Abstract
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 deathtoll of malaria. The human body is adept at keeping fungal pathogens at bay, but when ourimmune system is down, pathogenic yeast can infect and even kill us. One of the mostrecently 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 drugresistant. Development of antifungal drugs has been even less prioritized than the development of new bacterial antibiotics, but the need for novel strategies to combat fungalinfections is real. This thesis explores the antifungal mechanisms and potentials of Saccharomycopsis schoenii, a little-studied mycoparasitic yeast with the rare ability to attackand 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 onplants, 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 tosynthesise 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 againstfungal plant pathogens, have expanded gene families encoding for cell wall degrading enzymes, that they use when they antagonize or kill other fungi. Similarly, the yeastpathogen 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 aswell as live cell imaging techniques with the aim of genetically and functionallycharacterizing Saccharomycopsis yeasts. We first generated draft genomes of threeSaccharomycopsis yeasts, S. fodiens, S. fermentans and S. schoenii and found that thegenomic 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 alsofound 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.Through genomic analyses, we also found special tRNA-genes in the Saccharomycopsisyeasts, that suggested they belong to the CTG clade, a group of yeasts that translate the CTGcodon 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 assaysI set up, I was also able to determine that it is the lack of complex nitrogenous compounds,and not solely the sulfur amino acid methionine, as previously hypothesised, that is the maintrigger of predatory activity.To determine the genetic tools S. schoenii employ during its predatory activity, I performedextensive transcriptomic analyses. These revealed predation-specific upregulation of cellwall specific proteases, transposable elements and sulfur scavenging transporters. In total,my transcriptomic analysis suggested that S. schoenii breaks down the cell wall of its fungal prey through protein hydrolysis, and that methionine and other sulfur compounds wereacquired from the prey cells.To explore if the fungicidal properties of S. schoenii could be applied to medically important yeast pathogens, I set up an assay where I could analyse the susceptibility of yeast pathogensin the Candida genus. Indeed, I found that S. schoenii was able to kill clinical isolates of C.albicans, C. glabrata, C. parapsilopsis, C. tropicalis, and C. lutsitaniae as well as both sensitive and multi drug resistant isolates of C. auris.
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 deathtoll of malaria. The human body is adept at keeping fungal pathogens at bay, but when ourimmune system is down, pathogenic yeast can infect and even kill us. One of the mostrecently 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 drugresistant. Development of antifungal drugs has been even less prioritized than the development of new bacterial antibiotics, but the need for novel strategies to combat fungalinfections is real. This thesis explores the antifungal mechanisms and potentials of Saccharomycopsis schoenii, a little-studied mycoparasitic yeast with the rare ability to attackand 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 onplants, 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 tosynthesise 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 againstfungal plant pathogens, have expanded gene families encoding for cell wall degrading enzymes, that they use when they antagonize or kill other fungi. Similarly, the yeastpathogen 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 aswell as live cell imaging techniques with the aim of genetically and functionallycharacterizing Saccharomycopsis yeasts. We first generated draft genomes of threeSaccharomycopsis yeasts, S. fodiens, S. fermentans and S. schoenii and found that thegenomic 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 alsofound 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.Through genomic analyses, we also found special tRNA-genes in the Saccharomycopsisyeasts, that suggested they belong to the CTG clade, a group of yeasts that translate the CTGcodon 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 assaysI set up, I was also able to determine that it is the lack of complex nitrogenous compounds,and not solely the sulfur amino acid methionine, as previously hypothesised, that is the maintrigger of predatory activity.To determine the genetic tools S. schoenii employ during its predatory activity, I performedextensive transcriptomic analyses. These revealed predation-specific upregulation of cellwall specific proteases, transposable elements and sulfur scavenging transporters. In total,my transcriptomic analysis suggested that S. schoenii breaks down the cell wall of its fungal prey through protein hydrolysis, and that methionine and other sulfur compounds wereacquired from the prey cells.To explore if the fungicidal properties of S. schoenii could be applied to medically important yeast pathogens, I set up an assay where I could analyse the susceptibility of yeast pathogensin the Candida genus. Indeed, I found that S. schoenii was able to kill clinical isolates of C.albicans, C. glabrata, C. parapsilopsis, C. tropicalis, and C. lutsitaniae as well as both sensitive and multi drug resistant isolates of C. auris.
| Originalsprog | Engelsk |
|---|
| Forlag | Department of Biology, Faculty of Science, University of Copenhagen |
|---|---|
| Status | Udgivet - 2018 |
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