Abstract
The hybrid histidine kinase 3 (HHK3) is a highly conserved sensor kinase in fungi that regulates the downstream HOG/p38 mitogen-activated protein kinase (MAPK). In addition to its role in osmoadaptation, HHK3 is involved in hyphal morphogenesis, conidiation, virulence, and cellular adaptation to oxidative stress. However, the molecular mechanisms by which it controls these processes remain obscure. Moreover, HHK3 is a molecular target for antifungal agents such as fludioxonil, which thereby interferes with the HOG/p38 pathway, leading to the abnormal accumulation of glycerol and subsequent cell lysis. Here, we used a chemical genomics approach with the yeast Saccharomyces cerevisiae to better understand the fungicidal action of fludioxonil and the role of HHK3 in fungal growth and physiology. Our results indicated that the abnormal accumulation of glycerol is not the primary cause of fludioxonil toxicity. Fludioxonil appears to impair endosomal trafficking in the fungal cells. We found that the components of class C core vacuole/endosome tethering (CORVET) complex are essential for yeast viability in the presence of a subthreshold dose of fludioxonil and that their overexpression alleviates fludioxonil toxicity. We also noted that by impeding secretory vesicle trafficking, fludioxonil inhibits hyphal growth in the opportunistic fungal pathogen Candida albicans Our results suggest that HHK3 regulates fungal hyphal growth by affecting vesicle trafficking. Together, our results reveal an important role of CORVET complex in the fungicidal action of fludioxonil downstream of HHK3.
Highlights
The hybrid histidine kinase 3 (HHK3) is a highly conserved sensor kinase in fungi that regulates the downstream HOG/p38 mitogen-activated protein kinase (MAPK)
We found that the components of class C core vacuole/ endosome tethering (CORVET) complex are essential for yeast viability in the presence of a subthreshold dose of fludioxonil and that their overexpression alleviates fludioxonil toxicity
Fludioxonil treatment was shown to cause an increase in the intracellular glycerol content, hyphal swelling, 4 The abbreviations used are: HOG, high-osmolarity glycerol; MAPK, mitogenactivated protein kinase; HHK, hybrid histidine kinase; CORVET, class C core vacuole/endosome tethering; SD, synthetic defined; DSP, deletion-sensitive profile; SNARE, soluble N-ethylmaleimide–sensitive factor (NSF) attachment protein receptor; HOPS, homotypic fusion and vacuole protein sorting; GO, gene ontology; LY, Lucifer yellow; FRAP, fluorescence recovery after photobleaching; SSRE, synthetic Skn7 response element; aa, amino acids; DAPI, 4Ј,6-diamidino-2-phenylindole
Summary
The hybrid histidine kinase 3 (HHK3) is a highly conserved sensor kinase in fungi that regulates the downstream HOG/p38 mitogen-activated protein kinase (MAPK). Role of CORVET complex in fungicidal action of fludioxonil and metabolite leakage in the fungal cells [6, 7] Based on these observations, it is suggested that fludioxonil interferes with the osmosensing signal transduction pathway, leading to the abnormal accumulation of glycerol and subsequent cell lysis [6, 7]. The molecular target of fludioxonil toxicity is Nik orthologs or hybrid histidine kinase 3 (HHK3) [6, 12,13,14] Initial evidence in this regard came from analyzing laboratory-generated and naturally isolated fludioxonil-resistant mutants. Fungicidal action of fludioxonil involves inhibition of HHK3, resulting in the constitutive activation of the Hog1p MAPK in fungi. For the first time, an important role of CORVET complex in the fungicidal action of fludioxonil downstream of HHK3
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