Zurich Open Repository and Archive University of Zurich Main Library Strickhofstrasse 39 CH-8057 Zurich www.zora.uzh.ch Year: 2014 Structural characterization, bioactivity and biodegradation of cyanobacterial toxins Kohler, Esther Posted at the Zurich Open Repository and Archive, University of Zurich ZORA URL: https://doi.org/10.5167/uzh-105523 Dissertation Published Version Originally published at: Kohler, Esther. Structural characterization, bioactivity and biodegradation of cyanobacterial toxins. 2014, University of Zurich, Faculty of Science. STRUCTURAL CHARACTERIZATION, BIOACTIVITY AND BIODEGRADATION OF CYANOBACTERIAL TOXINS Dissertation zur Erlangung der naturwissenschaftlichen Doktorwürde (Dr. sc. nat.) vorgelegt der Mathematisch-naturwissenschaftlichen Fakultät der Universität Zürich von Esther Kohler von Schwaderloch AG Promotionskomitee Prof. Dr. Jakob Pernthaler (Vorsitz) Prof. Dr. Leo Eberl PD Dr. Judith F. Blom Zürich, 2015 Meiner Familie GLOSSARY Adda (2S,3S,8S,9S)-3-amino-9-methoxy-2,6,8-trimethyl-10-phenyldeca-4,6-dienoic acid AG 828A Aeruginosin 828A Ahp 3-amino-6-hydroxy-2-piperidone BMAA β-methyl-amino-L-alanine Choi 2-carboxy-6-hydroxyoctahydroindole CP 1020 Cyanopeptolin 1020 DNA Deoxyribonucleic acid DOPA Dihydroxyphenylalanine GC-MS Gas chromatography-mass spectrometry GDM Gravity-driven membrane filtration GSH Glutathione GST Glutathione-s-transferase (H)PLA (Hydroxyl)phenyllactic acid HPLC High-performance liquid chromatography IC50 Half maximal inhibitory concentration i.p. Intraperitoneal (injection) LC50 Median lethal concentration LC-ESI-MS Liquid chromatography coupled to electrospray ionization mass spectrometry LC-MS Liquid chromatography-mass spectrometry LPS Lipopolysaccharid MC Microcystin Mdha Methyl-dehydroalanine NRPS Nonribosomal peptide synthase OATPs Organic anion transport proteins PKS Polyketide synthase ROS Reactive oxygen species SAR Structure-activity relationship VTG Vitellogenin WHO World health organisation TABLE OF CONTENTS SUMMARY ..................................................................................................................................................... 1 ZUSAMMENFASSUNG ....................................................................................................................................... 3 PREFACE ........................................................................................................................................................ 6 INTRODUCTION ............................................................................................................................................... 7 1. CYANOBACTERIA – ECOLOGY AND ENVIRONMENTAL IMPACT .................................................................... 7 2. CYANOBACTERIAL SECONDARY METABOLITES .......................................................................................... 8 3. CYANOBACTERIAL PEPTIDES .................................................................................................................. 9 3.1. MICROCYSTINS – THE BEST STUDIED CYANOTOXINS ......................................................................... 9 3.2. MICROCYSTINS IN DRINKING WATER ........................................................................................... 11 3.3. AERUGINOSINS ......................................................................................................................... 12 3.4. CYANOPEPTOLINS ..................................................................................................................... 13 4. THE ECOLOGICAL ROLE OF CYANOBACTERIAL PEPTIDES .......................................................................... 14 4.1. MICROCYSTINS AS GRAZER DEFENCE ........................................................................................... 15 4.2. MICROCYSTIN-DEFICIENT STRAINS............................................................................................... 15 5. AIMS OF THE THESIS ......................................................................................................................... 17 CHAPTER 1 - DRINKING WATER ................................................................................................................... 20 MANUSCRIPT I ...................................................................................................................................... 20 CHAPTER 2 - STRUCTURE ELUCIDATION AND ENZYMATIC CHARACTERIZATION .................................................... 21 MANUSCRIPT II ..................................................................................................................................... 21 CHAPTER 3 - EFFECTS ON THE AQUATIC MODEL ORGANISMS ZEBRAFISH AND DAPHNIA MAGNA ......................... 22 MANUSCRIPT III .................................................................................................................................... 22 MANUSCRIPT IV ................................................................................................................................... 23 DISCUSSION ................................................................................................................................................. 54 6. TOXICOLOGICAL AND ECOTOXICOLOGICAL RELEVANCE OF CYANOBACTERIAL PEPTIDES ............................... 54 6.1. REMOVAL OF MICROCYSTINS FROM DRINKING WATER .................................................................. 56 6.2. SERINE PROTEASE INHIBITORS ..................................................................................................... 59 7. POSSIBLE FUNCTIONS OF CYANOBACTERIAL PEPTIDES ............................................................................ 60 7.1. GRAZER DEFENCE ..................................................................................................................... 61 7.2. ALLELOCHEMICALS .................................................................................................................... 62 7.3. PARASITE-DEFENCE MECHANISM ................................................................................................ 62 8. CONCLUSION .................................................................................................................................... 63 REFERENCES ................................................................................................................................................. 64 CURRICULUM VITAE ...................................................................................................................................... 74 ACKNOWLEDGEMENTS ................................................................................................................................... 76 SUMMARY Recurrent mass developments of cyanobacteria, so-called blooms, have become a matter of public concern as they are more and more frequently found in waters intended as recreational areas and drinking water. Many of the cyanobacterial species are known to produce toxins that have a variety of adverse effects, such as acute diarrhoea, skin irritation, liver damage, neurotoxicity, and even fatali- ties in humans a well as in domestic and wild animals. Yet, cyanobacterial impact on aquatic ecosystems can only be estimated, as knowledge about the effects of cyanobacterial bioactive compounds on aquatic organisms is rather limited. This thesis focused on three major topics: removal strategies for already known potentially toxic cyanobacterial peptides were evaluated; the chemical structure of a novel cyanobacterial toxin was elucidated; and, further studies included detailed characterizations of largely undescribed or novel toxins in order to evaluate their potential risk for humans and aquatic eco- systems in future. Drinking water purification in terms of the elimination of cyanobacterial toxins is of major im- portance. Particularly in developing countries, simple and inexpensive purification systems are required. Gravity-driven membrane (GDM) ultrafiltration was shown to be an effective tool for the removal of pathogens and suspended solids by retention. We evaluated the biodegradation potential of the bacte- rial biofilm forming on the GDM ultrafiltration membrane for a cyanotoxin, microcystin. Successful elim- ination of microcystins in a GDM system required a maturation process of the biofilm, e.g. enrichment of bacteria capable of degrading microcystins. Phylogenetic analysis of the microbial communities form- ing the biofilm on the GDM ultrafiltration membrane gave first hints about potential new microcystin degraders. Microcystins have long been considered as the most toxic and therefore most important cya- nobacterial peptide class. Moreover, the investment of high amounts of resources into the production of microcystins suggests a very distinct and important function of these peptides for Microcystis, Ana- baena and Planktothrix. Compounds being able to compensate the lack of microcystins were hypothe- sized. A screening of six Planktothrix strains with and without the ability to produce microcystins re- vealed the presence of a distinct peptide group, i.e. chlorinated and sulfated aeruginosins, in strains that lack microcystins. Structure elucidation of one representative of the chlorinated and sulfated aeru- ginosins, aeruginosin 828A, was conducted. Subsequent toxicological and enzymatic characterization revealed the discovery of a new cyanobacterial toxin. The toxicity of aeruginosin 828A towards the freshwater crustacean Thamnocephalus platyurus