CHARACTERIZATION OF CDC14 PHOSPHATASE BIOCHEMICAL MECHANISMS AND THEIR RELATIONSHIP TO FUNGAL PATHOGENESIS
Creators: name: Milholland, Kedric L
Publisher: name: Purdue University Graduate School
Year: 2023
Subjects: classificationCode: 310101
subject: Analytical biochemistry

description: The Cdc14 phosphatase family is highly conserved in fungi. In Saccharomyces cerevisiae, Cdc14 is essential for down-regulation of cyclin-dependent kinase activity at mitotic exit. However, this essential function is not broadly conserved and requires only a small fraction of normal Cdc14 activity. In general, few conserved functions of Cdc14 phosphatase have been defined. Here, I present mechanistic biochemical and phenotypic characterization of Cdc14 phosphatases in fungi. I have demonstrated that fungal Cdc14 phosphatases possess an invariant motif in the disordered C-terminal tail that is required for full enzymatic activity. This motif, termed substrate-like catalytic enhancer (SLiCE), functions during the rate-limiting step of Cdc14-directed catalysis, by binding to the active site and supporting phospho-enzyme hydrolysis. Adjacent to the SLiCE motif exists a conserved minimal Cdk consensus motif that likely serves a regulatory function as phosphorylation of this site inhibits Cdc14 activity in vitro. Vertebrate Cdc14 enzymes also possess a distinct, but mechanistically similar SLiCE motif, which may be the first described biochemical difference between Cdc14 enzymes. Moreover, the vertebrate SLiCE motif lacks an adjacent Cdk consensus motif, which may point to differences in how Cdc14 activity is regulated in higher eukaryotes.Mutation of this motif in vivo served as a tool to discover biological processes that require high Cdc14 activity. In S. cerevisiae strains expressing this hypomorphic mutant allele (cdc14hm), I discovered a novel sensitivity to cell wall stresses, including chitin-binding compounds and echinocandin antifungal drugs. This sensitivity was also observed in the distantly related fungi Schizosaccharomyces pombe deletion strain and the human fungal pathogen Candida albicans hypomorphic and deletion strains, suggesting that this phenotype reflects a conserved function of Cdc14 orthologs in mediating fungal cell wall integrity. I also revealed that high Cdc14 activity is required for C. albicans ability to develop hyphae, which is an important virulence trait. This led to our determination that high Cdc14 activity is critical for virulence in two animal models of invasive candidiasis. Together, these results argue that Cdc14 would be an excellent antifungal drug target for the treatment of invasive Candida infections and sensitization to existing antifungal drugs.Lastly, I implemented the auxin-inducible degradation system in C. albicans. Using this system, we were able to deplete Cdc14 and other target protein levels to &gt;95% within minutes. Depletion of Cdc14 was robust enough to phenocopy gene deletions, confirming previous results and demonstrating the utility of rapid target protein inactivation. This system will serve as a powerful tool for future functional characterization of Cdc14 in C. albicans and other pathogenic fungal species.