l’objet d’une étude. Dans ce contexte, le projet de thèse décrit dans ce manuscrit est focalisé sur le potentiel biotechnologique des champignons marins isolés d’éponges marines. Ces champignons sont caractérisés par une importante biodiversité et chimiodiversité susceptible de conduire à de nouvelles molécules bioactives. Il s’agit d’un projet pluridisciplinaire qui joint la mycologie, la chimie, la biochimie et les biotechnologies. Il couvre la stratégie complète de découverte de nouveaux produits naturels avec l'isolement et l'identification des souches fongiques à l’extraction et l'isolement des molécules ainsi que l’évaluation des propriétés biologiques. Le manuscrit est divisé en trois parties principales : - La première partie est dédiée à l’isolement des communautés fongiques cultivables associées à quatre éponges de l’océan Atlantique et trois éponges de Méditerranée. Nous avons obtenu au total 129 taxons parmi lesquels 84,5% ont pu être identifiées jusqu’au niveau de l’espèce via une approche polyphasique basée sur des techniques morphologiques, moléculaires et phylogénétiques. Parmi ces derniers, nous avons décrit pour la première fois deux espèces : Thelebolus balaustiformis et Thelebolus spongiae. Nos travaux ont permis de souligner la spécificité des communautés fongiques hébergées par chaque éponge ce qui laisse à penser que les éponges sont capables de recruter leur propre mycobiote. - La seconde partie est consacrée à l'étude de la diversité chimique des champignons marins associés à l'éponge Grantia compressa en utilisant l'approche OSMAC (une souche – de nombreux composés). Les résultats obtenus ont révélé les difficultés à obtenir des conditions de culture optimales. De façon générale et pour tous les champignons, les milieux riches en nutriments favorisent à la fois le développement du mycélium et la production de métabolites secondaires. Parmi les champignons isolés, Eurotium chevalieri MUT 2316 produits de nombreux métabolites, comparativement aux autres champignons. Dans ce contexte, nous avons pu isoler et caractériser dix composés. - La troisième partie est dédiée à l’évaluation des propriétés biologiques (pharmacologiques et environnementales) des différentes molécules isolées. Six composés ont montré des propriétés antibactériennes notamment l'isodihydroauroglaucine qui s’est avérée active vis-à-vis de la plupart des bactéries à Gram-positif testées et pour laquelle une activité bactéricide a pu également être décelée. La dihydroauroglaucine et le physcion inhibent complètement la réplication du virus de la grippe A tandis que la neoechinuline inhibe le virus de l'herpès simplex 1. Enfin, les différentes molécules ont été évaluées pour leurs propriétés antifoulings susceptibles de rentrer dans la composition de peintures plus respectueuses de l’environnement. Les molécules inhibent à de très faibles concentrations l'adhésion et la croissance de bactéries et de microalgues impliquées dans le biofouling. Par ailleurs, la combinaison de molécules isolées d’E. chevalieri MUT 2316 inhibe la totalité des bactéries et microalgues testées. Les travaux menés ont permis de mettre en avant l’importante biodiversité et chimiodiversité de champignons marins hébergés par les éponges. Les molécules isolées d’E. chevalieri MUT 2316 sont susceptibles de valorisation dans différents domaines de recherche tels que le développement de nouveaux médicaments ou de peintures antifoulings plus respectueuses de l’environnement