Filamentation protects Candida albicans from amphotericin B-induced programmed cell death via a mechanism involving the yeast metacaspase, MCA1

ABSTRACT Amphotericin B treatment was previously shown to inhibit Candida albicans reproduction and reduce the fluorescence of vitality-specific dyes without causing a corresponding increase in the fluorescence of the mortality-specific dyes bis-(1,3-dibutylbarbituric acid)trimethine oxonol and SYBR Green Ι. In the present study, we have confirmed these results and have shown that the numbers of CFU are reduced by 99.9% by treatment with 0.5 μg of amphotericin B per ml for 10 h at 35°C. This reduction was not due to fungal cell death. First, the level of reduction of the tetrazolium salt 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-2H-tetrazolium hydroxide increased in the presence of concentrations of amphotericin B that caused greater than 90% reductions in the numbers of CFU. Second, fungal cells treated with amphotericin B at a concentration of 0.5 μg/ml were resuscitated by further incubation at 22°C for 15 h in the continued presence of amphotericin B. Third, recovery of the ability to replicate was prevented by sequential treatment with 20 μg of miconazole per ml, which also increased the fluorescence of mortality-specific dyes to near the maximal levels achieved with 0.9 μg of amphotericin B per ml. Sequential treatment with fluconazole and flucytosine did not increase the levels of staining with the mortality-specific dyes. Itraconazole was less effective than ketoconazole, which was less effective than miconazole. The practice of equating the loss of the capacity of C. albicans to form colonies with fungal cell death may give incorrect results in assays with amphotericin B, and the results of assays with caution with other antifungal agents that are lipophilic or that possess significant postantifungal effects may need to be interpreted.

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Ras pathway signaling accelerates programmed cell death in the pathogenic fungus Candida albicans

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.0506405103 ◽

2006 ◽

Vol 103 (3) ◽

pp. 726-731 ◽

Cited By ~ 105

Author(s):

A. J. Phillips ◽

J. D. Crowe ◽

M. Ramsdale

Keyword(s):

Cell Death ◽

Candida Albicans ◽

Programmed Cell Death ◽

Pathogenic Fungus ◽

Ras Pathway

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Amphotericin B induces apoptosis-like programmed cell death in Naegleria fowleri and Naegleria gruberi

Microbiology ◽

10.1099/mic.0.000500 ◽

2017 ◽

Vol 163 (7) ◽

pp. 940-949 ◽

Cited By ~ 3

Author(s):

Roberto Cárdenas-Zúñiga ◽

Angélica Silva-Olivares ◽

José D' Artagnan Villalba-Magdaleno ◽

Virginia Sánchez-Monroy ◽

Jesús Serrano-Luna ◽

...

Keyword(s):

Cell Death ◽

Programmed Cell Death ◽

Amphotericin B ◽

Naegleria Fowleri ◽

Nægleria Fowleri ◽

Naegleria Gruberi

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A Genome‐wide Screen for Transcription Factors Involved in Programmed Cell Death in the Yeast, Candida albicans

The FASEB Journal ◽

10.1096/fasebj.25.1_supplement.943.2 ◽

2011 ◽

Vol 25 (S1) ◽

Author(s):

Vincent Cascio ◽

Richard Bennett ◽

Joachim Morschhauser ◽

Nicanor Austriaco

Keyword(s):

Cell Death ◽

Candida Albicans ◽

Transcription Factors ◽

Programmed Cell Death ◽

Genome Wide ◽

A Genome

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Caspofungin Kills Candida albicans by Causing both Cellular Apoptosis and Necrosis

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01366-12 ◽

2012 ◽

Vol 57 (1) ◽

pp. 326-332 ◽

Cited By ~ 79

Author(s):

Binghua Hao ◽

Shaoji Cheng ◽

Cornelius J. Clancy ◽

M. Hong Nguyen

Keyword(s):

Cell Death ◽

Candida Albicans ◽

Programmed Cell Death ◽

Membrane Integrity ◽

Annexin V ◽

Terminal Deoxynucleotidyl Transferase ◽

Dapi Staining ◽

Content Type ◽

Glucan Synthesis ◽

Apoptosis And Necrosis

ABSTRACTCaspofungin exerts candidacidal activity by inhibiting cell wall (1,3)-β-d-glucan synthesis. We investigated the physiologic mechanisms of caspofungin-inducedCandida albicanscell death. Apoptosis (programmed cell death) and necrosis were studied afterC. albicansSC5314 cells were exposed to caspofungin at 0.06, 0.125, and 0.5 μg/ml (0.5×, 1×, and 4× the MIC, respectively) for 3 h. Caspofungin at 0.125 and 0.5 μg/ml reduced cellular viability by >50%, as measured by colony counts and methylene blue exclusion. Apoptosis and necrosis were demonstrated by annexin V and propidium iodide staining for phosphatidylserine externalization and loss of membrane integrity, respectively. At all concentrations of caspofungin, 20 to 25% and 5 to 7% ofC. albicanscells exhibited early apoptosis and late apoptosis/necrosis, respectively (Pvalue was not significant [NS]). Necrosis, on the other hand, was significantly greater at 0.125 (43%) and 0.5 (48%) μg/ml than at 0.06 μg/ml (26%) (Pvalues of 0.003 and 0.003, respectively). The induction of apoptosis at concentrations less than or equal to the MIC was corroborated by dihydrorhodamine 123 (DHR-123) and dihydroethidium (DHE) staining (reactive oxygen species production), JC-1 staining (mitochondrial membrane potential dissipation), and terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) and 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI) staining (DNA damage and nuclear fragmentation). Moreover, electron microscopy of cells exposed to 0.125 μg/ml of caspofungin showed hallmark apoptotic features like chromatin margination and condensation and nuclear blebs. Apoptosis was associated with metacaspase 1 activation, as demonstrated by D2R staining. Caspofungin exerts activity againstC. albicansby directly killing cells (resulting in necrosis) and causing others to undergo programmed cell death (apoptosis). Apoptosis is initiated at subinhibitory concentrations, suggesting that strategies to target this process may augment the benefits of antifungal agents.

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Deletion of AIF1 but not of YCA1/MCA1 protects Saccharomyces cerevisiae and Candida albicans cells from caspofungin-induced programmed cell death

Microbial Cell ◽

10.15698/mic2014.01.119 ◽

2014 ◽

Vol 1 (2) ◽

pp. 58-63 ◽

Cited By ~ 4

Author(s):

Christopher Chin ◽

◽

Faith Donaghey ◽

Katherine Helming ◽

Morgan McCarthy ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Cell Death ◽

Candida Albicans ◽

Programmed Cell Death

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Human Salivary Histatin 5 Fungicidal Action Does Not Induce Programmed Cell Death Pathways in Candida albicans

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.48.1.110-115.2004 ◽

2004 ◽

Vol 48 (1) ◽

pp. 110-115 ◽

Cited By ~ 31

Author(s):

David Wunder ◽

Jin Dong ◽

Didi Baev ◽

Mira Edgerton

Keyword(s):

Cell Death ◽

Candida Albicans ◽

Programmed Cell Death ◽

Protein Carbonyl ◽

Direct Role ◽

Fungicidal Action ◽

Mean Cell Volume ◽

Histatin 5 ◽

Cell Death Pathways ◽

Intracellular Expression

ABSTRACT Salivary histatins (Hsts) are potent candidacidal proteins that induce a nonlytic form of cell death in Candida albicans accompanied by loss of mean cell volume, cell cycle arrest, and elevation of intracellular levels of reactive oxygen species (ROS). Since these phenotypes are often markers of programmed cell death and apoptosis, we investigated whether other classical markers of apoptosis, including generation of intracellular ROS and protein carbonyl groups, chromosomal fragmentation (laddering), and cytochrome c release, are found in Hst 5-mediated cell death. Increased intracellular levels of ROS in C. albicans were detected in cells both following exogenous application of Hst 5 and following intracellular expression of Hst 5. However, Western blot analysis failed to detect specifically increased protein carbonylation in Hst 5-treated cells. There was no evidence of chromosomal laddering and no cytochrome c release was observed following treatment of C. albicans mitochondria with Hst 5. Superoxide dismutase enzymes of C. albicans and Saccharomyces cerevisiae provide essential protection against oxidative stress; therefore, we tested whether SOD mutants have increased susceptibility to Hst 5, as expected if ROS mediate fungicidal effects. Cell survival of S. cerevisiae SOD1/SOD2 mutants and C. albicans SOD1 mutants following Hst 5 treatment (31 μM) was indistinguishable from the survival of wild-type cells treated with Hst 5. We conclude that ROS may not play a direct role in fungicidal activity and that Hst 5 does not initiate apoptosis or programmed cell death pathways.

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