Reduced Cdc14 phosphatase activity impairs septation, hyphal differentiation and pathogenesis and causes echinocandin hypersensitivity in Candida albicans

Abstract

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 a small fraction of normal Cdc14 activity. It remains unclear what fungal Cdc14 functions require high Cdc14 activity. We identified an invariant motif in the disordered C-terminal tail of fungal Cdc14 enzymes that is required for full enzyme activity. Mutation of this motif reduced Cdc14 catalytic rate and provided a tool for studying the biological significance of high Cdc14 activity. A S. cerevisiae strain expressing the reduced-activity hypomorphic mutant allele (cdc14hm) as the sole source of Cdc14 exhibited an unexpected sensitivity to cell wall stresses, including chitin-binding compounds and echinocandin antifungal drugs. Sensitivity to echinocandins was also observed in Schizosaccharomyces pombe and Candida albicans strains lacking CDC14, suggesting this phenotype reflects a conserved function of Cdc14 orthologs in mediating fungal cell wall integrity. In C. albicans, the orthologous cdc14hm allele was sufficient to elicit echinocandin hypersensitivity and perturb cell wall integrity signaling. It also caused striking abnormalities in septum structure and the same cell separation and hyphal differentiation defects previously observed with cdc14 gene deletions. Since hyphal differentiation is important for C. albicans pathogenesis, we assessed the effect of reducing Cdc14 activity on virulence in Galleria mellonella and mouse models of invasive candidiasis. Partial reduction in Cdc14 activity via cdc14hm mutation severely impaired C. albicans virulence in both assays. Our results reveal that high Cdc14 activity promotes fungal cell wall integrity and, in C. albicans, is needed to orchestrate septation and hyphal differentiation, and for pathogenesis. Cdc14 may therefore be worth future exploration as an antifungal drug target. AUTHOR SUMMARYInvasive fungal infections are a serious concern for the immune-compromised. Antifungal drugs to treat invasive infections are limited and pathogens are developing resistance to them. Novel targets for antifungal drug development are needed. In this study we developed a system to test if partial therapeutic reduction in activity of a protein phosphatase called Cdc14 could reduce virulence of the opportunistic human pathogen Candida albicans. This idea arose from prior studies in fungal pathogens of plants, where Cdc14 was unexpectedly required for host infection through an unknown mechanism. We found that successful C. albicans infections in two animal models of invasive candidiasis were dependent on high Cdc14 activity. Moreover, we made the surprising observation that integrity of the C. albicans cell wall is also dependent on high Cdc14 activity, with Cdc14-deficient cells becoming hypersensitive to cell wall-targeted antifungal drugs. We conclude that even modest reduction in Cdc14 activity could have therapeutic benefit for human fungal infections and possibly help overcome resistance to some antifungal drugs. Cdc14 structure and specificity are unique among phosphatases and highly conserved in pathogenic fungi, suggesting that highly selective inhibitors can be developed that would be useful against a broad range of fungal pathogens.