Phillips Andrew J, Sudbery Ian, Ramsdale Mark
Department of Molecular and Cell Biology, Institute of Medical Sciences, University of Aberdeen, Foresterhill, AB25 2ZD Aberdeen, Scotland.
Proc Natl Acad Sci U S A. 2003 Nov 25;100(24):14327-32. doi: 10.1073/pnas.2332326100. Epub 2003 Nov 17.
New antifungal agents are urgently required to combat life-threatening 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 microg.ml-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 G2/M 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.
迫切需要新型抗真菌药物来对抗由白色念珠菌等机会性真菌病原体引起的危及生命的感染。操纵内源性真菌程序性细胞死亡反应可为未来的治疗提供基础。在此，我们评估白色念珠菌在响应环境应激（乙酸和过氧化氢）和一种抗真菌药物（两性霉素B）时的死亡生理学。将白色念珠菌暴露于40 - 60 mM乙酸、5 - 10 mM过氧化氢或4 - 8 μg.ml-1两性霉素B会产生类似于哺乳动物细胞凋亡的细胞变化。排斥碘化丙啶的无活力细胞显示出凋亡标志物磷脂酰丝氨酸（通过膜联蛋白-V-异硫氰酸荧光素标记显示），是末端脱氧核苷酸转移酶介导的dUTP缺口末端标记（TUNEL）阳性（表明核酸酶介导的双链DNA断裂），并产生活性氧。凋亡细胞的超微结构变化包括染色质浓缩和边缘化、核膜分离以及核碎片化。用这些化合物的更高剂量处理的白色念珠菌细胞表现出坏死特征性的细胞变化。坏死细胞TUNEL染色减少、缺乏表面磷脂酰丝氨酸、活性氧产生受限且无法排斥碘化丙啶。坏死细胞缺乏明确的细胞核并显示出广泛的细胞内空泡化。白色念珠菌中的凋亡与细胞周期G2/M期细胞的积累相关，并且在某些诱导凋亡的条件下，相当比例的酵母细胞在死亡前转变为菌丝生长。这证明了一种医学上重要的真菌病原体中存在凋亡现象。
