The presence of Hog1p (lower panel, Hog1) was confirmed in all strains. Hog1p appears at approximately 50 kDa. Discussion We previously

showed that expression of the group III HK from the human fungal pathogen C. albicans, CaNIK1 in S. cerevisiae resulted in susceptibility of the transformants to the fungicides buy EPZ015666 fludioxonil, iprodione and ambruticin VS3 [25]. Moreover, the fungicidal activity was decreased by deletion of single or double pairs of the N-terminal HAMP domains [25]. For other group III HKs it was already shown that mutations in the conserved phosphate-accepting residues and partial deletion of the HAMP domains conferred fungicide resistance [23, 26]. This stimulated our interest to investigate the involvement of the HisKA, HATPase_c and REC domains from CaNik1p in the fungicide activity, as they are conserved in all HKs. To prevent the primary phosphorylation of the histidine residue and the subsequent His-Asp phosphate-transfer Selleckchem SBI-0206965 from the HisKA to the REC domains, respectively, the point mutations H510Q and D924N were introduced. The N627D mutation was supposed to inactivate the ATP binding site. The complete resistance of the strains H510 and D924 and the reduced

susceptibility of the strain N627 in comparison to the strain NIK clearly showed that the functionalities of the above mentioned domains were essential for the susceptibility of the transformed yeast to the tested fungicides. In agreement, similar patterns of Hog1p phosphorylation were obtained after treating the different S. cerevisiae transformants with fludioxonil. Phosphorylation of Hog1p was totally abolished in the strains H510 and D924 and partially inhibited in the strain N627, while in all strains expressing genes with point mutations Hog1p was phosphorylated in response to osmotic stress, but was not phosphorylated without external stimuli. These results are in agreement with earlier reports of reduced antifungal susceptibilities of strains, which expressed other group III HKs carrying point mutations in the HisKA and REC domains [26, 27]. However, the

correlation between the functionality of conserved HisKA, REC and HATPase_c domains of CaNik1p and both the fungicidal sensitivity and phosphorylation of Hog1p after fungicidal treatment was not shown before. Altogether, we present before clear evidences that the histidine kinase functionality of CaNik1p was essential for the fungicidal effect and that this effect correlated with the activation of the MAPK Hog1p after treatment with fungicides. The yeast histidine kinase Sln1p (group VI histidine kinase) is a negative regulator of the MAPK Hog1p, as its inhibition leads to activation of the MAPK. However, for group III HKs different effects were reported: Dic1p, the group III HK from Cochliobolus heterostrophus, was described as a positive regulator of Hog1p [24], whereas DhNik1p from Dabaryomyces selleck chemical hansenii was identified as a negative regulator [23].