Haynes M P, Chong P L, Buckley H R, Pieringer R A
Department of Biochemistry, Temple University School of Medicine, Philadelphia, Pennsylvania 19140, USA.
Biochemistry. 1996 Jun 18;35(24):7983-92. doi: 10.1021/bi952910c.
The molecular action of amphotericin B (AmB) on the cell membranes of both AmB-susceptible and AmB-resistant fungal cells was investigated through the use of the fluorescent membrane probe trimethylammonium diphenylhexatriene (TMA-DPH). AmB, the most effective drug for the treatment of systemic fungal infections, is known to interact specifically with membrane sterols, especially ergosterol (the major sterol in fungal cells). Treatment of AmB-susceptible Candida albicans and Cryptococcus neoformans cells with AmB induced a novel biphasic change in TMA-DPH fluorescence intensity over time. The initial decrease in fluorescence intensity results from energy transfer between AmB and TMA-DPH when AmB binds to the fungal cell membrane. The second phase of increasing fluorescence intensity is interpreted in terms of a combination of probe repartitioning and probe segregation as a result of the formation of membrane pores via the aggregation of AmB-ergosterol complexes. An AmB-resistant strain of C. neoformans, containing 94% of aberrant delta-8 double-bonded ergosterol precursors and only 4% of ergosterol (74% ergosterol in wild-type cells), exhibited the first phase of AmB binding but not the second phase of increasing fluorescence intensity. This result suggests that AmB's antifungal activity lies in its ability to form membrane pores due to aggregation of AmB-ergosterol complexes. The AmB-Induced biphasic fluorescence intensity profile may lead to further elucidation of the molecular action of AmB on fungal cells and may provide a sensitive method for screening the development of drug resistance in fungal cells.
通过使用荧光膜探针三甲基铵二苯基己三烯（TMA-DPH），研究了两性霉素B（AmB）对两性霉素B敏感和耐药真菌细胞膜的分子作用。AmB是治疗系统性真菌感染最有效的药物，已知它能与膜固醇，特别是麦角固醇（真菌细胞中的主要固醇）特异性相互作用。用AmB处理两性霉素B敏感的白色念珠菌和新生隐球菌细胞，随着时间的推移，TMA-DPH荧光强度出现了一种新的双相变化。荧光强度的初始下降是由于AmB与真菌细胞膜结合时，AmB与TMA-DPH之间的能量转移。荧光强度增加的第二阶段被解释为由于AmB-麦角固醇复合物的聚集形成膜孔，导致探针重新分配和探针分离的综合结果。一株对AmB耐药的新生隐球菌菌株，含有94%异常的δ-8双键麦角固醇前体和仅4%的麦角固醇（野生型细胞中为74%的麦角固醇），表现出AmB结合的第一阶段，但没有荧光强度增加的第二阶段。这一结果表明，AmB的抗真菌活性在于其通过AmB-麦角固醇复合物的聚集形成膜孔的能力。AmB诱导的双相荧光强度图谱可能会进一步阐明AmB对真菌细胞的分子作用，并可能为筛选真菌细胞耐药性的发展提供一种灵敏的方法。
