In VitroEffect of Malachite Green on Candida albicans Involves Multiple Pathways and Transcriptional RegulatorsUPC2andSTP2

Abstract

In this study, we show that a chemical dye, malachite green (MG), which is commonly used in the fish industry as an antifungal, antiparasitic, and antibacterial agent, could effectively kill Candida albicans and non-C. albicans species. We have demonstrated that Candida cells are susceptible to MG at a very low concentration (MIC that reduces growth by 50% [MIC 50 ], 100 ng ml ؊1 ) and that the effect of MG is independent of known antifungal targets, such as ergosterol metabolism and major drug efflux pump proteins. Transcriptional profiling in response to MG treatment of C. albicans cells revealed that of a total of 207 responsive genes, 167 genes involved in oxidative stress, virulence, carbohydrate metabolism, heat shock, amino acid metabolism, etc., were upregulated, while 37 genes involved in iron acquisition, filamentous growth, mitochondrial respiration, etc., were downregulated. We confirmed experimentally that Candida cells exposed to MG resort to a fermentative mode of metabolism, perhaps due to defective respiration. In addition, we showed that MG triggers depletion of intracellular iron pools and enhances reactive oxygen species (ROS) levels. These effects could be reversed by the addition of iron or antioxidants, respectively. We provided evidence that the antifungal effect of MG is exerted through the transcription regulators UPC2 (regulating ergosterol biosynthesis and azole resistance) and STP2 (regulating amino acid permease genes). Taken together, our transcriptome, genetic, and biochemical results allowed us to decipher the multiple mechanisms by which MG exerts its anti-Candida effects, leading to a metabolic shift toward fermentation, increased generation of ROS, labile iron deprivation, and cell necrosis.

Clotrimazole as a Potent Agent for Treating the Oomycete Fish Pathogen Saprolegnia parasitica through Inhibition of Sterol 14α-Demethylase (CYP51)

“…Malachite green-treated Saprolegnia had unaltered sterol profiles, indicating that the mode of action of malachite green was different from that of azole antifungals, with no inhibition of CYP51 function. This is in agreement with previous findings that malachite green acts independently of known antifungal targets, such as sterol biosynthesis and drug efflux pump proteins in C. albicans (MIC 50 , 0.1 g ml Ϫ1 ) (72). Malachite green was found to affect 207 genes in C. albicans, with the upregulation of 167 genes involved in oxidative stress, virulence, carbohydrate metabolism, The sterol compositions of untreated and prochloraz-treated S. ferax, S. parasitica, and S. diclina, in addition to clotrimazole-treated S. parasitica, were determined by extraction of the nonsaponifiable lipid fraction (50) followed by derivatization with BSTFA-TMS-pyridine and analysis by GC-MS (45).…”

“…heat shock, and amino acid metabolism and the downregulation of 37 genes involved in iron acquisition, filamentous growth, and mitochondrial respiration (72). Malachite green affects multiple mechanisms to exert its antifungal effect and leads to a shift in metabolism toward fermentation, increased generation of reactive oxygen species, iron depletion, and cell necrosis in C. albicans (72) and is likely to behave similarly in Saprolegnia species. There appear to be no published studies investigating the effectiveness of clotrimazole at combating Saprolegnia infection of salmonid eggs and fry in vivo.…”

Curcumin Targets Cell Wall Integrity via Calcineurin-Mediated Signaling in Candida albicans

“…For CUR treatment, cells at an OD of 0.1 were allowed to reach an OD of 0.4, and then 251 M (92.45 g/ml) CUR (MIC 50 ) was added to one of the cultures and the second culture was subjected to an equivalent mock treatment (water). After 14 h of CUR treatment, the cells were harvested and used for RNA isolation (13). Three independent cultures were prepared for biological repeats.…”

“…Each experimental condition was independently repeated three times, including a dye swap. C. albicans microarrays (batch C050G) containing a cDNA probe deposited in duplicate for 98% of Candida open reading frames (ORFs) (assembly 19) were obtained from Eurogentec (Seraing, Belgium) (13).…”

Ascorbic Acid Inhibition of Candida albicans Hsp90-Mediated Morphogenesis Occurs via the Transcriptional Regulator Upc2

“…Figure S4 in the supplemental material shows that the tolerance for H 2 O 2 of the mutants is absolutely comparable to that of the corresponding isogenic wildtype strain, thus ruling out the hypothesis that deletion of UPC2 could lower the anti-oxidative-stress potential of the cells. Different response of other upc2 mutants to oxidative stress may have different causes, such as a different genetic background (44). The results obtained with pharmacological inhibition of Hsp90 were confirmed using genetic depletion of Hsp90.…”