Stress response and expression of fluconazole resistance associated genes in the pathogenic yeast Candida glabrata deleted in the CgPDR16 gene.

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.

Stimulus rate increases lateralisation in linguistic and non-linguistic tasks measured by functional transcranial Doppler sonography.

Studies to date that have used fTCD to examine language lateralisation have predominantly used word or sentence generation tasks. Here we sought to further assess the sensitivity of fTCD to language lateralisation by using a metalinguistic task which does not involve novel speech generation: rhyme judgement in response to written words. Line array judgement was included as a non-linguistic visuospatial task to examine the relative strength of left and right hemisphere lateralisation within the same individuals when output requirements of the tasks are matched.

Mutation of the CgPDR16 gene attenuates azole tolerance and biofilm production in pathogenic Candida glabrata.

The PDR16 gene encodes the homologue of Sec14p, participating in protein secretion, regulation of lipid synthesis and turnover in vivo and acting as a phosphatidylinositol transfer protein in vitro. This gene is also involved in the regulation of multidrug resistance in Saccharomyces cerevisiae and pathogenic yeasts. Here we report the results of functional analysis of the CgPDR16 gene, whose mutation has been previously shown to enhance fluconazole sensitivity in Candida glabrata mutant cells.

Antibacterial activity of CTBT (7-chlorotetrazolo[5,1-c]benzo[1,2,4]triazine) generating reactive oxygen species.

CTBT (7-chlorotetrazolo[5,1-c]benzo[1,2,4]triazine) is an antifungal and chemosensitizing agent that induces oxidative stress in yeast and filamentous fungi and enhances the cytotoxic activity of 5-fluorocytosine and azole antimycotics. This study reports the effect of CTBT on bacterial cells. CTBT inhibited the growth of both Gram-positive and Gram-negative bacterial species.

Overexpression of the YAP1, PDE2, and STB3 genes enhances the tolerance of yeast to oxidative stress induced by 7-chlorotetrazolo[5,1-c]benzo[1,2,4]triazine.

7-chlorotetrazolo[5,1-c]benzo[1,2,4]triazine (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.

CTBT (7-chlorotetrazolo[5,1-c]benzo[1,2,4]triazine) producing ROS affects growth and viability of filamentous fungi.

CTBT (7-chlorotetrazolo[5,1-c]benzo[1,2,4]triazine) 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.

Oxidative stress response and virulence factors in Candida glabrata clinical isolates.

We determined the susceptibility to oxidative stress and assessed the four virulence factors of the 38 Candida glabrata clinical isolates originating from two teaching hospitals in Slovakia. All the isolates were susceptible to hydrogen peroxide, diamide, and 7-chlorotetrazolo[5,1-c]benzo[1,2,4]triazine (CTBT) inducing an increased formation of reactive oxygen species in fungal cells. The mean relative cell surface hydrophobicity (CSH) of isolates was 21.

A yeast cell-based system for screening Candida glabrata multidrug resistance reversal agents and selection of loss-of-function pdr1 mutants.

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.

[Opportunistic pathogen Candida glabrata and the mechanisms of its resistance to antifungal drugs].

Treatment of not only bacterial but also fungal infections is currently a growing concern. A major reason is the acquisition of multidrug resistance in both prokaryotic and human cells. The multidrug resistance phenotype is a cellular response to the presence of cytotoxic substances in the environment.

Molecular analysis of Candida glabrata clinical isolates.

Candida glabrata is an important human pathogen, and an understanding of the genetic relatedness of its clinical isolates is essential for the prevention and control of fungal infections. In this study, we determined the relatedness of 38 Candida glabrata clinical isolates originating from two teaching hospitals in Slovakia. The 14 different genotypes were found by using microsatellite marker analysis (RPM2, MTI and Cg6) and DNA sequencing for analysis of the entire ERG11 gene.

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.

Site-directed mutagenesis of Asp853 in Pdr3p transcriptional activator from Saccharomyces cerevisiae.

The PDR3 gene encodes one of the main transcriptional activators involved in the control of multidrug resistance in the yeast Saccharomyces cerevisiae. Recently, it has been demonstrated that a specific D853Y mutation results in the loss of transactivation activity of Pdr3p and its conversion to multicopy suppressor of multidrug resistance. In this study, the Asp853 in Pdr3p was replaced by eight different amino acids and the function of mutated proteins was analysed.

Molecular and phenotypic analysis of mutations causing anionic phospholipid deficiency in closely related yeast species.

The pel1 mutation in Saccharomyces cerevisiae and the Cgpgs1Delta mutation in Candida glabrata result in deficiency of mitochondrial phosphatidylglycerolphosphate synthase and lack of two anionic phospholipids, phosphatidylglycerol and cardiolipin. DNA sequence analysis of the PCR-amplified pel1 mutant allele revealed that the pel1 mutation resulted from a single amino-acid substitution (Glu(463)Lys) in the C-terminal part of encoded enzyme. The CgPGS1 gene cloned in a centromeric pFL38 vector functionally complemented the pel1 mutation in S.

Mutations in the CgPDR1 and CgERG11 genes in azole-resistant Candida glabrata clinical isolates from Slovakia.

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.

Transcriptional regulators of seven yeast species: comparative genome analysis. Review.

The regulation of gene transcription allows yeast cells to respond properly to changing environmental conditions. Several protein complexes take part in this process. They involve RNA polymerase complexes, chromatin remodeling complexes, mediators, general transcription factors and specific transcriptional regulators.

Functional characterization of the CgPGS1 gene reveals a link between mitochondrial phospholipid homeostasis and drug resistance in Candida glabrata.

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.

RPD3 and ROM2 are required for multidrug resistance in Saccharomyces cerevisiae.

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.

Loss-of-function pdr3 mutations convert the Pdr3p transcription activator to a protein suppressing multidrug resistance in Saccharomyces cerevisiae.

The PDR1 and PDR3 genes encode the main transcription activators involved in the control of multidrug resistance in Saccharomyces cerevisiae. To identify the amino acids essential for Pdr3p function, the loss-of-function pdr3 mutants were isolated and characterized. Two plasmid-borne pdr3 alleles, pdr3-E902Ter and pdr3-D853Y, which failed to complement drug hypersensitivity in the Deltapdr1Deltapdr3 mutant strain, were isolated.

Chemosensitisation of drug-resistant and drug-sensitive yeast cells to antifungals.

Multidrug resistance in yeast results from overexpression of genes encoding drug efflux transporters owing to gain-of-function mutations in transcription factors regulating their expression. We have screened a library of synthetic compounds for modulators of drug resistance using the multidrug-resistant Saccharomyces cerevisiae pdr3-9 mutant strain. One of the compounds, 7-chlorotetrazolo[5,1-c]benzo[1,2,4]triazine (CTBT), displayed weak antifungal activity and strongly inhibited the growth of yeast cells in combination with subinhibitory concentrations of other antifungals with a different mode of action.

Biology of the pathogenic yeast Candida glabrata.

The yeasts, being favorite eukaryotic microorganisms used in food industry and biotechnologies for production of biomass and various substances, are also used as model organisms in genetic manipulation, molecular and biological research. In this respect, Saccharomyces cerevisiae is the best-known species but current situation in medicine and industry requires the use of other species. Here we summarize the basic taxonomic, morphological, physiological, genetic, etc.

Resistance mechanisms in fluconazole-resistant Candida albicans isolates from vaginal candidiasis.

Candida albicans is the most frequently identified yeast species causing mycotic vaginitis. A significant number of vaginal yeast isolates are resistant to azole antifungal agents in vitro. Here we investigated the molecular mechanisms of resistance in 22 randomly selected fluconazole-resistant vaginal C.

Synthetic lethal interaction between the pel1 and op1 mutations in Saccharomyces cerevisiae.

The Saccharomyces cerevisiae mutant strain containing the op1 mutation affecting the function of a mitochondrial ATP/ADP translocator has been crossed to the pel1 and crd1 mutants deficient in the biosynthesis of mitochondrial phosphatidylglycerol (PG) and cardiolipin (CL). Using tetrad analysis of diploids issued from corresponding crosses a synthetic lethal interaction has been observed between the op1 and pel1 mutations resulting in the lack of growth of a corresponding double mutant on minimal medium containing glucose. The op1 pel1 double mutant also displayed a decreased susceptibility to fluconazole and a compromised growth even in complex medium containing glucose.

YAP1-mediated KNQ1 expression in Kluyveromyces lactis.

The b-Zip transcription factor Yap1p plays an important role in oxidative stress response and multidrug resistance in Saccharomyces cerevisiae. We have previously demonstrated that the KNQ1 gene, encoding a multidrug transporter of the major facilitator superfamily in Kluyveromyces lactis and containing two potential Yap1p response elements in its promoter, is a putative transcriptional target of KlYap1p, the structural and functional homologue of ScYap1p. In this work, we provide evidence that KlYAP1 controls the expression of the KNQ1 gene.

The KlPGS1 gene encoding phosphatidylglycerolphosphate synthase in Kluyveromyces lactis is essential and assigned to chromosome I.

The phosphatidylglycerolphosphate synthase (CDP-diacylglycerol:sn-glycerol-3-phosphate 3-phosphatidyltransferase, EC 2.7.8.

Yeast strains designed for screening of reversal agents and genetic suppressors of multidrug resistance.

Multidrug resistance in yeast results from over-expression of drug efflux transporter genes due to gain-of-function mutations in transcription factors. To suppress multidrug resistance at the level of gene expression, we have developed a yeast-based screening system for the detection of compounds down-regulating the major multidrug ABC transporter Pdr5p expressed under the control of Pdr3p transcription factor. Here, we report the construction and properties of the improved set of yeast strains designed along with such screening also for a global analysis of genetic suppressors of multidrug resistance.