Apoptosis induced by environmental stresses and amphotericin B inCandida albicans

Abstract

New antifungal agents are urgently required to combat lifethreatening infections caused by opportunistic fungal pathogens like Candida albicans. The manipulation of endogenous fungal programmed cell death responses could provide a basis for future therapies. Here we assess the physiology of death in C. albicans in response to environmental stresses (acetic acid and hydrogen peroxide) and an antifungal agent (amphotericin B). Exposure of C. albicans to 40 -60 mM acetic acid, 5-10 mM hydrogen peroxide, or 4 -8 g⅐ml ؊1 amphotericin B produced cellular changes reminiscent of mammalian apoptosis. Nonviable cells that excluded propidium iodide displayed the apoptotic marker phosphatidylserine (as shown by annexin-V-FITC labeling), were terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL)-positive (indicating nuclease-mediated double-strand DNA breakage), and produced reactive oxygen species. Ultrastructural changes in apoptotic cells included chromatin condensation and margination, separation of the nuclear envelope, and nuclear fragmentation. C. albicans cells treated at higher doses of these compounds showed cellular changes characteristic of necrosis. Necrotic cells displayed reduced TUNEL staining, a lack of surface phosphatidylserine, limited reactive oxygen species production, and an inability to exclude propidium iodide. Necrotic cells lacked defined nuclei and showed extensive intracellular vacuolization. Apoptosis in C. albicans was associated with an accumulation of cells in the G2͞M phase of the cell cycle, and under some apoptosis-inducing conditions, significant proportions of yeast cells switched to hyphal growth before dying. This is a demonstration of apoptosis in a medically important fungal pathogen.

The MAP kinase Mkc1p is activated under different stress conditions in Candida albicans

“…This activation would trigger self-protection, since the absence of the activity of these kinases in the macrophage leads to apoptosis in the presence of GSNO (Callsen & Brune, 1999). Since C. albicans undergoes apoptosis in the presence of oxidative stress (Phillips et al, 2003), Mkc1p phosphorylation could avoid apoptosis in the presence of NO radicals generated by macrophages. This activation could contribute to the survival rate of 80 % of C. albicans after macrophage endocytosis (Ibata-Ombetta et al, 2001), a process that increases the expression of ROS detoxifying genes and DNAdamage repair genes (Lorenz et al, 2004).…”

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

“…The features of apoptosis include externalization of phosphatidylserine, chromatin condensation, accumulation of ROS, DNA degradation, and caspase activation. 15,23,24 Candida spp undergoing apoptosis exhibit biochemical hallmarks similar to those of mammalian apoptosis in response to diverse stimuli, 15,25 in which mitochondria play a pivotal role in controlling both caspase-dependent and caspase-independent cell death pathways. ROS accumulation and mitochondrial dysfunction signal the onset of apoptosis.…”

“…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.…”

Apoptosis and the yeast actin cytoskeleton

“…For example excessive Ras signalling has also been shown to lead to apoptosis in the fungal pathogen Candida albicans. 80,81 The prevention of aggregate formation by addition of the actin monomer sequestering drug Lat-A was sufficient to prevent apoptosis. 82 This strengthens the proposal that the formation of stable F-actin structures, or aggregates, exerts an effect as the initiating factor within this apoptotic pathway.…”