The PDR5 gene encodes the major multidrug resistance efflux pump in Saccharomyces cerevisiae. In drug-resistant cells, the hyperactive Pdr1p or Pdr3p transcriptional activators are responsible for ...the PDR5 upregulation. In this work, it is shown that the RPD3 gene encoding the histone deacetylase that functions as a transcriptional corepressor at many promoters and the ROM2 gene coding for the GDP/GTP exchange protein for Rho1p and Rho2p participating in signal transduction pathways are required for PDR5 transcription under cycloheximide-induced and noninduced conditions. Transposon insertion mutations in ROM2, RPD3 and some other genes encoding specific subunits of the large Rpd3L protein complex resulted in enhanced susceptibility of mutant cells to antifungals. In the rpd3Δ and rom2Δ mutants, the level of PDR5 mRNA and the rate of rhodamine 6G efflux were reduced. Unlike rpd3Δ, in rom2Δ mutant cells the drug hypersensitivity and the defect in PDR5 expression were suppressed by PDR1 or PDR3 overexpressed from heterologous promoters and by the hyperactive pdr3-9 mutant allele. The results indicate that Rpd3p histone deacetylase participating in chromatin remodeling and Rom2p participating in the cell integrity pathway are involved in the control of PDR5 expression and modulation of multidrug resistance in yeast.
In yeasts, the PDR16 gene encodes a phosphatidylinositol transfer protein which belongs to the Sec14 homologue (SFH) family and localizes to lipid droplets, microsomes and at the cell periphery. The ...loss of its function alters the lipid droplet metabolism and plasma membrane properties, and renders yeast cells more sensitive to azole antimycotics. In this study, the entire chromosomal CgPDR16 ORF was replaced by the ScURA3 gene both in azole sensitive and azole resistant strains of Candida glabrata bearing a gain-of-function mutation in the CgPDR1 gene, and their responses to different stresses were assessed. The CgPDR16 deletion was found to sensitize the mutant strains to azole antifungals without changes in their osmo- and halotolerance. Fluconazole treated pdr16Δ mutant strains displayed a reduced expression of several genes involved in azole tolerance. The gain-of-function CgPDR1 allele as well as the cycloheximide and hydrogen peroxide treatments of cells enhanced the expression of the CgPDR16 gene. The results indicate that CgPDR16 belongs to genes whose expression is induced by chemical and oxidative stresses. The loss of its function can attenuate the expression of drug efflux pump encoding genes that might also contribute to the decreased azole tolerance in pdr16Δ mutant cells.
Abstract Candida glabrata is an important human pathogen that is naturally less susceptible to antimycotics compared with Candida albicans . Ten unmatched C. glabrata clinical isolates were selected ...from a collection of isolates exhibiting decreased susceptibilities to azole antifungals. Overexpression of the CgPDR1 gene, encoding the main multidrug resistance transcription factor, and its target genes CgCDR1 and CgCDR2 , coding for drug efflux transporters, was observed in six fluconazole-resistant isolates. Sequence analysis of the polymerase chain reaction (PCR)-amplified DNA fragments of each isolate's CgPDR1 gene was used to identify two novel L347F and H576Y mutations in CgPdr1p. These proved to be responsible for fluconazole resistance in transformants of the C. glabrata pdr1Δ mutant strain. Five isolates harbouring the H576Y mutation also contained the mutation E502V in CgErg11p 14C-lanosterol-demethylase. Heterologous expression of the CgERG11 mutant allele did not provide evidence for its involvement in azole resistance. In four fluconazole-sensitive isolates that were itraconazole-resistant, slightly enhanced CgCDR2 expression was observed. No upregulation of the CgERG11 gene was observed in any of the ten isolates. The results demonstrate that decreased susceptibilities of C. glabrata clinical isolates to azole antifungals mainly results from gain-of-function mutations in the gene encoding the CgPdr1p transcription factor.
Abstract
CTBT (7-chlorotetrazolo5,1-cbenzo1,2,4triazine) causes intracellular superoxide production and oxidative stress and enhances the susceptibility of Saccharomyces cerevisiae, Candida albicans, ...and C. glabrata cells to cycloheximide, 5-fluorocytosine, and azole antimycotic drugs. Here, we demonstrate the antifungal activity of CTBT against 14 tested filamentous fungi. CTBT prevented spore germination and mycelial proliferation of Aspergillus niger and the pathogenic Aspergillus fumigatus. The action of CTBT is fungicidal. CTBT increased the formation of reactive oxygen species in fungal mycelium as detected by 2′,7′-dichlorodihydrofluorescein diacetate and reduced the radial growth of colonies in a dose-dependent manner. Co-application of CTBT and itraconazole led to complete inhibition of fungal growth at dosages lower than the chemicals alone. Antifungal and chemosensitizing activities of CTBT in filamentous fungi may be useful in combination treatments of infections caused by drug-resistant fungal pathogens.
Abstract
7-chlorotetrazolo5,1-cbenzo1,2,4triazine (CTBT) is an antifungal agent that induces oxidative stress and enhances the activity of other antifungals with different modes of action. A ...genome-wide screening of Saccharomyces cerevisiae genomic library in the high-copy-number plasmid revealed three genes, yap1, PDE2, and STB3, which increased the CTBT tolerance of the parental strain. The yap1 gene is known to activate many genes in response to oxidants. The PDE2 and STB3 genes encode the high-affinity cAMP phosphodiesterase and the transcription factor recognizing the ribosomal RNA processing element in promoter sequences, respectively. The protective effects of their overexpression against CTBT toxicity was observed in the absence of certain proteins involved in stress responses, cell wall integrity signaling, and chromatin remodeling. The enhanced CTBT tolerance of the yap1, PDE2. and STB3 transformants was a consequence of their high antioxidant enzyme activities at the beginning of CTBT treatment in comparison with that of the parental strain, for that they inactivated the CTBT-induced reactive oxygen species. These results point to the complex interplay among the oxidant sensing, cAMP-protein kinase A signaling, and transcription reprogramming of yeast cells, leading to their better adaptation to the stress imposed by CTBT.
In the pathogenic yeast Candida glabrata, multidrug resistance is associated with the overexpression of drug efflux pumps caused by gain-of-function mutations in the CgPDR1 gene. CgPdr1p ...transcription factor, which activates the expression of several drug efflux transporter genes, is considered to be a promising target for compounds sensitizing the multidrug-resistant yeast cells. Here, we describe a cell-based screening system for detecting the inhibitory activity of compounds interfering with the CgPdr1p function in a heterologous genetic background of the hypersensitive Saccharomyces cerevisiae mutant strain. The screening is based on the ability to abrogate the growth defect of cells suffering from the galactose-induced and CgPdr1p-driven overexpression of a dominant lethal pma1(D378N) allele placed under the control of the ScPDR5 promoter. The system allows rapid identification of multidrug resistance reversal agents inhibiting the CgPdr1p activity or loss-of-function Cgpdr1 mutations, and is amenable to high-throughput screening on solid or liquid media.
Cardiolipin and its precursor phosphatidylglycerol are two anionic phospholipids that are essential for the biogenesis of functional mitochondria. To assess their role in mitochondrial and cellular ...functions in the pathogenic yeast
Candida glabrata
, a functional characterization of the
CgPGS1
gene encoding the phosphatidylglycerolphosphate synthase has been carried out. Transposon insertion mutation in
CgPGS1
resulted in the loss of phosphatidylglycerolphosphate synthase activity and in deficiency of both phosphatidylglycerol and cardiolipin. The Cgpgs1Δ mutant cells displayed reduced amounts of cytochrome
b
and cytochrome
a
, and had impaired growth on minimal media containing non-fermentable carbon and energy sources. They did not grow at elevated temperatures and failed to form colonies after induction of mitochondrial DNA deletions. The mutant cells also displayed a decreased susceptibility to fluconazole, ketoconazole, clotrimazole, voriconazole and posaconazole. In the Cgpgs1Δ mutant, a quantitative real time PCR revealed enhanced mRNA levels for multidrug resistance associated genes such as
CgPDR1
encoding transcriptional activator and
CgCDR1, CgPDH1
and
CgSNQ2
coding for drug efflux transporters. These results indicate that
CgPGS1
and anionic phospholipids are required for optimal mitochondrial functions and maintenance of yeast susceptibility to azole antifungals.
Candida glabrata is an important human pathogen that is naturally less susceptible to antimycotics compared with Candida albicans. Ten unmatched C. glabrata clinical isolates were selected from a ...collection of isolates exhibiting decreased susceptibilities to azole antifungals. Overexpression of the CgPDR1 gene, encoding the main multidrug resistance transcription factor, and its target genes CgCDR1 and CgCDR2, coding for drug efflux transporters, was observed in six fluconazole-resistant isolates. Sequence analysis of the polymerase chain reaction (PCR)-amplified DNA fragments of each isolate's CgPDR1 gene was used to identify two novel L347F and H576Y mutations in CgPdr1p. These proved to be responsible for fluconazole resistance in transformants of the C. glabrata pdr1Δ mutant strain. Five isolates harbouring the H576Y mutation also contained the mutation E502V in CgErg11p 14C-lanosterol-demethylase. Heterologous expression of the CgERG11 mutant allele did not provide evidence for its involvement in azole resistance. In four fluconazole-sensitive isolates that were itraconazole-resistant, slightly enhanced CgCDR2 expression was observed. No upregulation of the CgERG11 gene was observed in any of the ten isolates. The results demonstrate that decreased susceptibilities of C. glabrata clinical isolates to azole antifungals mainly results from gain-of-function mutations in the gene encoding the CgPdr1p transcription factor.
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