Mitochondrial Signs and Subcellular Imaging Provide Insight into the Antifungal Mechanism of Carabrone against Gaeumannomyces graminis var. tritici

Article abstract of DOI:10.1021/acs.jafc.7b03913

Carabrone, a botanical bicyclic sesquiterpenic lactone, has broad-spectrum antifungal activity and is particularly efficient against the devastating phytopathogen Gaeumannomyces graminis var. tritici (Ggt). The antifungal mechanism of carabrone against Ggt, however, remains unclear. The main objective of this study was to investigate the subcellular localization of carabrone in Ggt to gain a better understanding of its mechanism of action. When Ggt was exposed to carabrone (EC₅₀ value of 28.45 μg/mL) for 7 days, a decline in mitochondrial concentration together with some obvious alternations in mitochondrial structure, including hazy outlines, medullary transitions, excess accumulation of unclear settlings, and vacuolar degeneration, were observed, indicating that carbrone may act on the mitochondria directly. A fluorescent conjugate (TTY) was thus designed and synthesized as a surrogate of carabrone that possessed comparable antifungal activity against Ggt (EC₅₀ of 33.68 μg/mL). Additionally, a polyclonal antibody specific to carabrone and with a high titer (256?000) was also prepared by immunizing mice. Subsequently, two imaging techniques, the use of the fluorescent conjugate (FC) and immunofluorescence (IF), were applied to determine the subcellular localization of carabrone. Both FC and IF fluorescent signals demonstrated its mitochondrial localization with a Pearson's coefficient of 0.83 for FC and 0.86 for IF. These results imply that carabrone exerts its antifungal activity against Ggt by interfering with mitochondrial function.

Full text of DOI:10.1021/acs.jafc.7b03913

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Article
Mitochondrial Signs and Subcellular Imaging Imply the Antifungal
Mechanism of Carabrone against Gaeumannomyces graminis var. tritici
Lanying Wang, Yunfei Zhang, Delong Wang, Mei Wang, Yong Wang, and Juntao Feng
J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b03913 • Publication Date (Web): 12 Dec 2017
Downloaded from http://pubs.acs.org on December 18, 2017
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Mitochondrial Signs and Subcellular Imaging Imply the
Antifungal Mechanism of Carabrone against Gaeumannomyces
graminis var. tritici
†,‡
†
†
†
Lanying Wang, Yunfei Zhang, Delong Wang, Mei Wang, Yong
†
,†,#
Wang, and Juntao Feng*
†
Research and Development Center of Biorational Pesticide, Northwest A&F
University, Yangling 712100, Shaanxi, China
‡
Institute of Tropical Agriculture and Forestry, Hainan University, Haikou 570228,
Hainan, China
#
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Engineering and Research Center of Biological Pesticide of Shaanxi Province,
Yangling 712100, Shaanxi, China
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ABSTRACT: Carabrone, a botanical bicyclic sesquiterpenic lactone, has broadꢀspectrum
antifungal activities, and is particularly efficient against the devastating phytopathogen
Gaeumannomyces graminis var. tritici (Ggt). The antifungal mechanism of carabrone
against Ggt, however, remains unclear. The main objective of this study was to investigate
the subcellular localization of carabrone in Ggt to make a better understanding of the
mechanism of action. When Ggt was exposed to carabrone (EC50 value, 28.45 ꢁg/mL) for
7 days, the decline of mitochondrial concentration, together with some obvious alternations
in the mitochondrial structure including hazy outline, medullary transition, excess
accumulation of unclear settlings, and vacuolar degeneration were observed, indicating that
carbrone may act on the mitochondria directly. A fluorescent conjugate (TTY) was thus
designed and synthesized as a surrogate of carabrone, which possessed comparable
antifungal activity against Ggt (EC50, 33.68 ꢁg/mL). Meanwhile, polyclonal antibody
specific to carabrone with a high titer (256,000) was also prepared by immunizing mice.
Subsequently, two imaging techniques, fluorescent conjugate (FC) and
immunofluorescence (IF), were applied to determine the subcellular localization of
carabrone. Both FC and IF fluorescent signals demonstrated its mitochondrial localization
with a Pearson’s coefficient of 0.83 for FC, and 0.86 for IF. These results imply that
carabrone exerts its antifungal activity against Ggt by interference with mitochondrial
functions.
KEYWORDS:
carabrone,
fluorescent
conjugate,
polyclonal
antibody,
immunofluorescence, subcellular localization, mitochondria, Gaeumannomyces graminis
var. tritic
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INTRODUCTION
Takeꢀall, a worldwide soilꢀborne disease of wheat caused by Gaeumannomyces graminis
var. tritici (Ggt), severely affects global wheat production, which can bring about serious
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, 2
yield and economic losses in epidemic years. The traditional control methods are often
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, 3
limited in practical application by lack of resistant cultivar, inefficiency of crop rotation
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and tillage management measures, poor robustness of takeꢀall decline, as well as
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environmental concerns and fungicide resistance issues related to chemical fungicides.
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Biocontrol agents and botanical fungicides have been recognized as safer and more
effective alternatives than conventional strategies. To date, microorganisms such as
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, 9
10, 11
Pseudomonads
and Bacillus species
have been developed as biocontrol agents
against takeꢀall. However, natural chemical compounds effective against Ggt is rarely
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reported.
Carabrone (Figure 1), a bicyclic sesquiterpenic lactone isolated from the fruits of
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Carpesium abrotanoides, is widely distributed in feverfew and other plant species, and
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exhibits diverse bioactive properties including antibacterial,
and antitumor activities.
It has been reported that carabrone possessed a broadꢀspectrum antifungal activity, and that
it was more effective against Ggt than other phytopathogens tested, with a EC50 value of
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28.45 ꢁg/mL. Although carabrone has a great potential to be developed as an effective
antifungal agent for takeꢀall control, the antifungal mechanism of carabrone against Ggt
still remains unclear.
A vast array of sesquiterpene lactones depend on a αꢀmethyleneꢀγꢀbutyrolactone
structural motif to exert their biological activities, which may play a pivotal role in
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mediating the interaction with unidentified target molecule(s).
In addition, a range of
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natural lactones, such as britannin, arucanolide, parthenolide, costunolide, and
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isocostunolide have been proved to involve in intracellular mitochondriaꢀrelated
pathways to induce apoptosis of cancer cells (Figure 1). Given the identical
αꢀmethyleneꢀγꢀbutyrolactone substructure, we speculated that carabrone might also exert
its antifungal activity against Ggt via targeting the mitochondria (Figure 1). Our previous
studies also indicated that carabrone inhibited not only the intracellular oxidation process,
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but also the activity of respiratory chain complexes IꢀV in Ggt.
evidences supporting this hypothesis are still lacking.
Nonetheless, direct
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Imaging techniques such as immunofluorescence (IF)
and synthesis of fluorescent
are widely used to investigate the subcellular distribution of bioactive
molecules with high spatial and temporal resolution, and the action mechanisms of the
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conjugate (FC)
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corresponding molecules could be inferred based on these techniques.
In order to verify
our hypothesis (Figure 1) proposed above, we first examined the changes in mitochondrial
ultrastructure and concentration of Ggt hyphae exposed to carabrone. Subsequently, we
designed and synthesized a fluorescentꢀlabeled carabrone analog TTY, and generated the
polycolonal antibody against carabrone. After investigating their properties suitable for cell
imaging, they were used to image the subcellular localization of carabrone in Ggt by IF
and FC techniques.
MATERIALS AND METHODS
General Information. The carabrone compound characterized in this study was isolated
from Carpesium macrocephalu and purified (> 98% in purity) in our laboratory. NMR
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spectra were recorded on a Bruker AVANCE III 500 spectrometer (Germany) with
tetramethylsilane as internal standard. The mass spectra (MS) of new compounds were
obtained by a LCQ Fleet mass spectrometer (USA). NꢀMethyl anthranilic acid (NAA,
98%), carboxymethoxylamine hemihydrochloride (99%), NꢀHydroxysuccinimide (NHS,
98%), ethylenediamine (99%), 4ꢀdimethylaminopyridine (DMAP, 98%), N,
NꢀDicyclohexylcarbodiimide
(DCC,
97%),
1ꢀ(3ꢀDimethylaminopropyl)ꢀ3ꢀethylcarbodiimide hydrochloride (EDC, 98%), isobutyl
chloroformate (98%), tributylamine (97%), and tetramethylbenzidine (TMB, 98%) were
purchased from Aladdin Co. Ltd. (Shanghai, China). Bovine albumin (BSA), ovalbumin
(OVA), incomplete Freund’s adjuvant (IFA), complete Freund’s adjuvant (CFA), tweenꢀ20,
tritonXꢀ100, parafomaldehyde, HRPꢀlabeled Goat AntiꢀMouse IgG(H+L), antiꢀMouse IgG
(whole molecule)ꢀFITC, cellulase, driselase, and lyticase were purchased from
SigmaꢀAldrich (USA). MitoTrackerR Green FM (M7514), and MitoTrackerR Red
CMXRos (M7512) were purchased from Invitrogen (USA). RedDot™1 (200X) was
obtained from Biotium (USA). All organic solvents were commercial AR solvents and
were purified when necessary. Silica gels for TLC and column chromatography were
obtained from Qingdao Haiyang Chemical Co. Ltd (Qingdao, China).
Synthesis
of
Fluorescent-labeled
Carabrone
(TTY).
2-(((4-(5a-methyl-3-methylene-2-oxooctahydro-2H-cyclopropa[f]benzofuran-5-yl)butan-2-
ylidene) amino)oxy)acetic acid (HH1). HH1 was prepared according to the reported
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method with some modifications. For details, carboxymethoxylamine hydrochloride
(196.82 mg, 2.4 mmoL, 1.2 equiv) and sodium acetate (1.2 equiv) were successively added
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to a solution of carabrone (1.0 equiv) in 20 mL 95% ethanol. The reaction mixture was
stirred at room temperature under nitrogen for 12 h. After that, the resultant mixture was
filtered, evaporated, and purified by column chromatography on silica gel (80ꢀ100 mesh)
using petroleum ether/ethyl acetate/acetic acid (2/1/0.05, v/v/v) as the eluate to yield HH1.
1
Colorless oil: yield 90.3%. H NMR (500 MHz, CD
3
OD) δ 6.17 (d, J = 2.0 Hz, 1H), 5.68
(d, J = 2.0 Hz, 1H), 4.53 (d, J = 11.5 Hz, 2H), 3.30 – 3.19 (m, 1H), 2.50 (t, J = 7.6 Hz, 1H),
2.45 – 2.35 (m, 1H), 2.24–2.32 (m, 3H), 1.90 (d, J = 25.2 Hz, 3H), 1.66 – 1.56 (m, 1H),
1.51 (dq, J = 14.8, 7.5 Hz, 1H), 1.12 (dd, J = 10.8, 5.6 Hz, 4H), 0.99 (m, 2H), 0.62 – 0.52
13
(m, 1H), 0.51 – 0.39 (m, 1H); C NMR (125 MHz, MeOD) δ 171.31, 159.94, 159.44,
139.72, 121.69, 76.33, 69.43, 37.61, 36.82, 35.03, 33.95, 30.36, 25.60, 22.97, 16.55, 13.15;
+
ESIꢀMS, m/z 322.2 [M+H] .
N-(2-(2-(((4-(5a-methyl-3-methylene-2-oxooctahydro-2H-cyclopropa[f]
benzofuran-5-yl)butan-2-ylidene)amino)oxy)acetamido)ethyl)-2-(methylamino) benzamide
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(TTY). The synthetic procedures of TTY were modified according to the literature.
Nꢀmethyl anthranilic acid (TY, 302.3 mg, 2 mmoL, 1 equiv) was dissolved in 20 mL dry
CH Cl , to which NSH (1.5 equiv) and catalytic amount of DMAP (0.1 equiv) were added.
After cooling to 0°C, a solution of DCC (1.5 equiv) in 10 mL anhydrous CH Cl was
2
2
2
2
added dropwise into the mixture. The reaction mixture was stirred at 0°C for 45 min and
then stirred at room temperature for another 16 h. The mixture was filtered and the filtrate
was washed with 0.05 N HCl (10 mL × 2) and brine. The organic layer was dried over
anhydrous Na
2
SO
4
, filtered and evaporated by a rotary evaporator to yield 1. Compound 1
obtained was dissolved in 20 mL dry CH
2
Cl and the resulting solution was added
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2 2
dropwise to 10 mL anhydrous CH Cl with 41.33 mmoL of ethylenediamine (2.75 mL)
over 30 min. The mixture was stirred at room temperature under nitrogen for 24 h, and
then filtered and washed with brine (100 mL × 3). The organic layer was dried over
anhydrous Na SO , filtered, concentrated, and purified by chromatography on silica gel
2
4
(80ꢀ100 mesh) using dichloromethane/ethanol/ammonium hydroxide (30/1/0.05, v/v/v) as
the eluate to yield HH2. HH1 (250 mg, 0.78 mmoL, 1 equiv), EDC (1.2 equiv) and DMAP
(0.1 equiv) were dissolved in anhydrous CH
2
Cl
2
(30 mL) and stirred at 0°C for 30 min.
Cl (10 mL) was added dropwise to
Then, a solution of HH2 (1.5 equiv) in anhydrous CH
2
2
the mixture within 30 min. The reaction mixture was stirred at room temperature for
another 24 h and then filtered, concentrated, and purified by chromatography on silica gel
(80ꢀ100 mesh). Elution with petroleum ether/ethyl acetate/ammonium hydroxide (1/5/0.05,
v/v/v) gave the pure compound TTY. Slightly yellow oil: yield 61.7%. The fluorescent
properties of compound TTY in vitro were monitored using a Fꢀ4500 fluorescence
spectrophotometer (Hitachi, Japan) with the scanning mode, 250ꢀ400 nm for excitation
1
spectrum, and 415ꢀ800 nm for emission spectrum. H NMR (500 MHz, CDCl
3
) δ 7.56 (s,
1H), 7.41 (dd, J = 7.8, 1.1 Hz, 1H), 7.33 (t, J = 7.8 Hz, 1H), 7.29(s, 1H), 7.28(s, 1H), 6.67
(d, J = 8.4 Hz, 1H), 6.61 (t, J = 7.5 Hz, 1H), 6.25 (d, J = 2.3 Hz, 1H), 5.57 (d, J = 2.3 Hz,
1H), 4.85ꢀ4.69 (m, 1H), 4.52 (s, 2H), 3.56 (s, 3H), 3.16 (dt, J = 11.6, 9.0 Hz, 1H), 2.87 (s,
3H), 2.32 (ddd, J = 20.1, 13.9, 6.4 Hz, 2H), 2.25ꢀ2.18 (m, 2H), 1.93 (s, 3H), 1.54 (dt, J =
14.3, 7.1Hz, 1H), 1.48ꢀ1.37 (m, 1H), 1.28 (t, J = 7.1 Hz, 1H), 1.07 (s, 3H), 1.05ꢀ0.88 (m,
1
3
3
2H), 0.41 (dt, J = 11.2, 5.7 Hz, 1H), 0.38ꢀ0.32 (m, 1H); C NMR (125 MHz, CDCl ) δ
171.69, 170.72, 170.63, 160.08, 150.60, 139.09, 132.98, 127.59, 122.72, 114.69, 114.46,
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111.04, 75.72, 72.45, 40.26, 39.59, 37.52, 37.10, 35.64, 33.89, 30.61, 29.68, 25.87, 22.83,
+
18.36, 17.09, 14.56; ESIꢀMS, m/z 497.3 [M+H] .
Preparation of Immunogen and Coating Antigen. Immunogen (TꢀBSA) was
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synthesized according to the method described by Wang et al. with slight modification.
Hapten (HH1, 15.6 mg, 0.048 mmoL) was dissolved in 1.0 mL of dry DMF containing
0.28 mmoL of NSH. The mixture was stirred for 15 min at room temperature and 0.20
mmoL of DCC was added. After stirring in the dark for 5 h, the reaction mixture was
centrifuged (10,000 rpm, 10 min, 4°C) to remove the precipitate. The resulting supernatant
was added slowly to 5 mL carbonate buffer (50 mM, pH 9.6) with 50 mg BSA under
magnetic stirring at 0°C within 30 min. After dropping, the reaction continued for 6 h
under the same conditions and purified by dialyzing extensively with phosphate buffer
saline (PBS, 10 mM, pH 7.4) for 3 days at 4°C. The purified TꢀBSA solution was
lyophilized and kept at ꢀ20°C for use.
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The preparation of coating antigen (TꢀOVA) was according to Gendloff et al.’s method
with some revisions. Briefly, 15.6 mg of HH1 was dissolved in 1 mL dry DMF and cooled
to 0°C. To this, 77 ꢁL triꢀnꢀbutylamine and 22 ꢁL isobutyl chloroformate were added and
the mixture was stirred at room temperature for 5 h. After cooling to 0°C, the mixture was
added dropwise to a stirring mixture solution of DMF/water (10 mL, 1/4, v/v) containing
88 mg OVA within 30 min. Under the constant conditions, the reaction was maintained for
another 6 h. The subsequent procedures were identical to that of TꢀBSA described above.
Haptenꢀprotein conjugates, TꢀBSA and TꢀOVA, were characterized by SDSꢀPAGE, and
UV spectra. SDSꢀPAGE analysis of conjugates was performed by a 3% stacking gel and a
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10% separating gel with Coomassie blue staining. UV spectra were obtained by spectral
scanning (240ꢀ320 nm) of the proteins and their conjugates with a UVꢀ3310
spectrophotometer (Hitachi, Japan).
Preparation of Polyclonal Antibody. Female BALB/c mice were obtained from the
Experimental Animal Research Center, Fourth Military Medical University (Shaanxi,
China), with an average body weight of 20 g. They were housed under standard laboratory
conditions with free access to drinking water and a commercial pellet diet. Animal
manipulations were carried out in compliance with the Animal Management Rules of the
Ministry of Health of China (No. 55, 2001). Six female BALB/c mice of 6 weeks old were
immunized with antigen following the immunizing protocol described by Leenaars and
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Hendriksen . Before immunization, blood was collected by tail bleeding to prepare control
serum. Initial immunization was carried out by multiple subcutaneous injections with an
emulsion of physiological brine solution containing 50 ꢁg of TꢀBSA and complete Freund’s
adjuvant (1/1, v/v) for each mouse. Two weeks later, animals were boosted with 50 ꢁg of
TꢀBSA in incomplete Freund’s adjuvant per mouse for two times at one week intervals.
One week after the third inoculation, each mouse was given a single intraperitoneal
injection with 100 ꢁg of TꢀBSA in a physiological brine solution. After one week, the
blood samples were harvested by removing the eyeballs and titers of antisera were
monitored by indirect enzymeꢀlinked immunosorbent assays (ELISAs) described by
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Manclús and Montoya to evaluate the immune responses. Antiserum with highest titer
was purified by affinity column chromatography on Sepharoseꢀ4B gel to prepare
polyclonal antibody. Antiserum titer was defined as the reciprocal of the highest dilution
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which gave an absorbance of about 1.0. The concentration of the purified polyclonal
antibody was measured using a NanoVue Plus at 280 nm, and the sample was stored at
−20°C for further use.
Antifungal Bioassay. The antifungal activity of TTY against Ggt was determined by the
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agar dilution method described by Rios et al. Stocks of carabrone and TTY in DMSO
with different concentrations were prepared in advance. Stock solutions (50 ꢁL) were
added to molten PDA (50 mL) at low temperature (45ꢀ50°C), respectively. After sufficient
mixing, the solutions were poured into 90ꢀmm petri dishes immediately to a thickness of
2ꢀ3 mm, with different concentrations of carabrone or TTY (6.25, 12.50, 25.00, 50.00, and
100.00 ꢁg/mL). PDA supplemented with DMSO served as the control. After plating, a
4ꢀmm diameter mycelial disc from the actively growing colony front was then placed with
the inoculum side down in the center of each treatment plate, aseptically. Plates were then
incubated in the dark at a constant temperature of 25°C for 7 days. All experiments were
conducted in a sterile environment and performed in triplicate. Mean growth values were
measured and subsequently converted into an inhibition rate of mycelial growth in relation
to control treatment according to the following formula:
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c t c
Inhibition rate (%) = [(M −M )/ (M −0.4)] ×100
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c t
where M and M represent the mycelial growth diameter of control and treatment group,
respectively. The EC50 values (effective dose for 50% inhibition) were calculated
statistically by probit analysis using the probit package of SPSS 23.0 software (SPSS Inc.,
USA).
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Effects of Carabrone on Hyphal Morphology and Ultrastructure. The effects of
carabrone on hyphal morphology and ultrastructure were investigated using the reported
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method of Shao et al. A prepared mycelial agar disc from a 5ꢀdayꢀold culture was
inoculated in the center of PDA plate with EC50 value (28.45 ꢁg/mL) of carabrone and
incubated at 25°C for 7 days in dark. PDA plates without carabrone treatment were used as
the control.
For scanning electron microscope (SEM) observation, mycelial discs (3 mm × 5 mm × 5
mm) were fixed with 2.5% glutaraldehyde in 0.1 M phosphate buffer (pH 7.2) overnight at
4°C. After fixation, mycelial discs were gently washed with 0.1 M phosphate buffer (pH
7.2) for 6 times (15 min each). Then, the fixed samples were dehydrated in a graded
ethanol series (once at 30, 50, 70, 80, 90% and three times at 100%, v/v) for 15 min in each.
After the dehydration period, each specimen was dipped into pure isoamyl acetate for
replacement two times, each for 30 min. At last, the samples were dried by supercritical
carbon dioxide, goldꢀcoated by a E1010 sputter coating machine (Hitachi, Japan) for 60 s,
and then imaged using a JSMꢀ6360LV SEM (JEOL, Japan). No treated mycelial discs were
as the control.
For transmission electron microscopy (TEM) observation, the fixed mycelia for SEM
were postꢀfixed with 1% osmic acid for 2 h. Then, the samples were fully washed with 0.1
M phosphate buffer (pH 7.2) immediately followed by dehydration as described above.
Samples were dipped into epoxy propane two times (15 min each). After that, the
specimens were passed through the solution of epoxy resin and epoxy propane (1:1, v/v)
for 1 h and embedded in epoxy media at 55°C for 48 h. The ultrathin sections were
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prepared with a Ultramicrotome (LeicaꢀULTRACUT, Germany), contrasted with 2%
uranyl acetate followed by 2% lead citrate, and examined on a JEMꢀ1230 TEM (Hitachi,
Japan). At least three samples from the treated and control group were examined by TEM
and SEM, respectively.
Effect of Carabrone on the Mitochondrial concentration. The extraction of
mitochondria was according to the reported method by Tamura et.al. with some
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modifications. Fifteen prepared mycelial agar discs (4 mm) from a 7ꢀdayꢀold culture
were inoculated in 100 mL potato dextrose broth (potato infusion from 200 g/L, 20 g/L
dextrose) with EC50 (28.45 ꢁg/mL) or EC70 (49.97 ꢁg/mL) value of carabrone and
incubated at 25°C with 180 rpm for 7 days in dark. Mycelia were harvested through
filtration by sterile gauze and washed three times using 0.7% NaCl isotonic solution. After
freezeꢀdrying for 24 h, a certain quality of mycelium cells was grinded in liquid nitrogen
and the obtained powder was then resuspended in a fiveꢀfold volume of preꢀcooling
extraction buffer (10 mM KCl, 5 mM EDTA, 250 mM sucrose, 1.5 mg/mL BSA, 20 mM
HEPESꢀTris pH 7.2) for homogenization. The homogenate was centrifuged (1,500 rpm, 10
min, 4°C), and the supernatant was retained. The precipitate obtained was resuspended in
the same volume of extraction buffer and recentrifuged under the above conditions. The
supernatant was combined and centrifuged at 4°C (10,000 rpm, 20 min). The resulting
precipitate was washed with the extraction buffer without BSA. The final mitochondrial
fraction was suspended in a small volume of a cold buffer solution (250 mM sucrose, 20
mM HEPESꢀTris, pH 7.2) as the mitochondrial sample. All experiments were performed in
triplicate. The protein level of a mitochondrial sample was detected by the coomassie
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brilliant blue Gꢀ250 dyeꢀbinding method and used as the marker of the mitochondrial
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concentration .
Absorption Kinetics of TTY in Mycelial Cells. TTY was dissolved in an aqueous
solution (0.2% DMSO) to prepare the work solutions with different concentrations (0.3,
0.35, 0.4, and 0.8 ꢁM). Mycelial cells were inoculated in potatoꢀdextrose broth and
cultivated in darkness at 25°C on an Eberbach rotary shaker at 150 rpm for 72 h.
For absorption kinetics study, the prepared mycelia were washed three times with
ultrapure water and then surface water was removed by a filter paper. Mycelial cells (10
mg, wet weight) were placed on a glass slide and stained with the ready solution of TTY
(20 ꢁL) in the dark at room temperature for a designated time period (5, 10, 15, 30, 60, 90,
120, 150, 180, 210, and 240 min). After fully washing, the samples were observed with a
structured illumination microscopy (Observer Zl, Zeiss, Germany) excited at 365 nm with
the emission wavelength of 445 nm. NoꢀTTY treated sample was as control. Fluorescence
intensity per unit area of 90 hyphae in one visual field was counted by an Image J software
package and three visual fields were randomly selected for each treatment.
Stability of TTY in Mycelial Cells. Mycelial cells (500 mg, wet weight) were stained
with a TTY work sloution (1 mL, 0.4 ꢁM) in the dark at room temperature for 90 min.
After staining, the stained mycelial cells were fully washed to remove the free TTY and
then were kept under the same conditions for another 2 h. The obtained mycelial cells were
collected and grinded in liquid nitrogen. The sample was then extracted with methanol (2
mL, chromatographic grade) and centrifuged at 4°C (8,000 rpm, 5 min). The supernatant
was filtered by a millipore filter (0.22 ꢁm) for LCꢀHRMS analysis. Mycelial cells without
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TTY treatment were used as the control. The LCꢀHRMS analysis was performed on an AB
Sciex Triple TOF 5600+ System (USA) using a methanolꢀwater elution system (0ꢀ5 min,
10 vol% methanol in water; 5ꢀ10 min, 10 to 40% methanol; 10ꢀ15 min, 40 to 70%
methanol; 15ꢀ20 min, 70 to 90% methanol; and 20ꢀ25 min, 90 to 100% methanol). A C18
reversedꢀphase column was used for separation. The injection volume was 10 ꢁL, and the
flow rate was 1 mL/min.
Cell imaging. Stocks of carabrone (0.1 mM), TTY (0.4 mM), MitoTrackerR Green FM
(1 mM), and MitoTrackerR Red CMXRos (1 mM) were freshly prepared using DMSO as
solvent and diluted with ultrapure water before use. All the dyeing processes in this paper
were operated under dark condition. The Pearson’s colocalization coefficient was
calculated using a Colocalization Finder plugin of a free image processing software Image
J. The software and plugin are available on the website (https://imagej.nih.gov/ij/).
For FC imaging, mycelial cells (10 mg, wet weight) were washed three times with
ultrapure water, and then incubated with a mixture solution containing 0.4 ꢁM TTY and
100 nM MitoTrackerR Green FM at room temperature for 1 h. After rinsing with ultrapure
water to remove the free dyes, mycelial cells were reꢀdyed with RedDot™1 (1:200 dilution)
under a constant condition (30°C, 1 h). After fully washing, the sample was observed with
a structured illumination microscopy (Observer Zl, Zeiss, Germany) directly. TTY was
excited at 365 nm with the emission wavelength of 445 nm. Mitochondrial probe,
MitoTrackerR Green FM, was excited at 470 nm with the emission wavelength of 525 nm.
Excitation of RedDot™1 was carried out using 640 nm and the emission wavelength was
690 nm. Photos were collected using a Axio Visio Release 4.8.2 SP3 software.
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For IF imaging, mycelial cells prepared were first incubated with a carabrone solution
(0.1 ꢁM) at 25°C for 12 h. Then, the sample was washed with ultrapure water for three
times to drain off the free carabrone. After staining with MitoTrackerR Red CMXRos (100
nM) at 25°C for 2 h, mycelial cells were fixed with 2% paraformaldehyde for 40 min and
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the fixed cells were rinsed three times with MSB (PIPES, 50 mM; EGTA, 2 mM, MgSO ,
2 mM; pH 6.9) at 5 min intervels. The sample was then added to a mixed enzyme solution
(Cellulase/Driselase/Lyticase) for 5 min in the dark. After enzymolysis, the mycelial cells
were washed with PBS (10 mM, pH 7.2) for three times every 5 min and blocked in PBS
supplemented with BSA (5 mg/mL) for 30 min in the dark. The sample was stained
successively with primary antibody (1:1000 dilution in PBS, 37°C, 1 h), AntiꢀMouse IgG
(whole molecule)ꢀFITC (1:100 dilution in PBS, 37°C, 1 h), and washed with PBS as above
following each staining period. After immunofluorescence staining, the sample imaging
was performed on a confocal laser scanning microscopy equipped with Leica application
suite sdvanced fluorescence software (Leica TCS SP8, Germany) directly. MitoTrackerR
Red CMXRos: excitation wavelength, 561 nm; emission spectrum, 630 ꢀ 660 nm.
AntiꢀMouse IgG (whole molecule)ꢀFITC: excitation wavelength, 488 nm; emission
spectrum, 520 ꢀ 550 nm.
RESULTS AND DISCUSSION
Synthesis and Characterization. TTY. For fluorescence localization analysis, a
fluorescent carabrone conjugate, TTY, was designed and synthesized according to the
synthetic route illustrated in Scheme 1. Nꢀmethyl anthranilic acid (TY) was chosen as the
fluorogen, the carboxyl group of which was first activated by NSH to obtain Nꢀmethyl
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anthranilic acid succinimidyl ester (1). Compound 1 was further modified by
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ethylenediamine to give Nꢀ(2ꢀaminoethyl)ꢀ2ꢀ(methylamino)benzamide (HH2).
Meanwhile, carboxylated carabrone derivative, HH1, was prepared through the imidization
of carabrone with carboxymethoxylamine hydrochloride using sodium acetate as catalyst
3
4
under a nitrogen atmosphere. TTY was finally obtained through the condensation
reaction between HH1 and HH2, in the presence of EDC as coupling reagent and DMAP
as catalyst under mild conditions. The structures of the newly synthesized compounds were
1
13
characterized correctly by H NMR, C NMR, and ESIꢀMS (Figure S1ꢀS6). Moreover,
fluorescence spectrophotometer was also employed to investigate the fluorescent properties
of compound TTY with results shown in Figure S7. TTY exhibited excitation and emission
spectra maximum peaks at 381 nm (λex, max) and 445 nm (λem, max) in methanol, respectively.
Hapten-protein conjugates. As outlined in Scheme 1, immunogen (TꢀBSA) was prepared
3
7
by the active ester method , using HH1 as the hapten and BSA as the carrier for
polyclonal antibody production. Meanwhile, Coating antigen (TꢀOVA) was synthesized
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through coupling of HH1 with OVA by the mixed anhydride method for indirect ELISAs.
SDSꢀPAGE was used for mobility shift detection of haptenꢀprotein conjugates and the
results were shown in Figure S8. From SDSꢀPAGE, the bands of TꢀBSA and TꢀOVA
appeared at higher molecular weight positions with slower mobility relative to their
unreacted proteins, indicating successful protein conjugation.
The ultravioletꢀvisible spectral changes of haptenꢀprotein conjugates were also
monitored to confirm with their identities by UV spectroscopy. As shown in Figure S9,
TꢀBSA exhibited a 10ꢀnm blue shift from 278 nm to 268 nm relative to BSA, and TꢀOVA a
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7ꢀnm blue shift from 278 nm to 270 nm relative to OVA under the same conditions,
verifying that HH1 was successfully attached to the carrier proteins.
Production of Polyclonal Antibody. Antigen design plays a key role in polyclonal
antibody production. Due to weak antigenicity of carabrone with low molecular weight, it
46
must be coupled to a carrier protein to elicit an immune response. In order to generate
antibody with high sensitivity and specificity, the binding strategy was also designed
cautiously to ensure the minimum conformational changes of hapten relative to the targeted
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analyte even after conjugation with the carrier protein. Moreover, previous study revealed
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that antibodies produced by single antigens might have higher affinities to the targets.
Based on these information, HH1 retaining the core structure of carabrone was selected as
the hapten, and its BSA conjugate as the immunogen. After four rounds of immunization
with single TꢀBSA, polyclonal antisera produced by six female BALB/c mice were isolated
and their titers against carabrone were tested by indirect ELISAs (Table S1). Antisera
(TPAbs 1ꢀ6) exhibited different affinities to the coating antigen (TꢀOVA) with titers ranging
from 64,000 to 256,000, which were higher than those of previously reported antisera
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against various pesticides. TPAbsꢀ4 with the highest titer (256,000) was selected for
antibody purification. The concentration of the purified polyclonal antibody was about 4.52
mg/mL.
Antifungal Activity. Physicochemical properties of a parent compound will be
inevitably changed if it is modified with a fluorogen, including molecular weight, the
octanol/water partitioning coefficient value, etc., which may even affect its combination
with the target consequently. Previous studies of our group showed that carabone
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derivatives modified at Cꢀ4 position exhibited diverse antifungal activities against Botrytis
cinereal and Colletotrichum lagenarium through altering their affinities to the unclear
49, 50
target of carabrone.
Thus, to investigate the effect of substituent fluorogen on the
recognition of carabrone to its target, the effects of TTY and carabrone on the mycelial
growth of Ggt were evaluated in vitro. As illustrated in Table 1, TTY performed similar
EC50 value (33.68 ꢁg/mL) to that of carabrone (28.45 ꢁg/mL), verifying that TTY still
maintains the capacity of selectively binding to the target of carabrone. Thus, the
fluorescent conjugate (TY) does not affect the biological activity of carabrone.
Absorption Kinetics of TTY. To determine the potential of TTY to be used as a
fluorescent subcellular tracer, the absorption behavior of TTY was examined by
monitoring fluorescence intensity of mycelia stained with different concentrations of TTY
(0.3, 0.35, 0.4, and 0.8 ꢁM) over various time periods (0ꢀ240 min). As depicted in Figure 2,
TTY absorption exhibited a timeꢀ and doseꢀdependent manner. Adsorption equilibrium was
reached after 90 min and a saturated absorption was reached when the concentration of
TTY was over 0.4 ꢁM. We thus used the optimized staining condition (0.4 ꢁM TTY,
dyeing time: 90 min) for localization experiment. Under the optimal conditions obtained, a
fluorescence image with evident subcellular distribution was represented (Figure S10),
which indicated that TTY could enter the mycelial cells of Ggt for cell imaging.
Stability of TTY in Mycelial Cells. An ideal fluorescent tracer should be stable
throughout the imaging period. Thus, the TTY present in the mycelial cells at 2 h was
identified by LCꢀHRMS analysis. As shown in Figure S11, the chromatographic peak for
+
+
compound TTY (m/z, [M + H] 497.2751, and [M + Na] 519.2583) was found at 14.18
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min while no identical chromatographic peak of TTY was found in the control group
(Figure S12). Moreover, we have searched the possible decomposed products of TTY
(Figure S14) in the TOF MS spectrum from 6 min to 25 min using an Analyst TF 1.7.1
Software and there were no matched MS peaks for these products (Figure S13). Therefore,
TTY was relatively stable during the experimental period. These findings indicate that
TTY is a suitable fluorescent surrogate of carabrone and is believed to be capable of
revealing the subcellular localization of carabrone within Ggt.
Hyphal Morphology and Ultrastructure. Previous work of our group showed that
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carabrone possessed a remarkable inhibitory effect against the mycelial growth of Ggt.
To gain more evidence about the mode of antifungal action of carabrone treatment, the
hyphal morphology of Ggt was observed by SEM with results shown in Figure 3. The
control sample exhibited a normal morphology with uniform, smooth, uniseriate, and
robust hyphae with plump growing points (Figure 3a). The carabrone treated sample,
however, showed altered morphology characterized by twisted and irregular hyphae with
slight deformity at the growing points (Figure 3b) and even cellular collapse (Figure 3c).
TEM was employed to study the ultrastructural alterations of Ggt when treated with
carabrone and the results were illustrated in Figure 4. A typical fungal ultrastructure of
intact cell wall with normal thickness, regular and smooth cell membrane, evenly
distributed cellular cytoplasm, and regularly shaped organelles in the mycelia is clearly
represented by TEM images of the control treatment (Figure 4a, b). For the carabrone
treatment with EC50 value, no evident changes were viewed in the structures of cell wall,
cell membrane and the diaphragm of the mycelia. Interestingly, mitochondrial abnormality
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was observed in the treated mycelia which could be described as (i) the hazy outline of
mitochondria (Figure 4c), (ii) medullary transition of mitochondria (Figure 4d), (iii) excess
accumulation of unclear settlings in mitochondria (Figure 4e), and (iv) vacuolar
degeneration of mitochondria (Figure 4f). These findings suggested that the mitochondria
of Ggt might correlated with the action mechanism of carabrone.
Mitochondrial Concentration. To validate our ultrastructural findings, we sought to
test the changes of mitochondrial concentration of Ggt treated with carabrone. The effect
of carabrone on the mitochondrial concentration was evaluated by testing the protein
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content of mitochondria (Table 2). After exposure to carabrone for 7 days, the
mitochondrial concentration of Ggt decreased evidently (P < 0.05) along with the
increasing concentration of carabrone (EC50, 28.45 ꢁg/mL; EC70, 49.97 ꢁg/mL), in
agreement with the alterations of mitochondrial structure.
Subcellular Imaging. Given the fact that the aforementioned effects of carabrone on
Ggt (e.g. ultrastructural alterations, decreases in mitochondrial concentration) are closely
related to mitochondria, two imaging techniques (FC and IF) were employed to validate
the mitochondrial targeting of carabrone.
For FC imaging, three dyes with distinctive emission wavelengths (TTY, 445 nm;
MitoTrackerR Green FM, 525 nm; RedDot™1, 690 nm) were utilized. As depicted in
Figure 5, S15, TTY and MitoTrackerR Green FM (a mitochondrial dye) overlapped
perfectly while no overlay was observed between TTY and RedDot™ (a commercial
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nuclear probe). Pearson’s colocalization coefficient,
describing the correlation of the
intensity distribution between TTY and MitoTrackerR Green FM signal, was 0.83 (Figure
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5h). Thus, FC imaging strongly supports the mitochondrial localization of carabrone.
For IF imaging, polyclonal antibody specific to carabrone was used as the primary
antibody to recognize intracellular carabrone, and FITC labeled AntiꢀMouse IgG (whole
molecule) as the secondary antibody. Meanwhile, a commercially available dye,
MitoTrackerR Red CMXRos was employed for colocalization study. Again, excellent
colocalization between AntiꢀMouse IgG (whole molecule)ꢀFITC and the MitoTrackerR
Red CMXRos was observed (Figure 6, S16), with the Pearson’s colocalization coefficient
being 0.86 (Figure 6f). These results suggested that carabrone was predominantly present
in the mitochondria.
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α,βꢀunsaturated lactones, such as britannin,
arucanolide,
parthenolide,
2
3
24
costunolide, and isocostunolide exert their biologic functions via mitochondriaꢀrelated
pathways. Our previous study showed that carabrone could inhibit the intracellular
oxidation process in Ggt, and exert some impacts on the mitochondria, such as vacuolar
25
degeneration, which was consistent with the results obtained in this study. Additionally,
when treated with carabrone, the activity of respiratory chain complexes IꢀV was
determined to be significantly decreased, especially that of the complex III, and the
26
expression levels of the associated genes were upꢀregulated except GgCyc1,
demonstrating that carabrone involved in the respiratory electron transfer pathway either
directly or indirectly in Ggt. In this paper, we confirmed not only the effects of carabrone
on the mitochondrial structure and concentration, but also its mitochondrial targeting using
FC and IF imaging techniques. The excessive accumulation of reactive oxygen species
(ROS) in the mitochondria was also observed in Ggt when treated with carabrone (data not
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published). These results indicate that carabrone selectively distributes in the mitochondria
of Ggt and functions on the respiratory electronꢀtransport chain, especially the complex III,
which leads to ROS increase and the death of mycelial cells followed. However, the
detailed pathway of action on mitochondria is still unclear, and a further study of
mitochondriaꢀrelated pathway induced by carabrone in Ggt is in progress.
In conclusion, the major effects of carabrone as a potential botanical antifungal agent
against Ggt were the variations in mitochondrial structure and the decreased concentration.
A fluorescent conjugate of carabrone, TTY, was well designed and first obtained via
covalent coupling of a smart fluorophore with a soft linker. Antifungal activity and
absorption kinetics indicated that TTY was a reasonable fluorescent surrogate of carabrone
and suitable to reveal the subcellular distribution of carabrone analogues. Polyclonal
antibody specific to carabrone was also successfully prepared by immunizing mice with a
wellꢀdesigned immunogen according to a standard protocol. Subcellular imaging with FC
and IF provided identical and adequate evidences that the actual site(s) of action of
carabrone lay in the mitochondria of Ggt, orienting our followꢀup study for elucidating the
detailed mechanism of carabrone against Ggt. More importantly, the strategies established
for subcellular localization study of a natural substance in this paper could be also applied
in exploring the action mechanism of other drugs.
AUTHOR INFORMATION
Corresponding Author
*(Juntao Feng) Fax: +86ꢀ029ꢀ87092122. Eꢀmail: fengjt@nwafu.edu.cn.
Funding
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This work was financially supported by the National Natural Science Foundation of China
(No. 31272074).
Notes
The authors declare no competing financial interest.
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