Solasodine-3-O-β-D-glucopyranoside kills Candida albicans by disrupting the intracellular vacuole

Article abstract of DOI:10.1016/j.fct.2017.05.045

The increasing incidence of fungal infections and emergence of drug resistance underlie the constant search for new antifungal agents and exploration of their modes of action. The present study aimed to investigate the antifungal mechanisms of solasodine-3-O-β-D-glucopyranoside (SG) isolated from the medicinal plant Solanum nigrum L. In vitro, SG displayed potent fungicidal activity against both azole-sensitive and azole-resistant Candida albicans strains in Spider medium with its MICs of 32 μg/ml. Analysis of structure and bioactivity revealed that both the glucosyl residue and NH group were required for SG activity. Quantum dot (QD) assays demonstrated that the glucosyl moiety was critical for SG uptake into Candida cells, as further confirmed by glucose rescue experiments. Measurement of the fluorescence intensity of 2′,7'-dichlorofluorescin diacetate (DCFHDA) by flow cytometry indicated that SG even at 64 μg/ml just caused a moderate increase of reactive oxygen species (ROS) generation by 58% in C. albicans cells. Observation of vacuole staining by confocal microscopy demonstrated that SG alkalized the intracellular vacuole of C. albicans and caused hyper-permeability of the vacuole membrane, resulting in cell death. These results support the potential application of SG in fighting fungal infections and reveal a novel fungicidal mechanism.

Full text of DOI:10.1016/j.fct.2017.05.045

Food and Chemical Toxicology 106 (2017) 139e146
Contents lists available at ScienceDirect
Food and Chemical Toxicology
journal homepage: www.elsevier.com/locate/foodchemtox
Solasodine-3-O-
b
-D-glucopyranoside kills Candida albicans by
disrupting the intracellular vacuole
,
,
, *
Wenqiang Chang a 1, Ying Li a 1, Ming Zhang a, Sha Zheng a, Yan Li b, Hongxiang Lou a
a Department of Natural Product Chemistry, Key Lab of Chemical Biology of Ministry of Education, Shandong University, No. 44 West Wenhua Road, Jinan
City, Shandong Province, China
b Shandong Provincial Qianfoshan Hospital, Jinan City, Shandong Province, 250014, China
a r t i c l e i n f o
a b s t r a c t
Article history:
The increasing incidence of fungal infections and emergence of drug resistance underlie the constant
search for new antifungal agents and exploration of their modes of action. The present study aimed to
Received 8 December 2016
Received in revised form
15 May 2017
Accepted 22 May 2017
Available online 25 May 2017
investigate the antifungal mechanisms of solasodine-3-O-
medicinal plant Solanum nigrum L. In vitro, SG displayed potent fungicidal activity against both azole-
sensitive and azole-resistant Candida albicans strains in Spider medium with its MICs of 32 g/ml.
b-D-glucopyranoside (SG) isolated from the
m
Analysis of structure and bioactivity revealed that both the glucosyl residue and NH group were required
for SG activity. Quantum dot (QD) assays demonstrated that the glucosyl moiety was critical for SG
uptake into Candida cells, as further confirmed by glucose rescue experiments. Measurement of the
fluorescence intensity of 20,7'-dichlorofluorescin diacetate (DCFHDA) by flow cytometry indicated that SG
Keywords:
Solasodine-3-O-
Glycosides
Fungicide
b-D-glucopyranoside
even at 64 mg/ml just caused a moderate increase of reactive oxygen species (ROS) generation by 58% in
C. albicans cells. Observation of vacuole staining by confocal microscopy demonstrated that SG alkalized
the intracellular vacuole of C. albicans and caused hyper-permeability of the vacuole membrane, resulting
in cell death. These results support the potential application of SG in fighting fungal infections and reveal
a novel fungicidal mechanism.
Vacuole
© 2017 Published by Elsevier Ltd.
1. Introduction
extensive efforts to develop new antifungal agents and therapeutic
strategies.
The application of broad-spectrum antibiotics, implantable de-
vices and immunosuppressive agents in recent decades has led to
an increase in the incidence of fungal infections (Corti et al., 2009).
Invasive fungal infections are difficult to manage and are important
factors in morbidity and mortality among immunocompromised
patients (Gudlaugsson et al., 2003; Miller et al., 2001). A recent
epidemiological survey revealed that Candida species were the
third most common pathogen among hospital patients (Hidron
et al., 2008). Current clinically used antifungal agents are mainly
divided into four categories according to their modes of action:
polyenes, azoles, echinocandins and flucytosine (Pappas et al.,
2009). However, the latter three categories of therapeutics are
often associated with drug resistance, highlighting the need for
Natural products, particularly traditional Chinese medicine,
provide a rich pool for drug discovery due to the therapeutic effi-
cacy and versatile structures of secondary metabolites (Harvey,
2008). Solanum nigrum L. (SNL), which belongs to the nightshade
Solanaceae family, is traditionally used as a herbal plant, and its ripe
fruits are edible. SNL produces a class of steroidal alkaloids and
glycosides that possess a variety of biological activities, including
antifungal, antiviral, antibacterial, molluscicidal and anticancer
activities (Kumar et al., 2009; Simons et al., 2006; Sun et al., 2010;
Thorne et al., 1985). Due to these diverse bioactivities, the chemical
synthesis, biosynthesis and bioactivity of the steroidal alkaloids
ꢀ
have been the focus of several recent studies (Cardenas et al., 2015,
2016; Jiang et al., 2015; Sharpe and Johnson, 2016).
The glycosidic residues are considered crucial for the bioactivity
of glycosides. For example, the steroidal glycoalkaloid a-tomatine
produced by tomato exerts antimicrobial effects for self-defense.
However, removal of this glycosidic residue by the tomatinase of
Septoria lycopersici abolishes this antimicrobial activity (Bouarab
et al., 2002). Moreover, attachment of the glycosidic moiety
* Corresponding author. School of Pharmaceutical Sciences, Shandong University,
44 West Wenhua Road, Jinan, 250012, China.
E-mail address: louhongxiang@sdu.edu.cn (H. Lou).
1
These authors contributed equally to this work.
http://dx.doi.org/10.1016/j.fct.2017.05.045
0278-6915/© 2017 Published by Elsevier Ltd.

140
W. Chang et al. / Food and Chemical Toxicology 106 (2017) 139e146
glucose) for overnight culture at 30 ꢀC and 200 rpm.
Solasodine (1) and SG (2) were previously isolated from SNL and
several analogues of SG (3e8) were synthesized in our lab (Fig. 1)
(Cui et al., 2012; Zan et al., 2014). To synthesize these glycosides, we
first prepared glycosyl bromide as the glycosyl donor from the
corresponding benzoylated monosaccharide using a solution of 45%
HBr/HOAc in CH2Cl2. The glycosyl donors were condensed with
aglycones such as solasodine via silver trifluoromethanesulfonate
catalysis. The resultant intermediates were then hydrolyzed by
MeOH/MeONa or CH3NH2 to remove the benzoyl group, finally
providing the target compounds. Biotinylated-glucose (9) was
previously synthesized in our lab (Fig. 1) (Wang et al., 2011). The
vacuole-specific dyes Cell Tracker Blue CMAC Dye (CMAC) and N-
(3-triethylammoniumpropyl)-4-(6-(4-(diethylamino) phenyl)hex-
atrienyl)pyridinium dibromide (FM4-64) were purchased from
Invitrogen. Commercial quantum dots with streptavidin conjugate
605 nm (QD) were obtained from Invitrogen. The QD were
composed of streptavidin covalently attached to a fluorescent
nanocrystal (Qdot® nanocrystal). Qdot® nanocrystals are prepared
from a nanometer-scale crystal of a semiconductor material (CdSe),
which is coated with an additional semiconductor shell (ZnS) to
improve the optical properties of the material. Streptavidin has a
very high binding affinity to biotin or biotin conjugates, which
permits the specific detection of a variety of proteins. Quinacrine,
propidium iodide (PI), 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-
2H-tetrazolium-5-carboxanilide (XTT), 20,7'-dichlorofluorescin
diacetate (DCFH-DA) and amphotericin B (AMB) were purchased
from Sigma. Quinacrine, PI, DCFH-DA and AMB were prepared in
dimethyl sulfoxide (DMSO) at 10 mg/ml and stored at ꢁ20 ꢀC. In
each assay, the content of DMSO was less than 1%.
Abbreviations
AMB
CLSM
CMAC
CLSI
Amphotericin B
Confocal laser scanning microscopy
Cell Tracker Blue CMAC Dye
Clinical and Laboratory Standards Institute
DCFH- DA 20,7'-dichlorofluorescin diacetate
DMSO Dimethyl sulfoxide
MIC
Minimal inhibitory concentration
FM4-64 N-(3-triethylammoniumpropyl)-4-(6-(4-
(diethylamino) phenyl) hexatrienyl) pyridinium
dibromide
PI
Propidium iodide
QD
ROS
SNL
SG
Quantum dots with streptavidin conjugate 605 nm
Reactive oxygen species
Solanum nigrum L.
Solasodine-3-O-b-d-glucopyranoside
Spider-Glu Spider medium with glucose instead of mannitol
Spider-Rha Spider medium with rhamnose instead of
mannitol
XTT
2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-
tetrazolium-5-carboxanilide
YPD
Yeast-peptone-dextrose
increases the hydrophilicity of the molecule, which influences
pharmacokinetic properties such as circulation, elimination and
concentrations in body fluids (Kren and Martínkova, 2001).
ꢀ
We previously reported that solasodine-3-O-b-D-glucopyrano-
side (SG) displays anti-virulence activity against C. albicans by
inhibiting cell adhesion, morphological transition and biofilm for-
mation (Li et al., 2015b). However, little is known about the anti-
fungal activities of SG analogues and the essential functional
groups in the structure of SG. Moreover, the mode of action of SG
remains unclear.
2.2. Antifungal susceptibility test
The minimal inhibitory concentration (MIC) values of glycosides
against C. albicans were determined using the broth microdilution
method according to the Clinical and Laboratory Standards Institute
(CLSI) guidelines (M27-A3) (CLSI, 2008). Briefly, the antifungal
agents were 2-fold serially diluted in cell suspensions in RPMI 1640
medium or Spider medium (1% nutrient broth, 0.5% mannitol, 0.1%
We previously isolated solasodine (1) and its glycoside SG (2)
from SNL and synthesized several analogues of SG including sol-
asodine conjugated with
b
-L-rhamnopyranose (3),
b-D-xylopyr-
K2HPO4, pH 7.2). Then, 100-ml aliquots were added to 96-well flat-
bottomed microtitration plates. The plates were incubated at 35 ꢀC
for 24 h; zero visible growth was considered as the endpoint value.
anose (4), -arabinopyranose (5), or b-D
b
-
L
-ribopyranose (6), thio
group substituted SG (7), and oxic group substituted SG (8) (Fig. 1)
(Sun et al., 2010; Cui et al., 2012; Zan et al., 2014). In the present
study, the in vitro antifungal activities of SG and its analogues were
evaluated. SG exhibited fungicidal activity against C. albicans in
Spider medium, whereas the other analogues were inactive.
Structure-activity analysis revealed that both the NH group and
glucosyl residue were necessary for SG activity. Further investiga-
tion demonstrated that SG penetrated the C. albicans cell mem-
brane via the glucosyl residue. SG elicited the alkalization of
vacuoles, which resulted in increased vacuole permeability and,
consequently, triggered the death of C. albicans.
2.3. Time-killing kinetics
To investigate the fungicidal activity of SG, time-killing curves
were plotted by measuring the cell-survival rates. Overnight-
cultured SC5314 cells were diluted to a final concentration of
1 ꢂ 105 cells/ml and treated with 32 or 64
mg/ml SG in Spider
medium at 30 ꢀC. At different time intervals, an aliquot was
removed and plated on YPD agar plates to enumerate the surviving
colonies.
2. Materials and methods
2.4. QD visualization assay
2.1. Strains, culture and chemicals
Biotinylated-glucose (9) (2 mM) and QD (20 nM) were co-
incubated at 4 ꢀC for 40 min to form glucose-QD complexes
(Wang et al., 2011). Prepared SC5314 cells were diluted to
5 ꢂ 105 cells/ml and incubated with the glucose-QD complexes or
QD as a negative control at 30 ꢀC for 1 h. After washing twice, the
cells were imaged by confocal laser scanning microscopy (CLSM)
(Carl Zeiss, LSM700, Germany) using a 63 ꢂ objective lens. The
fluorescence of the QD upon excitation by a 555-nm laser was
recorded in the emission spectrum of 590e700 nm.
The C. albicans wild type strain SC5314 and ten clinical isolates
including five azole-sensitive and five azole-resistant isolates (Li
et al., 2015a) were used in this study. The strains were stored
routinely and propagated on yeast-peptone-dextrose (YPD) agar
plates (2% tryptone,1% yeast extract, 2% glucose and 2% agar) before
each experiment. Following incubation for 24 h at 30 ꢀC, the cells
were inoculated in YPD broth (2% tryptone, 1% yeast extract, 2%

W. Chang et al. / Food and Chemical Toxicology 106 (2017) 139e146
141
Fig. 1. The structures of the compounds used in this study: 1 solasodine; 2e6 solasodine glycosides; 9 biotinylated glucose. These molecules were obtained through isolation from
SNL or chemical synthesis in our lab (Cui et al., 2012; Wang et al., 2011; Zan et al., 2014).
2.5. Rescuing experiments using different carbohydrates
described (Raines et al., 2013). After staining, the cells were
collected, washed twice in YPD, and examined by CLSM with
excitation at 488 nm and emission in the range of 490e550 nm.
Overnight-cultured SC5314 cells were inoculated into modified
Spider medium containing different carbon sources (0.5% mannitol,
5% mannitol, 0.5% rhamnose, 5% rhamnose, 0.5% glucose, or 5%
glucose) and incubated for 4 h at 30 ꢀC and 200 rpm. The cells were
2.8. Vacuole permeability measurements
then exposed to different doses of SG (32, 64, and 128 mg/ml) in the
To visualize vacuole membrane permeabilization, we utilized
two independent staining dyes. For FM4-64 staining, log-phase
C. albicans cells were diluted to 1 ꢂ 105 cells/ml and grown in
corresponding culture medium. After incubation for12 h, an XTT
reduction assay (Ramage et al., 2001) was performed to determine
the inhibitory effect of SG in each treated group.
YPD medium with or without SG for 4 h. Next, 20 mg/ml of FM4-64
was added to the cell cultures and incubated for 1 h at 30 ꢀC. The
cells were then collected, resuspended in fresh YPD medium, and
incubated for 1 h at 30 ꢀC prior to CLSM observation. For CMAC
2.6. Measurement of ROS formation in C. albicans
C. albicans SC5314 was cultured in Spider medium and chal-
staining, cells were loaded with 5
mM CMAC and treated with SG for
lenged with SG. After 4 h of treatment, the cells were collected and
4 h, followed by staining with 5
mg/ml PI staining for an additional
stained with 40
m
g/ml DCFH-DA. After incubation for 30 min in the
30 min. The cells were then collected and washed prior to CLSM
observation. The fluorescence of FM4-64 was excited by a 555 nm
laser and recorded in an emission spectrum of 570e700 nm. CMAC
and PI were excited by 405 nm and 555 nm lasers, respectively, and
emission spectra of 420e490 nm for blue fluorescence and
570e700 nm for red fluorescence were recorded.
dark, the samples were washed twice and analyzed using a FACS-
Calibur flow cytometer (Becton Dickinson, San Jose, CA). The
green fluorescence of DCF was excited by a 488-nm laser and
collected in the FL1 channel (530 15 nm). A total of 10,000 events
in each sample were recorded for analysis. The resultant data were
processed using WinMDI 2.9 software (Joseph Trotter, The Scripps
Institute, La Jolla, CA).
2.9. The relationship between vacuole permeability and cell death
2.7. Vacuolar pH measurements
To determine if increased vacuole permeability was a late
consequence of cell death or a prerequisite event for cell death
Quinacrine accumulation, which exhibits green fluorescence
under acidic conditions, was used to qualitatively assess the vacu-
olar pH in live C. albicans cells. Cells (1 ꢂ 105 cells/ml) were exposed
to different concentrations of SG. After 4 h of incubation at 30 ꢀC,
caused by SG, C. albicans SC5314 cells were loaded with 5
a dye that specifically accumulates in normal vacuoles. The pre-
stained CMAC cells were treated with 64 g/ml SG; 5 g/ml PI
was added at the same time as a death indicator. After treatment for
3 h at 30 ꢀC, we chose typical cells with diffuse CMAC and without
mM CMAC,
m
m
the cells were stained with 200
mM quinacrine as previously

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W. Chang et al. / Food and Chemical Toxicology 106 (2017) 139e146
Table 2
PI staining for CLSM observation. These cells were imaged at 5-min
intervals. CMAC and PI were excited by 405 nm and 555 nm lasers,
respectively, and emission spectra of 420e490 nm for blue fluo-
rescence and 570e700 nm for red fluorescence were recorded.
The MIC values of SG against different C. albicans strains cultured in Spider medium.
Strains
MICs of fluconazole (
m
g/ml)a
MICs of SG (mg/ml)
Wild type strains
SC5314
YEM30
Clinical isolates
23C
23L
24F
28A
28I
18A
18B
2
2
32
32
2.10. Statistical analysis
>128
>128
>128
>128
>128
1
0.5
1
1
1
32
32
32
32
32
32
32
32
32
32
All experiments were performed three times on different days.
The results are represented as the means standard deviations
(SDs). Statistical significances were determined by Student's t-test
and a P value < 0.05 indicated statistical significance.
18C
11E
11F
3. Results
3.1. SG exerts fungicidal activity against C. albicans in vitro
a
The MICs of fluconazole against clinical isolates have been reported in our
previous publication.
The antifungal activities of SG and its derivatives (Fig. 1) were
assessed by the broth microdilution method. The susceptibility test
results showed that SG (2) exhibited antifungal activity against the
C. albicans wild type strain SC5314. However, when the glucosyl
residue was substituted by other monosaccharides (3e6) including
b
b
-
-
L
-rhamnopyranose,
-ribopyranose (Cui et al., 2012), no inhibitory effect on the
b-D-xylopyranose, b-L-arabinopyranose, and
D
growth of C. albicans was observed, even at concentrations of
1024 g/ml (Table 1). Moreover, the aglycone solasodine (1) did not
show any antifungal activity, even at 1024 g/ml (Table 1). These
m
m
data suggested an essential role of the glucosyl residue in anti-
fungal bioactivity.
Further investigation revealed that the activity of SG was
enhanced in Spider medium, with an MIC value of 32
mg/ml,
compared to 128 g/ml in RPMI 1640 medium (Table 1). Based on
m
its higher activity in the susceptibility test, SG was chosen for
subsequent study. Azoles often elicit drug resistance in C. albicans
(Canuto, 2002). We found that SG inhibited the growth of both
azole-sensitive and azole-resistant clinically derived strains in
Spider medium (Table 2). Moreover, time-killing curves demon-
strated that SG displayed potent fungicidal activity at the concen-
Fig. 2. The time-killing kinetics of SG against C. albicans SC5314. The wild type strain
SC5314 was diluted with Spider medium to 1 ꢂ 105 cells/ml and exposed to 32 or
64 mg/ml of SG. At specific time points, the survival rates were calculated based on
surviving colonies. The results are shown as the averages and standard deviations of
three independent experiments.
trations of 32
mg/ml and higher, with AMB serving as the positive
control (Fig. 2).
into C. albicans. To test this hypothesis, we utilized previously
synthesized biotinylated-glucose (9) as a molecular probe bound to
QD with bio-conjugated streptavidin to perform a binding assay
(Wang et al., 2011). Confocal imaging revealed that the
biotinylated-glucose preferentially bound to the cell membrane, as
indicated by QD fluorescence around the cytoplasmic membrane,
suggesting the possible existence of glucose lectins (Fig. 3). As
further confirmation, we compared the susceptibility to SG of
C. albicans cultured in Spider medium with mannitol as the carbon
source to that of C. albicans cultured in Spider medium with glucose
or rhamnose instead of mannitol (Spider-Glu or Spider-Rha). Pre-
incubation of C. albicans in Spider-Glu for 4 h greatly reduced the
susceptibility of the cells to SG (Fig. 4). However, when glucose was
3.2. The NH group is required for SG activity
NH is required for the bioactivity of some agents (Cheng et al.,
2007). When the NH group in SG was replaced with a thio or oxic
group, the antifungal activity of the resultant compounds 7 and 8
(Fig. 1) was abolished (Table 1), suggesting that NH is necessary for
the activity of SG.
3.3. Uptake of SG into C. albicans cells is mediated by the glucosyl
moiety
We hypothesized that glucose lectins mediate the uptake of SG
Table 1
The MIC values of different compounds against C. albicans wild type strain SC5314 when cultured in RPMI1640 medium or Spider medium.
Compound
MICs in RPMI1640 medium (
m
g/ml)
MICs in Spider medium (mg/ml)
1
2
3
4
5
6
7
8
>1024
128
>512
32
>1024
>1024
>1024
>1024
>1024
>1024
>512
>512
>512
>512
>512
>512

W. Chang et al. / Food and Chemical Toxicology 106 (2017) 139e146
143
Fig. 3. Confocal microscopic observation of the binding of glucose-QD to the cell surface of C. albicans. C. albicans SC5314 cells were diluted to 5 ꢂ 105 cells/ml and incubated with
preformed glucose-QD complexes or QD (negative control) for 1 h. The cellular localization of the QD was visualized by CLSM. Scale bar, 5 mm.
Fig. 4. The effect of different carbon sources on the fungicidal activity of SG. The wild
type strain SC5314 was pre-incubated in Spider medium with different concentrations
Fig. 5. ROS generation by C. albicans SC5314 when treated with SG. C. albicans cells
were cultured in Spider medium and treated with SG for 4 h. The intracellular ROS
contents were detected by flow cytometry based on the fluorescence intensity. Black
line, negative control in which C. albicans cells were not stained with DCFH-DA; red
of mannitol, rhamnose, or glucose as carbon sources for 4 h. The pre-incubated cells
were then exposed to different doses of SG. After 24 h, an XTT reduction assay was
performed to assess the growth-inhibitory effects of SG under different culture con-
ditions. The bars indicate standard deviations.
line, vehicle control; green line, group treated with 16
m
g/ml SG; blue line, group
g/ml SG. (For inter-
treated with 32 g/ml SG; purple line, group treated with 64
m
m
pretation of the references to colour in this figure legend, the reader is referred to the
substituted as the carbon source by other sugars such as rhamnose,
the susceptibility to SG was not significantly different from that in
Spider or Spider-Rha medium (Fig. 4). These results indicated that
the addition of rhamnose did not inhibit the uptake of glucose into
C. albicans cells, further confirming the existence of glucose lectins.
web version of this article.)
3.5. SG elicits the alkalization of vacuoles in C. albicans
The fungal vacuole is an acidic compartment that is involved in
hydrolysis, storage, osmoregulation, homeostasis and detoxifica-
tion (Teter and Klionsky, 2000). Normal vacuole function is critical
for fungal cell survival. We found that the vacuolar pH of C. albicans
became more alkaline upon treatment with SG, as assessed by
quinacrine staining (Fig. 6).
3.4. The fungicidal action of SG is independent of ROS generation
Fungicidal agents often cause ROS production, an important
factor in cell death (Kobayashi et al., 2002; Phillips et al., 2003). The
fluorescent probe DCFH-DA, which is an indicator of ROS formation
(Shirtliff et al., 2009), was utilized to evaluate the effect of SG on
intracellular ROS production in C. albicans. Flow cytometry detec-
tion showed that SG caused only a slight increase in ROS in
C. albicans cells compared with the control (Fig. 5), suggesting that a
mode of action distinct from ROS is responsible for the fungicidal
activity of SG.
3.6. SG increases vacuole permeability and cell death in C. albicans
FM4-64, a lipophilic dye that binds to the cellular membrane, is
internalized by endocytosis and accumulates within the vacuole
(Vida and Emr, 1995), whereas CMAC passively accumulates in the
vacuolar lumen (Rane et al., 2014). FM-64 staining revealed that the

144
W. Chang et al. / Food and Chemical Toxicology 106 (2017) 139e146
Fig. 6. The effect of SG on the vacuole acidification in C. albicans. The wild type strain SC5314 was treated with different concentrations of SG for 4 h and stained with quinacrine for
CLSM observation. Quinacrine stains acidic compartments and is quenched as the pH increases. Scale bar, 5 m.
m
vacuole membrane was disrupted by SG at a dose of 64
YPD medium (Fig. 7A). CMAC staining showed that the vacuole
contents leaked into the cytoplasm after exposure to 64 g/ml of
SG, resulting in cell death, as indicated by intracellular PI accu-
mulation (Fig. 7B).
To determine if the increased vacuole permeability was a late
consequence of cell death or a prerequisite caused by SG, C. albicans
cells were pre-stained with CMAC, a dye that specifically accumu-
lates in normal vacuoles. The pre-stained CMAC cells were treated
m
g/ml in
with SG, and PI was added as a cell death indicator. After 3 h of
treatment, typical cells with diffuse CMAC and without PI staining
were chosen for confocal microscopic observation. The time-
sequence images revealed that the increased vacuole perme-
ability occurred before cytoplasm membrane disruption, as indi-
cated by the penetration of PI. These observations suggest that
vacuole permeability was a prerequisite for cell death rather than a
late consequence of cell death caused by SG (Fig. 8).
m
Fig. 7. The effect of SG on the vacuole membrane permeability of C. albicans. (A) The wild type strain SC5314 was treated with different concentrations of SG for 4 h. SG-treated cells
were stained with FM4-64 for CLSM observation. (B) C. albicans cells pre-stained with CMAC, a dye that specifically stains vacuoles in fungi, were treated with SG for 4 h, followed by
PI staining to assess the live/dead state. The double-stained cells were then observed by CLSM. Scale bar, 5 mm.

W. Chang et al. / Food and Chemical Toxicology 106 (2017) 139e146
145
Fig. 8. Time-sequence observation of SG-treated cells. SC5314 cells were loaded with CMAC for 30 min. The preloaded cells were treated with 64
m
g/ml SG for 3 h. Typical cells with
diffuse CMAC and without PI staining were chosen for confocal microscopic observation. Images were obtained at 5-min intervals.
4. Discussion
vacuole permeability (Fig. 5). Alkalization is not a prerequisite
event for cell death because fluconazole, a fungistatic drug, also
elicits vacuole alkalization in C. albicans cells (Zhang et al., 2010).
We hypothesized that the increased vacuole permeability is a key
event resulting in cell death. Time-sequence images revealed that
the increased vacuole permeability occurred before cytoplasmic
membrane disruption (Fig. 8), suggesting that SG resulted in vac-
uole leakage, which mediated cell death. This conclusion is
consistent with findings in Saccharomyces cerevisiae (Kim et al.,
2012).
Taken together, we found that the glucosyl residue and NH
group are necessary for the activity of SG, which displays fungicidal
action by disrupting vacuole function in C. albicans cells. Moreover,
glucosylation might be employed as a strategy to transform some
natural products into active antifungal agents.
Steroidal glycoalkaloids have been reported to possess antimi-
crobial and antitumor activities (Nino et al., 2009; Sun et al., 2010).
~
In this study, we found that SG isolated from Solanum nigrum L.
displayed potent antifungal activity against C. albicans in vitro,
whereas its aglycon solasodine was inactive, suggesting a pivotal
role of the glycan in bioactivity. However, when the glucosyl res-
idue of SG was substituted by other monosaccharides, the anti-
fungal activity was abolished (Table 1), implying a necessary role of
the glucosyl moiety in SG. The QD-glucose binding assay also
revealed that the glucosyl residue is a key mediator of the intra-
cellular uptake of SG and thus probably determines the antifungal
activity of SG. Similarly, dihydrodehydrodiconiferyl alcohol 90-O-
b-
D-glucoside, which exhibits antifungal activity via disruption of the
cell membrane (Choi et al., 2012), does not exhibit antifungal ac-
tivity when the glucosyl moiety is removed to generate the agly-
cone (our unpublished data). Furthermore, when the NH group in
the SG structure was replaced by a thio or oxic group, the resultant
derivatives (7e8) also lacked the antifungal activity, suggesting an
essential role of NH in maintaining the antifungal activity of SG.
This finding is consistent with a previous study that indicated that
the presence of NH is critical for the DNA-damaging activity of
Conflict of interest
The authors declare that they have no conflict of interest.
Acknowledgements
We appreciate Dr Qi Qingguo from School of Stomatology in
Shandong University for offering the clinical strains. This work was
funded by National Natural Science Foundation (Nos. 81402804,
81273383, 81172956, 81102480), China Postdoctoral Science
Foundation (2014M551925).
~
solasodine (Kim et al., 1996; Nino et al., 2009). The presence of an
NH group is also important for the bioactivity of other steroidal
alkaloids (Yang et al., 2012).
Given the critical role of the glucosyl residue in SG, we utilized
biotinylated glucose as a mimic of SG to monitor its distribution in
C. albicans cells. The QD visualization test demonstrated that the
glucosyl group mediated the transport of QD through the cell
membrane in C. albicans, suggesting the possible existence of
glucose lectins (Fig. 3). Further experiments showed that the
addition of glucose retarded the efficacy of SG (Fig. 4), thus con-
firming the existence of glucose lectins.
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