Candida albicans augments endogenous reactive oxygen species production – a microarray analysis study Rong Mei Liang1,2, Xiao Lan Yong2, Yun Ping Jiang2, Yong Hong Tan3, Bao Di Dai1, Shi
Trang 1Candida albicans augments endogenous reactive oxygen species production – a microarray analysis study
Rong Mei Liang1,2, Xiao Lan Yong2, Yun Ping Jiang2, Yong Hong Tan3, Bao Di Dai1,
Shi Hua Wang3, Ting Ting Hu2, Xi Chen2, Nan Li2, Zhao Hui Dong2, Xiao Chun Huang2, Jun Chen2, Yong Bing Cao1and Yuan Ying Jiang1
1 Drug Development Center, School of Pharmacy, Second Military Medical University, Shanghai, China
2 Department of Clinical Pharmacy, General Hospital of Chengdu Military Command Region, Chengdu, China
3 Department of Pharmacy, General Hospital of Chengdu Military Command Region, Chengdu, China
Introduction
Candida infections have become a serious medical
problem, because of the high incidence and mortality
in AIDS patients, transplant recipients and other
immunosuppressed individuals [1–4] Despite
conti-nuous expansion of the arsenal of antifungal drugs,
the antifungal drugs available cannot meet the
increas-ing requirements for managincreas-ing infections in medically complex patients
2-Amino-nonyl-6-methoxyl-tetralin muriate (10b), a 2-aminotetralin derivative, was synthesized as an anti-fungal agent with a novel chemical structural (Fig 1) [5] Tetralin derivatives are known to be potentially
Keywords
10b; action mechanism; Candida albicans;
microarray analysis; ROS
Correspondence
Y B Cao and Y Y Jiang, Drug
Development Center, School of Pharmacy,
Second Military Medical University, 325
Guohe Road, Shanghai 200433, China
Fax: +86 021 6549 0641
Tel: +86 021 8187 1357;
+86 021 8187 1275
E-mail: jiangyysmmu@163.com;
ybcao@vip.sina.com
(Received 21 October 2010, revised 14
December 2010, accepted 18 January 2011)
doi:10.1111/j.1742-4658.2011.08021.x
Candidainfections have become an increasingly significant problem, mainly because of the widespread nature of Candida and drug resistance There is
an urgent need to develop new classes of drugs for the treatment of oppor-tunistic Candida infections, especially in medically complex patients Previ-ous studies have confirmed that 2-amino-nonyl-6-methoxyl-tetralin muriate (10b) possesses powerful antifungal activity in vitro against Candia albicans
To clarify the underlying action mechanism, an oligonucleotide microarray study was performed in C albicans SC5314 without and with 10b treat-ment The analytical results showed that energy metabolism-related genes, including glycolysis-related genes (PFK1, CDC19 and HXK2), fermenta-tion-related genes (PDC11, ALD5 and ADH1) and respiratory electron transport chain-related genes (CBP3, COR1 and QCR8), were downregu-lated significantly Functional analysis revealed that 10b treatment increased the generation of endogenous reactive oxygen species, and decreased mitochondrial membrane potential, ubiquinone–cytochrome c reductase (complex III) activity and intracellular ATP levels in C albicans SC5314 Also, addition of the antioxidant ascorbic acid reduced the anti-fungal activity of 10b significantly These results suggest that mitochondrial aerobic respiration shift and endogenous reactive oxygen species augmenta-tion might contribute to the antifungal activity of 10b against C albicans This information may prove to be useful for the development of new strategies to treat Candida infections
Abbreviations
AA, ascorbic acid; CFU, colony-forming unit; DCFH-DA, 2,7-dichlorofluorescein diacetate; FI, fluorescence intensity; MCZ, miconazole; ROS, reactive oxygen species; DWm, mitochondrial membrane potential; 10b, 2-amino-nonyl-6-methoxyl-tetralin muriate.
Trang 2applicable to psychiatry, and dialkylated tetralin
derivatives are the most effective [6] Aminotetralins,
including 8-hydroxy-2-(di-n-propylamino)-tetralin and
7-hydroxy-2-(di-n-propylamino)-tetralin, behave as
preferential agonists at serotonin
(5-hydroxytrypta-mine)1A and dopamine D3 and D2 receptors [7] The
former affects intracranial self-stimulation, and the
lat-ter possess anxiolytic properties However, there are
few reports from other laboratories describing the
anti-fungal activities of 2-aminotetralin derivatives Our
previous studies [5,8] indicated that 10b possessed high
antifungal activity In an oophorectomized female SD
rat model of Candida albicans infections, 10b
consis-tently exhibited better antifungal activity than
itraco-nazole [5] Also, 10b significantly reduced the ergostrol
content by inhibiting the activity of sterol C-14
reduc-tase, encoded by ERG24 in the ergosterol biosynthetic
pathway [8] However, C albicans ERG24 is not
required for growth The erg24 mutant of C albicans
is capable of growth under normal aerobic conditions
on standard defined and enriched media There is a
suggestion that the significant level of
ergosta-8,14,22-trienol accumulated by C albicans erg24 mutants may
be the element that allows growth under normal
condi-tions [9], implying that the major action mechanism of
10b against C albicans is not correlated with inhibition
of the activity of sterol C-14 reductase in the ergosterol
biosynthetic pathway
The present study was a continuation of our
previ-ous studies, in an attempt to clarify the mechanism of
action of 10b against C albicans through analyzing the
gene expression profiles of C albicans SC5314 without
and with 10b treatment, using oligonucleotide
micro-array assays and real-time RT-PCR assays It was
found that a series of differentially expressed genes
were involved in energy metabolism, oxidoreduction
and other biological functions In addition,
measure-ments of endogenous reactive oxygen species (ROS)
generation, mitochondrial membrane potential (DWm),
intracellular ATP concentration, respiratory electron
transport chain complex III (ubiquinone–cytochrome c
reductase) activity and the effect of antioxidant
ascor-bic acid on the antifungal activity of 10b suggested
that the antifungal activity of 10b against C albicans
might be related to mitochondrial aerobic respiration
shift and endogenous ROS augmentation
Results
Response of gene expression to 10b exposure
A total of 957 differentially expressed genes were found upon exposure to 10b; the expression of 457 genes was decreased, and the expression of 500 genes was increased Forty-five downregulated genes and nine upregulated genes were found to be related to energy metabolism Of the 45 downregulated genes, 34 were involved in glycolysis (e.g PFK1, CDC19 and HXK2), fermentation (e.g., PDC11, ALD5 and ADH1), the respiratory electron transport chain (e.g CBP3, COR1 and QCR8) and ROS scavenging (e.g GPX2) Of the nine upregulated genes, five were related to fermenta-tion (e.g ADH3 and ADH5) and ROS scavenging (e.g GPX1, SOD5 and SOD6) An additional 29 upregulated genes and 15 downregulated genes were concerned with lipid metabolism Of the 29 upregulated genes, nine were directly linked to ergosterol biosynthesis In addi-tion, 93 (20.35%) of the 457 downregulated genes were related to translation, whereas only two genes in this category were upregulated, suggesting that the transla-tion level might be lower in SC5314 cells exposed to 10b than in controls (Tables S1 and S2)
Validation of microarray data by real-time RT-PCR
Knowing that augmentation of endogenous ROS pro-duction was directly related to the antifungal activity
of some antifungal drugs [10–12], we were particularly interested in energy metabolism-related genes There-fore, real-time RT-PCR analysis was designed to detect genes related to energy metabolism Real-time RT-PCR reactions were performed in triplicate with independent RNA isolations As shown in Fig 2, the expression levels of glycosis-related genes, PFK1, PFK2, HXK2 and CDC19, decreased significantly by 30.30-fold, 43.48-fold, 20.83-fold and 20.41-fold, respectively; the expression levels of fermentation-related genes, ALD5, ADH1 and PDC11, decreased significantly by 15.63-fold, 2.20-fold and 83.33-fold respectively; and the expression levels of genes coding for mitochondrial respiratory chain complex III, CBP3 (333.33-fold), COR1 (9.62-fold), QCR2 (8.70-fold), QCR8 (5.43-fold), CYT1 (11.24-fold) and RIP1 (12.5-fold), were also markedly decreased Also, the expression level of GPX2 decreased significantly, by 12.05-fold, whereas the expression levels of SOD5 and GPX1 increased by 38.686-fold and 5.433-fold, respec-tively In addition, the expression levels of ADH3 and ADH5, two fermentation-related genes, also increased
CH3O
NH(CH2)8CH3.HCl
Fig 1 Chemical structure of 10b.
Trang 3significantly, by 10.382-fold and 3.212-fold,
respec-tively
ROS production in C albicans treated with 10b
Before measurement of the level of ROS production,
drug concentrations inhibiting growth to 80% of
con-trol levels were estimated by interpolation, and this
concentration for 10b was 0.5 lgÆmL)1 The level of
endogenous ROS production induced by 10b was
mea-sured with concentrations of 1, 3 and 9 lgÆmL)1
Because miconazole (MCZ) is known to be an
excel-lent endogenous ROS inducer [10], it was selected as
the positive control to investigate the effect of 10b on
endogenous ROS production in C albicans SC5314
As shown in Fig 3, after treatment with 9 lgÆmL)1
10b, the level of ROS production increased by
22.7-fold in a dose-dependent manner MCZ treatment also
augmented ROS production, but to a lesser extent
Effects on DWm, complex III activity and ATP
content in mitochondria of C albicans after 10b
treatment
Treatment with 10b caused DWm degradation in a
dose-dependent manner (Fig 4A), which was opposite
to the result obtained for endogenous ROS
produc-tion Although the generation of ROS is exponentially
dependent on DWm[13], dysfunction of proton pumps
can disrupt the positive correlation between
endoge-nous ROS production and DWm [14] Therefore, we
determined the activity of two important proton
pumps, complex III and complex I
(NADH–ubiqui-none reductase), which are the main sources of ROS in
mitochondria As was expected, complex III activity
decreased in a dose-dependent manner after 10b
treat-ment (Fig 4B) The inhibitory efficiencies of 10b at
3 lgÆmL)1 and 9 lgÆmL)1 were 25.43% and 57.26%, respectively, after 9 h of exposure However, no signifi-cant difference in complex I activity was observed between the control group and the 10b group (data not shown) It was therefore assumed that 10b treat-ment inhibited complex III activity, resulting in proton pump inactivation and a decrease in DWm Because intracellular ATP generation is known to be positively correlated with DWm in C albicans under normal cul-ture conditions, the intracellular ATP concentration in cells without and with 10b was also measured The results revealed a dose-dependent decrease in intracel-lular ATP generation, which was consistent with the result obtained for DWm(Fig 4C)
–10 –9 –8 –7 –6 –5 –4 –3 –2 –1 0 1 2 3 4 5 6 7
ADH1 ALD5PDC11CDC19 HXK2 PFK1 PFK2 CBP3COR1 RIP1 CYT1 QCR8 QCR2COX4 GPX2SOD5 GPX1 ADH3 ADH5
Fig 2 Changes in gene expression levels
of 19 energy metabolism-related genes in
10b-treated C albicans SC5314 The
con-centration of 10b was 3 lgÆmL)1 All genes
were examined by real-time RT-PCR with
gene-specific primers Relative fold change
was calculated with the C T value (see
details in Experimental procedures) Results
are the mean ± standard deviations for
three independent experiments.
0
5000
10 000
15 000
20 000
25 000
1 µg·mL –1
3 µg·mL –1
9 µg·mL –1
**
* *
**
*
*
Fig 3 Endogenous ROS generation in C albicans SC5314 cells without and with 10b and MCZ The concentrations of 10b and MCZ were 1, 3 and 9 lgÆmL)1 ROS levels represent the mean ± standard deviations for three independent experiments Statistically significant differences (as determined by Student’s t-test, as compared with control): *P < 0.01; **P < 0.05.
Trang 4Effects of ROS on the antifungal activity of 10b
against C albicans
To determine whether ROS production was directly
involved in the antifungal effect of 10b, the effect of
an antioxidant on the net level of ROS production and
antifungal activity was observed in 10b-treated cells
The net ROS production in cells induced by 10b
treat-ment was inhibited by addition of the antioxidant
ascorbic acid (AA) in a dose-dependent manner, with
complete inhibition occurring at 10 mm (Fig 5) We
then examined whether AA treatment interfered with
the antifungal effect of 10b In a colony formation
assay, 9 lgÆmL)1 10b caused a cytostatic effect
(approximately 72% inhibition) at 48 h AA treatment
prevented the colony-inhibitory effect induced by 10b
in a dose-dependent manner (Fig 6)
Discussion
To further investigate the mechanism of action of 10b
at a molecular level, an oligonucleotide microarray
study was performed in C albicans SC5314 without
and with 10b treatment The results showed that
differ-entially expressed genes were involved in multiple
bio-chemical functions Many experiments have confirmed
that ROS play a central role in yeast signaling and
apoptotic death [15–19] In addition, they can damage
a wide range of molecules, including nucleic acids, proteins and lipids, that are involved in a variety of key events leading to cell death [20–22] Damage to
0
20
40
60
80
100
120
C
**
*
*
0 20 40 60 80 100 120
*
**
0 100 200 300 400 500 600
**
*
*
Fig 4 Mitochondrial functional analysis in
C albicans SC5314 treated or untreated with 10b: (A) DWm (B) Complex III activity (C) Intracellular ATP level The concentra-tions of 10b were 1, 3 and 9 lgÆmL)1 DWm, complex III activity and ATP levels represent the mean ± standard deviations for three independent experiments Statistically signif-icant differences (as determined by Stu-dent’s t-test, as compared with control):
*P < 0.01; **P < 0.05.
–5000 0 5000
10 000
15 000
20 000
25 000
*
*
**
Fig 5 Effect of AA on ROS production in 10b-treated C albicans The concentrations of AA were 2.5, 5 and 10 m M , and that of 10b was 9 lgÆmL)1 Data represent the mean ± standard deviations for three independent samples Statistically significant differences (as determined by Student’s t-test, as compared with control):
*P < 0.01; **P < 0.05.
Trang 5mitochondrial macromolecules may lead to increased
ROS production and further damage to mitochondrial
components, thus causing a ‘vicious downward spiral’
in terms of ROS production and damage accumulation
[23] The antifungal activities of several compounds,
including ketoconazole, polygodial, histain 5, UK-2A
and UK-3A, are achieved by inhibiting the respiratory
activity of mitochondria [24–27]
Therefore, we were particularly interested in the
striking changes in the expression levels of energy
metabolism-related genes: glycolysis-related genes,
fer-mentation-related genes, respiratory electron transport
chain-related genes, and ROS scavenging-related genes
According to the Pasteur effect, by which aerobic
oxidation can inhibit glycolysis (alcohol-facient
fermentation) under aerobic circumstances, the
tricar-boxylic acid cycling process might be enhanced
follow-ing exposure to 10b, because of the marked decreases
in the expression levels of the genes participating in
glycolysis, including those coding for rate-limiting
enzymes (HXK2, PFK1, PFK2 and CDC19) Also,
sig-nificantly downregulated genes were also involved in
fermentation (e.g ADH1, ALD5 and PDC11) and
ROS scavenging (e.g GPX2) Together, these changes
mean that endogenous ROS generation might be
strongly augmented, as shown in Fig 7 Interestingly,
we also found that the expression levels of two ROS
scavenging-related genes (SOD5 and GPX1) and two
fermentation-related genes (ADH3 and ADH5) were
notably increased This might be the result of feedback
control in response to high ROS levels
Mitochondrial oxidative phosphorylation is a major ATP synthetic pathway in eukaryotes, where electrons liberated from reducing substrates are delivered to O2 via a chain of respiratory proton pumps These pumps (complexes I, III and IV) establish a proton electro-chemical gradient (proton concentration gradient and
DWm) across the inner mitochondrial membrane to store energy for the production of ATP Endogenous ROS are derived from mitochondrial respiratory chain electron leakage The main source of ROS in mitochon-dria is the ubisemiquinone radical intermediate (QHÆ), which is formed during the ubiquinone cycle at the Qo site of complex III [28–30] Complex I is also a source
of ROS, although the mechanism of generation is less clear than that of complex III In vitro, electrons enter-ing complex II can flow backwards through complex I
to make ROS [31] Experimentally, a large increase in ROS formation is often seen in the condition known as reverse electron flow [32] In this study, we found that 10b treatment promoted ROS generation and decreased
DWmand ATP production in mitochondria of C albi-cans The results of our further investigation showed that 10b inhibited complex III activity It is therefore presumed that 10b inhibited mitochondrial complex III activity, causing a reverse flow of electrons from com-plex II to comcom-plex I, resulting in ROS augmentation Simultaneously, 10b blocked electron transport in the mitochondrial respiratory chain and decreased DWm and ATP production (Fig 8)
AA is a known antioxidant, and interactions between AA and ROS may attenuate the oxidant effect
of ROS and alleviate ROS-induced damage to the organism [33] In this study, addition of AA signifi-cantly reduced the antifungal activity of 10b against
C albicans, indicating that the antioxidant could alle-viate the oxidative damage caused to the organism by endogenous ROS, allowing C albicans to survive 10b treatment These results also imply that endogenous ROS augmentation might be a major mechanism of the activity of 10b against C albicans
The results of the present study demonstrate that 10b treatment could augment the production of endogenous ROS via three different mechanisms in
C albicans: (a) providing more electrons for the mito-chondrial respiratory chain by enhancing the tricarbox-ylic acid cycle; (b) attenuating ROS scavenging; and (c) enhancing the reverse flow of electrons from complex II to complex I by inhibiting complex III activity Increased ROS production contributes to the antifungal effect by means of strong oxidative damage
to the organism This biochemical process might
be involved in the mechanism of action of 10b against
C albicans
0
20
40
60
80
100
120
**
*
*
Fig 6 Effect of AA on 10b-induced colony inhibition in C albicans.
The concentrations of AA were 2.5, 5 and 10 m M , and that of 10b
was 9 lgÆmL)1 Cells were incubated at 30 C under constant
shak-ing (200 r.p.m.) for 48 h, and the colonies were counted The rate
of cell survival is represented as a percentage of the survival rate
for cells not treated with 10b Data represent the mean ± standard
deviations for three independent experiments Statistically
signifi-cant differences (as determined by Student’s t-test, as compared
with 10b treatment alone): *P < 0.01; **P < 0.05.
Trang 6Experimental procedures
Strains and culture
C albicans SC5314 was used throughout this study The
antifungal reagents used in the present study included 10b
(Department of Medicinal Chemistry, School of Pharmacy
of the Second Military Medical University, Shanghai,
China), MCZ (Pfizer-Roerig Pharmaceuticals, New York,
NY, USA) and stock solutions of various concentrations
in dimethylsulfoxide C albicans cells were propagated in
yeast–peptone–dextrose) medium [1% (w⁄ v) yeast extract,
2% (w⁄ v) peptone, and 2% (w ⁄ v) dextrose]
RNA isolation and microarray hybridization
The number of cells was adjusted to 1.0· 106
colony-form-ing units (CFUs)ÆmL)1in yeast–peptone–dextrose medium,
divided into two parts: one was exposed to 3 lgÆmL)110b,
and the other was used as the control The cells were then
grown at 30C under constant shaking (200 r.p.m.) for an
additional 5 h for the control and 9 h for the 10b group,
until they reached 0.6–0.9· 107
CFUsÆmL)1, as described
by Hughes [34] Cells were then collected by centrifugation
at 3000 g for 5 min at room temperature, and frozen in liquid nitrogen We chose this 10b concentration because
it had an obvious inhibitory effect on C albicans and allowed for recovery of a sufficient cellular mass for RNA extraction
Total RNA was isolated by the hot phenol method, and purified with a NecleoSpin Extract II kit (Machery-Nagel Corp., Du¨ren, Germany) [35] A 7925 C albicans genome 70-mer oligonucleotide microarray was obtained from Capi-talBio Corporation (Beijing, China) A 1-lg sample of total RNA was used for preparing fluorescent dye-labeled cDNA
by linear mRNA amplification [36] A DNAÆDNA hybrid-ization protocol was used to replace RNAÆDNA hybridiza-tion, to reduce cross-hybridization [37] The labeled cDNAs were dissolved in 80 lL of hybridization solution [3· SSC, 0.2% (w⁄ v) SDS, 5 · Denhardt’s solution, 25% (v ⁄ v) form-amide], and denatured at 95C for 3 min before hybridiza-tion A sample of the mixed hybridization buffer was placed onto a microarray slide and covered with a glass coverslip Hybridization was performed with a BioMixer II
Glucose
HXK2
GPX2
GSSG
Glycolysis
PFK2
Fru6P
2 O or ROH + H 2 O
PFK1 Fru(1,6)P2
e –
e – e –
PFK2
GrnP Gri(1,3)P2
Pyruvate
Inner membrane
CX Τ
CX Φ
NADH
e –
GA3P
PEP
ENO1
GPM1
GA2P
Aldehyde
Acetic acid Ethanol
TCA cycle
Mitochondrion
y
Acetic acid
Cytoplasm
Fermentation
ROS
Fig 7 Central carbon metabolism in C albicans SC5314 The gray rectangles indicate low expression genes coding for metabolic enzymes
of participated in energy metabolic process after 10b treatment The black ellipse or circle indicate augmented the tricarboxylic acid (TCA) cycling process and endogenous ROS generation after 10b treatment Glc6P, glucose 6-phosphate; Fru6P, fructose 6-phosphate; Fru(1,6)P2, fructose 1,6-bisphosphate; GraP, glyceraldehyde 3-phosphate; GrnP, dihydroxyacetone phosphate; Gri(1,3)P2, 1,3-bisphosphoglycerate; GA3P, 3-phosphoglyceric acid; GA2P, 2-phosphoglyceric acid; PEP, phosphoenolpyruvate; LAC, lactic acid; CX I–V, complexes I–V; Q, ubiqui-none cycle; Cyt c, cytochrome c.
Trang 7(CapitalBio Corp.) After hybridization, the slides were
washed with washing solution 1 (2· SSC, 0.2% SDS) and
then with washing solution 2 (2· SSC) at 42 C for 4 min
Self-hybridization of the control sample was used to
evalu-ate the system noise
Microarray data processing
The microarrays were scanned with a LuxScan
10KAscan-ner (CapitalBio Corp.) at two wavelengths to detect
emis-sions from both Cy3 and Cy5 The images obtained were
analyzed with luxscan 3.0 software (CapitalBio Corp.)
Normalization was performed on the basis of a Lowess
program The Cy5⁄ Cy3 ratio was calculated for each
loca-tion on each microarray To minimize artefacts arising from
low expression values, only genes with raw intensity values
of > 800 counts for both Cy3 and Cy5 were chosen for
analysis Significance Analysis of Microarrays software
(sam) was used to identify significantly differentially
expressed genes Genes with a false discovery rate < 5%, a
q-value <1% and variation of at least two-fold in C
albi-cansSC5314 following 10b exposure were identified as
sig-nificantly differentially expressed genes in two independent
experiments
Differentially expressed genes were clustered
hierarchi-cally by gene cluster 3.0 (Stanford University) DNA
sequences were annotated on the basis of the results of
blastnand blastx searches with the sequencing database
of Stanford University (Palo Alto, CA, USA)
(http://www-sequence stanford.edu⁄ group ⁄ Candida), GenBank (http://
www.ncbi.nlm.nih.gov/BLAST/), and the CandidaDB
data-base (http://genolist.pasteur fr⁄ CandidaDB ⁄ ) All of the
array data have been deposited in the NCBI Gene
Expres-sion Omnibus (http://www.ncbi.nlm.nih.gov) and are acces-sible through Gene Expression Omnibus series accession number GSE19552
Microarray data analysis
We used cgd gene ontology slim mapper to cluster these differentially expressed genes into particular categories by choosing GO Set Name ‘Process’ (http://www.candidagenome org/cgi-bin/GO/go TermMapper) The specific functions
of individual genes were determined from the Candida Genome database (http://www.candidagenome.org/)
Quantitative real-time RT-PCR assay
Real-time RT-PCR was used to confirm the microarray results for changes in gene expression First-strand cDNAs were synthesized from 1 lg of total RNA in a 20-lL reac-tion volume, using the cDNA synthesis kit for RT-PCR (TaKaRa Biotechnology, Dalian, China) Real-time PCR reactions were performed with SYBR Green I (TaKaRa), using the ABI 7500 Real-Time PCR System (Applied Bio-systems, CA, USA) Gene-specific primers were designed with discovery studio gene software (Accelrys, San Diego, U.S.A) The thermal cycling conditions comprised
an initial step at 95C for 1 min, followed by 40 cycles at
95C for 10 s, 55 C for 20 s, and 72 C for 30 s Changes
in SYBR Green I fluorescence in every cycle were monitored
by the system software, and the threshold cycle (CT) was measured With 18S rRNA as the internal control, gene expression levels of C albicans SC5314 cells treated with 10b relative to those without treatment were calculated with the formula 2)CT Primer sequences are listed in Table 1
ΔΨm
H +
Cyt c
H +
H +
ROS
Intermembrane space/cytoplasm
Inner
e –
e – e –
e
-ROS
+ membrane C
CX Τ
F
Q
QH 2 Q
NADH
e –
e
-e –
CX V –
F 1
TCA
NAD + NADH
10b
O 2 TCA
cycle
10b
ATP
+
–
Fig 8 Proposed mechanism of action of ROS augmentation induced by 10b Stimulation of the tricarboxylic acid cycle (TCA) by 10b treat-ment enhances electron flow into the mitochondrial respiratory chain, and generates a reverse flow of electrons from complex II to complex I by inhibiting complex III, which inhibits electron transport and causes a collapse of the proton gradient across the mitochondrial inner membrane These events enhance ROS generation and decrease DW m and ATP production Dashed lines indicate the subdued meta-bolic process CX I–V, complexes I–V; Q, ubiquinone cycle; Cyt c, cytochrome c.
Trang 8Measurement of ROS production
The endogenous amount of ROS was measured by a
fluori-metric assay with 2,7-dichlorofluorescin diacetate
(DCFH-DA) (Molecular Probes, Eugene, OR, USA) Briefly, cells
were first adjusted to an attenuance at 600 nm of 1.0 in
40 mL of NaCl⁄ Pi, treated without and with 2.5, 5 and
10 mm AA, and grown at 30C under constant shaking
(200 r.p.m.) for 1 h After incubation with 1, 3 and
9 lg mL)1 10b and MCZ at 30C under constant shaking
(200 r.p.m.) for 2 h, cells were collected by centrifugation
at 3000 g for 5 min, suspended in NaCl⁄ Pi, and adjusted to
an attenuance at 600 nm of 1.0 in 10 mL of NaCl⁄ Pi DCFH-DA 20 lm in NaCl⁄ Piwas added and incubated at
30C under constant shaking (200 r.p.m.) for 9 h The flu-orescence intensity (FI) of the resuspended cells were detected on a Thermo Scientific Varioskan Flash (BMG, Labtech, Offenburg, Germany), with an excitation wave-length of 488 nm and an emission wavewave-length of 525 nm ROS production was calculated by subtracting the FI for cells not treated with DCFH-DA from the FI for cells treated with DCFH-DA
Functional analysis on mitochondria – DWm, complex III and complex I activity and intracellular ATP content
For DWm measurement, cells treated without and with 10b
at 30C under constant shaking (200 r.p.m.) for 9 h were washed and adjusted to 1· 106CFUsÆmL)1with NaCl⁄ Pi After being treated with 10 lgÆmL)1 5,5¢,6,6¢-tetrachloro-1,1¢,3,3¢-tetra-ethylbenzimidazolcarbocy-anine iodide (JC-1; Molecular Probes, Eugene, OR, USA) at 30C under con-stant shaking (200 r.p.m.) for 15 min [38], cells were washed with NaCl⁄ Piand analyzed on a Thermo Scientific Varioskan Flash with an excitation wavelength of 485 nm and an emission wavelength shifting from green ( 525 nm)
to red ( 590 nm) DWmwas determined from red FI⁄ green
FI ratio
For complex III and complex I activity evaluation, cells treated without and with 10b at 30C under constant shak-ing (200 r.p.m.) for 9 h were adjusted to 5· 108cells per
mL Mitochondria were isolated with the Yeast Mitochon-dria Isolation Kit (GenMed Scientifics INC., Arlington, U.S.A.) and lysed in EDTA buffer by ultrasound The mito-chondrial protein concentration was determined by the Brad-ford method [39] Complex III and complex I activity was determined by colorimetry, with the Mitochondria Com-plex III and ComCom-plex I Activity Assay Kit (GenMed Scienti-fics INC., Arlington, U.S.A.) The absorbance was measured
on the Thermo Scientific Varioskan Flash at a wavelength of
550 nm and two wavelengths of 340 nm and 380 nm One unit of complex III activity was defined as the amount of enzyme activity that oxidized 1 lmol of reduced ubiquinone per minute at 30C and pH 7.5, and one unit of complex I activity was defined as the amount of enzyme activity that oxidized 1 lmol of NADH per minute at 30C and pH 7.5 For intracellular ATP content determination, cells treated without and with 10b at 30C under constant shaking (200 r.p.m.) for 9 h were adjusted to 1· 106
CFUsÆmL)1
A 100-lL cell suspension was mixed completely with the same volume of BacTiter-Glo reagent (Promega Corpara-tion, Madison, WI, USA), and incubated for 10 min at room temperature Luminescent signals were determined on
a TD 20⁄ 20 luminometer (Turner Biosystem, Sunnyvale,
CA, USA), with a 1-s integration time per sample The
Table 1 List of primers used for real-time RT-PCR F, forward; R,
reverse.
Target
genes Primer pairs (5¢- to 3¢)
Amplicon size (bp)
R: TCGATAGTCCCTCTAAGAAGTG
150
R: AACTGGGTAATCCTTGTAG
184
R: ATTTCTCAACCGCACC
225
R: CTCTTGCCTTATCCTTT
162
R: CCAACCACCACAGGAT
197
R: AGATTCTGGGTCGTTTG
182
R: TCCAAGACTGGGAATGT
217
R: AATACGGGGAAAGTCAC
164
R: ATTACCTTGAGGAGCA
185
R: AATGGGTAGACACCTCTG
163
R: TTGGATGGATAAGAGGC
132
R: TTCGTAAACGGCATAA
142
R: CCAACCCTAATCTGTCG
177
R: TCAGGAGGCACAAACT
132
R: TTGGCAAAGTATCGTCT
160
R: TTGGCAAAGTATCGTCT
110
R: CTGGGAAGTAAGGGTT
280
R: AAATGGAATGGCAACA
183
R: CGAAAGTTCCACCTAAT
103
R: TTCACTGATTGGCATTT
137
Trang 9control tube without cells was used to obtain a value for
background luminescence The signal-to-noise ratio was
cal-culated: signal-to-noise ratio = (mean of signal) mean of
background)⁄ standard deviation of background A
stan-dard curve for ATP increments (from 1 lm to 10 pm) was
constructed Signals represented the mean of three separate
experiments, and the ATP content was calculated from the
standard curve
Colony formation assay
Drug sensitivity and the effect of AA on 10b fungicidal
activity were determined by a colony formation assay based
on the macrodilution reference method (M27-A) of the
Clinical and Laboratory Standards Institute (formerly the
National Committee for Clinical Laboratory Standards)
Briefly, cells were adjusted to 1· 106CFUsÆmL)1 in
RPMI-1640 medium buffered with Mops, treated without
and with 2.5, 5 and 10 mm of AA at 30C under constant
shaking (200 r.p.m.) for 1 h, and then incubated with 10b
for 48 h under the same conditions After incubation,
20 lL of appropriately diluted solution was used for colony
formation on Sabouraud dextrose agar (Becton Dickinson
Microbiology Systems, Oakvile, ON, Candida)
Statistical analysis
The statistical significance of differences was determined
with Student’s t-test A P-value of < 0.05 was considered
to indicate significance
Acknowledgements
We thank W A Fonzi for kindly offering the isolate
of C albicans SC5314, and Y.-J Zhou for synthesizing
the 10b used in this study.This work was supported by
the National Natural Science Foundation of China
(30825041, 30500628 and 30630071), the National
Basic Research Project (2005CB523105), National
High Technology Research, and Development
Pro-gram 863 of China (2008AA02Z302)
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Supporting information
The following supplementary material is available: Table S1 Selected genes that are downregulated in 10b-grown C albicans SC5314 as compared with growth without treatment, determined in two indepen-dent experiments