Cdc14 protein phosphatases play an important role in plant infection by several fungal pathogens. This and other properties of Cdc14 enzymes make them an intriguing target for development of new antifungal crop treatments. Active site architecture and substrate specificity of Cdc14 from the model fungus Saccharomyces cerevisiae (ScCdc14) are well-defined and unique among characterized phosphatases. Cdc14 appears absent from some model plants. However, the extent of conservation of Cdc14 sequence, structure, and specificity in fungal plant pathogens is unknown. We addressed this by performing a comprehensive phylogenetic analysis of the Cdc14 family and comparing the conservation of active site structure and specificity among a sampling of plant pathogen Cdc14 homologs. We show that Cdc14 was lost in the common ancestor of angiosperm plants but is ubiquitous in ascomycete and basidiomycete fungi. The unique substrate specificity of ScCdc14 was invariant in homologs from eight diverse species of dikarya, suggesting it is conserved across the lineage. A synthetic substrate mimetic inhibited diverse fungal Cdc14 homologs with similar low µM K i values, but had little effect on related phosphatases. Our results justify future exploration of Cdc14 as a broad spectrum antifungal target for plant protection. Plant pathogens pose a constant threat to agricultural productivity and global food security, with fungi and the fungal-like oomycetes being the most dangerous culprits 1-4. Despite the development of chemical pesticides and disease-resistant cultivars to curb crop infections over the past century, damage from fungal and other pathogens persists at nearly comparable levels 3. Estimates suggest more than 10% of the world agricultural harvest may be lost annually to fungal infections alone, equating to hundreds of billions of dollars and enough food to feed an estimated 600 million people 2-5. Post-harvest losses from fungal-induced spoilage and toxin accumulation further exacerbate the problem, especially in developing countries 6. A major challenge to effectively suppressing fungal crop diseases is the ability of fungi to rapidly develop resistance to pesticides and acquire mutations that counteract plant defenses in disease-resistant lines 2,3,7,8. Consequently, the continual battle against fungal pathogens requires a constant stream of new management strategies, including both the generation of new infection resistance mechanisms in crops along with identification of novel pesticide compounds and targets 1. The Cdc14 phosphatases, known best for roles in counteracting cyclin-dependent kinase activity during mitosis in model fungi like Saccharomyces cerevisiae and Schizosaccharomyces pombe 9,10 may be an attractive novel target for development of broad-acting antifungal agents. Deletion of the CDC14 gene in several plant pathogen species severely impairs virulence, demonstrating that Cdc14 function is important for host infection 11-13. Fusarium graminearum lacking CDC14 exhibited defective conidia and ascospore for...

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. Here, 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 proliferated like the wild-type parent strain but 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 novel and 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 reduced 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 is important for C. albicans cell wall integrity and pathogenesis and suggest that Cdc14 may be worth future exploration as an antifungal drug target.

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 phenotypes 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.

The target protein, Hcp1, was first described as part of the bacterial Type VI secretion system from Pseudomonas aeruginosa. The protein first self-assembles into a hexamer and then the hexamers further stack into a nanotubular structure. Hcp1 monomers were targeted for mutagenesis with two widely used photoactivatable amino acids: para-benzoyl phenylalanine or para-azidophenylalanine. The ability of these amino acids to form covalent adducts within the Hcp1 self-assembled system was investigated. Multiple residues, putatively of equal distance between the monomer-monomer interface were targeted. The efficiency of each amino acid to covalently link self-assembled hexamers was determined. The results demonstrate the choice and role of genetically encoded tools applied to complicated biological processes such as self-assembly and also suggested some structural dynamics of the Hcp-1 protein not obvious from crystallographic structures.

Cdc14 protein phosphatase is highly conserved across the eukaryotic kingdom, from single‐celled yeast and protozoa to multicellular organisms including mammals. In budding yeast, where it was first studied, Cdc14 is required for mitotic exit; however, this function is not widely conserved. In humans and mice, Cdc14A mutations cause deficiencies in hearing and male fertility due to defects in cilia formation and maintenance. There is evidence for Cdc14 localization to the basal bodies at the proximal end of cilia. The large number of cilia in the free‐living protozoan Tetrahymena thermophila make it an attractive model for molecular studies of cilia structure and function. Our goal is to determine the localization of Cdc14 isoforms to gain some insight into their cellular function and to test if Cdc14 cilia localization is broadly conserved. Interestingly, T. thermophilahas a larger number of Cdc14 isoforms than most organisms. Thus far, genes encoding three of the seven T. thermophilaCdc14 isoforms have been used to create C‐terminal gene fusions with YFP that are expressed in vivo. The constructs were inserted into the T. thermophilamacronuclear genome adjacent to the RPL29 gene and cell lines were selected due to the resulting conversion to cycloheximide resistance. Expression of the transgenes was regulated by a metallothionine promoter. Cells were examined via fluorescence microscopy in the presence and absence of cadmium. All three isoforms localize along ciliary rows and in the oral apparatus after induction during vegetative growth, consistent with basal body localization. No differences in localization have been noted between the three isoforms thus far. The presence of multiple isoforms with the same localization raises questions regarding the redundancy and/or functional specialization of Cdc14 in this highly ciliated organism.

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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The Cdc14 protein phosphatase family regulates diverse cellular processes, including mitotic exit, cytokinesis, and DNA repair. Cdc14 is a member of the dual‐specificity subfamily of protein tyrosine phosphatases (PTPs), but has evolved to selectively recognize specific phosphoserine sites deposited by cyclin‐dependent kinases. In humans, it has been linked to hearing loss and male infertility conditions. In several fungal species, Cdc14 has been linked to pathogenesis. The Hall lab identified a novel pseudosubstrate motif in the disordered region of all fungal Cdc14 orthologs. Studies with S. cerevisiae Cdc14 revealed this motif interacts with the Cdc14 active site to stimulate the rate‐limiting catalytic step and is functionally important for fungal cell wall integrity. The motif may allow cells to dynamically control Cdc14 activity. Metazoan Cdc14 orthologs do not share this sequence motif. However, we demonstrate here that the human Cdc14A (hCdc14A) C‐terminal region also contains a similar catalytic enhancer. AlphaFold structural predictions suggest that a conserved alpha helix in the otherwise disordered C‐terminus makes extensive contacts with the active site region. Using site‐directed mutagenesis, recombinant protein expression and purification, and steady‐state kinetic assays, we show that the hCdc14A pseudosubstrate motif accelerates phosphoenzyme hydrolysis by nearly two orders of magnitude, similar to the fungal motif. Using a mass spectrometry‐based kinetics assay, we also demonstrate that hCdc14A substrate specificity is nearly identical to fungal Cdc14s, and that the pseudosubstrate motif in hCdc14A does not influence specificity or substrate binding. Current work is 1) testing the hypothesis that the hCdc14A pseudosubstrate motif also binds directly to the active site to accelerate the rate‐limiting catalytic step and 2) identifying the specific amino acids required for active site binding. Future work will focus on characterizing the biological significance of this motif and testing its conservation in human Cdc14B. Our results suggest that fungi and metazoa independently evolved pseudosubstrate motifs to similarly regulate Cdc14 catalytic activity. The direct contribution of sequences outside the core phosphatase domain to catalysis appears to be unique among PTP enzymes.