A novel functional assay for fungal histidine kinases group III reveals the role of HAMP domains for fungicide sensitivity

Buschart, Anna; Gremmer, Katja; El‐Mowafy, Mohammed; Heuvel, Joop van den; Mueller, Peter P.; Bilitewski, Ursula

doi:10.1016/j.jbiotec.2011.09.017

Cited by 24 publications

(46 citation statements)

References 33 publications

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“…Samples were run on an SDS-PAGE gel followed by phosphorimaging and quantification of signal intensity via densitometry. 32 P and signal quantification are representative of at least five independent experiments. Signal quantification data are the means Ϯ SEM from two experimental replicates.…”

Section: Lawry Et Al Antimicrobial Agents and Chemotherapymentioning

confidence: 99%

“…yeast engenders fludioxonil sensitivity (29,31,32). In this model, both the group III HHK and HOG pathway are required for the drug's action.…”

mentioning

confidence: 99%

“…In this model, both the group III HHK and HOG pathway are required for the drug's action. Treatment causes constitutive Hog1 phosphorylation (32)(33)(34) and cell death, whereas deletion of pathway components engenders drug resistance (29,31,35). The drug's action also requires the N-terminal HAMP domain repeats that are a signature feature of group III HHKs (27,36).…”

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confidence: 99%

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Fludioxonil Induces Drk1, a Fungal Group III Hybrid Histidine Kinase, To Dephosphorylate Its Downstream Target, Ypd1

Lawry

Tebbets

Kean

et al. 2017

Antimicrob Agents Chemother

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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 (highosmolarity 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.

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Section: Lawry Et Al Antimicrobial Agents and Chemotherapymentioning

confidence: 99%

“…yeast engenders fludioxonil sensitivity (29,31,32). In this model, both the group III HHK and HOG pathway are required for the drug's action.…”

mentioning

confidence: 99%

mentioning

confidence: 99%

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Fludioxonil Induces Drk1, a Fungal Group III Hybrid Histidine Kinase, To Dephosphorylate Its Downstream Target, Ypd1

Lawry

Tebbets

Kean

et al. 2017

Antimicrob Agents Chemother

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“…In fungi, however, simpler high-throughput screens for HK inhibitors have been recently reported. One screen was based on the po- tential for growth inhibition due to activation of the HOG1 osmotic response MAPK pathway by inhibitors of group III HKs from C. albicans expressed heterologously in S. cerevisiae (77). In this study, several known antifungals were used successfully in proof-of-principle experiments (77).…”

Section: Prospects For Ypd1 As An Antifungal Drug Targetmentioning

confidence: 99%

“…One screen was based on the po- tential for growth inhibition due to activation of the HOG1 osmotic response MAPK pathway by inhibitors of group III HKs from C. albicans expressed heterologously in S. cerevisiae (77). In this study, several known antifungals were used successfully in proof-of-principle experiments (77). In a related screen, small molecules were tested for fungicidal activity against an S. cerevisiae reporter strain expressing a group III HK from Magnaporthe grisea.…”

Section: Prospects For Ypd1 As An Antifungal Drug Targetmentioning

confidence: 99%

Histidine Phosphotransfer Proteins in Fungal Two-Component Signal Transduction Pathways

Fassler

West

2013

Eukaryot Cell

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bThe histidine phosphotransfer (HPt) protein Ypd1 is an important participant in the Saccharomyces cerevisiae multistep twocomponent signal transduction pathway and, unlike the expanded histidine kinase gene family, is encoded by a single gene in nearly all model and pathogenic fungi. Ypd1 is essential for viability in both S. cerevisiae and in Cryptococcus neoformans. These and other aspects of Ypd1 biology, combined with the availability of structural and mutational data in S. cerevisiae, suggest that the essential interactions between Ypd1 and response regulator domains would be a good target for antifungal drug development. The goal of this minireview is to summarize the wealth of data on S. cerevisiae Ypd1 and to consider the potential benefits of conducting related studies in pathogenic fungi.

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Molecular mechanism of reduced biological fitness of fludioxonil‐resistant strains of Botrytis cinerea based on transcriptome analysis

Yin,

Wang,

Wang

et al. 2024

Pest Management Science

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BACKGROUNDFludioxonil is a fungicide used to control gray mold. However, the frequency of resistance in the field is low, and highly resistant strains are rarely isolated. The biological fitness of the resistant strain is lower than that of the wild strain. Therefore, the molecular mechanism underlying the decrease in the fitness of the fludioxonil‐resistant strain of Botrytis cinerea was explored to provide a theoretical basis for resistance monitoring and management.RESULTSTranscriptome analysis was performed on five different‐point mutant resistant strains of fludioxonil, focusing on mining and screening candidate genes that lead to reduced fitness of the resistant strains and the functional verification of these genes. The differentially expressed genes (DEGs) of the five point‐mutation resistant strains intersected with 1869 DEGs. Enrichment analysis showed that three downregulated genes (Bcin05g07030, Bcgad1, and Bcin03g05840) were enriched in multiple metabolic pathways and were downregulated in both domesticated strains. Bcin05g07030 and Bcin03g05840 were involved in mycelial growth and development, pathogenicity, and conidial yield, and negatively regulated oxidative stress and cell wall synthesis. Bcgad1 was involved in mycelial growth and development, conidial yield, oxidative stress, and cell wall synthesis. Furthermore, Bcin05g07030 was involved in osmotic stress and spore germination, whereas Bcin03g05840 and Bcgad1 negatively regulated osmotic stress and cell wall integrity.CONCLUSIONThese results enable us to further understand the molecular mechanism underlying the decrease in the biological fitness of B. cinerea fludioxonil‐resistant strains. © 2024 Society of Chemical Industry.

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A novel functional assay for fungal histidine kinases group III reveals the role of HAMP domains for fungicide sensitivity

Cited by 24 publications

References 33 publications

Fludioxonil Induces Drk1, a Fungal Group III Hybrid Histidine Kinase, To Dephosphorylate Its Downstream Target, Ypd1

Fludioxonil Induces Drk1, a Fungal Group III Hybrid Histidine Kinase, To Dephosphorylate Its Downstream Target, Ypd1

Histidine Phosphotransfer Proteins in Fungal Two-Component Signal Transduction Pathways

Molecular mechanism of reduced biological fitness of fludioxonil‐resistant strains of Botrytis cinerea based on transcriptome analysis

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