title: Fludioxonil Induces Drk1, a Fungal Group III Hybrid Histidine Kinase, To Dephosphorylate Its Downstream Target, Ypd1.
abstract: Novel antifungal drugs and targets are urgently needed. Group III hybrid histidine kinases (HHKs) represent an appealing new therapeutic drug target because they are widely expressed in fungi but absent from humans. We investigated the mode of action of the widely utilized, effective fungicide fludioxonil. The drug acts in an HHK-dependent manner by constitutive activation of the HOG (high-osmolarity glycerol) pathway, but its mechanism of action is poorly understood. Here, we report a new mode of drug action that entails conversion of the HHK from a kinase into a phosphatase. We expressed Drk1 (dimorphism-regulating kinase), which is an intracellular group III HHK from the fungal pathogen Blastomyces dermatitidis, in Saccharomyces cerevisiae Drk1 engendered drug sensitivity in B. dermatitidis and conferred sensitivity upon S. cerevisiae In response to fludioxonil, Drk1 behaved as a phosphatase rather than as a kinase, leading to dephosphorylation of its downstream target, Ypd1, constitutive activation of the HOG pathway, and yeast cell death. Aspartic acid residue 1140 in the Drk1 receiver domain was required for in vivo phosphatase activity on Ypd1, and Hog1 was required for drug effect, indicating fidelity in HHK-dependent drug action. In in vitro assays with purified protein, intact Drk1 demonstrated intrinsic kinase activity, and the Drk1 receiver domain exhibited intrinsic phosphatase activity. However, fludioxonil failed to induce intact Drk1 to dephosphorylate Ypd1. We conclude that fludioxonil treatment in vivo likely acts on an upstream target that triggers HHK to become a phosphatase, which dephosphorylates its downstream target, Ypd1.
authors.affiliation: Department of Pediatrics, University of Wisconsin School of Medicine and Public Health, Madison, Wisconsin, USA.|Department of Pathology, University of Wisconsin School of Medicine and Public Health, Madison, Wisconsin, USA.|Cellular and Molecular Pathology Program, University of Wisconsin-Madison, Madison, Wisconsin, USA.
authors.forename: Stephanie M
authors.initials: SM
authors.lastname: Lawry
authors.affiliation: Department of Pediatrics, University of Wisconsin School of Medicine and Public Health, Madison, Wisconsin, USA.|Department of Medical Microbiology and Immunology, University of Wisconsin School of Medicine and Public Health, Madison, Wisconsin, USA.
authors.forename: Brad
authors.initials: B
authors.lastname: Tebbets
authors.affiliation: Department of Pediatrics, University of Wisconsin School of Medicine and Public Health, Madison, Wisconsin, USA.
authors.forename: Iain
authors.initials: I
authors.lastname: Kean
authors.affiliation: Department of Pediatrics, University of Wisconsin School of Medicine and Public Health, Madison, Wisconsin, USA.
authors.forename: Douglas
authors.initials: D
authors.lastname: Stewart
authors.affiliation: Department of Pediatrics, University of Wisconsin School of Medicine and Public Health, Madison, Wisconsin, USA.|Department of Medical Microbiology and Immunology, University of Wisconsin School of Medicine and Public Health, Madison, Wisconsin, USA.
authors.forename: Joel
authors.initials: J
authors.lastname: Hetelle
authors.affiliation: Department of Pediatrics, University of Wisconsin School of Medicine and Public Health, Madison, Wisconsin, USA bsklein@pediatrics.wisc.edu.|Department of Medical Microbiology and Immunology, University of Wisconsin School of Medicine and Public Health, Madison, Wisconsin, USA.|Department of Internal Medicine, University of Wisconsin School of Medicine and Public Health, Madison, Wisconsin, USA.
authors.forename: Bruce S
authors.initials: BS
authors.lastname: Klein
mesh_terms: D000935:Antifungal Agents / Q000494:pharmacology; D001758:Blastomyces / Q000187:drug effects* / Q000201:enzymology / Q000235:genetics; D002176:Candida albicans / Q000187:drug effects; D004149:Dioxoles / Q000494:pharmacology*; D000071677:Histidine Kinase / Q000235:genetics / Q000378:metabolism*; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D000072417:Protein Domains; D011494:Protein Kinases / Q000235:genetics / Q000378:metabolism*; D011758:Pyrroles / Q000494:pharmacology*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: D000935:Antifungal Agents; D004149:Dioxoles; D047908:Intracellular Signaling Peptides and Proteins; D011758:Pyrroles; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D011494:Protein Kinases; C102035:YPD1 protein, S cerevisiae; D000071677:Histidine Kinase; C108339:fludioxonil
keywords: HOG signaling; antifungal mechanism; antimicrobial agents; fungi; kinase