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1.
Oxid Med Cell Longev ; 2013: 704654, 2013.
Article in English | MEDLINE | ID: mdl-24078861

ABSTRACT

This study demonstrates a role for superoxide dismutases (Sods) in governing tolerance of Candida albicans biofilms to amphotericin B (AmB). Coincubation of C. albicans biofilms with AmB and the Sod inhibitors N,N'-diethyldithiocarbamate (DDC) or ammonium tetrathiomolybdate (ATM) resulted in reduced viable biofilm cells and increased intracellular reactive oxygen species levels as compared to incubation of biofilm cells with AmB, DDC, or ATM alone. Hence, Sod inhibitors can be used to potentiate the activity of AmB against C. albicans biofilms.


Subject(s)
Amphotericin B/pharmacology , Biofilms/drug effects , Superoxide Dismutase/antagonists & inhibitors , Ditiocarb/pharmacology , Molybdenum/pharmacology , Reactive Oxygen Species/metabolism
2.
J Infect Dis ; 206(11): 1790-7, 2012 Dec 01.
Article in English | MEDLINE | ID: mdl-22984120

ABSTRACT

In this study, we demonstrated that in vitro Candida albicans biofilms grown in the presence of diclofenac showed increased susceptibility to caspofungin. These findings were further confirmed using a catheter-associated biofilm model in rats. C. albicans-inoculated catheters retrieved from rats that were treated with both diclofenac and caspofungin contained significantly fewer biofilm cells and showed no visible biofilms inside the catheter lumens, as documented by scanning electron microscopy, as compared to catheters retrieved from rats receiving only caspofungin or diclofenac. This report indicates that diclofenac could be useful in combination therapy with caspofungin to treat C. albicans biofilm-associated infections.


Subject(s)
Biofilms/drug effects , Candida albicans/drug effects , Diclofenac/pharmacology , Echinocandins/pharmacology , Animals , Anti-Inflammatory Agents, Non-Steroidal/administration & dosage , Anti-Inflammatory Agents, Non-Steroidal/pharmacology , Antifungal Agents/administration & dosage , Antifungal Agents/pharmacology , Candidiasis/drug therapy , Candidiasis/microbiology , Caspofungin , Catheters/microbiology , Diclofenac/administration & dosage , Drug Resistance, Fungal , Drug Synergism , Echinocandins/administration & dosage , Lipopeptides , Rats
3.
Mol Microbiol ; 84(1): 166-80, 2012 Apr.
Article in English | MEDLINE | ID: mdl-22384976

ABSTRACT

The antifungal plant defensin RsAFP2 isolated from radish interacts with fungal glucosylceramides and induces apoptosis in Candida albicans. To further unravel the mechanism of RsAFP2 antifungal action and tolerance mechanisms, we screened a library of 2868 heterozygous C. albicans deletion mutants and identified 30 RsAFP2-hypersensitive mutants. The most prominent group of RsAFP2 tolerance genes was involved in cell wall integrity and hyphal growth/septin ring formation. Consistent with these genetic data, we demonstrated that RsAFP2 interacts with the cell wall of C. albicans, which also contains glucosylceramides, and activates the cell wall integrity pathway. Moreover, we found that RsAFP2 induces mislocalization of septins and blocks the yeast-to-hypha transition in C. albicans. Increased ceramide levels have previously been shown to result in apoptosis and septin mislocalization. Therefore, ceramide levels in C. albicans membranes were analysed following RsAFP2 treatment and, as expected, increased accumulation of phytoC24-ceramides in membranes of RsAFP2-treated C. albicans cells was detected. This is the first report on the interaction of a plant defensin with glucosylceramides in the fungal cell wall, causing cell wall stress, and on the effects of a defensin on septin localization and ceramide accumulation.


Subject(s)
Candida albicans/growth & development , Cell Wall/metabolism , Defensins/metabolism , Glucosylceramides/metabolism , Plant Proteins/metabolism , Septins/metabolism , Candida albicans/ultrastructure , Cell Wall/ultrastructure , Hyphae/growth & development , Microscopy, Electron, Transmission , Raphanus
4.
J Med Microbiol ; 61(Pt 6): 813-819, 2012 Jun.
Article in English | MEDLINE | ID: mdl-22422573

ABSTRACT

We investigated the molecular basis of the tolerance of Candida albicans biofilms to antifungals using the miconazole as a model compound, and translated the resulting data to other antifungals. Sessile cells of C. albicans Δefg1, lacking the transcription factor Efg1, showed increased susceptibility to miconazole, amphotericin B and caspofungin, whereas these sessile cells were equally resistant to fluconazole. The increased sensitivity to miconazole was, at least, partly due to an increased accumulation of miconazole in the cells as compared to wild-type or reintegrant Δefg1(EFG1) sessile cells. By using a rat biofilm model, we further confirmed the role of Efg1 in the tolerance of C. albicans biofilms to miconazole when grown in vivo.


Subject(s)
Antifungal Agents/pharmacology , Biofilms/drug effects , Candida albicans/drug effects , Candida albicans/physiology , DNA-Binding Proteins/metabolism , Drug Resistance, Fungal , Fungal Proteins/metabolism , Transcription Factors/metabolism , Animals , Biofilms/growth & development , Candida albicans/growth & development , DNA-Binding Proteins/genetics , Fungal Proteins/genetics , Gene Deletion , Miconazole/pharmacology , Microbial Sensitivity Tests , Rats , Transcription Factors/genetics
5.
Antimicrob Agents Chemother ; 56(5): 2290-4, 2012 May.
Article in English | MEDLINE | ID: mdl-22354293

ABSTRACT

Previous research has shown that 1% to 10% of sessile Candida albicans cells survive treatment with high doses of miconazole (a fungicidal imidazole). In the present study, we investigated the involvement of sphingolipid biosynthetic intermediates in this survival. We observed that the LCB4 gene, coding for the enzyme that catalyzes the phosphorylation of dihydrosphingosine and phytosphingosine, is important in governing the miconazole resistance of sessile Saccharomyces cerevisiae and C. albicans cells. The addition of 10 nM phytosphingosine-1-phosphate (PHS-1-P) drastically reduced the intracellular miconazole concentration and significantly increased the miconazole resistance of a hypersusceptible C. albicans heterozygous LCB4/lcb4 mutant, indicating a protective effect of PHS-1-P against miconazole-induced cell death in sessile cells. At this concentration of PHS-1-P, we did not observe any effect on the fluidity of the cytoplasmic membrane. The protective effect of PHS-1-P was not observed when the efflux pumps were inhibited or when tested in a mutant without functional efflux systems. Also, the addition of PHS-1-P during miconazole treatment increased the expression levels of genes coding for efflux pumps, leading to the hypothesis that PHS-1-P acts as a signaling molecule and enhances the efflux of miconazole in sessile C. albicans cells.


Subject(s)
Antifungal Agents/pharmacology , Candida albicans/genetics , Drug Resistance, Fungal/genetics , Gene Expression Regulation, Bacterial/drug effects , Miconazole/pharmacology , Sphingosine/analogs & derivatives , Candida albicans/drug effects , Candida albicans/metabolism , Cell Membrane/drug effects , Cell Membrane/genetics , Cell Membrane/metabolism , Drug Resistance, Fungal/drug effects , Membrane Transport Proteins/genetics , Membrane Transport Proteins/metabolism , Mutation , Phosphorylation , Phosphotransferases (Alcohol Group Acceptor)/genetics , Phosphotransferases (Alcohol Group Acceptor)/metabolism , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolism , Signal Transduction/genetics , Sphingosine/metabolism , Sphingosine/pharmacology
6.
Antimicrob Agents Chemother ; 55(9): 4033-7, 2011 Sep.
Article in English | MEDLINE | ID: mdl-21746956

ABSTRACT

We investigated the cellular mechanisms responsible for the occurrence of miconazole-tolerant persisters in Candida albicans biofilms. Miconazole induced about 30% killing of sessile C. albicans cells at 75 µM. The fraction of miconazole-tolerant persisters, i.e., cells that can survive high doses of miconazole (0.6 to 2.4 mM), in these biofilms was 1 to 2%. Since miconazole induces reactive oxygen species (ROS) in sessile C. albicans cells, we focused on a role for superoxide dismutases (Sods) in persistence and found the expression of Sod-encoding genes in sessile C. albicans cells induced by miconazole compared to the expression levels in untreated sessile C. albicans cells. Moreover, addition of the superoxide dismutase inhibitor N,N'-diethyldithiocarbamate (DDC) to C. albicans biofilms resulted in an 18-fold reduction of the miconazole-tolerant persister fraction and in increased endogenous ROS levels in these cells. Treatment of biofilms of C. albicans clinical isolates with DDC resulted in an 18-fold to more than 200-fold reduction of their miconazole-tolerant persister fraction. To further confirm the important role for Sods in C. albicans biofilm persistence, we used a Δsod4 Δsod5 mutant lacking Sods 4 and 5. Biofilms of the Δsod4 Δsod5 mutant contained at least 3-fold less of the miconazole-tolerant persisters and had increased ROS levels compared to biofilms of the isogenic wild type (WT). In conclusion, the occurrence of miconazole-tolerant persisters in C. albicans biofilms is linked to the ROS-detoxifying activity of Sods. Moreover, Sod inhibitors can be used to potentiate the activity of miconazole against C. albicans biofilms.


Subject(s)
Antifungal Agents/pharmacology , Biofilms/drug effects , Candida albicans/drug effects , Candida albicans/enzymology , Fungal Proteins/metabolism , Miconazole/pharmacology , Superoxide Dismutase/metabolism , Candida albicans/metabolism , Ditiocarb/pharmacology , Drug Resistance, Fungal/genetics , Fungal Proteins/antagonists & inhibitors , Fungal Proteins/genetics , Microbial Sensitivity Tests , Reactive Oxygen Species/metabolism , Superoxide Dismutase/antagonists & inhibitors , Superoxide Dismutase/genetics
7.
FEMS Yeast Res ; 10(7): 812-8, 2010 Nov.
Article in English | MEDLINE | ID: mdl-20726898

ABSTRACT

To unravel the working mechanism of the fungicidal piperazine-1-carboxamidine derivative BAR0329, we found that its intracellular accumulation in Saccharomyces cerevisiae is dependent on functional lipid rafts. Moreover, BAR0329 induced caspase-dependent apoptosis in yeast, in which the mitochondrial fission machinery consisting of Fis1 (Whi2), Dnm1 and Mdv1 is involved. Our data are consistent with a prosurvival function of Fis1 (Whi2) and a proapoptotic function of Dnm1 and Mdv1 during BAR0329-induced yeast cell death.


Subject(s)
Antifungal Agents/toxicity , Apoptosis , Mitochondria/drug effects , Piperazines/toxicity , Saccharomyces cerevisiae/drug effects , Adaptor Proteins, Signal Transducing/metabolism , GTP Phosphohydrolases/metabolism , Membrane Microdomains/metabolism , Mitochondrial Proteins/metabolism , Saccharomyces cerevisiae Proteins/metabolism
8.
J Biol Chem ; 284(47): 32680-5, 2009 Nov 20.
Article in English | MEDLINE | ID: mdl-19783660

ABSTRACT

Azoles inhibit ergosterol biosynthesis, resulting in ergosterol depletion and accumulation of toxic 14alpha-methylated sterols in membranes of susceptible yeast. We demonstrated previously that miconazole induces actin cytoskeleton stabilization in Saccharomyces cerevisiae prior to induction of reactive oxygen species, pointing to an ancillary mode of action. Using a genome-wide agar-based screening, we demonstrate in this study that S. cerevisiae mutants affected in sphingolipid and ergosterol biosynthesis, namely ipt1, sur1, skn1, and erg3 deletion mutants, are miconazole-resistant, suggesting an involvement of membrane rafts in its mode of action. This is supported by the antagonizing effect of membrane raft-disturbing compounds on miconazole antifungal activity as well as on miconazole-induced actin cytoskeleton stabilization and reactive oxygen species accumulation. These antagonizing effects point to a primary role for membrane rafts in miconazole antifungal activity. We further show that this primary role of membrane rafts in miconazole action consists of mediating intracellular accumulation of miconazole in yeast cells.


Subject(s)
Membrane Microdomains/metabolism , Miconazole/pharmacokinetics , Saccharomyces cerevisiae/metabolism , Antifungal Agents/pharmacokinetics , Drug Resistance, Fungal , Endocytosis , Ergosterol/metabolism , Gene Deletion , Gene Expression Regulation, Fungal , Genome, Fungal , Membrane Microdomains/drug effects , Miconazole/pharmacology , Phosphodiesterase Inhibitors/pharmacology , Phospholipid Ethers/pharmacology , Reactive Oxygen Species
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