A Novel Functional Assay for Fungal Histidine Kinases Group III Reveals the Role of HAMP Domains for Fungicide Sensitivity

J Biotechnol. 2012 Jan;157(1):268-77. doi: 10.1016/j.jbiotec.2011.09.017. Epub 2011 Sep 22.

Abstract

Signal transduction systems comprising histidine kinases are suggested as new molecular targets of antibiotics. The important human fungal pathogen Candida albicans possesses three histidine kinases, one of which is the type III histidine kinase CaNik1, which activates the MAP kinase Hog1. We established a screening system for inhibitors of this class of histidine kinases by functional expression of the CaNIK1 gene in S. cerevisiae. This transformant was susceptible to fungicides to which the wild type strain was resistant, such as fludioxonil and ambruticin. Growth inhibition correlated with phosphorylation of Hog1 and was dependent on an intact Hog1 pathway. At the N-terminus the histidine kinase CaNik1 has four amino acid repeats of 92 amino acids each and one truncated repeat of 72 amino acids. Within these repeats we identified 9 HAMP domains with a paired structure. We constructed mutants in which one or two pairs of these domains were deleted. S. cerevisiae transformants expressing the full-length CaNIK1 showed the highest sensitivity to the fungicides, any truncation reduced the susceptibility of the transformants to the fungicides. This indicates that the HAMP domains are decisive for the mode of action of the antifungal compounds.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Motifs / genetics
  • Amino Acid Sequence
  • Antifungal Agents / pharmacology*
  • Candida albicans / drug effects
  • Candida albicans / enzymology
  • Candida albicans / genetics
  • Dioxoles / pharmacology
  • Drug Resistance, Fungal / genetics*
  • Fungal Proteins / chemistry*
  • Fungal Proteins / drug effects
  • Fungal Proteins / genetics
  • Histidine Kinase
  • Microbial Sensitivity Tests
  • Mitogen-Activated Protein Kinases / chemistry
  • Mitogen-Activated Protein Kinases / metabolism
  • Molecular Sequence Data
  • Phosphorylation
  • Protein Kinase Inhibitors / pharmacology
  • Protein Kinases / chemistry*
  • Protein Kinases / drug effects
  • Protein Kinases / genetics
  • Pyrans / pharmacology
  • Pyrroles / pharmacology
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / drug effects
  • Recombinant Proteins / genetics
  • Saccharomyces cerevisiae / genetics
  • Saccharomyces cerevisiae / metabolism
  • Saccharomyces cerevisiae Proteins / chemistry
  • Saccharomyces cerevisiae Proteins / metabolism
  • Sequence Alignment
  • Sequence Deletion
  • Signal Transduction / genetics
  • Stress, Physiological

Substances

  • Antifungal Agents
  • Dioxoles
  • Fungal Proteins
  • Protein Kinase Inhibitors
  • Pyrans
  • Pyrroles
  • Recombinant Proteins
  • Saccharomyces cerevisiae Proteins
  • Protein Kinases
  • HOG1 protein, S cerevisiae
  • Mitogen-Activated Protein Kinases
  • Histidine Kinase
  • fludioxonil
  • ambruticin