Phenylpyrrole fungicides act on triosephosphate isomerase to induce methylglyoxal stress and alter hybrid histidine kinase activity

T Tristan Brandhorst,Iain R L Kean,Darin L Wiesner,Bruce S Klein,Stephanie M Lawry

doi:10.1038/s41598-019-41564-9

T Tristan Brandhorst, Iain R L Kean + Show 3 more

Open Access

https://doi.org/10.1038/s41598-019-41564-9

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Journal: Scientific Reports	Publication Date: Mar 25, 2019
Citations: 37	License type: open-access

Affiliation: University of Wisconsin–Madison

Abstract

Fludioxonil, a natural product of pyrrolnitrin, is a potent fungicide used on crops worldwide. Drug action requires the presence of a group III hybrid histidine kinase (HHK) and the high osmolarity glycerol (HOG) pathway. We have reported that the drug does not act directly on HHK, but triggers the conversion of the kinase to a phosphatase, which dephosphorylates Ypd1 to constitutively activate HOG signaling. Still, the direct drug target remains unknown and mode of action ill defined. Here, we heterologously expressed a group III HHK, dimorphism-regulating kinase 1 (Drk1) in Saccharomyces cerevisae to delineate fludioxonil’s target and action. We show that the drug interferes with triosephosphate isomerase (TPI) causing release of methylglyoxal (MG). MG activates the group III HHK and thus the HOG pathway. Drug action involved Drk1 cysteine 392, as a C392S substitution increased drug resistance in vivo. Drug sensitivity was reversed by dimedone treatment, indicating Drk1 responds in vivo to an aldehydic stress. Fludioxonil treatment triggered elevated cytosolic methylglyoxal. Likewise, methylglyoxal treatment of Drk1-expressing yeast phenocopied treatment with fludioxonil. Fludioxonil directly inhibited TPI and also caused it to release methylglyoxal in vitro. Thus, TPI is a drug target of the phenylpyrrole class of fungicides, inducing elevated MG which alters HHK activity, likely converting the kinase to a phosphatase that acts on Ypd1 to trigger HOG pathway activation and fungal cell death.

Highlights

Fungicides are used worldwide to combat fungal infection in agriculture and in human and animal disease
Due to the necessity of group III hybrid histidine kinase (HHK) for sensitivity to fludioxonil, it was initially posited that these kinases fulfilled the role of direct target; this model went unchallenged for decades
Producers of the fungicide have since advanced a mode of action suggesting inhibition of transport-associated glucose phosphorylation[52], a model that is not consonant with prior assumptions on direct action on HHKs

Summary

Introduction

Fungicides are used worldwide to combat fungal infection in agriculture and in human and animal disease. Ypd[1] dephosphorylation leads to Hog[1] phosphorylation[10] This finding revealed how group III HHKs act to inappropriately activate the HOG pathway in response to fludioxonil. We showed that fludioxonil does not induce purified Drk[1] protein to produce the same effect in vitro[17], suggesting fludioxonil may not act directly on group III HHKs. We showed that fludioxonil does not induce purified Drk[1] protein to produce the same effect in vitro[17], suggesting fludioxonil may not act directly on group III HHKs This discrepancy between fludioxonil induced Drk[1] behavior in vivo and in vitro led us to investigate whether group III sensor kinases alter their activity in response to a stress condition elicited by exposure to fludioxonil rather than by the direct action of the fungicide itself upon the HHK

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