Fungal pathogens are a growing threat to human health and global food security as they are becoming increasingly resistant to the few existing antifungal treatments. Cdc14 is a protein phosphatase primarily known for its essential role in controlling mitotic exit in the model yeast Saccharomyces cerevisiae. However, in other fungal species Cdc14 is non-essential and the reasons for its strict conservation across Dikarya remain unclear. Recently, Cdc14 was shown to be required for host infection by several plant pathogenic fungi. Here, we show that Cdc14 is also required for virulence in the human pathogen, Candida albicans.These observations, coupled with our structural and biochemical knowledge of Cdc14 phosphatases, suggest it could be a useful target for antifungal development. In our search for differences between fungal and animal Cdc14 enzymes that could be exploited for antifungal design, we discovered an invariant motif in the otherwise disordered C-terminal tail of fungal Cdc14 orthologs. The motif resembles the recognition sequence of optimal Cdc14 substrates. We provide evidence from molecular modeling and enzyme kinetic studies that this motif is a "pseudosubstrate" that binds the Cdc14 active site and functions to accelerate the rate-limiting catalytic step, providing a plausible mechanism for dynamic control of intracellular Cdc14 activity. The turnover rate for Cdc14 lacking a functional pseudosubstrate motif is reduced by an order of magnitude compared to the wild-type enzyme. Both S. cerevisiaeand C. albicanscells expressing Cdc14 variants with point mutations in this motif exhibit a specific and pronounced sensitivity to cell wall stresses, including treatment with echinocandin antifungal drugs that inhibit cell wall synthesis. Moreover, signaling through the cell wall integrity pathway is chronically elevated in these cells, indicative of a cell wall structural defect. Preliminary evidence in synchronized S. cerevisiaecultures with reduced Cdc14 activity suggests that cell wall integrity is compromised around the time of cytokinesis and septation, consistent with its known functions in these processes. We are currently testing if the pseudosubstrate motif is required for virulence of C. albicans.Our findings reveal 1) a novel contribution to catalysis of a structural element outside the conserved catalytic core of Cdc14 enzymes and 2) novel roles of Cdc14 in promoting cell wall integrity and pathogenesis. More detailed characterization of the mechanisms by which Cdc14 contributes to cell wall integrity and host infection in human pathogenic fungi should help assess its future value for antifungal development.

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Journal Article

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Milholland KL, Naughton NH, DeMarco AG, Baker KM, Briggs SD, Correa-Bordes J, Hall MC

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