Dissertation
Promoter regulation of WOR1 and commitment to the opaque phase in Candida albicans
University of Iowa
Doctor of Philosophy (PhD), University of Iowa
Summer 2020
DOI: 10.17077/etd.005516
Abstract
Candida albicans is a prevalent human commensal and opportunistic pathogen. Its success as both a commensal and pathogen is due, in part, to its ability to undergo reversible phenotypic transitions and inducible developmental programs that are key to colonization and survival in the disparate host niches that it encounters. The work described in this thesis is focused on the regulation of and commitment to the opaque phenotype in the white-opaque switching system of C. albicans. In the past, a strategy for temperature-induced opaque-to-white mass conversion has been used to study gene regulation and phenotypic commitment in the transition from the opaque phase to the white phase, but the white-to-opaque transition had not previously been studied in the same detail due to the lack of a strategy for white-to-opaque mass conversion. Here, we describe a strategy devised for the induction of white-to-opaque mass conversion, which incorporates synchrony and temporal parallelism and allows one to analyze commitment to the opaque phase and its association with structural and molecular events that occur during daughter cell formation. We demonstrate that upon release from stationary phase under conditions that induce white-to-opaque mass conversion, opaque commitment occurs simultaneously with the initiation of daughter cell formation, as it exhibits a strong positive correlation with bud evagination and the simultaneous appearance of a chitin-rich ring at the bud-mother cell junction that marks the future site of septation. We also analyzed the transcriptional dynamics and regulation of the master regulator of the opaque phase, WOR1. It was found that WOR1 transcription is upregulated hours before commitment to the opaque phase, suggesting the cellular process that triggers commitment to the opaque phase occurs downstream of WOR1 upregulation. In addition to the investigation of phenotypic commitment in the white-to-opaque switch, the functions of transcription factor binding regions in the WOR1 promoter that were previously identified by ChIP-chip experiments were analyzed. By generating deletion derivatives of transcription factor binding regions of the WOR1 promoter and assessing the effects of each deletion derivative on white-to-opaque switching frequencies, WOR1 transcriptional activation, and opaque commitment, it is demonstrated that four transcription factor binding regions upstream of the WOR1 open reading frame (ORF) function mainly as cis-acting enhancers of WOR1 transcription and a single binding region downstream of the WOR1 3’UTR functions as a cis-acting silencer of WOR1. It is further demonstrated that while no individual binding region is essential for the formation of opaque cells, three of the four binding regions upstream of the WOR1 ORF are essential for heritable commitment to the opaque phenotype, indicating that these binding regions may be involved in changes in chromatin architecture associated with opaque commitment.
Details
- Title: Subtitle
- Promoter regulation of WOR1 and commitment to the opaque phase in Candida albicans
- Creators
- Thomas P. Conway
- Contributors
- David R Soll (Advisor)Bryan Phillips (Committee Member)Veena Prahlad (Committee Member)Scott Moye-Rowley (Committee Member)Anna Malkova (Committee Member)
- Resource Type
- Dissertation
- Degree Awarded
- Doctor of Philosophy (PhD), University of Iowa
- Degree in
- Integrated Biology
- Date degree season
- Summer 2020
- DOI
- 10.17077/etd.005516
- Publisher
- University of Iowa
- Number of pages
- xvi, 261 pages
- Copyright
- Copyright 2020 Thomas P. Conway
- Language
- English
- Description illustrations
- color illustrations
- Description bibliographic
- Includes bibliographical references (pages 240-261).
- Public Abstract (ETD)
The reversible transition from the default white phenotype to the opaque phenotype is central to the basic biology of the common human commensal and opportunistic pathogen, Candida albicans, as it is a prerequisite to mating and has been implicated in the efficient colonization of the host. Due to the lack of a strategy for studying the white-to-opaque transition, past investigations of white-opaque switching have involved comparisons of the initial and terminal phenotypes, but have neglected the temporal changes accompanying the transition from white to opaque. Here, we describe a strategy for white-to-opaque mass conversion in C. albicans and the analysis of phenotypic commitment in populations undergoing synchronized white and opaque daughter cell formation. Our results indicate that the event that triggers commitment to the opaque phase is closely associated with the initiation of daughter cell formation and occurs several hours after upregulation of the master regulator gene of the opaque phase, WOR1. In addition to our analysis of opaque commitment, we also assessed the functionality of previously identified transcription factor binding regions implicated in the regulation of WOR1 transcription. By deleting transcription factor binding regions individually, and in combination, and assessing the effects on opaque induction, commitment, and WOR1 transcript levels, we determined that the transcription factor binding regions function in both the dynamics of WOR1 transcriptional regulation as well as the commitment to the opaque phenotype. Thus, our results provide new insight into the regulation of the opaque phenotype and developmental gene regulation in eukaryotes.
- Academic Unit
- Biology
- Record Identifier
- 9983988197202771
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