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

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.

LFA-1 Ligation by High-Density ICAM-1 Is Sufficient To Activate IFN-γ Release by Innate T Lymphocytes.

By binding to its ligand ICAM-1, LFA-1 is known to mediate both adhesion and costimulatory signaling for T cell activation. The constitutively high LFA-1 cell surface expression of invariant NKT (iNKT) cells has been shown to be responsible for their distinctive tissue homing and residency within ICAM-rich endothelial vessels. However, the functional impact of LFA-1 on the activation of iNKT cells and other innate T lymphocyte subsets has remained largely unexplored. In particular, it is not clear whether LFA-1 contributes to innate-like pathways of T cell activation, such as IFN-γ secretion in response to IL-12. Using a recombinant ICAM-1-Fc fusion protein to stimulate human iNKT cells in the absence of APCs, we show that LFA-1 engagement enhances their IL-12-driven IFN-γ production. Surprisingly, exposure to high densities of ICAM-1 was also sufficient to activate iNKT cell cytokine secretion independently of IL-12 and associated JAK/STAT signaling. LFA-1 engagement induced elevated cytoplasmic Ca2+ and rapid ERK phosphorylation in iNKT cells, and the resulting IFN-γ secretion was dependent on both of these pathways. Analysis of freshly isolated human PBMC samples revealed that a fraction of lymphocytes that showed elevated LFA-1 cell surface expression produced IFN-γ in response to plate-bound ICAM-1-Fc. A majority of the responding cells were T cells, with the remainder NK cells. The responding T cells included iNKT cells, MAIT cells, and Vδ2+ γδ T cells. These results delineate a novel integrin-mediated pathway of IFN-γ secretion that is a shared feature of innate lymphocytes.

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

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.

Bacterioopsin-mediated regulation of bacterioruberin biosynthesis in Halobacterium salinarum.

Integral membrane protein complexes consisting of proteins and small molecules that act as cofactors have important functions in all organisms. To form functional complexes, cofactor biosynthesis must be coordinated with the production of corresponding apoproteins. To examine this coordination, we study bacteriorhodopsin (BR), a light-induced proton pump in the halophilic archaeon Halobacterium salinarum. This complex consists of a retinal cofactor and bacterioopsin (BO), the BR apoprotein. To examine possible novel regulatory mechanisms linking BO and retinal biosynthesis, we deleted bop, the gene that encodes BO. bop deletion resulted in a dramatic increase of bacterioruberins, carotenoid molecules that share biosynthetic precursors with retinal. Additional studies revealed that bacterioruberins accumulate in the absence of BO regardless of the presence of retinal or BR, suggesting that BO inhibits bacterioruberin biosynthesis to increase the availability of carotenoid precursors for retinal biosynthesis. To further examine this potential regulatory mechanism, we characterized an enzyme, encoded by the lye gene, that catalyzes bacterioruberin biosynthesis. BO-mediated inhibition of bacterioruberin synthesis appears to be specific to the H. salinarum lye-encoded enzyme, as expression of a lye homolog from Haloferax volcanii, a related archaeon that synthesizes bacterioruberins but lacks opsins, resulted in bacterioruberin synthesis that was not reduced in the presence of BO. Our results provide evidence for a novel regulatory mechanism in which biosynthesis of a cofactor is promoted by apoprotein-mediated inhibition of an alternate biochemical pathway. Specifically, BO accumulation promotes retinal production by inhibiting bacterioruberin biosynthesis.