Genetic analysis of yeasts of clinical and biotechnological importance

Fungi play important roles across many areas of life. Some fungi can cause infections in humans with high levels of morbidity and mortality but are often under- represented in research due in part to infections mainly occurring within the subpopulations of individuals with weak or compromised immune systems. Fungi are also important economically, with a new wave of interest currently being directed at brewing due to a newly emergent yeast in the sector. In this thesis I will be investigating two yeasts, one of clinical and one of biotechnological importance. The yeast species Candida parapsilosis is a human commensal and an opportunistic fungal pathogen. C. parapsilosis is one of the most commonly isolated species from nosocomial Candida infections, particularly in neonates. Resistance to antifungal treatments in this species has been on the rise worldwide. In Chapter 2, the role of multi-drug resistant (MDR) transporters in antifungal resistance in C. parapsilosis is reassessed using automated pinning and image analysis to generate quantifiable measurements. The MDR transporters investigated in this study all belong to a single branch of a family of transporters known as DHA1. This branch of the DHA1 family of transporters can be further divided into 6 clades. The results confirmed those of earlier analysis; the disruption of one gene from the TPO1 clade was found to result in increased susceptibility to mycophenolic acid, and the disruption of one gene from the MDR1 clade was found to increase susceptibility to fluconazole, voriconazole, cycloheximide and brefeldin A. The combined disruption of two MDR1 genes was confirmed to result in increased sensitivity to 4-NQO. Investigating the genes involved in antifungal resistance contributes to ongoing research to better understand the mechanisms of resistance and the search for novel antifungal drug targets. This chapter utilises methodologies that result in quantifiable measurements to avoid the subjectivity of visual assessment, which can be prone to human error and interpretation. This chapter also highlights the importance of ensuring that results are reproducible and investigates the effect temperature and media can have on observed results. In Chapter 3, a genome wide association study was conducted to investigate the genetic variation associated with alternate nitrogen metabolism in >300 C. parapsilosis isolates. Three variants potentially associated with poor growth on lysine as a sole nitrogen source were identified. However, CRISPR-Cas9 editing showed that introducing the variants either individually or in combination did not recapitulate the phenotype. In addition, a variant that resulted in truncation of the Dal4 transcription factor was identified that was associated with inefficient allantoin metabolism. CRISPR-Cas9 editing showed that the introduction of this variant contributed to reduced growth on allantoin. Nitrogen metabolism is vital for the survival and propagation of most fungal species, and in pathogenic species the ability to utilise a wide variety of nitrogen sources can be considered a virulence factor. Investigating the genetic background of nitrogen utilisation contributes to the understanding of the capabilities of this opportunistically pathogenic species. This chapter also highlights the importance of experimental testing to validate the results of bioinformatic analysis. Saccharomyces eubayanus is one of the parental species of the hybrid S. pastorianus, which is the yeast used in the brewing of lager. Irish S. eubayanus isolates are closely related to the S. eubayanus component of S. pastorianus, but are unable to metabolise maltose, the most abundant sugar in wort. In Chapter 4, the mechanisms underlying maltose metabolism in one of the original Irish S. eubayanus isolates (UCD650) and a newly identified isolate, (UCD926) is investigated. The isolates were adapted to grow in the presence of maltose over 52 and 67 generations respectively. Genome sequencing identified copy number variations in the MAL operon, containing the genes responsible for the utilisation of maltose. In addition, homozygous variants that resulted in amino acid substitutions in the transcriptional regulator MalR were identified. Finally, uracil auxotrophic isolates of the Irish S. eubayanus isolates were also generated using UV mutagenesis. Eight mutant strains were investigated by genome sequencing. Missense and nonsense Single nucleotide polymorphisms (SNPs) were identified in the URA5 gene in 7 isolates, and a frameshift mutation in the URA3 gene was identified in one isolate. Improved maltose metabolism in the Irish S. eubayanus isolates could potentially lead to the production of a novel lager and the identification of MalR variants could be further investigated to better understand the mechanisms behind maltose utilisation in this species. The generation of auxotrophic Irish S. eubayanus strains could be used to create novel brewing hybrids with other yeast species, furthering the potential for the production of a novel lager.