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Thesis Format

Monograph

Degree

Master of Science

Program

Anatomy and Cell Biology

Supervisor

Dr. Patrick Lajoie

Abstract

In eukaryotes, the Unfolded Protein Response (UPR) maintains proteostasis in the endoplasmic reticulum (ER). In yeast, the UPR is activated by the ER-resident kinase/RNase sensor protein; Inositol requiring enzyme 1 (Ire1). During ER stress, Ire1 oligomerizes and splices the premature HAC1 mRNA. The Hac1 transcription factor which binds to the unfolded protein response element (UPRE) and upregulate genes to mitigate the ER stress. Although poorly understood, the UPR is thought to play an essential role in antifungal resistance of pathogenic species. Therefore, with the highly characterized Saccharomyces cerevisiae model organism, I characterize the function of Ire1 upon azole treatment. For the first time in S. cerevisiae, I found that while the Ire1 is required for azole resistance, the UPR transcriptional response is dispensable as HAC1 splicing and Ire1 oligomerization upon azole treatment do not occur. This suggests a requirement for Ire1 in azole resistance which is Hac1 and UPR independent. Moreover, I have found that the kinase and luminal domains of Ire1 are dispensable during azole resistance. I also show that the upregulation of the UPRE-regulated ERAD proteins; KAR2 and HRD1 are dispensable, suggesting the absence of proteotoxic stress upon azole treatment. Interestingly, I found that in the absence of Ire1, ergosterol synthesis genes which are regulated independently of the UPR are downregulated, which may explain the increased azole resistance in ∆ire1 mutants. Overall, our data suggest that the S. cerevisiae Ire1 has a UPR independent function in the regulation of ergosterol synthesis genes which confers greater resistance to azoles.

Summary for Lay Audience

Antifungal drug resistance is increasingly becoming a more frequent cause of death by infection, especially in immunocompromised individuals. This is because, like the increase of antibiotic resistance in bacteria which require stronger and stronger antibiotics, the emergence of antifungal drug resistance in yeast have diminished the effectiveness of the limited selection of antifungals available today. Therefore, understanding the mechanisms used by yeast to develop resistance is vital for the creation of effective drugs. The current understanding of antifungal drug resistance is that stress adaptation pathways of these species are among the main contributing factors for the emergence of drug resistance in yeast. The day-to-day functions of the cell can often result in the formation of damaged proteins which must be corrected through stress adaptation pathways to resume normal cellular function. Unfortunately, upon treatment with antifungal drugs, these same pathways are activated to provide yeast cells drug resistance. Therefore, studying these pathways and how they help yeast become resistant antifungals will help develop new treatments and preserve the existing selection of drugs. In this study, with the use of baker’s yeast, also known as Saccharomyces cerevisiae, I found for the first time that azole antifungal drug resistance requires the presence of a stress sensor protein, yet it does not activate its associated stress adaptation pathway. This suggests that this protein has an unknown function which confers yeast cells with drug resistance. Furthermore, I have found evidence to suggest that in the absence of this sensor protein, the synthesis of a yeast membrane component is reduced. Overall, our findings using S. cerevisiae suggest that azole resistance requires this sensor protein in a new way than previously described in literature.

Creative Commons License

Creative Commons Attribution 4.0 License
This work is licensed under a Creative Commons Attribution 4.0 License.

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