Identifying antifungal resistance mechanisms and modes of action using SAturated Transposon Analysis in Yeast (SATAY)

Thesis

Fungal pathogens are a serious burden for public health and global food security. This problem is exacerbated by the rapid emergence of resistance to our limited armamentarium of antifungal compounds. The future control of fungal diseases therefore depends on the discovery of antifungals with novel modes of action and the development of robust resistance management strategies. These strategies require a comprehensive understanding of the molecular mechanisms governing antifungal resistance, yet our knowledge is incomplete.

Recently, a powerful transposon sequencing method was developed in Saccharomyces cerevisiae, called SAturated Transposon Analysis in Yeast (SATAY). SATAY couples saturated transposon mutagenesis to next-generation sequencing, allowing the genome-wide identification of growth-affecting loci in a straightforward and time-efficient manner. Importantly, SATAY can identify loss- and gain-of-function mutations conferring antifungal resistance, as well as the direct targets of antifungal compounds.

This thesis applies SATAY to uncover novel antifungal resistance mechanisms and to examine the modes of action of nine antifungal compounds. These screens reveal antifungal drug targets, transporters responsible for drug uptake or efflux, as well as metabolic, signalling and trafficking pathways affecting antifungal susceptibility. The most novel and interesting findings in these screens are characterised further. This includes the discovery of Hol1 as the transporter that concentrates the potent antifungal ATI-2307 within yeast, a finding that unveils a straightforward evolutionary path to ATI-2307 resistance with minimal fitness cost. SATAY is also performed in drug-sensitive strains to examine antifungals that lack activity against conventional laboratory strains. This approach is utilised to test a previously proposed model for the mode of action of Fludioxonil, and identifies cell wall mannosylphosphate as the target for the natural antifungal Chitosan. Together, this thesis improves our understanding of several antifungal modes of action and unveils a diverse array of resistance mechanisms, supporting efforts to address the increasing threats posed by multidrug-resistant fungal pathogens.

Authors: Karadzas, M

• DOI: 10.5287/ora-6rkq6jyr1
• Type of award: DPhil
• Level of award: Doctoral
• Awarding institution: University of Oxford
• Language: English
• Keywords: yeast; chemogenomic screening; genome-wide transposon mutagenesis; antifungal resistance mechanisms; SATAY
• Subjects: Molecular biology; Biochemistry; Microbiology; Antifungal agents; Drug resistance in microorganisms; Yeast; Genetics