The Absence of PDR16 Gene Restricts the Overexpression of CaSNQ2 Gene in the Presence of Fluconazole in Candida albicans.

In yeast, the PDR16 gene encodes one of the PITP proteins involved in lipid metabolism and is regarded as a factor involved in clinical azole resistance of fungal pathogens. In this study, we prepared Candida albicans CaPDR16/pdr16Δ and Capdr16Δ/Δ heterozygous and homozygous mutant strains and assessed their responses to different stresses. The CaPDR16 deletion strains exhibited increased susceptibility to antifungal azoles and acetic acid. The addition of Tween80 restored the growth of Capdr16 mutants in the presence of azoles. However, the PDR16 gene deletion has not remarkable influence on sterol profile or membrane properties (membrane potential, anisotropy) of Capdr16Δ and Capdr16Δ/Δ mutant cells. Changes in halotolerance of C. albicans pdr16 deletion mutants were not observed. Fluconazole treatment leads to increased expression of ERG genes both in the wild-type and Capdr16Δ and Capdr16Δ/Δ mutant cells, and the amount of ergosterol and its precursors remain comparable in all three strains tested. Fluconazole treatment induced the expression of ATP-binding cassette transporter gene CaSNQ2 and MFS transporter gene CaTPO3 in the wild-type strain but not in the Capdr16Δ and Capdr16Δ/Δ mutants. The expression of CaSNQ2 gene markedly increased also in cells treated with hydrogen peroxide irrespective of the presence of CaPdr16p. CaPDR16 gene thus belongs to genes whose presence is required for full induction of CaSNQ2 and CaTPO3 genes in the presence of fluconazole in C. albicans.

Sulfide (Na2S) and Polysulfide (Na2S2) Interacting with Doxycycline Produce/Scavenge Superoxide and Hydroxyl Radicals and Induce/Inhibit DNA Cleavage.

Doxycycline (DOXY) is an antibiotic routinely prescribed in human and veterinary medicine for antibacterial treatment, but it has also numerous side effects that include oxidative stress, inflammation, cancer or hypoxia-induced injury. Endogenously produced hydrogen sulfide (H2S) and polysulfides affect similar biological processes, in which reactive oxygen species (ROS) play a role. Herein, we have studied the interaction of DOXY with H2S (Na2S) or polysulfides (Na2S2, Na2S3 and Na2S4) to gain insights into the biological effects of intermediates/products that they generate. To achieve this, UV-VIS, EPR spectroscopy and plasmid DNA (pDNA) cleavage assay were employed. Na2S or Na2S2 in a mixture with DOXY, depending on ratio, concentration and time, displayed bell-shape kinetics in terms of producing/scavenging superoxide and hydroxyl radicals and decomposing hydrogen peroxide. In contrast, the effects of individual compounds (except for Na2S2) were hardly observable. In addition, DOXY, as well as oxytetracycline and tetracycline, interacting with Na2S or other studied polysulfides reduced the •cPTIO radical. Tetracyclines induced pDNA cleavage in the presence of Na2S. Interestingly, they inhibited pDNA cleavage induced by other polysulfides. In conclusion, sulfide and polysulfides interacting with tetracyclines produce/scavenge free radicals, indicating a consequence for free radical biology under conditions of ROS production and tetracyclines administration.

Deletion of the PDR16 gene influences the plasma membrane properties of the yeast Kluyveromyces lactis.

The plasma membrane is the first line of cell defense against changes in external environment, thus its integrity and functionality are of utmost importance. The plasma membrane properties depend on both its protein and lipid composition. The PDR16 gene is involved in the control of Kluyveromyces lactis susceptibility to drugs and alkali metal cations. It encodes the homologue of the major K. lactis phosphatidylinositol transfer protein Sec14p. Sec14p participates in protein secretion, regulation of lipid synthesis, and turnover in vivo. We report here that the plasma membrane of the Klpdr16Δ mutant is hyperpolarized and its fluidity is lower than that of the parental strain. In addition, protoplasts prepared from the Klpdr16Δ cells display decreased stability when subjected to hypo-osmotic conditions. These changes in membrane properties lead to an accumulation of radiolabeled fluconazole and lithium cations inside mutant cells. Our results point to the fact that the PDR16 gene of K. lactis (KlPDR16) influences the plasma membrane properties in K. lactis that lead to subsequent changes in susceptibility to a broad range of xenobiotics.

Isolation and functional analysis of the KlPDR16 gene.

The fight against multidrug-resistant pathogens requires an understanding of the underlying cellular mechanisms. In this work, we isolate and characterize one of the multidrug resistance determinants in Kluyveromyces lactis, the KlPDR16 gene. We show that KlPdr16p (345 aa), which belongs to the KlPdr1p regulon, is a functional homologue of the Saccharomyces cerevisiae Pdr16p. Deletion of KlPDR16 resulted in hypersensitivity of K. lactis cells to antifungal azoles, oligomycin, rhodamine 6G, 4-nitroquinoline-N-oxide and alkali metal cations. The Klpdr16∆ mutation led to a decreased content of ergosterol in whole-cell extract. In spite of the hypersensitivity of Klpdr16∆ mutant cells to rhodamine 6G and oligomycin, the transcript level of the KlPDR5 gene and the rhodamine 6G efflux in the mutant was the same as in the parental strain. Increased accumulation of rhodamine 6G in Klpdr16∆ cells indicates that KlPDR16 limits the rate of passive drug diffusion across the membrane, without affecting the glucose-induced drug export. The results obtained show that KlPDR16, similar to its orthologues in other yeast species, influences the passive drug diffusion into the yeast cell.

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. 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.

Acute Exposure to Bisphenol A Causes Oxidative Stress Induction with Mitochondrial Origin in Saccharomyces cerevisiae Cells.

Bisphenol A (BPA) is a major component of the most commonly used plastic products, such as disposable plastics, Tetra Paks, cans, sport protective equipment, or medical devices. Due to the accumulation of excessive amounts of plastic waste and the subsequent release of BPA into the environment, BPA is classified as a pollutant that is undesirable in the environment. To date, the most interesting finding is the ability of BPA to act as an endocrine disrupting compound due to its binding to estrogen receptors (ERs), and adverse physiological effects on living organisms may result from this action. Since evidence of the potential pro-oxidizing effects of BPA has accumulated over the last years, herein, we focus on the detection of oxidative stress and its origin following BPA exposure using pulsed-field gel electrophoresis, flow cytometry, fluorescent microscopy, and Western blot analysis. Saccharomyces cerevisiae cells served as a model system, as these cells lack ERs allowing us to dissect the ER-dependent and -independent effects of BPA. Our data show that high concentrations of BPA affect cell survival and cause increased intracellular oxidation in yeast, which is primarily generated in the mitochondrion. However, an acute BPA exposure does not lead to significant oxidative damage to DNA or proteins.

Screening for the Key Proteins Associated with Rete Testis Invasion in Clinical Stage I Seminoma via Label-Free Quantitative Mass Spectrometry.

Rete testis invasion (RTI) is an unfavourable prognostic factor for the risk of relapse in clinical stage I (CS I) seminoma patients. Notably, no evidence of difference in the proteome of RTI-positive vs. -negative CS I seminomas has been reported yet. Here, a quantitative proteomic approach was used to investigate RTI-associated proteins. 64 proteins were differentially expressed in RTI-positive compared to -negative CS I seminomas. Of them, 14-3-3γ, ezrin, filamin A, Parkinsonism-associated deglycase 7 (PARK7), vimentin and vinculin, were validated in CS I seminoma patient cohort. As shown by multivariate analysis controlling for clinical confounders, PARK7 and filamin A expression lowered the risk of RTI, while 14-3-3γ expression increased it. Therefore, we suggest that in real clinical biopsy specimens, the expression level of these proteins may reflect prognosis in CS I seminoma patients.

DNA damage measured in blood cells predicts overall and progression-free survival in germ cell tumour patients.

Germ cell tumour (GCT) patients who fail to respond to chemotherapy or who relapse have a poor prognosis. Timely and accurately stratifying such patients could optimise their therapy. We identified endogenous DNA damage levels as a prognostic marker for progression-free (PFS) and overall (OS) survival in chemotherapy-naïve GCT patients. In the present study, we have extended our previous results and reviewed the prognostic power of DNA damage level in GCTs. Endogenous DNA damage levels were measured with the comet assay. Receiver operator characteristic analysis was applied to determine the optimal cut-off value and to evaluate its prognostic accuracy. PFS and OS were estimated by the Kaplan-Meier method and compared using the log-rank test. Hazard ratio (HR) estimates were calculated by Cox regression analysis. A cut-off value of 6.34 provided the highest sensitivity and specificity, with area under curve values of 0.813 and 0.814 for disease progression and mortality, respectively. A % DNA in tail > 6.34 was significantly associated with shorter PFS (HR = 9.54, 95 % confidence interval [CI]: 3.43-26.55, p < 0.001) and OS (HR = 14.62, 95 % CI: 3.14-67.95, p = 0.001) by univariate analysis. The prognostic value of DNA damage measurement was confirmed by multivariate models (HR = 6.45, 95 % CI: 2.22-18.75, p = 0.001 for PFS and HR = 9.40, 95 % CI: 1.70-52.09, p = 0.010 for OS), when HR was adjusted for relevant clinical categories. The added prognostic value of DNA damage in combination with International Germ Cell Cancer Collaborative Group (IGCCCG) risk groups has been revealed. Endogenous DNA damage is an independent prognosticator for PFS and OS in GCT patients and its clinical use, particularly in combination with IGCCCG risk groups, may help in stratifying these patients.

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. The Cgpgs1 Delta 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 Cgpgs1 Delta 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.