The role of phase separation in a transcriptional repression mechanism relevant to Candida albicanspathogenesis.

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Project Summary: Candida albicans is an opportunistic fungal pathogen capable of causing mucosal and systemic disease. Oropharyngeal candidiasis (OPC) is a frequent fungal disease in humans, and can potentially spread to other locations in the body causing systemic infections. Even with clinical intervention, systemic candidiasis has a mortality rate of ~40%. OPC involves the formation of biofilms in the oral mucosa that contain multiple C. albicans morphotypes, including both yeast and hyphal cells. The ability of C. albicans to form different cell types is crucial for virulence and contributes to its capacity to colonize different host tissues. The transition between yeast and hyphal cells is regulated by a network of transcription factors (TFs) that include the co-repressor Ssn6. Deletion of Ssn6 blocks formation of hyphal cells and attenuates C. albicans virulence. Ssn6 is also integral to a second TF network that regulates transitions between ‘white’ and ‘opaque’ states that exhibit different tissue tropisms in the host. Importantly, the Bennett lab has uncovered that multiple TFs (including Ssn6) in both networks contain prion-like domains (PrLDs) and that these can promote phase separation of the corresponding proteins. Phase separation refers to the process of liquid demixing that can generate protein condensates with liquid-like properties. This phenomenon is now implicated in the formation of many cellular structures including nucleoli, Cajal bodies, stress granules, and transcriptional complexes. While a number of studies on TF phase separation have been conducted, these studies primarily focus on transcriptional activators and not on transcriptional co-repressors such as Ssn6. Previous work in our lab has shown that C. albicans TFs containing PrLDs readily form phase-separated condensates when purified or when expressed in mammalian cells. Moreover, deletion or mutation of PrLDs can eliminate the ability of these TFs to function in C. albicans cells. My preliminary data shows Ssn6 is also able to form condensates, both individually and with other fungal TFs. I hypothesize that phase separation plays a key role in the ability of Ssn6 to act as a co-repressor in regulating cell morphology, and that it will contribute to virulence during OPC. In Aim 1, I will examine the biochemical properties of Ssn6 condensates. In Aim 2, I will address how changes to Ssn6 properties impact C. albicans morphotypes in vitro as well as virulence using a mouse model of OPC. Additionally, the goals of this fellowship include developing skill sets in order to lead a successful research career. Attending conferences, presenting to the department and graduate program, and regular meetings with the sponsor are planned to this end. The training plan will be carried out in an environment that benefits from the Molecular Biology, Cell Biology, and Biochemistry Graduate Program. Completion of the proposed goals will advance both the field of Candida biology and the ability of the recipient to become an effective independent scientific researcher.