The incidence of Candida glabrata infections increases every year due to its higher resistance to commonly used antifungal drugs. We characterized the antifungal mechanism of action of eight new styrylpyridinium derivatives, with various N-alkyl chains (-C6H13, -C8H17, -C10H21, -C12H25) and different substituents, on C. glabrata strains differing in their drug resistance due to the presence or absence of two major drug-efflux pumps. We found that the tested styrylpyridinium compounds affected the growth of C. glabrata cells in a compound- and strain-dependent manner, and apparently they were substrates of CgCdr1 and CgCdr2 pumps. Further, we determined the impact of the tested compounds on plasma membrane integrity. The ability to cause damage to a plasma membrane depended on the compound, its concentration and the presence of efflux pumps, and corresponded well with the results of growth and survival tests. We also tested possible synergism with three types of known antifungal drugs. Though we did not observe any synergism with azole drugs, styrylpyridinium compounds 5 and 6 together with FK506 demonstrated excellent antifungal properties, whereas compounds 2, 3, 5, and 6 exhibited a significant synergistic effect in combination with terbinafine. Based on our results, derivatives 2 and 6 turned out to be the most promising antifungal drugs. Moreover, compound 6 was not only able to effectively permeabilize the yeast plasma membrane, but also exhibited significant synergism with FK506 and terbinafine. Finally, we also characterized the spectroscopic properties of the tested styrylpyridinium compounds. We measured their absorption and fluorescence spectra, determined their localization in yeast cells and found that their fluorescence characteristics differ from the properties of current commercial vacuolar styrylpyridinium markers and allow multi-color staining. Compounds 1, 3, 7, and 8 were able to accumulate in plasma and vacuolar membranes, and compounds 2, 5, and 6 stained the whole interior of dead cells. In summary, of the eight tested compounds, compound 6 is the most promising antifungal drug, compound 8, due to its minimal toxicity, is the best candidate for a new vacuolar-membrane probe or new benchmark substrate of C. glabrata Cdr pumps, and derivative 5 for a new vital dye.

• Keywords
• Candida glabrata, diS-C3(3) assay, membrane potential, multidrug resistance, styrylpyridinium derivatives, vacuolar marker, yeast,
• Publication type
• Journal Article MeSH

Saccharomyces species, which are mostly used in the food and beverage industries, are known to differ in their fermentation efficiency and tolerance of adverse fermentation conditions. However, the basis of their difference has not been fully elucidated, although their genomes have been sequenced and analyzed. Five strains of four Saccharomyces species (S. cerevisiae, S. kudriavzevii, S. bayanus, and S. paradoxus), when grown in parallel in laboratory conditions, exhibit very similar basic physiological parameters such as membrane potential, intracellular pH, and the degree to which they are able to quickly activate their Pma1 H+-ATPase upon glucose addition. On the other hand, they differ in their ability to proliferate in media with a very low concentration of potassium, in their osmotolerance and tolerance to toxic cations and cationic drugs in a growth-medium specific manner, and in their capacity to survive anhydrobiosis. Overall, S. cerevisiae (T73 more than FL100) and S. paradoxus are the most robust, and S. kudriavzevii the most sensitive species. Our results suggest that the difference in stress survival is based on their ability to quickly accommodate their cell size and metabolism to changing environmental conditions and to adjust their portfolio of available detoxifying transporters.

• Keywords
• Intracellular pH, Membrane potential, Saccharomyces, Stress tolerance,
• MeSH
• Fermentation MeSH
• Fungal Proteins genetics   metabolism   MeSH
• Stress, Physiological MeSH
• Glucose metabolism   MeSH
• Proton-Translocating ATPases genetics   metabolism   MeSH
• Saccharomyces classification   genetics   growth & development   physiology   MeSH
• Publication type
• Journal Article MeSH
• Comparative Study MeSH
• Names of Substances
• Fungal Proteins MeSH
• Glucose MeSH
• Proton-Translocating ATPases MeSH

We investigated the impact of the deletions of genes from the final steps in the biosynthesis of ergosterol (ERG6, ERG2, ERG3, ERG5, ERG4) on the physiological function of the Saccharomyces cerevisiae plasma membrane by a combination of biological tests and the diS-C3(3) fluorescence assay. Most of the erg mutants were more sensitive than the wild type to salt stress or cationic drugs, their susceptibilities were proportional to the hyperpolarization of their plasma membranes. The different sterol composition of the plasma membrane played an important role in the short-term and long-term processes that accompanied the exposure of erg strains to a hyperosmotic stress (effect on cell size, pH homeostasis and survival of yeasts), as well as in the resistance of cells to antifungal drugs. The pleiotropic drug-sensitive phenotypes of erg strains were, to a large extent, a result of the reduced efficiency of the Pdr5 efflux pump, which was shown to be more sensitive to the sterol content of the plasma membrane than Snq2p. In summary, the erg4Δ and erg6Δ mutants exhibited the most compromised phenotypes. As Erg6p is not involved in the cholesterol biosynthetic pathway, it may become a target for a new generation of antifungal drugs.

• MeSH
• ATP-Binding Cassette Transporters genetics   metabolism   MeSH
• Antifungal Agents pharmacology   MeSH
• Biosynthetic Pathways genetics   MeSH
• Cell Membrane chemistry   physiology   MeSH
• Ergosterol biosynthesis   chemistry   MeSH
• Fluconazole pharmacology   MeSH
• Microscopy, Fluorescence MeSH
• Hydrogen-Ion Concentration MeSH
• Membrane Potentials physiology   MeSH
• Methyltransferases genetics   metabolism   MeSH
• Drug Resistance, Multiple, Fungal drug effects   genetics   physiology   MeSH
• Molecular Structure MeSH
• Mutation MeSH
• Saccharomyces cerevisiae Proteins genetics   metabolism   MeSH
• Saccharomyces cerevisiae chemistry   genetics   physiology   MeSH
• Salt Tolerance genetics   physiology   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• ATP-Binding Cassette Transporters MeSH
• Antifungal Agents MeSH
• delta 24-sterol methyltransferase MeSH Browser
• Ergosterol MeSH
• Fluconazole MeSH
• Methyltransferases MeSH
• PDR5 protein, S cerevisiae MeSH Browser
• Saccharomyces cerevisiae Proteins MeSH
• SNQ2 protein, S cerevisiae MeSH Browser

Carbocyanine dye diS-C3(3) was repeatedly employed in monitoring the plasma membrane potential of yeast and other living cells. Four methods of measuring and evaluating probe fluorescence signal were used in different studies, based on following fluorescence parameters: fluorescence intensity emitted within a certain spectral interval, F(580)/F(560) fluorescence emission ratio, wavelength of emission spectrum maximum, and the ratio of respective fluorescence intensities corresponding to the diS-C3(3) bound to cytosolic macromolecules and remaining dissolved in the aqueous cell medium (i.e., unbound, or free). Here we show that data corresponding to the three latter spectral assessments of diS-C3(3) accumulation in cells is mutually convertible, which means that their alternative use cannot lead to ambiguities in the interpretation of the results of biological experiments. On the other hand, experiments based on the effortless measurements of fluorescence intensities should be interpreted cautiously because controversial results can be obtained, depending on the particular choice of cell-to-dye concentration ratio and emission wavelength.

• Keywords
• Fluorescent probe, Plasma membrane potential, Saccharomyces cerevisiae, Spectral analysis, Yeast,
• MeSH
• Fluorescent Dyes chemistry   MeSH
• Spectrometry, Fluorescence methods   MeSH
• Carbocyanines chemistry   MeSH
• Membrane Potentials * MeSH
• Saccharomyces cerevisiae chemistry   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• 3,3'-dipropylthiacarbocyanine MeSH Browser
• Fluorescent Dyes MeSH
• Carbocyanines MeSH

Cationic amphipathic drugs, such as amiodarone, interact preferentially with lipid membranes to exert their biological effect. In the yeast Saccharomyces cerevisiae, toxic levels of amiodarone trigger a rapid influx of Ca(2+) that can overwhelm cellular homeostasis and lead to cell death. To better understand the mechanistic basis of antifungal activity, we assessed the effect of the drug on membrane potential. We show that low concentrations of amiodarone (0.1-2 microm) elicit an immediate, dose-dependent hyperpolarization of the membrane. At higher doses (>3 microm), hyperpolarization is transient and is followed by depolarization, coincident with influx of Ca(2+) and H(+) and loss in cell viability. Proton and alkali metal cation transporters play reciprocal roles in membrane polarization, depending on the availability of glucose. Diminishment of membrane potential by glucose removal or addition of salts or in pma1, tok1Delta, ena1-4Delta, or nha1Delta mutants protected against drug toxicity, suggesting that initial hyperpolarization was important in the mechanism of antifungal activity. Furthermore, we show that the link between membrane hyperpolarization and drug toxicity is pH-dependent. We propose the existence of pH- and hyperpolarization-activated Ca(2+) channels in yeast, similar to those described in plant root hair and pollen tubes that are critical for cell elongation and growth. Our findings illustrate how membrane-active compounds can be effective microbicidals and may pave the way to developing membrane-selective agents.

• MeSH
• Amiodarone pharmacology   MeSH
• Fluorescence MeSH
• Immunoprecipitation MeSH
• Ion Transport MeSH
• Humans MeSH
• Membrane Proteins * MeSH
• Saccharomyces cerevisiae drug effects   physiology   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, N.I.H., Extramural MeSH
• Names of Substances
• Amiodarone MeSH
• Membrane Proteins * MeSH

• MeSH
• Yeasts * MeSH
• Publication type
• Congress MeSH
• Overall MeSH

The rate and extent of uptake of the fluorescent probe diS-C3(3) reporting on membrane potential in S. cerevisiae is affected by the strain under study, cell-growth phase, starvation and by the concentration of glucose both in the growth medium and in the monitored cell suspension under non-growth conditions. Killer toxin K1 brings about changes in membrane potential. In all types of cells tested, viz. in glucose-supplied stationary or exponential cells of the killer-sensitive strain S6/1 or a conventional strain RXII, or in glucose-free exponential cells of both strains, both active and heat-inactivated toxin slow down the potential-dependent uptake of diS-C3(3) into the cells. This may reflect "clogging" of pores in the cell wall that hinders, but does not prevent, probe passage to the plasma membrane and its equilibration. The clogging effect of heat-inactivated toxin is stronger than that exerted by active toxin. In susceptible cells, i.e. in exponential-phase glucose-supplied cells of the sensitive strain S6/1, this phase of probe uptake retardation is followed by an irreversible red shift in probe fluorescence maximum lambda max indicating damage to membrane integrity and cell permeabilization. A similar fast red shift in lambda max signifying lethal cell damage was found in heat-killed or nystatin-treated cells.

• MeSH
• Fluorescent Dyes metabolism   MeSH
• Fungal Proteins pharmacology   MeSH
• Carbocyanines metabolism   MeSH
• Killer Factors, Yeast MeSH
• Membrane Potentials drug effects   MeSH
• Mycotoxins pharmacology   MeSH
• Nystatin pharmacology   MeSH
• Saccharomyces cerevisiae physiology   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• 3,3'-dipropylthiacarbocyanine MeSH Browser
• Fluorescent Dyes MeSH
• Fungal Proteins MeSH
• K1 killer toxin MeSH Browser
• Carbocyanines MeSH
• Killer Factors, Yeast MeSH
• Mycotoxins MeSH
• Nystatin MeSH

Changes in the membrane potential of Saccharomyces cerevisiae were monitored by the electrochromic probe 3-(4-(2-(6-(dibutylamino)-2-naphthyl)-trans- ethenyl)pyridinium)propanesulfonate (di-4-ANEPPS) that should incorporate into the plasma membrane. The probe had suitable spectral characteristics and exhibited an electrochromic shift upon a change in membrane potential but the magnitude of the response increased with time. The presence and properties of the cell wall affected the extent of cell staining. The time dependence of the fluorescent response indicated that the probe was not incorporated solely into the plasma membrane but spread gradually into the whole cell; this was confirmed by confocal microscopy. The probe is therefore suitable for assessing membrane potential changes only over time intervals up to 30 min. Longer monitoring will require either a modified staining protocol or a derivatization of the probe molecule. As found by using the dioctyl derivative di-8-ANEPPS, extending the aliphatic chains of the di-4-ANEPPS molecule does not prevent the dye from penetrating into the cell or liposome interior and, in addition, impairs staining.

• MeSH
• Staining and Labeling MeSH
• Cell Membrane metabolism   physiology   MeSH
• Cell Wall metabolism   MeSH
• Time Factors MeSH
• Fluorescence MeSH
• Microscopy, Confocal MeSH
• Liposomes metabolism   MeSH
• Membrane Potentials * MeSH
• Pyridinium Compounds metabolism   MeSH
• Saccharomyces cerevisiae metabolism   physiology   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• 1-(3-sulfonatopropyl)-4-(beta-(2-(di-n-octylamino)-6-naphthyl)vinyl)pyridinium betaine MeSH Browser
• 1-(3-sulfonatopropyl)-4-(beta)(2-(di-n-butylamino)-6-naphthylvinyl)pyridinium betaine MeSH Browser
• Liposomes MeSH
• Pyridinium Compounds MeSH