Key words
polygodial - fungicidal activity - vacuole -
Saccharomyces cerevisiae
The fungicidal activity of amphotericin B (AmB; [Fig. 1 A]), a polyene macrolide, is attributed mainly to its ability to form a molecular complex
with plasma membrane-embedded ergosterol [1]. This complex functions as an ion channel, enhancing the leakage of intracellular
K+ and other ionic substances. AmB-mediated lethality is alternatively attributed to
the induction of oxidative stress via the enhanced generation of superoxide anions
by this macrolide, as well as to its ability to extract ergosterol from the plasma
membrane phospholipid bilayer [2], [3]. In previous studies, for the first time, we found that AmB is incorporated across
the plasma membrane of fungal cells, resulting in vacuolar membrane disruption with
lethal effects [4], [5], [6]. Such vacuole-targeting fungicidal activity is also exerted by niphimycin from Streptomyces sp., as well as by polymyxin B from Bacillus polymyxa, with both selective and effective lethal effects against fungal cells [4]. To date, however, vacuole-disrupting activity has been reported only for microbial
metabolites, not for plant-derived compounds.
Fig. 1 Chemical structures of AmB (A) and polygodial (B).
Polygodial ([Fig. 1 B]), a bicyclic sesquiterpene dialdehyde, was first isolated as a pungent principle
from the sprout of Polygonum hydropiper L. (Polygonaceae), known as “tade”, which is used as a food spice in Japan [7], [8]. The biological activity of polygodial has been reported to include antifungal effects
[9], [10], [11], [12]. The biological mechanism of action of polygodial involves structural and functional
damage to the plasma membrane, which is accompanied by the leakage of intracellular
components. Polygodial-induced plasma membrane damage is attributed to the enhanced
induction of cellular oxidative stress by this compound, as reflected by a marked
increase in the level of mitochondrial reactive oxygen species (ROS) production [13]. It is still not known whether polygodial-induced lethality is dependent on plasma
membrane damage and ROS production.
In this study, we examined the fungicidal activity of polygodial under conditions
in which cells were protected from oxidative stress and plasma membrane damage. Vacuole
disruption was identified as the most likely mechanism by which polygodial exerts
lethal effects against Saccharomyces cerevisiae and other pathogenic fungal strains.
We first examined the relationship between lethality and ROS production by polygodial
treatment of S. cerevisiae cells in 2.5 % malt extract medium. As shown in [Fig. 2 A], polygodial was not lethal to S. cerevisiae cells at a concentration of 2 µM; however, as is the case with AmB, this sesquiterpene
dialdehyde was found to markedly reduce the viable cell number to around 0.1 % of
the original level at 10 µM over the course of a 2-h incubation. The lethality of
polygodial was far lower in nutrient-rich medium consisting of 2.0 % (w/v) glucose,
2.0 % (w/v) peptone, and 1.0 % (w/v) yeast extract (YPD); in the latter, the viable
cell number was reduced to 15 % of the original level, even at a polygodial concentration
of 100 µM, during 2 h of incubation [13].
Fig. 2 Effects of polygodial on cell viability, ROS production, and vacuole disruption.
Cells were incubated in 2.5 % malt extract medium containing polygodial at the indicated
concentration, at 30 °C, with vigorous shaking for measurement of the viable cell
number (A) and cellular ROS production (B). Data are shown as means ± SD of triplicate experiments (A, B). Cells were additionally used for microscopic observation of vacuole morphology
(C); bar, 2 µm. AmB (A, C) and farnesol (B) were used as positive controls. (Color figure available online only.)
In YPD medium, polygodial, at a concentration of 100 µM, reduced the viable cell number
via an increase in mitochondrial ROS production in cells of the parent strain, but
not in the respiration-deficient mutant with polygodial-resistant phenotype [13]. We therefore examined the level of ROS production in polygodial-treated cells in
malt extract medium. In agreement with a previous finding, polygodial, at a concentration
of 100 µM, was found to markedly increase the level of ROS production in the medium.
The observed increase was comparable to that induced by an isoprenoid farnesol due
to hyperpolarization of the mitochondrial transmembrane potential [14], as shown in [Fig. 2 B]. At a concentration of 10 µM, polygodial did not elicit an elevation in cellular
ROS production levels; however, lethal effects were observed at this concentration,
suggesting that polygodial exhibits lethal activity against fungal cells via modes
of action other than the induction of oxidative stress.
We therefore examined the possibility that polygodial damages the vacuole, a recently
identified fungicidal target, resulting in the disruption of vacuolar membrane architecture.
As shown in [Fig. 2 C], the yeast vacuole was observed to possess a swollen, rounded architecture in untreated
cells. In addition, no structural alteration was observed in cells treated with polygodial
at the nonlethal concentration of 2 µM. However, even under nonlethal conditions,
the entire luminal space exhibited abnormal staining with the fluorescent dye FM4–64,
suggesting the impairment of normal transport function across the vacuolar membrane
([Fig. 2 C]). In cells treated with polygodial at the lethal concentration of 10 µM, the dye
was found to be scattered throughout the cytoplasm, in which normal vacuolar membrane
architecture could not be observed clearly, reflecting the disruption of the vacuolar
membrane into smaller fragments. The observed mode of vacuolar membrane disruption
by polygodial appeared to be similar to that of AmB-treated cells ([Fig. 2 C]).
The simultaneous addition of K+ and Mg2+ is known to protect S. cerevisiae cells against AmB lethality, suggesting their maintenance effects on vacuole membrane
integrity in addition to the compensatory effect against intracellular K+ leakage (see [Fig. 3 A, B]) [15], [16]. The putative contribution of the vacuole membrane-disrupting effect to the fungicidal
activity of polygodial was thus investigated by examining the protective effects of
these ions against the loss of viability of polygodial-treated cells. As shown in
[Fig. 3 C, D], polygodial was found to exert a marked lethal effect with serious vacuolar membrane
disruptive damage similarly in the absence as well as in the presence of both ions.
The result suggests that the vacuole-targeting action of polygodial contributed to
cell death more significantly than that of AmB in the malt extract medium.
Fig. 3 Cell viability and vacuole morphology of S. cerevisiae cells in the absence and presence of 85 mM KCl and 45 mM MgCl2. Cells were incubated in 2.5 % malt extract medium containing AmB (A, B) or polygodial (C, D) at the indicated concentrations, at 30 °C for 2 h, with vigorous shaking, for measurement
of viable cell numbers (A, C) and microscopic observation of the vacuoles (B, D). Data are shown as means ± SD of triplicate experiments (A, C); bar, 2 µm (B, D). (Color figure available online only.)
We finally examined whether polygodial elicits disruption of the vacuole membrane
in the pathogenic fungal strains Candida albicans and Aspergillus niger. The vacuole was found to exhibit a swollen, spherical architecture in untreated
C. albicans cells; however, the organelles were observed as small discrete dots in the cytoplasm
of cells treated with polygodial at a lethal concentration ([Fig. 4 A, B]). Polygodial was also found to cause vacuole disruption in the filamentous cells
of A. niger at the higher concentration of 150 µM ([Fig. 4 C]). This concentration is equivalent to the value at which fungal spore germination
is inhibited (data not shown).
Fig. 4 Vacuole morphology (A, C) and viability (B) of C. albicans cells, and the mycelial cells of the filamentous fungus A. niger. In A and B, cells were incubated in RPMI 1640 medium containing polygodial at the indicated
concentrations, at 30 °C for 2 h. In C, cells were incubated in 2.5 % malt extract medium containing polygodial at the indicated
concentration, at 30 °C for 2 h. Data are represented as means ± SD of triplicate
experiments (B); bars, 2 µm (A, C). (Color figure available online only.)
Vacuoles are organelles that are involved in osmoregulation, ion and pH homeostasis,
metabolite accumulation, and macromolecular degradation in fungal cells [17]. Vacuoles are additionally involved in autophagic processes required for the degradation
of cytoplasmic components as well as the cellular stress response [18], [19]. Therefore, structural and functional damage to this organelle is considered to
trigger cell death [4], [5], [6]. The antifungal property of polygodial has been attributed to its disruptive effects
against the phospholipid bilayers of the plasma membrane, which is thought to occur
via the induction of cellular oxidative stress. Our findings suggest that the vacuole-targeting
fungicidal activity of polygodial may represent a novel mechanism of action of the
compound, which is distinct from those previously reported. Our findings are additionally
considered to be of potential therapeutic significance: the selective enhancement
of the vacuole-targeting fungicidal activity of polygodial, via the structural modification
of this compound, should enable its application in clinical settings.
Materials and Methods
Measurement of cell growth and viability
S. cerevisiae W303-1A and C. albicans IFO 1061 cells were grown overnight in malt extract medium (2.5 % malt extract, pH 5.0)
at 30 °C with vigorous shaking. Then, cells were inoculated to 1 × 107 cells/mL in freshly prepared malt extract medium (S. cerevisiae) or RPMI 1640 medium (C. albicans) containing polygodial or AmB at various concentrations. Cell suspensions were then
incubated with vigorous shaking at 30 °C, and plated on YPD medium containing 1.8 %
(w/v) agar. Cell viability was determined as the number of colonies formed after a
48-h incubation at 30 °C.
Vacuole staining
Vacuoles were stained with the fluorescent probe FM4–64 [N-(3-triethylammoniumpropyl)-4-(6-(4-(diethylamino)phenyl)hexatrienyl)pyridinium
dibromide] according to previously described methods [20], [21], with some modifications, depending on the fungal strain used, as follows. In the
case of S. cerevisiae and C. albicans, cells were grown overnight in malt extract medium and harvested by centrifugation.
Cells were suspended in fresh malt extract medium to a density of 1 × 107 cells/mL and incubated with 8 µM FM4–64. After collection by centrifugation, FM4–64-stained
cells were resuspended in fresh malt extract medium (S. cerevisiae) or RPMI 1640 medium (C. albicans) to obtain a density of 1 × 107 cells/mL. These cell suspensions were further incubated in the absence or presence
of polygodial, at the indicated concentrations, with vigorous shaking at 30 °C for
2 h, and then washed and suspended in phosphate buffered saline (PBS). Cells were
then observed under a phase-contrast microscope and fluorescence microscope with the
excitation at 520–550 nm and emission at 580 nm. In the case of A. niger ATCC 6275, the mycelial suspension was directly prepared by inoculation from the
agar slant into 2.5 % malt extract medium for FM4–64-treatment of the fungal cells.
The effect of polygodial on fungal vacuole morphology was examined by resuspending
the mycelial cells with an equal volume of freshly prepared malt extract medium, followed
by incubation at 30 °C for 2 h. Polygodial-treated mycelial cells were washed and
suspended with PBS. Cells were then observed by phase-contrast microscopy and fluorescence
microscopy, as described above.
Assay of cellular ROS production
The effect of polygodial on cellular ROS production was assayed using a previously
described method [22], [23]. Cells from an overnight culture were inoculated into freshly prepared YPD medium
to a density of approximately 1 × 107 cells/mL. Cells were then incubated with 40 µM 2′,7′-dichlorodihydrofluorescein diacetate
(DCFH-DA) at 30 °C for 60 min, collected by centrifugation, and suspended in an equal
volume of 2.5 % malt extract medium. Cell suspensions were further treated with or
without polygodial at various concentrations for 20 min, and then washed and resuspended
in 50 mM succinate buffer (pH 6.0). Fluorescence from the cells was then measured
with the excitation at 485 nm and emission at 530 nm.
Chemicals
Polygodial (97 % purity) and farnesol (89 % purity) were purchased from Wako Pure
Chemical Industries, Ltd. AmB (80 % purity) and FM4–64 was obtained from Sigma-Aldrich.
DCFH-DA was purchased from Molecular Probes. All other reagents were of analytical
grade. Malt extract medium was purchased from Oriental Yeasts Co.
Statistical analysis
Statistical evaluation was performed using Studentʼs t-test; p < 0.05 was considered to represent statistical significance.
Acknowledgements
The authors are grateful to M. Kawabata for providing support with experiments.
