Chemogenomic and transcriptome analysis identifies mode of action of the chemosensitizing agent CTBT (7-chlorotetrazolo[5,1-c]benzo[1,2,4]triazine).
BACKGROUND
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.


RESULTS
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.


CONCLUSIONS
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.
Batova
Monika
M
Comenius University in Bratislava, Department of Microbiology and Virology, Bratislava, Slovak Republic.
Klobucnikova
Vlasta
V
Oblasova
Zuzana
Z
Gregan
Juraj
J
Zahradnik
Pavol
P
Hapala
Ivan
I
Subik
Julius
J
Schüller
Christoph
C
D000935:Antifungal Agents / Q000494:pharmacology; D004271:DNA, Fungal / Q000187:drug effects; D004272:DNA, Mitochondrial / Q000187:drug effects; D020869:Gene Expression Profiling; D008826:Microbial Sensitivity Tests; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism; D020411:Oligonucleotide Array Sequence Analysis; D018384:Oxidative Stress / Q000187:drug effects*; D012441:Saccharomyces cerevisiae / Q000187:drug effects* / Q000235:genetics; D017384:Sequence Deletion; D013481:Superoxides / Q000378:metabolism; D014227:Triazines / Q000494:pharmacology*
C518245:7-chlorotetrazolo(5,1-c)benzo(1,2,4)triazine; D000935:Antifungal Agents; D004271:DNA, Fungal; D004272:DNA, Mitochondrial; D014227:Triazines; D013481:Superoxides