Treatment options for serious fungal infections are currently limited. New antifungal drugs are urgently needed, especially compounds with a fungicidal mode of action. In this study, we used flow cytometric analysis, single-cell-sorting techniques, and standard microbiological methods to investigate the activity of LY303366 against C. albicans
and S. cerevisiae
. Our results indicate that LY303366 is a highly potent and rapidly acting fungicidal compound. While our methods are novel, our results are consistent with an accumulating body of knowledge regarding the potency and broad-spectrum fungicidal activity of this promising antifungal compound (11
Advancements in flow cytometry instrumentation have pushed the technology into microbiology laboratories worldwide. A thorough review of flow cytometric applications for microbiology was published recently (4
). Flow cytometry offers unique advantages for studying the fungicidal activities of selected compounds against yeasts, providing the ability to examine the effects of compounds on thousands of individual cells within seconds. This type of analysis facilitates the understanding of the heterogeneous nature of yeast populations with respect to drug sensitivity. In a previous study (10
), members of our group showed how flow cytometric measurement of PI fluorescence could distinguish the activity of fungicidal agents, such as cilofungin, from that of a fungistatic compound such as fluconazole. The PI method was particularly well suited for determining the extent of fungal cell killing by cilofungin; more than 90% of the cells treated with cilofungin became PI+
within 3 h of treatment at the MFC. In the present study, we applied this method to investigate the antifungal effects of a more potent ECB analog, LY303366. Using this approach, we demonstrated an approximately 40-fold increase in potency of LY303366 over cilofungin. Other investigators, using standard in vitro and in vivo microbiology assays, have demonstrated that LY303366 has potent, broad-spectrum fungicidal activity (11
The additional cell-sorting experiments described in this paper supported our hypothesis that LY303366-treated, PI+
yeast cells were indeed nonviable (Fig. ). This confirmation is important because others have shown that certain bacteria may exhibit staining with selected viability dyes and yet be able to recover and grow (4
). In addition, other studies have indicated that treatment of yeast with the fungistatic agent fluconazole can increase PI fluorescence slightly, yet these cells resume their growth and exhibit normal PI-staining patterns after removal from compound (19
Results from the concentration response and time course experiments (Fig. and ) demonstrate that the PI assay and single-cell sorting for determination of SCG% complement each other. In the LY303366 concentration response experiments, PI staining correlated (r > 0.99) with decreased SCG%, further validating the PI method for measuring fungicidal activity of LY303366 against C. albicans and S. cerevisiae.
After confirming the lethal effects of a 3-h treatment with LY303366, we then focused on determining the minimal exposure period sufficient to kill C. albicans
by comparing pulsed-compound exposures to continuous 3- or 5-h incubations. Using this approach, we determined (Table ) that binding of LY303366 to C. albicans
is rapid; exposure to the MFC (0.01 μg/ml) for 5 min killed >99% of the cells. These data demonstrate that chemical modifications of the fatty acid side chains of the ECB nucleus (6
) incorporated in the synthesis of LY303366 resulted in an agent with much greater potency than cilofungin. The rapid killing of yeast cells by LY303366 after a short exposure period may contribute to its potent efficacy in vivo (3
The rapid action and enhanced potency of LY303366 compare favorably with those of AMB, a polyene antifungal that has been used clinically since the 1950s (8
). AMB has been reported to bind ergosterol in fungal cell membranes within 30 min (27
). Cell membrane permeability is increased, first for potassium ions and then for other cell constituents, resulting in cell death within hours after exposure. Binding of AMB was recently shown to decrease yeast plasma membrane potential within 30 min of AMB exposure (18
). Flow cytometric analysis of PI staining has been used to study AMB activity against yeasts (22
) and to investigate serum effects (15
) or synergy (21
) or antagonism (14
) between antifungals. Our studies also demonstrated that a 30-min incubation with AMB has an effect on C. albicans
, an effect measured by increased PI fluorescence (Table ). Although AMB binds rapidly, our data indicate that AMB does not increase yeast cell membrane permeability to PI as early as LY303366 does. C. albicans
cultures treated with AMB for 5 h display only one-fifth of the PI+
cells displayed by cultures treated with LY303366 for 3 h (Table ). Some investigators have used deoxycholate to enhance PI entry into AMB-treated cells (14
). AMB is highly effective against most clinical fungal isolates (17
); however, nephrotoxicity limits its clinical utility. Among its effects, LY303366 targets a fungus-specific enzyme, glucan synthase (5
), which may contribute to an improved toxicity profile.
Because LY303366 binds rapidly to yeast cells and has an inhibitory effect on cell wall synthesis, its antifungal effects would be expected to occur over time in a growing culture. One paradox emerged from the pulsed-exposure experiments: while a 5-min pulsed exposure to LY303366 was shown to be sufficient to eventually kill target cells (Table ), deposition of single cells into microwells following 30 min of incubation with LY303366 resulted in a smaller-than-expected decrease in SCG% (Fig. ). We hypothesized that active-growth conditions are important for maximal killing activity of LY303366. To test this idea, we conducted experiments in which cells were treated with LY303366 for 5 min, with or without an active-growth period (incubation in a shaker water bath) for 175 min in compound-free medium (Table ). The PI staining, CFU, and SCG% assays all indicated that a 5-min pulse with compound without the active-growth period resulted in a significantly reduced fungicidal effect compared to the effect on cultures that were actively grown in a shaking water bath for an additional 175 min. To reconcile these results, we considered the fact that for each of the three fungicidal activity assays, cells were suspended in drug-free medium following the drug pulse. It is possible that when LY303366-treated cells are suspended in drug-free broth, some of the drug will come off of the cells. We hypothesize that if yeast cells remain in log-phase growth, as promoted by the shaker bath, most will begin cell division and, therefore, die before much drug comes off. With static culture conditions following the drug pulse, such as the microtiter well or agar plate, drug release could become a more significant factor because fewer cells will divide while a lethal-threshold level of drug remains bound to the cell. Our results imply that yeasts growing at lower rates may require somewhat longer exposure periods in order to be killed by this agent.
In summary, we have utilized flow cytometric analysis, cell sorting, and standard microbiological plating methods to study the fungicidal activities of selected antifungals against S. cerevisiae and C. albicans. Our data indicate that cell sorting may be a valuable tool for studying and differentiating various antifungal activities. The PI fluorescence and cell-sorting methods provide tools for evaluating certain modes of action that are not easily accomplished by standard microbiological techniques. In addition, our data indicate that LY303366 is a potent, rapidly acting antifungal agent. In our experiments, we confirmed that LY303366-treated PI+ yeast cells are nonviable. Our data also suggest that LY303366 works best against actively growing yeasts. The potency and rapid action of LY303366 suggest that it may be useful for antifungal therapy of human disease.