UNIVERSITY OF COPENHAGEN DEPARTMENT OF BIOLOGY PhD thesis Klara Junker Prospecting a Predator: Exploration of the biocontrol potential of the mycoparasitic yeast Saccharomycopsis schoenii and an integrated microscopic and multiomic characterization of its predatory behaviour Supervisor: Birgitte Regenberg, University of Copenhagen Co-supervisor: Jürgen Wendland, Vrije Universiteit, Brussel This thesis has been submitted to the PhD School of The Faculty of Science, University of Copenhagen on the 6th of July, 2018 Name of department: Department of Biology Author: Klara Junker Title and subtitle: Prospecting a predator: Exploration of the biocontrol potential of the mycoparasitic yeast Saccharomycopsis schoenii and an integrated microscopic and multiomic characterization of its predatory behaviour Supervisor: Assoc. Prof. Birgitte Regenberg Department of Biology, University of Copenhagen, Copenhagen, Denmark Co-supervisor: Prof. Jürgen Wendland Research Group of Microbiology, Vrije Universiteit Brussel, Brussels, Belgium Submitted on: The 6th of July, 2018 Assessment committee: Assoc. Prof. Rasmus Kjøller Department of Biology, University of Copenhagen, Copenhagen, Denmark Dr. Alexandra Brand MRC Centre for Medical Mycology, University of Aberdeen, Aberdeen, Scotland Dr. Gianni Liti Institute of Research on Cancer and Ageing of Nice, CNRS, INSERM, Université de Nice, Nice, France ”Klara klarar allt” ~ Siri Johansson ~ In dedication to my grandmother Siri Johansson, 26/4 1916 ~ 14/11 2017 Det här är resultatet av möjligheter jag önskar du också fått Prospecting a Predator | Klara Junker Table of contents Preface ............................................................................................... 7 Summary .......................................................................................... 8 Resumé ............................................................................................ 10 List of papers ................................................................................. 12 Aim & objectives ........................................................................... 13 Introduction .................................................................................... 14 1. Introduction to fungi ............................................................................................. 14 1.1. Fungal nutrition ................................................................................................ 17 1.2. Nutrient acquisition tactics ........................................................................... 18 1.3. Mycoparasitism ................................................................................................ 19 1.4. Biocontrol fungi .............................................................................................. 20 1.5. The fungal cell wall ....................................................................................... 22 1.6. Fungal parasite genomics ........................................................................... 23 1.7. Human fungal pathogens ........................................................................... 24 2. Yeasts ....................................................................................................................... 25 2.1. Saccharomyces cerevisiae: the model yeast organism .................... 25 2.2. Human yeast pathogens in the Candida genus .................................. 26 2.3. Candida albicans: a commensal yeast in CTG clade ......................... 26 2.4. Candida auris: an emerging multidrug resistant pathogen ............. 28 2.5. Saccharomycopsis yeasts: necrotrophic mycoparasites .................. 29 5 Klara Junker | Prospecting a Predator Materials & methods ................................................................. 32 1. Strains & culture conditions .............................................................................. 32 2. Live cell microscopy ............................................................................................. 34 3. Genome sequencing & assembly .................................................................... 36 4. Genome annotation ............................................................................................. 37 5. Proteomic analyses ............................................................................................. 39 6. Transcriptomic analyses .................................................................................... 40 7. GO term analysis ................................................................................................... 41 8. Genetic manipulations of S. schoenii .............................................................. 42 Summary of papers .................................................................... 43 Results & Discussion .................................................................. 46 Perspectives for further research ............................................................................ 55 Conclusions .................................................................................... 57 Acknowledgements .................................................................... 58 References ..................................................................................... 62 Papers ............................................................................................. 68 Paper I ............................................................................................................................. 69 Paper II ............................................................................................................................. 73 Paper III ............................................................................................................................ 77 Paper IV ......................................................................................................................... 103 6 Prospecting a Predator | Klara Junker Preface This work was funded by the Marie Curie Initial Training Network “FUNGIBRAIN”, Grant agreement no. 607963, from the European Union. The majority of research was carried out in the Yeast and Fermentation group at the Carlsberg Research Laboratory, Copenhagen, Denmark. Work with clinical isolates of Candida species, S. cerevisiae and S. pombe was performed in collaboration with Neil Gow, Alexander Lorenz and Gustavo Bravo of the Aberdeen Fungal Group, at the University of Aberdeen, Aberdeen, UK. 7 Klara Junker | Prospecting a Predator Summary Fungal infections have in the recent years gone from being associated to minor annoyances, to being recognized as a diverse group of emerging killers, now even outnumbering the death toll of malaria. The human body is adept at keeping fungal pathogens at bay, but when our immune system is down, pathogenic yeast can infect and even kill us. One of the most recently emerging pathogens, the yeast Candida auris, was first described in Japan in 2009, but has already spread across the globe. In addition, several strains of C. auris are multi drug resistant. Development of antifungal drugs has been even less prioritized than the development of new bacterial antibiotics, but the need for novel strategies to combat fungal infections is real. This thesis explores the antifungal mechanisms and potentials of Saccharomycopsis schoenii, a little-studied mycoparasitic yeast with the rare ability to attack and kill other fungi including, as I demonstrate, multi drug resistant C. auris. Fungi are diverse in the way they feed themselves. Some filamentous fungi parasitize on plants, whereas yet others are mycoparasites, meaning that they can parasitize on other fungi. A common feature of these parasites is that they have often lost the genes that allow them to synthesise essential nutrients that they instead are able to acquire from their hosts. In parallel, they have increased the copy numbers of genes that allow them to parasitize. For instance, filamentous fungi in the mycoparasitic genus Trichoderma, used as biocontrol agents against fungal plant pathogens, have expanded gene families encoding for cell wall degrading enzymes, that they use when they antagonize or kill other fungi. Similarly, the yeast pathogen C. albicans also use an expanded set of proteases when it infects humans. In this thesis I made use of state-of-the-art next generation sequencing and bioinformatics as well as live cell imaging techniques with the aim of genetically and functionally characterizing Saccharomycopsis yeasts. We first generated draft genomes of three Saccharomycopsis yeasts, S. fodiens, S. fermentans and S. schoenii and found that the genomic reason that these yeasts, unlike all other yeasts, are unable to assimilate inorganic sulfate is because they lack all genes in the sulfate assimilation pathway. In parallel, we also found that they, analogous to filamentous mycoparasitic fungi, have expanded their sets of genes encoding proteases, chitinases and glucanases that can break down fungal cell walls. 8 Prospecting a Predator | Klara Junker Through genomic analyses, we also found special tRNA-genes in the Saccharomycopsis yeasts, that suggested they belong to the CTG clade, a group of yeasts that translate the CTG codon to serine instead of leucine. We were able to confirm the translation of CTG to serine, through a proteomic assay. By means of live microscopy, I provided spatial and temporal visualization of how the S. schoenii attacks and kills S. cerevisiae, a model prey cell. Through microscopy-based