The uptake of amino acids by<i>erg</i>mutants of<i>Candida albicans</i>

Ansari, Shehnaz; Gupta, Poonam; Mahanty, Sanjoy K.; Prasad, R.

doi:10.1080/02681219380000481

Cited by 19 publications

(20 citation statements)

References 21 publications

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“…In order to ascertain the physical state of the membrane, we used steady-state fluorescence polarization to examine the membrane order (fluidity) of erg2, erg3, erg4, and erg6 mutants and the WT strain. A specific plasma membrane fluorescent probe, DPH, was used for such measurements, as described earlier (1,19). All the erg mutant strains showed decreases in fluorescence polarization, thus implying decreased membrane order or enhanced fluidity of the membranes of these mutants compared to that for the WT strain ( Fig.…”

Section: Resultsmentioning

confidence: 99%

“…Both ergosterol and 24(28)-dehydroergosterol (DHE) absorb at 281.5 nm, whereas only DHE absorbs at 230 nm. The ergosterol content was calculated as a percentage of the wet weight of the cell by the following equations (2) Ϫ1 , which had been dissolved in petroleum ether and added directly into the medium during inoculation (1). Sterols from supplemented cells were then extracted and analyzed by recording the wavelength scan between 200 and 320 nm as described above.…”

Section: Methodsmentioning

confidence: 99%

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Drug Susceptibilities of Yeast Cells Are Affected by Membrane Lipid Composition

Mukhopadhyay

Kohli

Prasad

2002

Antimicrob Agents Chemother

187

164

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In the present study we have exploited isogenic erg mutants of Saccharomyces cerevisiae to examine the contribution of an altered lipid environment on drug susceptibilities of yeast cells. It is observed that erg mutants, which possess high levels of membrane fluidity, were hypersensitive to the drugs tested, i.e., cycloheximide (CYH), o-phenanthroline, sulfomethuron methyl, 4-nitroquinoline oxide, and methotrexate. Most of the erg mutants except mutant erg4 were, however, resistant to fluconazole (FLC). By using the fluorophore rhodamine-6G and radiolabeled FLC to monitor the passive diffusion, it was observed that erg mutant cells elicited enhanced diffusion. The addition of a membrane fluidizer, benzyl alcohol (BA), to S. cerevisiae wild-type cells led to enhanced membrane fluidity. However, a 10 to 12% increase in BA-induced membrane fluidity did not alter the drug susceptibilities of the S. cerevisiae wild-type cells. The enhanced diffusion observed in erg mutants did not seem to be solely responsible for the observed hypersensitivity of erg mutants. In order to ascertain the functioning of drug extrusion pumps encoding the genes CDR1 (ATP-binding cassette family) and CaMDR1 (MFS family) of Candida albicans in a different lipid environment, they were independently expressed in an S. cerevisiae erg mutant background. While the fold change in drug resistance mediated by CaMDR1 remained the same or increased in erg mutants, susceptibility to FLC and CYH mediated by CDR1 was increased (decrease in fold resistance). Our results demonstrate that between the two drug extrusion pumps, Cdr1p appeared to be more adversely affected by the fluctuations in the membrane lipid environment (particularly to ergosterol). By using 6-[(7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino-hexanoyl] sphingosyl phosphocholine (a fluorescent analogue of sphingomyelin), a close interaction between membrane ergosterol and sphingomyelin which appears to be disrupted in erg mutants is demonstrated. Taken together it appears that multidrug resistance in yeast is closely linked to the status of membrane lipids, wherein the overall drug susceptibility phenotype of a cell appears to be an interplay among drug diffusion, extrusion pumps, and the membrane lipid environment.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Drug Susceptibilities of Yeast Cells Are Affected by Membrane Lipid Composition

Mukhopadhyay

Kohli

Prasad

2002

Antimicrob Agents Chemother

187

164

View full text Add to dashboard Cite

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“…Steady-state fluorescence anisotropy was measured at 37°C with a spectrofluorimeter (model 650-60; Hitachi) with excitation at 360 nm and emission at 430 nm ( 5-and 5-nm slits, respectively). Fluorescence anisotropy was calculated as described by Ansari et al [2]. The degree of fluorescence polarization (p) was calculated as follows:…”

Section: Measurement Of Fluorescence Anisotropymentioning

confidence: 99%

Lactobacillus casei combats acid stress by maintaining cell membrane functionality

Zhang

Wang

et al. 2012

Journal of Industrial Microbiology and Biotechnology

161

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Lactobacillus casei strains have traditionally been recognized as probiotics and frequently used as adjunct culture in fermented dairy products where lactic acid stress is a frequently encountered environmental condition. We have investigated the effect of lactic acid stress on the cell membrane of L. casei Zhang [wild type (WT)] and its acid-resistant mutant Lbz-2. Both strains were grown under glucose-limiting conditions in chemostats; following challenge by low pH, the cell membrane stress responses were investigated. In response to acid stress, cell membrane fluidity decreased and its fatty acid composition changed to reduce the damage caused by lactic acid. Compared with the WT, the acid-resistant mutant exhibited numerous survival advantages, such as higher membrane fluidity, higher proportions of unsaturated fatty acids, and higher mean chain length. In addition, cell integrity analysis showed that the mutant maintained a more intact cellular structure and lower membrane permeability after environmental acidification. These results indicate that alteration in membrane fluidity, fatty acid distribution, and cell integrity are common mechanisms utilized by L. casei to withstand severe acidification and to reduce the deleterious effect of lactic acid on the cell membrane. This detailed comparison of cell membrane responses between the WT and mutant add to our knowledge of the acid stress adaptation and thus enable new strategies to be developed aimed at improving the industrial performance of this species under acid stress.

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“…C. albicans strains ATCC 44829 (wild type, 33 ERG ϩ ade Ϫ ), ATCC 44830 (erg16 ade Ϫ ), and ATCC 44831 (erg2 ade Ϫ ) were procured from the American Type Culture Collection. The strains were maintained as described previously (2). The Saccharomyces cerevisiae strain used was PSCDR1-GFP (an AD1-8u Ϫ derivative expressing Cdr1p-green fluorescent protein [GFP]) (36).…”

Section: Methodsmentioning

confidence: 99%

Membrane Sphingolipid-Ergosterol Interactions Are Important Determinants of Multidrug Resistance in Candida albicans

Mukhopadhyay

Prasad

Saini

et al. 2004

Antimicrob Agents Chemother

157

155

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In this study, we examined the importance of membrane ergosterol and sphingolipids in the drug susceptibilities of Candida albicans. We used three independent methods to test the drug susceptibilities of erg mutant cells, which were defective in ergosterol biosynthesis. While spot and filter disk assays revealed that erg2 and erg16 mutant cells of C. albicans became hypersensitive to almost all of the drugs tested (i.e., 4-nitroquinoline oxide, terbinafine, o-phenanthroline, itraconazole, and ketoconazole), determination of the MIC at which 80% of the cells were inhibited revealed more than fourfold increase in susceptibility to ketoconazole and terbinafine. Treatment of wild-type C. albicans cells with fumonisin B1 resulted in 45% inhibition of sphingolipid biosynthesis and caused cells to become hypersensitive to the above drugs. Although erg mutants displayed enhanced membrane fluidity and passive diffusion, these changes alone were not sufficient to elicit the observed hypersusceptibility phenotype of erg mutants. For example, the induction in vitro of a 12% change in the membrane fluidity of C. albicans cells by a membrane fluidizer, benzyl alcohol, did not affect the drug susceptibilities of Candida cells. Additionally, the surface localization of green fluorescent protein-tagged Cdr1p, a major drug efflux pump protein of C. albicans, revealed that any disruption in ergosterol and sphingolipid interactions also interfered with its proper surface localization and functioning. A 50% reduction in the efflux of the Cdr1p substrate, rhodamine 6G, in erg mutant cells or in cells with a reduced sphingolipid content suggested a strong correlation between these membrane lipid components and this major efflux pump protein. Taken together, the results of our study demonstrate for the first time that there is an interaction between membrane ergosterol and sphingolipids, that a reduction in the content of either of these two components results in a disruption of this interaction, and that this disruption has deleterious effects on the drug susceptibilities of C. albicans cells.

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The uptake of amino acids byergmutants ofCandida albicans

Cited by 19 publications

References 21 publications

Drug Susceptibilities of Yeast Cells Are Affected by Membrane Lipid Composition

Drug Susceptibilities of Yeast Cells Are Affected by Membrane Lipid Composition

Lactobacillus casei combats acid stress by maintaining cell membrane functionality

Membrane Sphingolipid-Ergosterol Interactions Are Important Determinants of Multidrug Resistance in Candida albicans

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