Journal of Medical Microbiology (2014), 63, 988–996
DOI 10.1099/jmm.0.073890-0
Synergistic mechanism for tetrandrine on
fluconazole against Candida albicans through the
mitochondrial aerobic respiratory metabolism
pathway
Hui Guo,1,2,33 Si Ming Xie,3,43 Shui Xiu Li,1 Yan Jun Song,1 Xia Lin Lv1
and Hong Zhang1
Correspondence
1
Hong Zhang
2
[email protected]
First Affiliated Hospital and Institute of Mycology, Jinan University, Guangdong, PR China
Clinical Medicine Postdoctoral Mobile Station, Jinan University, Guangdong, PR China
3
Guangdong Province Key Laboratory of Molecule Immunology and Antibody Engineering,
Guangdong, PR China
4
Scholl of Medicine, Jinan University, Guangdong, PR China
Received 30 November 2013
Accepted 17 April 2013
We found that tetrandrine (TET) can reverse the resistance of Candida albicans to fluconazole
(FLC) and that this interaction is associated with the inhibition of drug efflux pumps. Mitochondrial
aerobic respiration, which plays a major role in C. albicans metabolism, is the primary source of
ATP for cellular processes, including the activation of efflux pumps. However, it was unclear if TET
exerts its synergistic action against C. albicans via its impact on the mitochondrial aerobic
respiratory metabolism. To investigate this mechanism, we examined the impact of FLC in the
presence or absence of TET on two C. albicans strains obtained from a single parental source
(FLC-sensitive strain CA-1 and FLC-resistant strain CA-16). We analysed key measures of
energy generation and conversion, including the activity of respiration chain complexes I and III
(CI and CIII), ATP synthase (CV) activity, and the generation of reactive oxygen species (ROS),
and studied intracellular ATP levels and the mitochondrial membrane potential (DYm), which has a
critical impact on energy transport. Mitochondrial morphology was observed by confocal
microscopy. Our functional analyses revealed that, compared with strains treated only with FLC,
TET+FLC increased the ATP levels and decreased DYm in CA-1, but decreased ATP levels and
increased DYm in CA-16 (P,0.05). Additionally, CI, CIII and CV activity decreased by 23–48 %.
The production of ROS increased by two- to threefold and mitochondrial morphology was altered
in both strains. Our data suggested that TET impacted mitochondrial aerobic respiratory
metabolism by influencing the generation and transport of ATP, reducing the utilization of ATP,
and resulting in the inhibition of drug efflux pump activity. This activity contributed to the
synergistic action of TET on FLC against C. albicans.
INTRODUCTION
In the 1990s, the widespread use and abuse of the first-line
drug fluconazole (FLC) led to treatment failure for the
fungal pathogen Candida albicans, which had developed
resistance to the drug (White et al., 1998) via several
mechanisms, including reduced access of the drug because
of the action of multidrug resistance efflux pumps (Odds,
1996; Hiller et al., 2006), mutation-mediated overexpression
3These authors contributed equally to this work.
Abbreviations: AA, Antimycin A; CI, complex I; CIII, complex III;
CRC, classical respiratory chain; CV, complex V (ATP synthase); FI,
fluorescence intensity; FLC, fluconazole; ROS, reactive oxygen species;
RT, reverse transcription; TET, tetrandrine.
988
of genes that code for proteins targeted by azole drugs
(Prasad & Kapoor, 2004) and the formation of the biomembrane (Mukhopadhyay et al., 2004). This resistance to
FLC is responsible for the high morbidity and mortality
among immunocompromised patients (Goffeau, 2008;
Monk & Goffeau, 2008).
Tetrandrine (TET) is the major alkaloid component
extracted from the plant Stephania tetrandra S. Moore,
and has been used widely in the treatment of rheumatoid
arthritis (Xu et al., 2008) and hepatic fibrosis (Feng et al.,
2008). Our previous studies found that TET could reverse
the resistance of C. albicans to FLC in vitro (Li & Zhang,
2006) and during the treatment of C. albicans-infected
mice (Wang et al., 2007). The mechanism of action may be
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Synergistic mechanism for tetrandrine on fluconazole
related to the inhibition of the drug efflux system, and the
expression of the drug efflux pump genes CDR1, CDR2,
MDR1 and FLU1 (Zhang et al., 2009).
22DDCt. Each strain in the absence of drug was used as the baseline
control strain and the expression levels of the treated strains were
quantified relative to that of the control strain.
Mitochondrial aerobic respiratory metabolism plays a
major role in C. albicans metabolism and the fermentation
pathway plays a minor role (Zeuthen et al., 1988). The
mitochondrion is the primary source of energy (ATP) for
cellular growth, multiplication, apoptosis and other processes, such as drug efflux. To further explore the synergistic
impact of TET and FLC on C. albicans, and the changes in
mitochondrial aerobic respiration, we studied the FLCsensitive C. albicans strain CA-1 and FLC-resistant strain
CA-16 following exposure to FLC and/or TET. We examined
key measures of energy production and conversion,
including the activity of two respiration chain complexes
[complexes I and III (CI and CIII)] and the ATP synthase
(CV), and the generation of endogenous reactive oxygen
species (ROS). Intracellular ATP levels and mitochondrial
membrane potential (DYm), which both have a critical
impact on energy transport and the mitochondrial morphology, were also observed.
Rh123 accumulation/efflux experiments. The percentage of
METHODS
Strains. The FLC-sensitive C. albicans strain CA-1 and FLC-resistant
strain CA-16 (generously provided by Theodore C. White, University
of Washington, USA) were confirmed previously to be derived from
the same parental source (Pfaller et al., 1994; White et al., 1997). The
responses of the strains were evaluated by the chequerboard
microdilution method in the preliminary study according to the
Clinical and Laboratory Standards Institute M27-A3 guidelines (CLSI,
2008). The MICs of FLC against the C. albicans strains CA-1 and CA16 were 0.25 and 64 mg ml21, respectively, prior to treatment with
TET; following treatment with TET (30 mg ml21), the FLC MICs for
the two strains were 0.0625 and 8 mg ml21, respectively. The maximum
non-cytotoxic concentration of TET (30 mg ml21) was consistent with
the existing literature (Li & Zhang, 2006). For the following
experiments, strains were grown in YPD (yeast extract peptone
dextrose) at 2.5–5.06106 c.f.u. ml21 for 6 h at 37 uC, shaking at
200 r.p.m., in the presence or absence of FLC (750 mg ml21; CIPLA,
purity 99 %) and in the presence or absence of TET (30 mg ml21;
Huico Plant, purity 99.6 %).
Real-time reverse transcription (RT)-PCR. C. albicans cultures
incubated with or without drugs for 6 h were diluted to a
concentration of 1.56107 c.f.u. ml21. Total RNA was extracted from
the cells using the E.Z.N.A. Yeast RNA kit (Omega Bio-tek) according
to the manufacturer’s protocol. cDNA was synthesized according to
the manufacturer’s instructions (TaKaRa, Biotechnology, Dalian, P. R.
China). Real-time RT-PCR was performed using SYBR Green I to
visualize and monitor the amplified product in a MiniOpticon Realtime PCR System (Bio-Rad). Gene-specific primers were designed as
described previously (Park & Perlin, 2005; Yan et al., 2008). The PCR
protocol (Wang et al., 2006) consisted of a denaturation step (95 uC for
10 s), and 40 cycles of amplification and quantification (95 uC for 10 s,
55 uC for 20 s and 72 uC for 15 s, with a single fluorescence
measurement). The change in the fluorescence of the SYBR Green I
dye in every cycle was monitored using the LightCycler system software
(Roche Diagnostics) and the threshold cycle (Ct) above the background
for each reaction was calculated. The Ct value of 18S rRNA was
subtracted from that of the gene of interest to obtain a DCt value. The
gene expression level relative to that of the calibrator was expressed as
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Rh123-positive cells in C. albicans strains in the presence or absence
of drugs was determined as described previously (Clark et al., 1996;
Zhang et al., 2009). During the exponential growth phase, recovered
C. albicans was suspended in 5 ml sorbitol (2.56106 c.f.u. ml21).
Then 150 U lyticase and 125 ml b-mercaptoethanol (0.28 mol l21)
were added to the cells, and the suspension was incubated at 37 uC for
2 h. Cells were resuspended in RPMI 1640 (Gibco) broth and divided
into five equal volumes (A–E) in separate test tubes following two
washes with pre-cooled PBS buffer (4 uC). Rh123 was added to tubes
A–D at a final concentration of 2 mg ml21. FLC, TET or TET+FLC
was added to tubes B, C and D, respectively. Neither drugs nor
Rh123 were added to tube E (control strain for background). For
accumulation experiments, the tubes were then incubated at 37 uC for
45 min. For efflux experiments, the tubes were incubated at 37 uC
for an additional 45 min. Samples were examined by flow cytometry
(FACS Aria; BD Company) and 10 000 cells were counted in the
FITC channel (lexcitation5488 nm, lemission5530 nm) to construct
single-parameter histograms. Data were processed using Cellques
software and the percentage of Rh123-positive cells was calculated.
Intracellular Rh123 fluorescence intensity (FI; lexcitation5488 nm,
lemission5530 nm) before and after drug administration and after the
accumulation/efflux experiments was measured at the same time.
Measurement of intracellular ATP levels. C. albicans strains
incubated in the presence or absence of Antimycin A (AA; 2, 4 or
8 mg ml21; Enzo Life Sciences), FLC and/or TET were adjusted to a
concentration of 16107 c.f.u. ml21. A total of 100 ml cell suspension
was mixed with the same volume of BacTiter-Glo reagent (Promega)
and incubated for 5 min at room temperature as described previously
(Zhang et al., 2013). Luminescent signals were determined on a
full wavelength multifunctional enzyme mark instrument (Safire2;
Tecan). A control tube without cells was used to obtain a value for
background luminescence. A standard curve of incremental ATP
concentrations (from 1 mM to 10 pM) was constructed. Signals
represented the mean of three separate experiments and the ATP
content was calculated from the standard curve.
Assessment of DYm. DYm was measured using a Yeast Cell
Mitochondria Membrane Potential Fluorescent Assay kit (GenMed
Scientifics) as described previously (Misra et al., 2009). C. albicans
strains (16107 c.f.u. ml21) incubated in the presence or absence
of drugs for 6 h were stained with 10 mg ml21 JC-1 (5,59,6,69tetrachloro-1,19,3,39-tetraethylbenzimidazol-carbocyanine iodide) at
30 uC for 20 min in the dark. Cells were washed twice, resuspended in
PBS buffer, and measured on the full wavelength multifunctional
enzyme mark instrument with an excitation wavelength of 490 nm
and emission wavelength shifting from green (~530 nm) to red
(~590 nm). DYm was determined as a ratio of red to green FI.
Measurement of endogenous ROS production. ROS production
was measured using a Yeast Cell Oxidative Stress Active Oxygen
Fluorescence Assay kit (GenMed Scientifics) as described previously
(Xiao et al., 2010). C. albicans strains (16107 c.f.u. ml21) incubated
in the presence or absence of drugs for 6 h were stained with 10 mg
ml21 CM-H2DCFDA (6-chloromethyl- 29,79-dichlorofluorescein
diacetate, acetyl ester) at 30 uC for 20 min. Following staining, the
cells were washed twice, resuspended in PBS buffer, and measured on
the full wavelength multifunctional enzyme mark instrument with an
excitation wavelength of 490 nm and emission wavelength of 530 nm.
ROS production was calculated by subtracting the FI value of cells in
the absence of CM-H2DCFDA from that of cells with CM-H2DCFDA.
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989
H. Guo and others
Isolation of mitochondria and assay for CI/CIII/CV activity. C.
albicans incubated in the presence or absence of drugs for 6 h were
washed and a total of 56108 cells were prepared for each sample.
Mitochondria were isolated using the Yeast Cell Mitochondria
Isolation kit (GenMed Scientifics) as described previously (Xu et al.,
2009). The isolated mitochondria were lysed using the Yeast
Mitochondria Lysis kit (GenMed Scientifics) and the mitochondrial
protein concentration was determined by the bicinchoninic acid
method using a BCA Protein Concentration Quantification kit
(GenMed Scientifics) (Bradford, 1976).
Complex activity was determined by colorimetric assay using
the Fungus Mito Respiration Chain CI/CIII/CV Activity Assay
kit (GenMed Scientifics) as described previously (Xu et al., 2009).
Optical density was measured on an UV-visible spectrophotometer at
wavelengths of 340–380/550/340 nm. One unit of CI/CIII/CV activity
was defined as the amount of enzyme activity that oxygenated 1 mM
NADH/CoQH2/NADH min21 at 30 uC and pH 7.5.
Mitochondria morphology. C. albicans cells incubated in the
presence or absence of drugs for 24 h were washed and a total of
16 107 cells were prepared for each sample. Mitochondria were
stained with 1 mM MitoTracker Red (1 : 750) for 20 min in the dark
as described previously (Dagley et al., 2011). Following staining, the
cells were washed twice, resuspended in PBS buffer and mounted 1 : 1
using 1 % low-melt agarose. A laser scanning confocal microscope
(510 META DuoScan; Carl Zeiss) equipped with a 6100 oilimmersion objective was used to image the cells. Confocal images
were compiled using the Zeiss LSM510 software.
Statistics. Experiments were performed three times.
SPSS 13.0 was
used for the analyses and the results were expressed as the mean±SD.
Differences between groups of the same strain were measured using
one-way ANOVA. Statistical significance was defined as P,0.05.
both strains compared with treatment with TET (P,0.05).
These results suggested that TET+FLC could downregulate
the expression of the energy-related drug resistance genes
CDR1 and CDR2 (Table 1, Fig. 1).
Effects of TET on C. albicans Rh123 accumulation
and efflux
Results of Rh123 experiments as determined by flow
cytometry. Compared with strains treated with FLC alone, the
mean percentages of Rh123-positive cells (A1) increased
significantly for both CA-1 and CA-16 strains treated with
TET or TET+FLC following the accumulation experiments,
but decreased significantly following treatment with TET or
TET+FLC for the efflux experiments (P,0.05). In the
accumulation/efflux experiments, the mean percentages of
Rh123-positive cells were not significantly different between
TET and TET+FLC treatments (P.0.05) (Table 2).
Results of Rh123 experiments as determined by
fluorescent spectrophotometry. Compared with strains
treated with FLC, the mean intracellular Rh123 FIs increased
markedly for both CA-1 and CA-16 strains following treatment with TET+FLC following accumulation experiments,
but decreased significantly following efflux experiments
(P,0.05). These results indicated that TET could inhibit
drug efflux pump activity in C. albicans, with decreasing
drug efflux activity correlating with increasing intracellular
Rh123 accumulation (Table 2).
Effects of TET on mitochondrial energy
conversion
RESULTS
Effects of TET on the expression levels of genes
and drug efflux pump activity
Relative expression levels of drug efflux pump genes in
C. albicans strains. In both the FLC-sensitive strain CA-1
and FLC-resistant strain CA-16, mRNA levels for CDR1,
CDR2, MDR1 and FLU1 all decreased (P,0.05) upon
treatment with TET+FLC compared with treatment with
FLC alone. Additionally, TET+FLC treatment decreased
the expression levels of CDR1, CDR2, MDR1and FLU1 in
TET can inhibit CI and CIII activity. Compared with FLC
treatment, mitochondrial respiratory chain CI and CIII
activity was inhibited upon treatment with TET+FLC for
both CA-1 and CA-16 strains (P,0.05; 40 % decrease in CI
activity compared with FLC in CA-1 and 48 % decrease in
CIII activity; 38 % decrease in CI activity compared with
FLC in CA-16 and 45 % decrease in CIII activity). Compared
with drug-free/FLC-treated cells, the enzymic activities of CI
and CIII were decreased significantly for both strains upon
treatment with TET+FLC (P,0.05) (Table 3, Fig. 2a, b).
Table 1. Relative expression levels of genes CDR1, CDR2, MDR1 and FLU1 in C. albicans strains CA-1 and CA-16
Gene
CA-1
FLC
CDR1
CDR2
MDR1
FLU1
1.19±0.01
1.73±0.15
1.29±0.06
1.57±0.11
CA-16
TET
TET+FLC
FLC
2.16±0.13
2.28±0.13
4.29±0.16
2.55±0.45
1.08±0.03
1.34±0.44
1.16±0.06
1.33±0.22
0.65±0.02
0.74±0.05
1.76±0.14
0.7±0.03
TET
0.53±0.06
0.52±0.04
1.49±0.08
0.73±0.02
TET+FLC
0.32±0.01
0.44±0.04
1.44±0.04
0.61±0.02
Relative expression levels of genes in C. albicans strain CA-1/CA-16 treated with FLC (750 mg ml21) and/or TET (30 mg ml21) were quantified
relative to the control strain (CA-1/CA-16 untreated with drug). Values represent mean±SEM of three independent LightCycler reactions.
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Journal of Medical Microbiology 63
Synergistic mechanism for tetrandrine on fluconazole
(a)
CA-1
CA-1+FLC
CA-1+TET
CA-1+TET+FLC
(b)
CA-16+FLC
CA-16+TET
CA-16+TET+FLC
2.0
Relative gene expression (fold)
5
Relative gene expression (fold)
CA-16
4
3
2
1
0
1.5
1.0
0.5
0
CDR1
CDR2
MDR1
FLU1
CDR1
CDR2
MDR1
FLU1
Fig. 1. Expression levels of CDR1, CDR2, MDR1 and FLU1 in C. albicans (a) FLC-sensitive strain CA-1 or (b) FLC-resistant
strain CA-16 treated with FLC (750 mg ml”1) and/or TET (30 mg ml”1) were normalized against control cells (untreated with
drug), and the relative expression levels were quantified relative to the control strain CA-1/CA-16.
These results suggested that TET could decrease the
production of ATP by inhibiting CI and CIII activity.
TET can inhibit CV activity. Compared with FLC-treated
cells, CV activity was decreased significantly in both CA-1
and CA-16 treated with TET+FLC (P,0.05, 41 % decrease
compared with FLC-treated cells for CA-1 and 23 % decrease
for CA-16). Compared with drug-free/TET-treated cells, the
enzymic activity of CV decreased significantly in both strains
upon treatment with TET+FLC (P,0.05) (Table 3, Fig. 2c).
These results demonstrated that TET could further decrease
ATP levels by inhibiting CV activity.
Combination of TET and FLC augments endogenous
ROS production. Compared with FLC-treated cells, the
concentration of endogenous ROS in both CA-1 and
CA-16 cells treated with TET+FLC increased significantly
(P,0.05, threefold increase compared with FLC alone for
CA-1 and twofold increase for CA-16). Compared with
drug-free/TET-treated cells, TET+FLC treatment resulted
in a significant increase in the production of endogenous
ROS in both strains (P,0.05) (Fig. 3). These results
indicated that the combination of TET and FLC could
increase endogenous ROS in C. albicans.
Effects of TET on mitochondrial energy transport
TET can regulate the intracellular ATP content in C.
albicans. Compared with drug-free cells, treatment with
the suppressive agent AA (2, 4 or 8 mg ml21) caused a
significant decrease in the intracellular ATP content of cells
of both strains (P,0.05). However, the differences were
Table 2. Percentages of Rh123-positive cells and intracellular Rh123 FI values for C. albicans strains CA-1 and CA-16
Strain
B1
CA-1
CA-1+FLC
CA-1+TET
CA-1+TET+FLC
B2
CA-16
CA-16+FLC
CA-16+TET
CA-16+TET+FLC
A1 (%)
FI1
E1 (%)
FI2
0.1
57.1
60.8
76.6
78.9
0.2
17.8
23.1
47.1
47.6
203
1531
1620
3420
3665
107
1258
1480
2640
2624
0.1
12.4
10.8
42.1
48.9
0.2
5.5
4
19.8
19.1
189
482
463
1595
1899
112
383
351
696
764
E2 (%)
78.46
82.4
45.17
38.15
70.22
83.55
58.39
60.29
B1 and B2, background controls for CA-1 and CA-16, respectively. A1, percentage of Rh123-positive cells (background subtracted) after the
accumulation experiment; E1, percentage of Rh123-positive cells (background subtracted) after the efflux experiment; E2, percentage of efflux
Rh123-positive cells; FI1 and FI2, FIs after the accumulation and efflux experiments. FLC, 750 mg ml21; TET, 30 mg ml21.
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H. Guo and others
suggested that TET treatment could alter the efficiency of
mitochondrial aerobic respiration by adjusting the DYm.
Table 3. Relative activity of mitochondrial respiratory chain CI/
CIII/CV in C. albicans strains CA-1 and CA-16
Relative activity (% of control)
CA-1+FLC
CA-1+TET
CA-1+TET+FLC
CA-16+FLC
CA-16+TET
CA-16+TET+FLC
Effects of TET on mitochondrial morphology
CI
CIII
CV
37.60
30.09
15.00
68.55
54.51
30.80
34.19
34.19
16.27
84.96
53.10
31.86
37.50
36.11
15.28
67.78
71.97
15.48
A large number of mitochondria were observed in both
CA-1 and CA-16 drug-free cells, and the mitochondria
were granular and dispersed. However, mitochondrial
morphology changed in both strains following treatment
with drugs for 24 h. Compared with FLC-treated cells,
both the strains treated with TET+FLC exhibited an
extensive tubular network of mitochondria, which were
elongated and fused (Fig. 6).
These results were compared with the results in the control
strain (CA-1/CA-16 untreated with drug). FLC, 750 mg ml21; TET,
30 mg ml21.
DISCUSSION
TET is known to reverse FLC drug resistance in C. albicans
by inhibiting the function of drug efflux pumps and this
activity is related to energy metabolism (Pfaller et al., 1994);
however, the mechanism of this synergistic interaction of
TET against FLC-resistant C. albicans remained unknown.
Mitochondrial aerobic respiratory metabolism, which
consists of energy generation (liberation), conversion and
utilization (consumption), plays a key role in C. albicans
energy metabolism (White et al., 1997; Kusch et al., 2008).
The inhibitory activity of TET against drug efflux pump
function in C. albicans is related to one or more aspects of
its influence on cellular mitochondrial energy generation,
conversion and utilization or to its impact on the structure
and activity of the efflux pump. This model serves as the
foundation of our research design.
not significant between the different AA concentrations for
the same strain (P.0.05) (Fig. 4a).
Compared with FLC-treated cells, the intracellular ATP
content of cells treated with TET+FLC increased significantly in CA-1 cells, but decreased in CA-16 cells (P,0.05).
Compared with the drug-free cells, TET+FLC treatment
increased the intracellular ATP content of CA-1 cells, but
decreased the intracellular ATP content of CA-16 cells
(P,0.05) (Fig. 4b). These results suggested that TET could
alter the production of intracellular ATP in C. albicans.
TET can regulate DYm. Compared with FLC-treated cells,
TET+FLC treatment resulted in a significant decrease
in DYm for CA-1, but a significant increase for CA-16
(P,0.05). Compared with drug-free cells, DYm increased
significantly for FLC-treated cells, but decreased significantly
for TET+FLC-treated cells for CA-1 (P,0.05). However,
DYm did not change significantly for FLC-treated CA-16
cells (P.0.05), but was decreased significantly in TET+
FLC-treated CA-16 cells (P,0.05) (Fig. 5). These results
(a)
(b)
25
20
#
## #
##
15
10
5
0
(c)
500
* *
** **
CA-1
#
*
CA-16
CIII activity (U g–1)
CI activity (U g–1)
In this study, we demonstrated that compared with
FLC-treated cells, TET+FLC treatment can influence the
production and conversion of ATP and promote cellular
apoptosis by significantly inhibiting the enzymic activity
of the respiration chain complex (CI, CIII and CV) and
increasing endogenous ROS production. TET+FLC
400
##
300
200
* *
** **
#
*
100
0
#
##
CA-1
Control
CV activity (×10–3 U g–1)
Strain
1500
1200
900
600
300
CA-16
FLC
TET
#
#
##
##
0
* *
** **
#
*
CA-1
CA-16
TET+FLC
Fig. 2. (a) CI activity, (b) CIII activity and (c) CV activity in C. albicans strains CA-1 and CA-16 untreated or treated with FLC
(750 mg ml”1) and/or TET (30 mg ml”1). *P,0.05 versus control for CA-1, **P,0.05 versus CA-1+TET+FLC, #P,0.05
versus control for CA-16, ##P,0.05 versus CA-16+TET+FLC.
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Journal of Medical Microbiology 63
Synergistic mechanism for tetrandrine on fluconazole
10 000
*
ROS (FI)
8000
#
6000
#
##
4000
**
**
Fig. 3. Intracellular ROS generation in C.
albicans strains CA-1 and CA-16 untreated or
treated with FLC (750 mg ml”1) and/or TET
(30 mg ml”1) for 6 h. *P,0.01 versus control
for CA-1, **P,0.01 versus CA-1+TET+FLC,
#P,0.01 versus control for CA-16, ##P,0.01
versus CA-16+TET+FLC.
##
2000
0
CA-1
Control
CA-16
FLC
TET
TET+FLC
treatment alters mitochondrial morphology and affects the
level of ATP transport from the mitochondrial matrix to
the cytoplasm, which results in a decrease in available ATP
by regulating intracellular ATP content and DYm. These
biochemical processes may impinge on the mechanism
of synergistic action between TET and FLC against C.
albicans.
generating an electrochemical proton gradient between
the intramembrane space and outer membrane. DYm is
then used by the inner mitochondrial membrane CV to
synthesize ATP, which completes mitochondrial energy
conversion. In this way, the CI, CIII and CV components
of electron flow in the CRC are important targets for
energy generation and conversion (Li et al., 2011).
We found that CI, CIII and CV activity was inhibited
significantly in both FLC-sensitive CA-1 and FLC-resistant
CA-16 cells treated with TET+FLC (Fig. 2), suggesting
that TET+FLC can reduce the production and conversion
of mitochondrial ATP by inhibiting the activities of these
key complexes (CI, CIII and CV) in C. albicans. AA, a
known inhibitor of CIII (Milani et al., 2001), can decrease
significantly the cellular ATP content in both strains, and
treatment with TET+FLC can similarly decrease significantly cellular ATP content in the FLC-resistant strain
CA-16. These results indicate that TET+FLC has a similar
We sought to determine how TET affects mitochondrial
energy generation and conversion in C. albicans. The
classical respiratory chain (CRC) is the major source of
respiratory activity and O2 consumption in C. albicans.
The CRC provides the largest amount of cellular ATP by
transmitting electrons through CI, CIII and CIV; subsequently, the electrons and O2 fuse together, releasing
energy (Alonso-Monge et al., 2009; Li et al., 2011). In the
CRC pathway, CI, CIII and CIV oxidoreductases on the
inner mitochondrial membrane produce H+ ions that are
pumped into the intermembrane space (Li et al., 2011),
(b)
600
400
**
*
200
*
*
**
**
ATP concentration (nM)
ATP concentration (nM)
(a) 800
2000
*
*
**
1600
1200
#
##
800
#
##
#
400
**
0
0
CA-1
Control
CA-1
CA-16
A2
A4
A8
Control
FLC
CA-16
TET
TET+FLC
Fig. 4. (a) Intracellular ATP content in C. albicans strains CA-1 and CA-16 untreated or treated with AA [2 (A2), 4 (A4) and 8
(A8) mg ml”1, respectively]. *P,0.01 versus control for CA-1, **P,0.01 versus control for CA-16. (b) Intracellular ATP content
in C. albicans strains CA-1 and CA-16 untreated or treated with FLC (750 mg ml”1) and/or TET (30 mg ml”1). *P,0.05 versus
control for CA-1, **P,0.05 versus CA-1+TET+FLC, #P,0.05 versus control for CA-16, ##P,0.05 versus CA16+TET+FLC.
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993
H. Guo and others
ΔΨm (Ratio of red to green FI)
12
*
**
#
**
10
*
##
##
8
6
4
2
0
CA-1
Control
FLC
CA-16
TET
TET+FLC
Fig. 5. gYm in C. albicans strains CA-1 and CA-16 treated or
untreated with FLC (750 mg ml”1) and/or TET (30 mg ml”1) for 6 h.
*P,0.05 versus control for CA-1, **P,0.05 versus CA-1
+TET+FLC, #P,0.05 versus control for CA-16, ##P,0.05
versus CA-16+TET+FLC.
inhibitory action against CIII as an inhibitor, i.e. AA,
and this inhibitory action maybe part of the synergistic
mechanism of TET and FLC against FLC-resistant C.
albicans.
Treatment with TET also alters the level of mitochondrial
energy transport during its synergistic action against C.
albicans. The transportation of ATP from the mitochondrial
(a)
(e)
(b)
(f)
matrix to the cytoplasm is affected by the cell’s DYm and
ATP/ADP translocase (Fig. 7). This study demonstrated
that the intracellular ATP content and level of DYm changes
dramatically in both strains treated with TET+FLC,
although the changes were not identical (Fig. 5). The
FLC-sensitive strain CA-1 treated with FLC or TET+FLC
was in a stringent state. Thus, mitochondrial metabolism
was feedback activated, resulting in the mitochondria
releasing more ATP. However, the DYm of CA-1 cells
decreased and the transport of ATP to the cytoplasm was
reduced, resulting in a reduction in the available energy.
For FLC-resistant CA-16 cells, the DYm increased and
the production of ATP decreased upon treatment with
TET+FLC. This reflects differences in energy metabolism
between the two strains and may be correlated with the
dose of FLC. These results suggest that treatment with
TET+FLC may influence the transport of ATP and alter
the efficiency of aerobic respiration by adjusting the DYm.
These changes may result in the synergistic activity of TET
against C. albicans by reducing available ATP.
The synergistic effect of TET against C. albicans is associated
with the production of endogenous ROS. ROS is the
byproduct of electron transfer in mitochondria, and is
produced primarily by CI and CIII. Excessive production
and leakage of ROS disrupts the redox equilibrium of
mitochondria, which can lead to oxidative stress in the cell.
Excessive ROS was confirmed to be correlated closely with
cellular aging, apoptosis and the selective degradation of
mitochondria (Drakulic et al., 2005; Wilhelm et al., 2006;
Soengas, 2012). Li et al. (2011) and Ruy et al. (2006)
have demonstrated that oxidative stress and enhanced
(c)
(g)
(d)
(h)
Fig. 6. Mitochondria were stained with MitoTracker Red and viewed by confocal microscopy: (a) CA-1, (b) CA-1+FLC, (c)
CA-1+TET, (d) CA-1+TET+FLC, (e) CA-16, (f) CA-16+FLC, (g) CA-16+TET, (h) CA-16+TET+FLC. Bar, 5 mm.
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Synergistic mechanism for tetrandrine on fluconazole
Energy
transport
Energy generation
Apoptosis
Mitophagy
H+
Cytoplasm
H+
H+
ROS
+
CI e–
Inner membrane
CIII
Q
1
2
CIV e–
e–
C
e–
CV 3
ΔΨm 4
F0
–
NADH
NAD+
ROS 5
F1
ROS
ADP+Pi
FLC/TET/FLC+TET
ATP 6
Mitochondrial matrix
Fig. 7. Relation of drugs and the mitochondria aerobic respiration chain (CRC): (1) CI activity, (2) CIII activity, (3) CV activity,
(4) DYm, (5) ROS and (6) ATP were altered following treatment with drugs (TET+FLC).
production of ROS occur following the disruption of CI
and/or CIII or when CIII is partially inhibited, which leads
to cellular apoptosis in C. albicans. Our data also confirm
that following combination treatment with TET+FLC,
endogenous ROS production increases significantly in both
CA-1 and CA-16 (Fig. 3). This suggests that TET can
influence aerobic respiratory metabolism in mitochondria
by increasing the production of endogenous ROS, leading
to cellular apoptosis and exerting a synergistic effect on C.
albicans.
We also found that mitochondria morphology was altered
after treatment of C. albicans with TET. Under normal
conditions, C. albicans cells contain numerous mitochondria,
which are dispersed as granules throughout the cytoplasm.
After treatment with FLC/TET for 24 h, the mitochondria
became elongated and fused, particularly following combined treatment with TET+FLC, and the mitochondria
appeared as a tubular network structure (Fig. 6). This change
in mitochondrial morphology is closely related to cellular
energy metabolism, autophagy and cell death (Gomes et al.,
2011). Under external pressure, the elongated, fused or
tubular network of mitochondria can increase ATP synthesis
to recover from damage. However, once the elongation of
mitochondria is blocked, either due to heredity or pathological changes, ATP is consumed in an uncontrolled
manner, leading to cellular death. We hypothesize that
prolonged exposure to TET+FLC further alters mitochondrial morphology, and that this is accompanied by a decrease
in the number of mitochondria and excessive consumption
of ATP. This ultimately leads to autophagy and degradation
of C. albicans because of an insufficient energy supply.
Taken together, our data indicate that treatment with TET
may inhibit the metabolic processes of mitochondrial
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energy conversion or transportation and decrease the
synthesis or transport ATP within mitochondria, thus
inhibiting the excretory activity of drug efflux pumps
and increasing the production of endogenous ROS.
Subsequently, the morphology of mitochondria is altered,
which leads to apoptosis. In this manner, TET may exert
synergistic activity with FLC against C. albicans. However,
the synergistic action of TET against C. albicans is a
complicated process with multiple factors and mechanisms. Therefore, we will investigate further whether there
are alternate methods for TET to exert its synergistic action
against C. albicans and clarify their mechanisms.
ACKNOWLEDGEMENTS
We thank Professor Theodore C. White (University of Washington,
and the Seattle Biomedical Research Institute, USA) for generously
providing C. albicans isolates. This work was supported by grants
from the National Natural Science Foundation of China (30972660/
81171542), the Natural Science Foundation of Guangdong Province,
China (10151008901000131), Guangzhou Key Technology R&D
Program, China (10A32070407) and the Fundamental Research
Funds for the Central Universities (2010/2011).
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