Distinguishing features of C. albicans pseudouridylation and their roles in pathogenesis

Ball State University, Muncie IN

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Abstract

Project Summary The human fungal pathogens C. albicans and C. auris are members of the CTG fungal clade which diverged roughly 200 million years ago from the Saccharomyces lineage. Work from previous funding periods suggests pseudouridylation and pseudouridine degradation are not conserved between CTG and Saccharomyces lineages. The Bernstein Lab will continue to characterize these processes in C. albicans and expand investigations to C. auris, an important emerging fungal pathogen with isolates that are resistant to all known antifungal therapeutics. This work will reveal fundamental differences in RNA modification processes between fungal lineages. Furthermore, the processes of pseudouridylation and pseudouridine degradation are not conserved in mammals, but some appear to be conserved in additional fungal pathogens. Pus1 is a pseudouridine synthase predicted to modify a variety of RNA substrates. In Aim 1, experiments will explore how heavy metals affect C. albicans Pus1 localization, determine if Pus1 A and B alleles interact with distinct cohorts of proteins, identify Candida nuclear targeting sequences, and identify Pus1 substrates. This work will be carried out by undergraduate and master’s students and will answer long standing questions regarding allele heterogeneity and stress response in C. albicans. Pug1 degrades pseudouridine. Aim 2 experiments will determine if abrogation of pseudouridine degradation affects C. auris growth, biofilm formation, or antifungal tolerance; identify Pug1 interactors; and determine if PUG1 deletion leads to a defect in virulence using a wax moth and planaria model system. This will be the first characterization of pseudouridine degradation in C. auris and one of the first characterizations of RNA metabolism in C. auris. Undergraduate and master’s students will be involved in all aspects of the proposed experiments providing excellent training opportunities for a diverse team of students. In addition to these two Aims, two of the classes designed and taught by the PI each year will be leveraged to provide the undergraduate and master’s students taking those classes an opportunity to examine important appropriate aspects of molecular and fungal biology. As part of their in-class laboratory activities, between 25 and 50 undergraduates a year will participate in focused research projects, working on important research questions using innovative approaches under direct supervision of the PI. The 2022 NIAID strategic plan states one of the Institute’s priorities is to “support basic and clinical research to better understand, diagnose, treat, and prevent infectious diseases and immune-mediated disorders—many of which have far-reaching global consequences—including fungal diseases.” Consistent with this vision, our research investigates RNA modifications in two important human fungal pathogens, C. albicans and C. auris. Our work seeks to better understand how these pathogens differ from humans at the molecular level. As such, this research has the potential to identify novel antifungal targets.