Caspofungin Kills Candida albicans by Causing both Cellular Apoptosis and Necrosis

Abstract

Caspofungin exerts candidacidal activity by inhibiting cell wall (1,3)-␤-D-glucan synthesis. We investigated the physiologic mechanisms of caspofungin-induced Candida albicans cell death. Apoptosis (programmed cell death) and necrosis were studied after C. albicans SC5314 cells were exposed to caspofungin at 0.06, 0.125, and 0.5 g/ml (0.5؋, 1؋, and 4؋ the MIC, respectively) for 3 h. Caspofungin at 0.125 and 0.5 g/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% of C. albicans cells exhibited early apoptosis and late apoptosis/necrosis, respectively (P value was not significant [NS]). Necrosis, on the other hand, was significantly greater at 0.125 (43%) and 0.5 (48%) g/ml than at 0.06 g/ml (26%) (P values 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 g/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 against C. albicans by 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.C aspofungin and other agents in the echinocandin class of antifungals have assumed an increasingly important role in the therapy of invasive candidiasis (1). These agents are nontoxic and exert potent fungicidal activity against Candida albicans and other Candida spp. Their antifungal activity is achieved through inhibition of (1,3)-␤-D-glucan synthase (2), an enzyme that synthesizes a major constituent of the fungal cell wall. Although the mechanism of activity for the echinocandins is known, the physiological mechanisms by which they cause cell death are not defined. At least two types of mammalian cell death, necrosis and apoptosis, have been described (3). Necrosis is death resulting from direct cellular injury, which is best defined by cell and organelle swelling and lysis (4). Apoptosis, on the other hand, is programmed cell death, the principal morphological feature of which is shrinkage of the cell and its nucleus (3, 4).Over the last decade, there have been a number of reports on apoptosis in yeas...

Sodium Ascorbate Kills Candida Albicans in Vitro Via Iron-Catalyzed Fenton Reaction: Importance of Oxygenation and Metabolism

“…However, when conditions are more favorable they can revert to a state of colony-forming competence [38]. It is possible that C. albicans cells exposed to ascorbate in PBS without shaking may have moved toward a state of 'early apoptosis' as there was no overt loss of viability by CFU determination, but only a delayed formation of colonies [39].…”

Micafungin triggers caspase-dependent apoptosis inCandida albicansandCandida parapsilosisbiofilms, including caspofungin non-susceptible isolates

“…Metacaspase activation, a distinct biochemical marker of apoptosis, has been previously characterized in Saccharomyces cerevisiae and C. albicans. 16,19,23 Recently, other research groups have demonstrated metacaspasedependent apoptosis in planktonic and sessile cells of C. albicans. [23][24][25] In conclusion, we provide evidence that MICA triggers apoptotic features in C. albicans and C. parapsilosis biofilms including in CAS-NS strains.…”

“…17 Biofilms pretreated with MICA (0.25-4.0 mg/mL) for 3 h at 37 C were fixed with 3.7% formaldehyde for 30 min on ice and were digested with a lysing enzyme mixture (0.25 mg/ mL of chitinase, 15 U of lyticase, and 20 mg/mL lysing enzyme; Sigma-Aldrich) for 3 h at 30 C. The enzyme-digested cells were used to detect DNA fragmentation by means of the TUNEL assay as previously described. 16 The cells were observed for fluorescence as described above, with excitation and emission wavelengths of 488 nm and 520 nm, respectively.…”

“…14 Recent studies have demonstrated that planktonic and sessile C. albicans cells undergo apoptosis when exposed to amphotericin B and caspofungin, via activation of cysteine-dependent, aspartate-specific proteases (caspases). 13,15,16 However, to our knowledge, no study has assessed in a biofilm environment, the mechanisms of cell death in Candida albicans and Candida parapsilosis induced by the echinocandin micafungin (MICA), an echinocandin that inhibits (1, 3)-b-D glucan synthesis in the cell wall. Elucidation of novel targets or mechanisms for enhancing the activity of current antifungals against Candida biofilms is critical for improving outcomes of biofilm associated Candida infection.…”

Stimulation of Eryptosis by Caspofungin

“…of Candida albicans [28]. The substance may be toxic as well for host cells and for host mitochondria [29].…”