Marine fungi from sponges : biodiversity, chemodiversity and biotechnological applications Elena Bovio To cite this version: Elena Bovio. Marine fungi from sponges : biodiversity, chemodiversity and biotechnological applica- tions. Other. COMUE Université Côte d’Azur (2015 - 2019); Università degli studi (Torino, Italia), 2019. English. NNT : 2019AZUR4009. tel-02514804 HAL Id: tel-02514804 https://tel.archives-ouvertes.fr/tel-02514804 Submitted on 23 Mar 2020 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. University of Turin Department of Life Sciences and Systems Biology University of Côte d’Azur Institute of Chemistry of Nice, UMR 7272 CNRS Ph.D. Program in Biology and Applied Biotechnologies Doctoral School of Sciences Fondamentales et Appliquées Doctoral School of Sciences Fondamentales et Appliquées Marine fungi from sponges: biodiversity, chemodiversity and biotechnological applications Elena Bovio Ph.D. Coordinators: Prof. Silvia Perotto Prof. Elisabeth Taffin de Givenchy Academic years: 2015-2018 Academic disciplines: BIO/02 - Chemistry University of Turin Ph.D. Programme in Biology and Applied Biotechnologies University of Côte d’Azur Doctoral School of Sciences Fondamentales et Appliquées Marine fungi from sponges: biodiversity, chemodiversity and biotechnological applications Elena Bovio Tutors: Prof. Giovanna Cristina Varese Prof. Mohamed Mehiri XXXI Cycle: 2015 – 2018 The sea, once it casts its spell, holds one in its net of wonder forever Jacques Y. Cousteau Table of contents List of Abbreviations i List of Figures and Tables iv 1. INTRODUCTION 1 1.1 Marine environment 1 1.2 Marine fungi 2 1.2.1 Origin 2 1.2.2 Diversity and occurrence of fungi in the marine environment 5 1.2.3 Ecology 8 1.3 Marine organisms and microorganisms: an unlimited source of novel compounds 12 1.3.1 The Mycotechnology 13 1.4 The sponge holobiont 16 1.4.1 Sponges 166 1.4.1.1 Origins, anatomy and physiology 166 1.4.1.2 Sponges, as producers of secondary metabolites 18 1.4.2 Fungi associated with sponges 19 1.4.3 Secondary metabolites from marine fungi associated with sponges 23 1.5 Classical and epigenetic approach to diversify the production of secondary metabolites in marine fungi 25 2. AIM OF THE WORK 30 2.1 Project outline 32 3. The culturable mycobiota associated with 4 Atlantic sponges 33 3.1 MATERIAL AND METHODS 34 3.1.1 Sampling sites and axenic isolation 34 3.1.2 Fungal identification 36 3.1.3 Statistical analyses 38 3.2 RESULTS AND DISCUSSION 38 3.2.1 Influence of isolation techniques on the fungal community 38 3.2.2 Fungal diversity 41 3.2.3 Fungal diversity among the sponges 58 3.3 CONCLUSION 63 4. Thelebolus balaustiformis and Thelebolus spongiae: two new fungal species from the Atlantic sponge D. fragilis 65 4.1 MATERIAL AND METHODS 65 4.1.1 Thelebolus spp. growth conditions and molecular study 65 4.2 RESULTS AND DISCUSSION 65 4.2.1 Thelebolus balaustiformis and Thelebolus spongiae sp. nov. 65 4.2.2 Taxonomy 67 4.2.2.1 Description of Thelebolus balaustiformis 67 4.2.2.2 Description of Thelebolus spongiae 72 4.2.2.3 Ecology and peculiar features of Thelebolus spp. 77 5. The culturable fungal communities inhabiting the Mediterranean sponges Aplysina cavernicola, Crambe crambe and Phorbas tenacior 82 5.1 MATERIAL AND METHODS 83 5.1.1 Sampling sites and fungal isolation 83 5.1.2 Fungal identification 84 5.1.3 Statistical analyses 85 5.2 RESULTS AND DISCUSSION 85 5.2.1 Influence of isolation techniques on the fungal community 85 5.2.2 Fungal diversity 86 5.2.3 Fungal culturable diversity among the sponges 96 5.2.4 Mycobiotas vs metabolome 99 5.2.5 Fungi from A. cavernicola isolated by direct plating 100 5.3 CONCLUSION 105 6. Chemical diversity of the fungal community associated with the Atlantic sponge G. compressa 106 6.1 MATERIALS AND METHODS 106 6.1.1 Small scale fermentation of the fungal community and OSMAC approach 106 6.1.1.1 Multi-well culture and co-culture conditions 106 6.1.1.2 Extraction procedure and chemical analysis 107 6.1.2 Scale-up of E. chevalieri MUT 2316 and molecules purification 108 6.1.2.1 Solid media culture condition and secondary metabolites extraction and purification from E. chevalieri 108 6.1.2.2 Liquid media culture condition and secondary metabolites extraction from E. chevalieri 110 6.1.2.3 High Resolution Mass Spectrometry analysis and molecular networking 111 6.2 RESULTS AND DISCUSSION 112 6.2.1 OSMAC approach 112 6.2.1.1 Effect of media on fungal development and chemical fingerprint 112 6.2.1.2 Effect of salts on the fungal development and the chemical fingerprint 117 6.2.1.3 Effect of co-culture on the fungal development and the chemical fingerprint 119 6.2.1.4 General remarks on the OSMAC approach 120 6.2.2 Scale up of Eurotium chevalieri MUT 2316 121 6.2.2.1 E. chevalieri MUT 2316 metabolites from solid culture condition 122 6.2.2.2 E. chevalieri MUT 2316 metabolites from liquid culture condition 127 6.2.2.3 Molecular networking of E. chevalieri MUT 2316 in solid and liquid culture condition 130 6.3 CONCLUSION 134 7. The antibacterial activity of the molecules produced by E. chevalieri MUT 2316 135 7.1 INTRODUCTION 135 7.1.1 The Golden Age of antibiotics, the antibiotic crisis and the need of new medicines 135 7.1.2 Developing of AMR 139 7.1.3 What determines a bacterium to become MDR? 141 7.2 MATERIAL AND METHODS 142 7.2.1 Bacterial growth conditions and inoculum preparation 142 7.2.2 Stock solutions of the molecules isolated from E. chevalieri MUT 2316 143 7.2.3 Determination of the MIC 144 7.2.4 Determination of the Minimal Bactericidal Concentration (MBC) 146 7.3 RESULTS AND DISCUSSION 146 7.3.1 E. chevalieri MUT 2316 derived compounds show antibacterial activity 146 7.3.2 Structure-activity relationship 151 7.4 CONCLUSION AND FUTURE PERSPECTIVES 151 8. The antiviral activity of the molecules produced by E. chevalieri MUT 2316 153 8.1 INTRODUCTION 153 8.1.1 Viruses and antivirals 153 8.1.2 Influenza A virus (IAV) 155 8.1.3 Herpes Simplex virus 1 (HSV-1) 156 8.2 MATERIAL AND METHODS 158 8.2.1 Cell culture conditions and viruses titre determination 158 8.2.2 Cytotoxic assays 159 8.2.3 Antiviral assay 159 8.3 RESULTS AND DISCUSSION 161 8.3.1 Cytotoxic activity of E. chevalieri MUT 2316 compounds 161 8.3.2 E. chevalieri MUT 2316-derived compounds show antiviral activity against HSV-1 and IAV 162 8.3.3 Structure-activity relationship 164 8.4 CONCLUSION AND FUTURE PERSPECTIVES 164 9. The antifouling activity of the molecules produced by E. chevalieri MUT 2316 166 9.1 INTRODUCTION 166 9.1.1 The biofouling: definition and impact 166 9.1.2 Biofouling formation 166 9.1.3 Antifouling methods 168 9.1.4 Why sponges and their associated microorganisms as candidates for the production of new antifouling? 171 9.2 MATERIAL AND METHODS 172 9.2.1 Marine Antibacterial Assays 173 9.2.1.1 Growth inhibition 174 9.2.1.2 Adhesion inhibition 175 9.2.2 Microalgal assay 176 9.2.2.1 Growth inhibition 176 9.2.2.2 Adhesion inhibition 177 9.2.3 Inhibition of blue mussel M. edulis settlement – tyrosinase assay 177 9.3 RESULTS AND DISCUSSION 178 9.3.1 The molecules produced by E. chevalieri MUT 2316 display antifouling activity 178 9.4 CONCLUSION AND FUTURE PERSPECTIVES 184 10. GENERAL CONCLUSION AND FUTURE PERSPECTIVES 186 References 190 Annexe 226 Annexe 2 242 Ph.D. activities 245 PUBLICATIONS 247 ACKNOWLEDGEMENTS 250 List of Abbreviations AC Algobank of Caen ACT Actin gene AIC Akaike Information Criterion AMR Antimicrobial resistance ANOVA Analysis of variance ATCC American type culture collection BHI Brain-Heart Infusion BPP Bayesian posterior probabilities CA Carrot Agar CAL Calmodulin gene CAP Canonical analysis of principal coordinates CFU Colony forming unit CLSI Clinical and laboratory standards institute CMASW Corn meal agar seawater cRNP Ribonucleoprotein DAAs Directly acting antivirals DBIOS Department of life sciences and system biology DDD 1,1-dichloro-2,2-bis-(4-chlorophenyl)ethane DMEM Dulbecco’s Modified Eagle Medium DMSO Dimethyl sulfoxide DNA Deoxyribonucleic acid DNMT DNA methyltransferase dNTPs Deoxynucleotides EPS Extracellular polymeric substances FA Formic acid FBS Fetal bovine serum FDA Food and Drug Administration GA Gelatin agar GAPDH Glyceraldehyde-3-phosphate dehydrogenase gene GAS Gelatin agar added with 3% NaCl GASW Gelatin agar seawater i GNPS Global natural products social molecular networking HAAs Host-acting antivirals HBV Hepatitis B virus HCV Hepatitis C virus HDAC Histone deacethylase HIV Human immunodeficiency virus HPLC High Performance Liquid Chromatography HR-MS/MS High-resolution tandem mass spectrometry HSV1 Herpes simplex virus 1 HSV2 Herpes simplex virus 2 IAV Influenza A virus IBV Influenza virus B IC Inhibitor concentration ITS Internal transcribed spacer regions LBCM Laboratoire de Biotechnologie et Chimie Marines LC-MS Liquid chromatography - mass spectrometry LOEC Low observable effect concentration LSU Large subunit MBC Minimal bactericidal concentration MBM Marine bacterial medium MCMC Markov chains monte carlo MDCK Madin darby canine kidney cells MDR Multi drug resistant