Chemogenomic and transcriptome analysis identifies mode of action of the chemosensitizing agent CTBT (7-chlorotetrazolo[5,1-c]benzo[1,2,4]triazine)
1 Comenius University in Bratislava, Department of Microbiology and Virology, 842 15 Bratislava, Slovak Republic
2 Slovak Academy of Sciences, Institute of Animal Genetics and Biochemistry, 90028 Ivanka pri Dunaji, Slovak Republic
3 University of Vienna, Max F Perutz Laboratories, Department of Chromosome Biology, A-1030 Vienna, Austria
4 Comenius University in Bratislava, Department of Organic Chemistry, 512 15 Bratislava, Slovak Republic
5 University of Vienna, Max F Perutz Laboratories, Department of Biochemistry and Cell Biology, A-1030 Vienna, Austria
BMC Genomics 2010, 11:153 doi:10.1186/1471-2164-11-153Published: 4 March 2010
CTBT (7-chlorotetrazolo [5,1-c]benzo[1,2,4]triazine) increases efficacy of commonly used antifungal agents by an unknown mechanism. It increases the susceptibility of Saccharomyces cerevisiae, Candida albicans and Candida glabrata cells to cycloheximide, 5-fluorocytosine and azole antimycotic drugs. Here we elucidate CTBT mode of action with a combination of systematic genetic and transcriptome analysis.
To identify the cellular processes affected by CTBT, we screened the systematic haploid deletion mutant collection for CTBT sensitive mutants. We identified 169 hypersensitive deletion mutants. The deleted genes encode proteins mainly involved in mitochondrial functions, DNA repair, transcription and chromatin remodeling, and oxidative stress response. We found that the susceptibility of yeast cells to CTBT depends on molecular oxygen. Transcriptome analysis of the immediate early response to CTBT revealed rapid induction of oxidant and stress response defense genes. Many of these genes depend on the transcription factors Yap1 and Cin5. Yap1 accumulates rapidly in the nucleus in CTBT treated cells suggesting acute oxidative stress. Moreover, molecular calculations supported a superoxide generating activity of CTBT. Superoxide production in vivo by CTBT was found associated to mitochondria as indicated by oxidation of MitoSOX Red.
We conclude that CTBT causes intracellular superoxide production and oxidative stress in fungal cells and is thus enhancing antimycotic drug effects by a secondary stress.