FLUORESCENCE STUDIES ON THE MOLECULAR ACTION OF AMPHOTERICIN-B ON SUSCEPTIBLE AND RESISTANT FUNGAL CELLS

The molecular action of amphotericin B (AmB) on the cell membranes of both AmB-susceptible and AmB-resistant fungal cells was investigated t hrough the use of the fluorescent membrane probe trimethylammonium dip henylhexatriene (TMA-DPH). AmB, the most effective drug for the treatm ent of systemic fungal infections, is known to interact specifically w ith membrane sterols, especially ergosterol (the major sterol in funga l cells). Treatment of AmB-susceptible Candida albicans and Cryptococc us neoformans cells with AmB induced a novel biphasic change in TMA-DP H fluorescence intensity over time. The initial decrease in fluorescen ce 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 pr obe 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 delt a-8 double-bonded ergosterol precursors and only 4% of ergosterol (71% ergosterol in wild-type cells), exhibited the first phase of AmB bind ing but not the second phase of increasing fluorescence intensity. Thi s result suggests that AmB's antifungal activity lies in its ability t o form membrane pores due to aggregation of AmB-ergosterol complexes. The AmB-induced biphasic fluorescence intensity profile may lead to fu rther elucidation of the molecular action of AmB on fungal cells and m ay provide a sensitive method for screening the development of drug re sistance in fungal cells.