Authors:

Leila Paltrowitz

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About Paper:

Candida albicans is the most common fungal pathogen encountered in the clinic and the primary cause of fungal disease in humans. Typically, C. albicans lives as a commensal member of the gut microbiome, yet it can become pathogenic and cause a variety of life-threatening infections. In contrast to bacterial members of the gut microbiome, the role of C. albicans has been far less studied, and there is interest in understanding how different aspects of Candida biology affect its behavior as a member of the microbiome. This fungus can occur in a variety of morphological forms; two important forms are yeast and hyphae. This study aimed to design a system for the inducible expression of multiple Candida genes in order to understand their respective roles. While certain gene regulation systems have already been established in C. albicans, such as the frequently used Tet-On and Tet-Off systems, each has limitations. No current system enables simultaneous control of multiple genes without any toxicity to Candida, bacterial cells, or the mammalian cells of the gut microbiome. This research project aimed to create a collection of inducible gene regulation programs for use in C. albicans. Several such systems have been established for use in the fungal species Saccharomyces cerevisiae; here, we adapted them for use in C. albicans. One such regulatable system is Small Molecule-Assisted Shutoff (SMASh-tag), which involves the fusion of target proteins to a degron that is removed in the absence of a drug, allowing for drug-induced degradation of target proteins. Here, we utilized the SMASh-tag system in C. albicans to effectively induce filamentation by tagging NRG1, a negative regulator of filamentation. Additionally, I used the Golden Gate Assembly (GGA) cloning approach to construct a set of hormone-based systems for inducible gene expression in Candida albicans. The systems encode mammalian nuclear receptors containing ligand binding, DNA binding, and transactivation domains. In the presence of specific hormone ligands, these nuclear receptors translocate to the nucleus, bind to the corresponding DNA binding domains upstream of a minimal promoter and a gene of interest, and activate transcription. Further, the efficacy of each of these systems was tested in controlling the expression of a mNeon reporter gene in C. albicans. Together, these systems expand the tools available for C. albicans gene regulation, providing potential new strategies for studying the role and behavior of C. albicans in the mouse gut environment. 80 Lilly Nguyen:

Source:

Brown / SPRINT|Undergraduate Teaching and Research Awards (UTRA)

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Co-authors:

Leila Paltrowitz