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Methyl anthranilate: A novel quorum sensing inhibitor and anti-biofilm agent against Aeromonas sobria
Journal Pre-proof Methyl anthranilate: a novel quorum sensing inhibitor and anti-biofilm agent against Aeromonas sobria Tingting Li, Xiaojia Sun, Haitao Chen, Binbin He, Yongchao Mei, Dangfeng Wang, Jianrong Li PII:
S0740-0020(19)30966-9
DOI:
https://doi.org/10.1016/j.fm.2019.103356
Reference:
YFMIC 103356
To appear in:
Food Microbiology
Received Date: 10 July 2019 Revised Date:
20 September 2019
Accepted Date: 23 October 2019
Please cite this article as: Li, T., Sun, X., Chen, H., He, B., Mei, Y., Wang, D., Li, J., Methyl anthranilate: a novel quorum sensing inhibitor and anti-biofilm agent against Aeromonas sobria, Food Microbiology, https://doi.org/10.1016/j.fm.2019.103356. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Elsevier Ltd. All rights reserved.
1
Methyl anthranilate: a novel quorum sensing inhibitor and
2
anti-biofilm agent against Aeromonas sobria
3
Tingting Lia, Xiaojia Sunb, Haitao Chenc, Binbin Heb, Yongchao Meib,
4
DangfengWangb, Jianrong Li b*,
5
a
6
University), Ministry of Education, Dalian, Liaoning, 116600. China
7
b
8
Liaoning Province; National & Local Joint Engineering Research Center of Storage,
9
Processing and Safety Control Technology for Fresh Agricultural and Aquatic
Key Laboratory of Biotechnology and Bioresources Utilization (Dalian Minzu
College of Food Science and Technology, Bohai University; Food Safety Key Lab of
10
Products; Jinzhou, Liaoning, 121013, China
11
c
12
University (BTBU), Beijing 100048, China
Beijing Key Laboratory of Flavor Chemistry, Beijing Technology and Business
13 14 15 16 17 18 19 20 21
*Correspondence:
22
Professor Jianrong Li, College of Food Science and Technology, Bohai University,
23
Jinzhou, Liaoning, China. Tel: +86-416-3400008; Email: [email protected] 1
24
Abstract: Quorum sensing (QS), bacterial cell-to-cell communication, is a gene regulatory
25
mechanism that regulates virulence potential and biofilm formation in many pathogens.
26
Aeromonas sobria, a common aquaculture pathogen, was isolated and identified by our laboratory
27
from the deteriorated turbot, and its potential for virulence factors and biofilm production was
28
regulated by QS system. In view of the interference with QS system, this study was aimed to
29
investigate the effect of methyl anthranilate at sub-Minimum Inhibitory Concentrations (sub-MICs)
30
on QS-regulated phenotypes in A. sobria. The results suggested that 0.5 µL/mL of methyl
31
anthranilate evidently reduced biofilm formation (51.44%), swinging motility (74.86%), swarming
32
motility (71.63%), protease activity (43.08%), and acyl-homoserine lactone (AHL) production.
33
Furthermore, the real-time quantitative PCR (RT-qPCR) and in silico analysis showed that methyl
34
anthranilate might inhibit QS system in A. sobria by interfering with the biosynthesis of AHL, as
35
well as competitively binding with receptor protein. Therefore, our data indicated the feasibility of
36
methyl anthranilate as a promising QS inhibitor and anti-biofilm agent for improving food safety.
37 38
Keywords: Aeromonas sobria; Biofilm formation; Foodborne pathogen; Methyl anthranilate;
39
Quorum sensing
40 41
1. Introduction
42
Aeromonas sobria is a Gram-negative, highly active, facultative anaerobic bacterium of the
43
family Aeromonadaceae (Janda and Abbott, 2010). It is a common zoonotic pathogen, can cause
44
furunculosis and septicemia in fish, as well as gastroenteritis and wound infection in humans.
45
Meanwhile, as a emerging foodborne pathogen, its virulence secretion and biofilm formation
2
46
caused serious food safety problems, and considerable economic loss in food industry (Chen et al.,
47
2013; Nagar et al., 2011). Recently, some studies have found that the expression of virulence
48
factors and biofilm formation of A. sobria are regulated by quorum sensing (QS) system
49
(Beaz-Hidalgo and Figueras, 2013; Kirov et al., 2004).
50
QS is a system in which bacterial cells communicate with each other to monitor the changes
51
in their population density through signal molecules (called autoinducers, AIs), and activate
52
specific sets of genes when a threshold is reached (Whiteley et al., 2017). Among them,
53
acyl-homoserine lactone (AHL) are the most common autoinducers of Gram-negative bacteria
54
(Papenfort and Bassler, 2016). LuxI/R-type QS system was the model system in Gram- negative
55
bacteria, containing two integrators, AHL synthase (I-protein), encoded by LuxI homologue
56
synthase, and a cognate receptor (R-protein), activated by AHL, which have been shown to
57
regulate multiplex phenotypic characteristics, such as expression of virulence factors and biofilm
58
formation (Ng and Bassler, 2009; Parsek and Greenberg, 2005; Rutherford and Bassler, 2012).
59
Recently, many studies have focused on interfering bacterial QS system with natural compounds,
60
known as QS inhibitors (QSIs), due to the ability of reducing virulence, and not imposing strong
61
selective pressures on development (Defoirdt, 2018).
62
Plant food extracts are commonly considered as attractive sources of anti-QS compounds,
63
due to their capacity to interfere with QS system, and many studies have reported that there are
64
three different ways to modulate QS of Gram-negative bacteria, by inhibition of AHL synthesis,
65
by degrading signaling molecules, and by targeting the signal receptor, LuxR (Defoirdt et al., 2013;
66
Truchado et al., 2009; Truchado et al., 2015). Methyl anthranilate, a plant spice extract, occurs
67
naturally in grapes and strawberries, and has been widely employed for the preparation of edible
3
68
flavor and food additives in food processing industries. It has been reported that biofilm formation
69
and elastase activity in Pseudomonas aeruginosa were significantly inhibited after treatment with
70
methyl anthranilate and its analogues (Calfee et al., 2001; Li et al., 2018). However, there is still
71
limited information about methyl anthranilate as a QSI in Gram-negative bacteria, especially in A.
72
sobria.
73
Furthermore, molecular docking and dynamic simulation methods have gained considerable
74
importance while analyzing the binding ability of ligands to the receptor proteins and the
75
conformational changes in receptor proteins after binding. The potential anti-QS activity of
76
cinnamaldehyde was revealed using molecular docking via interaction with hydrogen bond of
77
LuxR-type protein in P. fluorescens (Li et al., 2018). The conformational changes in the LasR
78
receptor protein of P. aeruginosa due to the binding of signaling molecule or quercetin were
79
predicted by Gopu et al. (2015). In this study, we analyzed the inhibitory effect of methyl
80
anthranilate on the expression of QS-related genes and QS-regulated phenotypes in A. sobria, such
81
as biofilm formation and AHL production, in order to evaluate its potential as a QS inhibitor. To
82
further elucidate the QS inhibitory mechanism of methyl anthranilate, in silico analysis was
83
performed, including molecular docking and dynamic simulation.
84
2. Materials and Methods
85
2.1. Materials and bacterial strains
86
Methyl anthranilate (≥ 98% purity) was purchased from Sigma-Aldrich (St. Louis, MO,
87
USA). AHL standards, including C4-HSL, C6-HSL, C8-HSL, C10-HSL, C12-HSL, and C14-HSL,
88
were purchased from the same company. A. sobria was originally isolated and identified from
89
deteriorated turbot. Chromobacterium violaceum CV026 was generously provided by Dr. Yang
4
90
from Xinjiang Shihezi University. It was used as a reporter strain for evaluating anti-QS activity.
91
Due to the resistance, the culture medium of CV026 was supplemented with 20 µg/mL kanamycin.
92
2.2. Determination of minimal inhibitory concentration (MIC) of methyl anthranilate
93
The MIC for methyl anthranilate was determined against CV026 and A. sobria by Oxford
94
cup method (Diao et al., 2013). Sterilized Oxford cups were firstly put into the plates, and then, 20
95
mL LB nutrient agar containing CV026 or A. sobria overnight cultures (OD595 nm= 1.0, 200 µL)
96
was poured into plates. 200 µL of varying concentrations of methyl anthranilate (0.125 to 5
97
µL/mL) were added to the wells and cells were incubated (160 rpm at 28 °C for 24 h). Equal
98
amount of sterile water was used as the negative control. The lowest concentration of methyl
99
anthranilate, which inhibited visible growth, was selected as MIC. Further experiments were
100
performed only at the sub-MIC level.
101
2.3. Determination of cell membrane integrity of A. sobria
102
The cell membrane integrity was determined according to the method described by Chakotiya
103
et al. (2017), with slight modification. Briefly, 200 mL LB broth containing the sub-MICs (0.5
104
µL/mL, 0.25 µL/mL, 0.125 µL/mL, and 0.0625 µL/mL) of methyl anthranilate were mixed with
105
200 µL of A. sobria overnight cultures, incubated at 28 °C until the density reached 106 CFU/mL.
106
Equivalent amount of sterile water was used as negative control and each experiment was done in
107
triplicate. The mixtures were centrifuged at 9000 ×g for 5 min and the supernatant was discarded.
108
The pellets were resuspended in 1 mL of PBS (pH 7.4), and then, fixed with 70% ethanol at 4 °C
109
for 30 min. After washing thrice by PBS, the bacterial cells were stained with PI stain (at final
110
concentration of 10 mg/mL) in dark for 30 min at 37 °C, then measured in Flow Cytometer (BD
111
AccuriTM C6 Plus). The autofluorescence was observed in the cells without methyl anthranilate
5
112
and PI staining.
113
2.4. QS inhibitory test
114
2.4.1. Violacein inhibition assay
115
CV026 overnight cultures (200 µL) were added to LB nutrient agar (20 mL) supplemented
116
with 20 µg/mL C4-HSL, following the method of Packiavathy et al. (2012). The mixtures were
117
poured into the plates containing sterilized Oxford cups. Then, 200 µL of sub-MICs (0.5 µL/mL,
118
0.25 µL/mL, 0.125 µL/mL, and 0.0625 µL/mL) of methyl anthranilate were added to the wells,
119
equivalent amount of sterile water was served as control group. The plates were incubated at
120
28 °C for 24 h, and QS inhibitory activity was assessed by evaluating the inhibitory zones.
121
2.4.2. Quantitative assay of violacein production
122
The violacein production was quantitatively determined by the method of Zhang et al. (2018).
123
LB broth (10 mL) containing CV026 overnight cultures (100 µL) was mixed with the sub-MICs
124
(0.5 µL/mL, 0.25 µL/mL, 0.125 µL/mL, and 0.0625 µL/mL) of methyl anthranilate as well as 20
125
µg/mL C4-HSL, then incubated at 28 °C at 160 rpm for 48 h. After incubation, the mixtures (300
126
µL) were transferred to the centrifuge tubes, and mixed with 10% sodium dodecyl sulfate (150 µL)
127
and butyl alcohol (600 µL) using a vortex mixer. After centrifugation at 9000 ×g for 6 min, the
128
violacein supernatant collected in the organic layer was added into a 96-well microplate to
129
determine OD595 nm values using microplate reader (Bio-tek, Vermont, USA).
130
2.4.3. Determination of biofilm formation
131
The assay for biofilm formation was performed according to the method of Zhang et al.
132
(2014). Overnight cultures (1 mL) of A. sobria were diluted with LB broth (100 mL) and
133
transferred to the centrifuge tubes. The centrifuge tubes were supplemented with different
6
134
sub-MICs of methyl anthranilate or 20 µg/mL C4-HSL (AHL treatment group). Equivalent amount
135
of sterile water was used as control group. After cultivation at 28 °C for 48 h, 200 µL of bacterial
136
cultures were transferred to a 96-well plate to measure the absorbance at 595 nm. Then, the
137
cultures were discarded. The centrifuge tubes were rinsed gently with sterile water, air dried,
138
stained with 0.1% crystal violet (w/v) for 20 min, and rinsed again. Biofilm was solubilized with
139
33% acetic acid and added into the 96-well microplate to determine OD595
140
microplate reader (Bio-tek, Vermont, USA).
141
2.4.4. Visualization of biofilm by scanning electron microscopy (SEM) and light microscopy
nm
values using
142
Biofilm visualization was performed with the method described by Zhou et al. (2018). The
143
polished zinc pieces (7 mm × 7 mm × 0.2 mm) were immersed in 10 mL of LB broth in sterile
144
plates. To the plates, A. sobria overnight cultures (100µL), the different sub-MICs of methyl
145
anthranilate, sterile water (control group), and 20 µg/mL C4-HSL (AHL treatment group) was
146
added. After incubation at 28 °C for 48 h, the biofilm formed on zinc piece was rinsed twice and
147
fixed with 2.5% glutaraldehyde for 4 h. The zinc piece was dehydrated in graded ethanol for 15
148
min. After air drying, the biofilm was visualized with SEM.
149
Method operations as described above, the biofilm formed on glass slides was rinsed thrice
150
with sterile water, and stained with 1mL of 0.1% crystal violet for 15 min. Then, the biofilm was
151
visualized by optical microscope.
152
2.4.5. Determination of bacterial motility
153
Motility was evaluated as described by Gutierrez-Pacheco et al. (2018), with slight
154
modifications. Swimming medium (0.5% NaCl, 0.3% agar, and 1% tryptone) or swarming
155
medium (0.5% NaCl, 0.6% agar, 0.5% D-fructose, and 1% peptone) was mixed with the different
7
156
sub-MICs of methyl anthranilate, sterile water (control group) and 20 µg/mL C4-HSL (AHL
157
treatment group), and then, poured into plates. Five microliters of A. sobria overnight cultures
158
were point inoculated at the center of plates, and each test was done in triplicate. After incubation,
159
the migration distance was determined using a vernier caliper to assess swimming and swarming
160
motility.
161
2.4.6. Determination of extracellular protease activity
162
Extracellular protease activity was assessed according to the method described by Li et al.
163
(2018). 10 mL of 15 % skim milk (115 °C, 30 min, 0.06 MPa) was mixed with 90 mL of LB
164
nutrient agar, and poured into plates. Overnight cultures of A. sobria treated with the different
165
sub-MICs of methyl anthranilate treatment, sterile water (control group), and 20 µg/mL C4-HSL
166
(AHL treatment group). After incubation, the mixtures were centrifugated at 9000 ×g for 10 min.
167
The supernatant was filtered using 0.22 µm filter; then, 100 µL of the filtrate was transferred into
168
the plates. After incubation at 28 °C for 48 h, the diameter of the transparent zone was measured
169
to assess protease activity using a vernier caliper.
170
2.4.7. Determination of AHL production
171
Overnight cultures of A. sobria with or without the different sub-MICs of methyl anthranilate
172
were incubated at 28 °C for 48 h, and then, centrifugated at 9000 ×g for 15 min. The supernatant
173
was extracted by acidified ethyl acetate (containing 0.1% acetic acid) for four times, and then,
174
concentrated by rotary evaporation. The extract was dissolved with 1 mL methanol and filtered
175
through 0.22 µm filter for GC-MS analysis.
176
AHL analysis was performed by GC-MS Agilent 7890 N/5975 (Agilent, USA). Briefly, all
177
samples were injected into HP-5 MS capillary column in the split mode (50:1), along with helium
8
178
as the carrier gas at a flow rate of 1 mL/min. The injector temperature, oven temperature program,
179
and mass spectrometry conditions were set following the method of Li et al. (2016). Data were
180
acquired by full-scan mode (m/z 35-800) and selected ion monitoring (SIM) mode (m/z 143).
181
2.5. RT-qPCR analysis
182
Overnight cultures of A. sobria with or without the different sub-MICs of methyl anthranilate
183
were incubated at 28 °C, until OD595 nm values reached 1.0. Total RNA was extracted by Trizol
184
(Thermo Fisher Scientific) from each cell pellet. cDNA synthesis was performed using the
185
RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific). RT-PCR reaction was
186
done by using the Power SYBR™ Green Master Mix (Thermo Fisher Scientific) and a CFX
187
Connect Real-Time PCR Detection System (Bio-Rad Laboratories). The primer sequences were
188
designed using Primer 5.0, and listed in Table S1. cDNA was served as the template and 16S
189
rRNA was served as the internal control. Specificity of product amplification was confirmed by
190
melting curve. The 2-(∆∆CT) method was used to calculate the expression levels of target genes, as
191
previously described by Li et al. (2019).
192
2.6. Molecular Docking analysis
193
The protein sequences of LuxI (WP_101529659.1) and LuxR (WP_005335753.1) of A.
194
sobria were obtained from National Centre for Biotechnology Information (NCBI) database for
195
further computer-simulated investigation. The three dimensional (3D) structures of the
196
homologous
197
(https://swissmodel.expasy.org). Ramachandran plot was used to predict whether the amino acid
198
residues in the protein were in the favored region, and reflected the rationality of conformation.
199
The preparation and optimization process of 3D protein structures were performed by Discovery
protein
were
predicted
and
9
assessed
by
SWISS-MODEL
200
Studio (DS). The structures of methyl anthranilate, halogenated furanone C30, and C4-HSL were
201
got from ZINC (http://zinc.docking.org/) and optimized in the DS for obtaining the lowest-energy
202
conformations. According to previous research (Li et al., 2018), the Libdock algorithm was used
203
to analyze the interactions between protein and ligand.
204
2.7. Molecular dynamics simulation analysis
205
Molecular dynamics simulation was performed to analyze the stability of the receptor
206
protein-signaling molecule and receptor protein-methyl anthranilate complexes using the
207
GROMACS 2018.2 package, as previously described by Zuo et al. (2017). The pdb structure of
208
complex was used as the beginning for MD simulations. The protein topology was generated by
209
Gromos54a7 force field, and ligand topology was constructed by PRODRG online server. The
210
whole complex was solvated in a cubic period box, and the minimum distance between the
211
complex and box edge was set at 1 nm. The charge of protein was neutralized by addition of a
212
sufficient number of chloride ions. Then, the energy minimization of entire system was performed
213
using 10000 steps of conjugate gradient algorithm. The entire system was equilibrated for 50 ps
214
each time for position restrains of NVT (constant number of particles, volume, and temperature)
215
and NPT (constant number of particles, pressure, and temperature). Linear Constraint Solver
216
algorithm was used for constraining the bond length during the simulation. Isothermal and isobaric
217
coupling constant were kept at 0.1 ps and 2 ps, respectively, bringing the system to a stable
218
environment of 300 K temperature and 1 bar pressure. Finally, the system at equilibrium for each
219
complex was subjected to 50 ns MD simulation with the time step of 2 fs, and the trajectory was
220
saved after every 10 ps.
221
2.8. Statistical analysis
10
222
All experiments were performed in triplicates and the data was expressed as mean ± standard
223
deviation (SD). Statistical analysis was performed by one-way analysis of variance (ANOVA)
224
using SPSS software (version18.0). Differences at P < 0.05 were considered statistically
225
significant.
226
3. Results
227
3.1. MIC of methyl anthranilate and the effect on cell membrane integrity of A. sobria
228
The MIC of methyl anthranilate was measured with concentrations ranging from 0.125
229
µL/mL to 5 µL/mL. Determination of MIC for methyl anthranilate against CV026 and A. sobria
230
was 2.0 µL/mL and 5.0 µL/mL, respectively. Consequently, the sub-MICs (0.5 µL/mL, 0.25
231
µL/mL, 0.125 µL/mL, and 0.0625 µL/mL) of methyl anthranilate were used for further
232
experiments. In addition, propidium iodide (PI) uptake assay was performed by flow cytometry to
233
assess the effect of sub-MICs of methyl anthranilate on cell membrane integrity of A. Sobria. PI, a
234
cationic dye, could not enter an intact cell membrane but could easily enter into the damaged
235
membrane and intercalate the nucleic acids. PI stain was retained in 4.58±1.009% of bacterial
236
cells when treated with 0.0625 µL/mL methyl anthranilate (Fig. 1A, B). Besides, 6.85±0.007% of
237
cells retained the stain after treatment with maximum sub-MIC of methyl anthranilate (0.5 µL/mL).
238
The negative control showed 6.43±0.005% of PI uptake, and 0% of auto fluorescence was shown
239
by bacterial cells without methyl anthranilate and PI stain treatment.
240
3.2. Effect of methyl anthranilate on violacein production
241
The QS inhibitory activity of methyl anthranilate was evaluated by determining its effect on
242
purple pigment violacein of CV026. As shown in Fig. 1C, a clear inhibitory zone was formed after
243
treatment with methyl anthranilate. In the quantitative assay of violacein production, the sub-MICs
11
244
of methyl anthranilate exhibited a dose-dependent inhibitory effect on violacein production.
245
Maximum inhibitory rate of 41.01% was observed at a concentration of 0.5 µL/mL, with no
246
inhibition on the growth of CV026 cells (Fig. 1D).
247
3.3. Effect of methyl anthranilate on biofilm formation
248
The assessment of anti-biofilm activity of methyl anthranilate against A. sobria was shown in
249
Fig. 2A. The group treated with sub-MICs (0.5 µL/mL, 0.25 µL/mL, 0.125 µL/mL, and 0.0625
250
µL/mL) of methyl anthranilate led to a significant reduction in biofilm content of A. sobria to the
251
level of 31.67%, 34.01%, 41.27%, and 51.44%, respectively. In addition, there was no significant
252
difference in the biofilm biomass after treatment with methyl anthranilate (Fig. 2B).
253
The biofilm structure of A. sobria was visualized by optical microscopy and SEM. As shown
254
in Fig. 2C, the deep purple clustered appearance of biofilm was exhibited in AHL treatment group.
255
However, the density of biofilm was visibly reduced in the methyl anthranate treatment group, and
256
the appearance was sparse. In the SEM images (Fig. 2D), the biofilm matrices treated with methyl
257
anthranilate were clearly decreased, and exhibited a small number of microcolonies, pores, and
258
channels, whereas the biofilm treated with C4-HSL was mature and dense.
259
3.4. Effect of methyl anthranilate on motility and extracellular protease activity
260
The migration ability of A. sobria was assayed in the presence and absence of methyl
261
anthranilate. As shown in Fig. 3 (A, B), swimming and swarming motility zones of A. sobria
262
gradually reduced. In presence of 0.5 µL/mL methyl anthranilate, the maximum inhibitory rates of
263
74.84% and 71.63%, respectively, were obtained (Table S2). In addition, the migration ability of
264
cells in AHL treatment group was distinctly enhanced.
265
The ability of methyl anthranilate to reduce extracellular protease activity of A. sobria was
12
266
evaluated by measuring the diameter of transparent zone. As shown in Fig. 3C, the protease
267
produced by A. sobria was significantly inhibited. In presence of 0.5 µL/mL methyl anthranilate,
268
the protease activity decreased by 43.08 % (Table S3). However, the addition of exogenous
269
C4-HSL enhanced its protease activity.
270
3.5. Effect of methyl anthranilate on QS-related gene expression and AHL production
271
To elucidate the effect of methyl anthranilate on QS-related genes in A. sobria, the expression
272
levels of luxI and luxR genes were examined using RT-qPCR. As shown in Fig. 4A, the expression
273
levels of luxI and luxR were downregulated when treated with methyl anthranilate. The minimum
274
levels of luxI (0.160) and luxR (0.064) were observed after treatment with 0.5 µL/mL methyl
275
anthranilate. However, the expression levels of genes were significantly upregulated in AHL
276
treatment group. Moreover, the effect of methyl anthranilate on the AHL production was
277
quantitatively analyzed by GC-MS. The retention time of the six AHL standards were determined,
278
and the ratio of the peak area of the sample to the peak area of the internal standard (C14-HSL)
279
was used to evaluate the relative production of AHL (Fig. 4B). C4-HSL was the main signal
280
molecule secreted by A. sobria. The minimal concentration of C4-HSL was decreased to 0.865
281
µg/mL after treatment with 0.5 µL/mL methyl anthranilate.
282
3.6. Homology modeling and assessment
283
The 3D model of homologous proteins was predicted and assessed by SWISS-MODEL,
284
using the sequences of LuxI (acyl-homoserine-lactone synthase, WP_101529659.1) and LuxR
285
(transcriptional regulators, WP_005335753.1). Global Model Quality Estimation (GMQE) and
286
sequence similarities were used to select the best 3D model (Table S4). QMEAN scores
287
represented the degree of agreement of the structural features between the predicted model and
13
288
experimental structures of similar size. Lower QMEAN scores indicated high model quality.
289
Thereby, the models LuxI 1ro5.1.A (-1.98) and LuxR 4lfu.1.A (-2.26) were selected for further
290
analysis. Furthermore, the presence of more than 90% of the residues in the favored region of
291
Ramachandran plot indicated the high rationality of protein structure. The results revealed that
292
90.2% (LuxI) and 94.7% (LuxR) residues existed in the favored region (Fig. S2). Consequently,
293
the models of LuxI 1ro5.1.A and LuxR 4lfu.1.A exhibited great reliability, the 3D structures of
294
LuxI and LuxR proteins in A. sobria were successfully predicted.
295
3.7. Molecular docking and dynamic simulation
296
The interaction of methyl anthranilate, the natural ligand C4-HSL, and the classical QS
297
inhibitor, halogenated furanone C30, into the receptor active site was investigated by molecular
298
docking analysis. As shown in Fig. 5C and c, methyl anthranilate interacted with Ala143, Ser145,
299
Met33, and Leu147 residues of LuxR protein via the H-bonds (green dashed lines), and underwent
300
hydrophobic interactions (pink dashed lines) with Tyr35, Leu149, and Leu147 residues. In
301
addition, the C4-HSL was found to interact through H-bonds with Tyr90, Asn119, and Leu147 (Fig.
302
5A,a). The furanone C30 formed hydrogen bonds with Leu147, hydrophobic interactions with
303
Ile154 and Leu149, and halogen bonds (blue dashed lines) with Ala143 (Fig. 5B, b). For LuxI
304
protein, methyl anthranilate interacted via the H-bonds with Ala85, Gln166, Leu162, and Gly175,
305
and formed hydrophobic interactions with Val168, Val176, and Ala87 (Fig. 5D, d).
306
To further investigate the conformational changes in receptor protein in the presence of
307
C4-HSL and methyl anthranilate, 50 ns of molecular dynamics simulation was performed, and the
308
stability of two complexes was assessed by the values of root mean square deviation (RMSD) and
309
root mean square fluctuation (RMSF). Their lowest values indicated that the complex was more
14
310
stable. RMSD is a measure of the average distance between the backbone atoms of superimposed
311
proteins. As shown in the Fig. 6A, the RMSD values of LuxR-methyl anthranilate complex
312
reached a stable state at 0.62 nm after 20,000 ps, which suggested that its conformation was
313
relatively stable over the time period of 50 ns. However, the RMSD values of LuxR-C4-HSL
314
complex displayed wide fluctuations during the entire simulation process. RMSF represents the
315
position deviation of amino acid residues with respect to the reference position. LuxR receptor
316
protein is composed of A and B chains; no significant difference was observed in the two curves
317
of RMSF profile of A chain (Fig. S3); however, the two curves of B chain were markedly different.
318
The results showed that the interaction on B chain was consistent with the results of molecular
319
docking analysis. As shown in Fig. 6B, the RMSF values of LuxR-methyl anthranilate complex
320
were relatively low, indicating that it had high stability. Notably, the RMSF values of
321
LuxR-methyl anthranilate complex decreased gradually in the region of binding site (residues
322
135-150), while the RMSF values of LuxR-C4-HSL complex increased gradually in this region.
323
4. Discussion
324
Recently, many studies have shown that the interference of plant food extracts on bacterial
325
QS system could be an effective strategy to alleviate the virulence of foodborne pathogens (Zhou
326
et al., 2019). We reported a promising QS inhibitor, methyl anthranilate, which exhibited a strong
327
inhibitory activity on the QS-regulated phenotypes of A. sobria, such as biofilm formation,
328
motility, protease activity, and AHL production. In the present study, the sub-MIC levels of methyl
329
anthranilate were selected for subsequent experiments. To evaluate the effect of sub-MICs of
330
methyl anthranilate on cell viability of A. sobria, the integrity of cell membrane was characterized
331
by flow cytometry with PI uptake as an indicator. Cell membrane integrity plays a crucial role in
15
332
maintaining stable metabolism and resistance against harsh environment in bacterial cells. There
333
was no significant difference in the fluorescence intensity and the percentage of PI-positive cells
334
between the methyl anthranilate treatment group and negative control group. This indicated that
335
the sub-MICs of methyl anthranilate exhibited no effect on the cell membrane integrity and no
336
damage to the bacterial cells.
337
C. violaceum CV026, a reporter strain, produced purple pigment violacein upon induction
338
with the exogenous addition of short-chain autoinducer. It was widely used to monitor the
339
production of acyl-homoserine lactone or QS inhibitory activity (Ding et al., 2019). In this study,
340
the anti-QS activity of methyl anthranilate was assessed by observing the formation of inhibitory
341
zone using the reporter strain, CV026. The results showed that the production of characteristic
342
violacein was significantly inhibited by methyl anthranilate, suggesting a high QS inhibitory
343
activity. To further evaluate the extent of inhibition on the violacein, the quantitative assay of
344
violacein production was performed. This result demonstrated the sub-MICs of methyl
345
anthranilate reduced the levels of violacein in a concentration-dependent manner without affecting
346
CV026 growth. Hence, it was confirmed that the decrease in violacein production was due to the
347
interference with QS system, rather than the inhibition of bacterial growth, which was consistent
348
with above result. Consistently, Zhang et al. (2014) found that Rosa rugosa tea polyphenol extract
349
inhibited QS-controlled violacein production in CV026 without affecting its growth.
350
In order to adapt to the environmental stress, the microbial communities adhere to the contact
351
surfaces and enmesh themselves into matrices, namely the biofilm. Biofilm is widely found on the
352
surface of food machinery and equipment made of various materials, such as metal, glass, and
353
plastic. Compared with planktonic bacteria, biofilm is more resistant and difficult to remove,
16
354
which easily cause serious contamination of food and medical equipment (Li et al., 2019). QS
355
system mediated by AHL is known to play an essential role in the process of biofilm formation
356
(Rudrappa et al., 2008). Therefore, interference of QS system could be used as a novel strategy to
357
regulate the biofilm formation (Zhou et al., 2018). This result demonstrated the sub-MICs of
358
methyl anthranilate reduced the biofilm content, with no inhibition on the growth of A. sobria
359
cells. Moreover, the addition of C4-HSL promoted the biofilm formation, which suggested that
360
biofilm formation was regulated by QS system. In this study, the effect of methyl anthranilate on
361
the biofilm microstructure of A. sobria was visualized by optical microscopy and SEM. In the
362
optical microscopy images, the biofilm formed in the presence of methyl anthranilate was sparse,
363
and the density was visibly reduced. In the AHL treatment group, the biofilm showed continuous
364
and dense appearance. The SEM images revealed that the biofilm matrices treated with C4-HSL
365
were clearly increased, and large amounts of microcolonies were connected and formed a dense
366
biofilm structure. However, the biofilm treated with methyl anthranilate exhibited a small number
367
of microcolonies, as well as the pores and channels. This suggested that methyl anthranilate might
368
cause the leakage of nutrients and reduction in biofilm matrices through these pores and channels,
369
thereby inhibiting the biofilm formation. The results of biofilm microscopy supported the
370
quantitative data. Similarly, Packiavathy et al. (2014) found that the sub-MICs of Cuminum
371
cyminum could cause the loosening of biofilm architecture and inhibit biofilm formation in
372
Pseudomonas aeruginosa, Proteus mirabilis, and Serratia marcescens.
373
The effect of methyl anthranilate on the expression of QS-regulated phenotypes of A. sobria,
374
such as motility and extracellular protease activity, were also evaluated in this study. The bacterial
375
flagella-mediated motility, such as swimming and swarming, plays a major role in the process of
17
376
biofilm formation regulated by AHL-mediated QS system (Du et al., 2018). Thus, the reduction of
377
bacterial migration ability might affect the ability of biofilm formation (Gutierrez-Pacheco et al.,
378
2018). In this research, the zones of swimming and swarming motility in methyl
379
anthranilate-treated group were observed to be markedly reduced, indicating that the motility of A.
380
sobria was significantly inhibited by methyl anthranilate. However, the swimming and swarming
381
motility of bacteria after treatment with C4-HSL was significantly promoted, suggesting that
382
exogenous AHL could promote bacterial migration ability. These results were associated with the
383
inhibition of methyl anthranilate on the biofilm formation of A. sobria. Consistent with our results,
384
Lou et al. (2017) found that burdock leaf components decreased the aggregation ability, thus
385
inhibiting the biofilm formation of P. aeruginosa. Furthermore, extracellular protease is one of the
386
primary virulence factors regulated by QS system in many pathogens. Extracellular proteases
387
secreted by microorganisms could decompose the proteins and free amino acids in food and
388
produce volatile and stimulating compounds containing nitrogen and sulfur, thus accelerating the
389
spoilage process and leading to deterioration of food quality (Ding et al., 2017). Thus, the
390
inhibitory effect of methyl anthranilate on the protease activity of A. sobria was evaluated. The
391
results showed that the sub-MICs of methyl anthranilate significantly reduced protease activity.
392
Besides, the addition of exogenous C4-HSL enhanced the protease activity of A. sobria. These
393
observations were similar with those reported by Husain et al. (2017), who reported that mango
394
extract significantly inhibited the production of virulence factors of P. aeruginosa, such as
395
protease, pyocyanin, and swarming motility.
396
Due to the interference ability of methyl anthranilate on QS system, biofilm formation,
397
migration ability, and protease activity was significantly reduced in A. sobria, along with
18
398
down-regulation of QS-related genes. The inhibition of methyl anthranilate on the QS system of A.
399
sobria was further characterized by determining the expression levels of AHL synthetase (luxI
400
gene) and receptor protein (luxR gene) using RT-qPCR technology. The results showed that the
401
expression levels of luxI and luxR were significantly down-regulated after treatment with methyl
402
anthranilate, indicating that the gene expression was interfered by methyl anthranilate.
403
Furthermore, due to the vital role of AHL in QS system of Gram-negative bacteria, the effect of
404
methyl anthranilate on the AHL (C4-HSL) production in A. sobria was analyzed using GC-MS.
405
Gradual reduction of the AHL production was observed in the methyl anthranilate-treated group,
406
which suggested that AHL production of A. sobria was effectively inhibited. Our study found that
407
the inhibition of methyl anthranilate on the QS system of A. sobria might involve interference
408
with AHL biosynthesis. These results were consistent with the reports of Li et al. (2019), who
409
reported that different sub-MICs of vanillin significantly down-regulated the expression levels of
410
QS-related genes halI/halR and the production of AHL in Hafnia alvei.
411
To reveal the anti-QS mechanism of methyl anthranilate, in silico analysis was performed.
412
The interaction between methyl anthranilate and receptor protein was evaluated using molecular
413
docking analysis. Hydrogen bonds can maintain the stability of complex molecules and play a key
414
role in molecular recognition. Here, we found that methyl anthranilate and furanone C30
415
interacted with the same residues, Leu147 and Leu149, via H-bond and hydrophobic bond,
416
respectively, revealing that two compounds had common binding sites. Moreover, methyl
417
anthranilate and C4-HSL formed H-bond interactions with LuxR protein at the common site
418
Leu147, and methyl anthranilate also exhibited hydrophobic interactions at this site. Compared
419
with furanone C30 and C4-HSL, methyl anthranilate formed more H-bond interactions with LuxR
19
420
protein. These results demonstrated that the binding affinity of methyl anthranilate bound to LuxR
421
receptor protein was higher than that of its cognate ligand C4-HSL. Furthermore, molecular
422
docking studies are not competent enough to reveal the conformational changes caused by the
423
interactions between receptors and ligands (Gopu et al., 2016). Therefore, molecular dynamics
424
simulation was performed to investigate the conformational changes of receptor protein in the
425
presence of signaling molecule and methyl anthranilate. RMSD profile showed that the
426
LuxR-methyl anthranilate complex was more stable than the LuxR-C4-HSL complex during the
427
whole simulation. This might be because the interactions between the receptor protein and methyl
428
anthranilate made the conformational changes of the receptor protein relatively small, and thus, its
429
complex was more stable. RMSF profile further demonstrated that the instability of LuxR-C4-HSL
430
complex might be related to the activation of protein conformation caused by the interactions of
431
C4-HSL, while this conformation in LuxR-methyl anthranilate complex was stabilized by
432
interactions with methyl anthranilate. The in silico results suggested that methyl anthranilate acted
433
as a potential competitive inhibitor of signaling molecules that bind to LuxR receptor protein.
434
Combined with RT-qPCR results, the QS inhibitory mechanism of methyl anthranilate might
435
involve two pathways, interfering with AHL biosynthesis as well as competitively binding with
436
receptor proteins.
437
To summarize, the inhibitory effect of methyl anthranilate against A. sobria was evaluated for
438
the first time in order to determine its possible use as a novel QS inhibitor. Our results have
439
demonstrated that methyl anthranilate exhibited a remarkable inhibition on the QS-regulated
440
phenotypes in A. sobria, including biofilm formation, motility, and protease activity. Besides, the
441
results of RTq-PCR and GC-MS showed that the expression levels of QS-related genes were
20
442
down-regulated and the biosynthesis level of AHL was reduced, when treated with the different
443
sub-MICs of methyl anthranilate. To expound QS inhibitory mechanism, the interaction between
444
methyl anthranilate and receptor protein and the conformational changes in receptor protein after
445
binding were shown by molecular docking and dynamics simulation. These results demonstrated
446
that methyl anthranilate might inhibit QS system in A. sobria by interfering with AHL
447
biosynthesis, as well as competitively binding with receptor protein. Our findings strongly
448
suggested that methyl anthranilate could be used as a novel QS inhibitor and anti-biofilm agent for
449
alleviation of the damage caused by foodborne pathogens.
450
Acknowledgements
451
This research was financially supported by the National Natural Science Foundation of China
452
and
453
(2018YFD0400601, 2017YFD0400106) and the Open Project Program of Beijing Key Laboratory
454
of Flavor Chemistry, Beijing Technology and Business University (SPFW2019YB08).
455
Conflict of interest
456
National
Key
Research
(No.
31471639),
Development
Programme
of
China
All authors declare no conflict of interest.
457 458 459
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A
B
C
D
Fig. 1. Quantitative assessment of cell membrane integrity, Cells were stained with PI and analyzed by flow cytometry (A). (Con fcs) control fluorescence, (Auto fcs) Auto fluorescence, (0.0625, 0.125, 0.25, and 0.5) methyl anthranilate fluorescence. Cell membrane integrity was evaluated by PI- positive cell rate (B). Inhibitory activity of the sub-MICs of methyl anthranilate on the violacein production in CV026 (C). Quantitative analysis of violacein production and CV026 biomass after treatment with the sub-MICs of methyl anthranilate (0.0625, 0.125, 0.25, and 0.5 µL/mL) (D).
26
565 566 567 568 569 570
A
C
B
D
Fig. 2. Quantitative analysis of biofilm content (A) and biofilm biomass (B) with sub-MICs of methyl anthranilate or C4-HSL treatment. Optical microscopic images (C) and SEM images (D) of the biofilm of A. sobria with sub-MICs of methyl anthranilate or C4-HSL treatment. (a) 20 µg/mL C4-HSL, (b) control, (c–f) 0.0625, 0.125, 0.25, and 0.5 µL/mL methyl anthranilate. A
B
C
571 572 573 574
Fig. 3. Inhibitory effect of the sub-MICs of methyl anthranilate on swimming (A) and swarming (B) motility, as well as proteinase activity (C) of A. sobria. (a) 20 µg/mL C4-HSL, (b) control group, (c–f) 0.0625, 0.125, 0.25, and 0.50 µL/mL methyl anthranilate.
27
A
B
575 576 577 578 579
580 581 582 583 584 585 586
Fig. 4. Effect of the sub-MICs of methyl anthranilate on the relative expression of luxI and luxR genes (A) was assessed by RTq-PCR, and the AHL production (B) of A. sobria was evaluated by GC-MS. A
a
B
b
C
c
D
d
Fig. 5. Molecular docking of the native ligands C4-HSL, halogenated furanone C30, and methyl anthranilate with the LuxR and LuxI protein models are shown as a 3D diagram (A–D) and 2D diagram (a–d). (A,a) native ligands C4-HSL, (B,b) halogenated furanone C30, (C,c) methyl anthranilate, (D,d) methyl anthranilate combined with LuxI.
28
A
587 588 589 590 591 592
B
Fig. 6. RMSD profile (A) and RMSF profile (B) of LuxR-C4-HSL and LuxR-methyl anthranilate complex during 50 ns of molecular dynamics simulation. Black line represents the complex of LuxR protein and signaling molecule while red line indicates the complex of LuxR protein and methyl anthranilate.
29
Highlights
Methyl anthranilate significantly inhibited the QS-regulated phenotypes in A. sobria, such as biofilm formation, motility and protease activity. Methyl anthranilate down-regulated the expression levels of QS-related genes and reduced the biosynthesis level of AHL in A. sobria. The QS inhibitory mechanism of methyl anthranilate might involve two pathways, interfering with AHL biosynthesis as well as competitively binding with receptor protein. Methyl anthranilate could be as a promising QS inhibitor and anti-biofilm agent for improving food safety.
S0740-0020(19)30966-9
DOI:
https://doi.org/10.1016/j.fm.2019.103356
Reference:
YFMIC 103356
To appear in:
Food Microbiology
Received Date: 10 July 2019 Revised Date:
20 September 2019
Accepted Date: 23 October 2019
Please cite this article as: Li, T., Sun, X., Chen, H., He, B., Mei, Y., Wang, D., Li, J., Methyl anthranilate: a novel quorum sensing inhibitor and anti-biofilm agent against Aeromonas sobria, Food Microbiology, https://doi.org/10.1016/j.fm.2019.103356. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Elsevier Ltd. All rights reserved.
1
Methyl anthranilate: a novel quorum sensing inhibitor and
2
anti-biofilm agent against Aeromonas sobria
3
Tingting Lia, Xiaojia Sunb, Haitao Chenc, Binbin Heb, Yongchao Meib,
4
DangfengWangb, Jianrong Li b*,
5
a
6
University), Ministry of Education, Dalian, Liaoning, 116600. China
7
b
8
Liaoning Province; National & Local Joint Engineering Research Center of Storage,
9
Processing and Safety Control Technology for Fresh Agricultural and Aquatic
Key Laboratory of Biotechnology and Bioresources Utilization (Dalian Minzu
College of Food Science and Technology, Bohai University; Food Safety Key Lab of
10
Products; Jinzhou, Liaoning, 121013, China
11
c
12
University (BTBU), Beijing 100048, China
Beijing Key Laboratory of Flavor Chemistry, Beijing Technology and Business
13 14 15 16 17 18 19 20 21
*Correspondence:
22
Professor Jianrong Li, College of Food Science and Technology, Bohai University,
23
Jinzhou, Liaoning, China. Tel: +86-416-3400008; Email: [email protected] 1
24
Abstract: Quorum sensing (QS), bacterial cell-to-cell communication, is a gene regulatory
25
mechanism that regulates virulence potential and biofilm formation in many pathogens.
26
Aeromonas sobria, a common aquaculture pathogen, was isolated and identified by our laboratory
27
from the deteriorated turbot, and its potential for virulence factors and biofilm production was
28
regulated by QS system. In view of the interference with QS system, this study was aimed to
29
investigate the effect of methyl anthranilate at sub-Minimum Inhibitory Concentrations (sub-MICs)
30
on QS-regulated phenotypes in A. sobria. The results suggested that 0.5 µL/mL of methyl
31
anthranilate evidently reduced biofilm formation (51.44%), swinging motility (74.86%), swarming
32
motility (71.63%), protease activity (43.08%), and acyl-homoserine lactone (AHL) production.
33
Furthermore, the real-time quantitative PCR (RT-qPCR) and in silico analysis showed that methyl
34
anthranilate might inhibit QS system in A. sobria by interfering with the biosynthesis of AHL, as
35
well as competitively binding with receptor protein. Therefore, our data indicated the feasibility of
36
methyl anthranilate as a promising QS inhibitor and anti-biofilm agent for improving food safety.
37 38
Keywords: Aeromonas sobria; Biofilm formation; Foodborne pathogen; Methyl anthranilate;
39
Quorum sensing
40 41
1. Introduction
42
Aeromonas sobria is a Gram-negative, highly active, facultative anaerobic bacterium of the
43
family Aeromonadaceae (Janda and Abbott, 2010). It is a common zoonotic pathogen, can cause
44
furunculosis and septicemia in fish, as well as gastroenteritis and wound infection in humans.
45
Meanwhile, as a emerging foodborne pathogen, its virulence secretion and biofilm formation
2
46
caused serious food safety problems, and considerable economic loss in food industry (Chen et al.,
47
2013; Nagar et al., 2011). Recently, some studies have found that the expression of virulence
48
factors and biofilm formation of A. sobria are regulated by quorum sensing (QS) system
49
(Beaz-Hidalgo and Figueras, 2013; Kirov et al., 2004).
50
QS is a system in which bacterial cells communicate with each other to monitor the changes
51
in their population density through signal molecules (called autoinducers, AIs), and activate
52
specific sets of genes when a threshold is reached (Whiteley et al., 2017). Among them,
53
acyl-homoserine lactone (AHL) are the most common autoinducers of Gram-negative bacteria
54
(Papenfort and Bassler, 2016). LuxI/R-type QS system was the model system in Gram- negative
55
bacteria, containing two integrators, AHL synthase (I-protein), encoded by LuxI homologue
56
synthase, and a cognate receptor (R-protein), activated by AHL, which have been shown to
57
regulate multiplex phenotypic characteristics, such as expression of virulence factors and biofilm
58
formation (Ng and Bassler, 2009; Parsek and Greenberg, 2005; Rutherford and Bassler, 2012).
59
Recently, many studies have focused on interfering bacterial QS system with natural compounds,
60
known as QS inhibitors (QSIs), due to the ability of reducing virulence, and not imposing strong
61
selective pressures on development (Defoirdt, 2018).
62
Plant food extracts are commonly considered as attractive sources of anti-QS compounds,
63
due to their capacity to interfere with QS system, and many studies have reported that there are
64
three different ways to modulate QS of Gram-negative bacteria, by inhibition of AHL synthesis,
65
by degrading signaling molecules, and by targeting the signal receptor, LuxR (Defoirdt et al., 2013;
66
Truchado et al., 2009; Truchado et al., 2015). Methyl anthranilate, a plant spice extract, occurs
67
naturally in grapes and strawberries, and has been widely employed for the preparation of edible
3
68
flavor and food additives in food processing industries. It has been reported that biofilm formation
69
and elastase activity in Pseudomonas aeruginosa were significantly inhibited after treatment with
70
methyl anthranilate and its analogues (Calfee et al., 2001; Li et al., 2018). However, there is still
71
limited information about methyl anthranilate as a QSI in Gram-negative bacteria, especially in A.
72
sobria.
73
Furthermore, molecular docking and dynamic simulation methods have gained considerable
74
importance while analyzing the binding ability of ligands to the receptor proteins and the
75
conformational changes in receptor proteins after binding. The potential anti-QS activity of
76
cinnamaldehyde was revealed using molecular docking via interaction with hydrogen bond of
77
LuxR-type protein in P. fluorescens (Li et al., 2018). The conformational changes in the LasR
78
receptor protein of P. aeruginosa due to the binding of signaling molecule or quercetin were
79
predicted by Gopu et al. (2015). In this study, we analyzed the inhibitory effect of methyl
80
anthranilate on the expression of QS-related genes and QS-regulated phenotypes in A. sobria, such
81
as biofilm formation and AHL production, in order to evaluate its potential as a QS inhibitor. To
82
further elucidate the QS inhibitory mechanism of methyl anthranilate, in silico analysis was
83
performed, including molecular docking and dynamic simulation.
84
2. Materials and Methods
85
2.1. Materials and bacterial strains
86
Methyl anthranilate (≥ 98% purity) was purchased from Sigma-Aldrich (St. Louis, MO,
87
USA). AHL standards, including C4-HSL, C6-HSL, C8-HSL, C10-HSL, C12-HSL, and C14-HSL,
88
were purchased from the same company. A. sobria was originally isolated and identified from
89
deteriorated turbot. Chromobacterium violaceum CV026 was generously provided by Dr. Yang
4
90
from Xinjiang Shihezi University. It was used as a reporter strain for evaluating anti-QS activity.
91
Due to the resistance, the culture medium of CV026 was supplemented with 20 µg/mL kanamycin.
92
2.2. Determination of minimal inhibitory concentration (MIC) of methyl anthranilate
93
The MIC for methyl anthranilate was determined against CV026 and A. sobria by Oxford
94
cup method (Diao et al., 2013). Sterilized Oxford cups were firstly put into the plates, and then, 20
95
mL LB nutrient agar containing CV026 or A. sobria overnight cultures (OD595 nm= 1.0, 200 µL)
96
was poured into plates. 200 µL of varying concentrations of methyl anthranilate (0.125 to 5
97
µL/mL) were added to the wells and cells were incubated (160 rpm at 28 °C for 24 h). Equal
98
amount of sterile water was used as the negative control. The lowest concentration of methyl
99
anthranilate, which inhibited visible growth, was selected as MIC. Further experiments were
100
performed only at the sub-MIC level.
101
2.3. Determination of cell membrane integrity of A. sobria
102
The cell membrane integrity was determined according to the method described by Chakotiya
103
et al. (2017), with slight modification. Briefly, 200 mL LB broth containing the sub-MICs (0.5
104
µL/mL, 0.25 µL/mL, 0.125 µL/mL, and 0.0625 µL/mL) of methyl anthranilate were mixed with
105
200 µL of A. sobria overnight cultures, incubated at 28 °C until the density reached 106 CFU/mL.
106
Equivalent amount of sterile water was used as negative control and each experiment was done in
107
triplicate. The mixtures were centrifuged at 9000 ×g for 5 min and the supernatant was discarded.
108
The pellets were resuspended in 1 mL of PBS (pH 7.4), and then, fixed with 70% ethanol at 4 °C
109
for 30 min. After washing thrice by PBS, the bacterial cells were stained with PI stain (at final
110
concentration of 10 mg/mL) in dark for 30 min at 37 °C, then measured in Flow Cytometer (BD
111
AccuriTM C6 Plus). The autofluorescence was observed in the cells without methyl anthranilate
5
112
and PI staining.
113
2.4. QS inhibitory test
114
2.4.1. Violacein inhibition assay
115
CV026 overnight cultures (200 µL) were added to LB nutrient agar (20 mL) supplemented
116
with 20 µg/mL C4-HSL, following the method of Packiavathy et al. (2012). The mixtures were
117
poured into the plates containing sterilized Oxford cups. Then, 200 µL of sub-MICs (0.5 µL/mL,
118
0.25 µL/mL, 0.125 µL/mL, and 0.0625 µL/mL) of methyl anthranilate were added to the wells,
119
equivalent amount of sterile water was served as control group. The plates were incubated at
120
28 °C for 24 h, and QS inhibitory activity was assessed by evaluating the inhibitory zones.
121
2.4.2. Quantitative assay of violacein production
122
The violacein production was quantitatively determined by the method of Zhang et al. (2018).
123
LB broth (10 mL) containing CV026 overnight cultures (100 µL) was mixed with the sub-MICs
124
(0.5 µL/mL, 0.25 µL/mL, 0.125 µL/mL, and 0.0625 µL/mL) of methyl anthranilate as well as 20
125
µg/mL C4-HSL, then incubated at 28 °C at 160 rpm for 48 h. After incubation, the mixtures (300
126
µL) were transferred to the centrifuge tubes, and mixed with 10% sodium dodecyl sulfate (150 µL)
127
and butyl alcohol (600 µL) using a vortex mixer. After centrifugation at 9000 ×g for 6 min, the
128
violacein supernatant collected in the organic layer was added into a 96-well microplate to
129
determine OD595 nm values using microplate reader (Bio-tek, Vermont, USA).
130
2.4.3. Determination of biofilm formation
131
The assay for biofilm formation was performed according to the method of Zhang et al.
132
(2014). Overnight cultures (1 mL) of A. sobria were diluted with LB broth (100 mL) and
133
transferred to the centrifuge tubes. The centrifuge tubes were supplemented with different
6
134
sub-MICs of methyl anthranilate or 20 µg/mL C4-HSL (AHL treatment group). Equivalent amount
135
of sterile water was used as control group. After cultivation at 28 °C for 48 h, 200 µL of bacterial
136
cultures were transferred to a 96-well plate to measure the absorbance at 595 nm. Then, the
137
cultures were discarded. The centrifuge tubes were rinsed gently with sterile water, air dried,
138
stained with 0.1% crystal violet (w/v) for 20 min, and rinsed again. Biofilm was solubilized with
139
33% acetic acid and added into the 96-well microplate to determine OD595
140
microplate reader (Bio-tek, Vermont, USA).
141
2.4.4. Visualization of biofilm by scanning electron microscopy (SEM) and light microscopy
nm
values using
142
Biofilm visualization was performed with the method described by Zhou et al. (2018). The
143
polished zinc pieces (7 mm × 7 mm × 0.2 mm) were immersed in 10 mL of LB broth in sterile
144
plates. To the plates, A. sobria overnight cultures (100µL), the different sub-MICs of methyl
145
anthranilate, sterile water (control group), and 20 µg/mL C4-HSL (AHL treatment group) was
146
added. After incubation at 28 °C for 48 h, the biofilm formed on zinc piece was rinsed twice and
147
fixed with 2.5% glutaraldehyde for 4 h. The zinc piece was dehydrated in graded ethanol for 15
148
min. After air drying, the biofilm was visualized with SEM.
149
Method operations as described above, the biofilm formed on glass slides was rinsed thrice
150
with sterile water, and stained with 1mL of 0.1% crystal violet for 15 min. Then, the biofilm was
151
visualized by optical microscope.
152
2.4.5. Determination of bacterial motility
153
Motility was evaluated as described by Gutierrez-Pacheco et al. (2018), with slight
154
modifications. Swimming medium (0.5% NaCl, 0.3% agar, and 1% tryptone) or swarming
155
medium (0.5% NaCl, 0.6% agar, 0.5% D-fructose, and 1% peptone) was mixed with the different
7
156
sub-MICs of methyl anthranilate, sterile water (control group) and 20 µg/mL C4-HSL (AHL
157
treatment group), and then, poured into plates. Five microliters of A. sobria overnight cultures
158
were point inoculated at the center of plates, and each test was done in triplicate. After incubation,
159
the migration distance was determined using a vernier caliper to assess swimming and swarming
160
motility.
161
2.4.6. Determination of extracellular protease activity
162
Extracellular protease activity was assessed according to the method described by Li et al.
163
(2018). 10 mL of 15 % skim milk (115 °C, 30 min, 0.06 MPa) was mixed with 90 mL of LB
164
nutrient agar, and poured into plates. Overnight cultures of A. sobria treated with the different
165
sub-MICs of methyl anthranilate treatment, sterile water (control group), and 20 µg/mL C4-HSL
166
(AHL treatment group). After incubation, the mixtures were centrifugated at 9000 ×g for 10 min.
167
The supernatant was filtered using 0.22 µm filter; then, 100 µL of the filtrate was transferred into
168
the plates. After incubation at 28 °C for 48 h, the diameter of the transparent zone was measured
169
to assess protease activity using a vernier caliper.
170
2.4.7. Determination of AHL production
171
Overnight cultures of A. sobria with or without the different sub-MICs of methyl anthranilate
172
were incubated at 28 °C for 48 h, and then, centrifugated at 9000 ×g for 15 min. The supernatant
173
was extracted by acidified ethyl acetate (containing 0.1% acetic acid) for four times, and then,
174
concentrated by rotary evaporation. The extract was dissolved with 1 mL methanol and filtered
175
through 0.22 µm filter for GC-MS analysis.
176
AHL analysis was performed by GC-MS Agilent 7890 N/5975 (Agilent, USA). Briefly, all
177
samples were injected into HP-5 MS capillary column in the split mode (50:1), along with helium
8
178
as the carrier gas at a flow rate of 1 mL/min. The injector temperature, oven temperature program,
179
and mass spectrometry conditions were set following the method of Li et al. (2016). Data were
180
acquired by full-scan mode (m/z 35-800) and selected ion monitoring (SIM) mode (m/z 143).
181
2.5. RT-qPCR analysis
182
Overnight cultures of A. sobria with or without the different sub-MICs of methyl anthranilate
183
were incubated at 28 °C, until OD595 nm values reached 1.0. Total RNA was extracted by Trizol
184
(Thermo Fisher Scientific) from each cell pellet. cDNA synthesis was performed using the
185
RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific). RT-PCR reaction was
186
done by using the Power SYBR™ Green Master Mix (Thermo Fisher Scientific) and a CFX
187
Connect Real-Time PCR Detection System (Bio-Rad Laboratories). The primer sequences were
188
designed using Primer 5.0, and listed in Table S1. cDNA was served as the template and 16S
189
rRNA was served as the internal control. Specificity of product amplification was confirmed by
190
melting curve. The 2-(∆∆CT) method was used to calculate the expression levels of target genes, as
191
previously described by Li et al. (2019).
192
2.6. Molecular Docking analysis
193
The protein sequences of LuxI (WP_101529659.1) and LuxR (WP_005335753.1) of A.
194
sobria were obtained from National Centre for Biotechnology Information (NCBI) database for
195
further computer-simulated investigation. The three dimensional (3D) structures of the
196
homologous
197
(https://swissmodel.expasy.org). Ramachandran plot was used to predict whether the amino acid
198
residues in the protein were in the favored region, and reflected the rationality of conformation.
199
The preparation and optimization process of 3D protein structures were performed by Discovery
protein
were
predicted
and
9
assessed
by
SWISS-MODEL
200
Studio (DS). The structures of methyl anthranilate, halogenated furanone C30, and C4-HSL were
201
got from ZINC (http://zinc.docking.org/) and optimized in the DS for obtaining the lowest-energy
202
conformations. According to previous research (Li et al., 2018), the Libdock algorithm was used
203
to analyze the interactions between protein and ligand.
204
2.7. Molecular dynamics simulation analysis
205
Molecular dynamics simulation was performed to analyze the stability of the receptor
206
protein-signaling molecule and receptor protein-methyl anthranilate complexes using the
207
GROMACS 2018.2 package, as previously described by Zuo et al. (2017). The pdb structure of
208
complex was used as the beginning for MD simulations. The protein topology was generated by
209
Gromos54a7 force field, and ligand topology was constructed by PRODRG online server. The
210
whole complex was solvated in a cubic period box, and the minimum distance between the
211
complex and box edge was set at 1 nm. The charge of protein was neutralized by addition of a
212
sufficient number of chloride ions. Then, the energy minimization of entire system was performed
213
using 10000 steps of conjugate gradient algorithm. The entire system was equilibrated for 50 ps
214
each time for position restrains of NVT (constant number of particles, volume, and temperature)
215
and NPT (constant number of particles, pressure, and temperature). Linear Constraint Solver
216
algorithm was used for constraining the bond length during the simulation. Isothermal and isobaric
217
coupling constant were kept at 0.1 ps and 2 ps, respectively, bringing the system to a stable
218
environment of 300 K temperature and 1 bar pressure. Finally, the system at equilibrium for each
219
complex was subjected to 50 ns MD simulation with the time step of 2 fs, and the trajectory was
220
saved after every 10 ps.
221
2.8. Statistical analysis
10
222
All experiments were performed in triplicates and the data was expressed as mean ± standard
223
deviation (SD). Statistical analysis was performed by one-way analysis of variance (ANOVA)
224
using SPSS software (version18.0). Differences at P < 0.05 were considered statistically
225
significant.
226
3. Results
227
3.1. MIC of methyl anthranilate and the effect on cell membrane integrity of A. sobria
228
The MIC of methyl anthranilate was measured with concentrations ranging from 0.125
229
µL/mL to 5 µL/mL. Determination of MIC for methyl anthranilate against CV026 and A. sobria
230
was 2.0 µL/mL and 5.0 µL/mL, respectively. Consequently, the sub-MICs (0.5 µL/mL, 0.25
231
µL/mL, 0.125 µL/mL, and 0.0625 µL/mL) of methyl anthranilate were used for further
232
experiments. In addition, propidium iodide (PI) uptake assay was performed by flow cytometry to
233
assess the effect of sub-MICs of methyl anthranilate on cell membrane integrity of A. Sobria. PI, a
234
cationic dye, could not enter an intact cell membrane but could easily enter into the damaged
235
membrane and intercalate the nucleic acids. PI stain was retained in 4.58±1.009% of bacterial
236
cells when treated with 0.0625 µL/mL methyl anthranilate (Fig. 1A, B). Besides, 6.85±0.007% of
237
cells retained the stain after treatment with maximum sub-MIC of methyl anthranilate (0.5 µL/mL).
238
The negative control showed 6.43±0.005% of PI uptake, and 0% of auto fluorescence was shown
239
by bacterial cells without methyl anthranilate and PI stain treatment.
240
3.2. Effect of methyl anthranilate on violacein production
241
The QS inhibitory activity of methyl anthranilate was evaluated by determining its effect on
242
purple pigment violacein of CV026. As shown in Fig. 1C, a clear inhibitory zone was formed after
243
treatment with methyl anthranilate. In the quantitative assay of violacein production, the sub-MICs
11
244
of methyl anthranilate exhibited a dose-dependent inhibitory effect on violacein production.
245
Maximum inhibitory rate of 41.01% was observed at a concentration of 0.5 µL/mL, with no
246
inhibition on the growth of CV026 cells (Fig. 1D).
247
3.3. Effect of methyl anthranilate on biofilm formation
248
The assessment of anti-biofilm activity of methyl anthranilate against A. sobria was shown in
249
Fig. 2A. The group treated with sub-MICs (0.5 µL/mL, 0.25 µL/mL, 0.125 µL/mL, and 0.0625
250
µL/mL) of methyl anthranilate led to a significant reduction in biofilm content of A. sobria to the
251
level of 31.67%, 34.01%, 41.27%, and 51.44%, respectively. In addition, there was no significant
252
difference in the biofilm biomass after treatment with methyl anthranilate (Fig. 2B).
253
The biofilm structure of A. sobria was visualized by optical microscopy and SEM. As shown
254
in Fig. 2C, the deep purple clustered appearance of biofilm was exhibited in AHL treatment group.
255
However, the density of biofilm was visibly reduced in the methyl anthranate treatment group, and
256
the appearance was sparse. In the SEM images (Fig. 2D), the biofilm matrices treated with methyl
257
anthranilate were clearly decreased, and exhibited a small number of microcolonies, pores, and
258
channels, whereas the biofilm treated with C4-HSL was mature and dense.
259
3.4. Effect of methyl anthranilate on motility and extracellular protease activity
260
The migration ability of A. sobria was assayed in the presence and absence of methyl
261
anthranilate. As shown in Fig. 3 (A, B), swimming and swarming motility zones of A. sobria
262
gradually reduced. In presence of 0.5 µL/mL methyl anthranilate, the maximum inhibitory rates of
263
74.84% and 71.63%, respectively, were obtained (Table S2). In addition, the migration ability of
264
cells in AHL treatment group was distinctly enhanced.
265
The ability of methyl anthranilate to reduce extracellular protease activity of A. sobria was
12
266
evaluated by measuring the diameter of transparent zone. As shown in Fig. 3C, the protease
267
produced by A. sobria was significantly inhibited. In presence of 0.5 µL/mL methyl anthranilate,
268
the protease activity decreased by 43.08 % (Table S3). However, the addition of exogenous
269
C4-HSL enhanced its protease activity.
270
3.5. Effect of methyl anthranilate on QS-related gene expression and AHL production
271
To elucidate the effect of methyl anthranilate on QS-related genes in A. sobria, the expression
272
levels of luxI and luxR genes were examined using RT-qPCR. As shown in Fig. 4A, the expression
273
levels of luxI and luxR were downregulated when treated with methyl anthranilate. The minimum
274
levels of luxI (0.160) and luxR (0.064) were observed after treatment with 0.5 µL/mL methyl
275
anthranilate. However, the expression levels of genes were significantly upregulated in AHL
276
treatment group. Moreover, the effect of methyl anthranilate on the AHL production was
277
quantitatively analyzed by GC-MS. The retention time of the six AHL standards were determined,
278
and the ratio of the peak area of the sample to the peak area of the internal standard (C14-HSL)
279
was used to evaluate the relative production of AHL (Fig. 4B). C4-HSL was the main signal
280
molecule secreted by A. sobria. The minimal concentration of C4-HSL was decreased to 0.865
281
µg/mL after treatment with 0.5 µL/mL methyl anthranilate.
282
3.6. Homology modeling and assessment
283
The 3D model of homologous proteins was predicted and assessed by SWISS-MODEL,
284
using the sequences of LuxI (acyl-homoserine-lactone synthase, WP_101529659.1) and LuxR
285
(transcriptional regulators, WP_005335753.1). Global Model Quality Estimation (GMQE) and
286
sequence similarities were used to select the best 3D model (Table S4). QMEAN scores
287
represented the degree of agreement of the structural features between the predicted model and
13
288
experimental structures of similar size. Lower QMEAN scores indicated high model quality.
289
Thereby, the models LuxI 1ro5.1.A (-1.98) and LuxR 4lfu.1.A (-2.26) were selected for further
290
analysis. Furthermore, the presence of more than 90% of the residues in the favored region of
291
Ramachandran plot indicated the high rationality of protein structure. The results revealed that
292
90.2% (LuxI) and 94.7% (LuxR) residues existed in the favored region (Fig. S2). Consequently,
293
the models of LuxI 1ro5.1.A and LuxR 4lfu.1.A exhibited great reliability, the 3D structures of
294
LuxI and LuxR proteins in A. sobria were successfully predicted.
295
3.7. Molecular docking and dynamic simulation
296
The interaction of methyl anthranilate, the natural ligand C4-HSL, and the classical QS
297
inhibitor, halogenated furanone C30, into the receptor active site was investigated by molecular
298
docking analysis. As shown in Fig. 5C and c, methyl anthranilate interacted with Ala143, Ser145,
299
Met33, and Leu147 residues of LuxR protein via the H-bonds (green dashed lines), and underwent
300
hydrophobic interactions (pink dashed lines) with Tyr35, Leu149, and Leu147 residues. In
301
addition, the C4-HSL was found to interact through H-bonds with Tyr90, Asn119, and Leu147 (Fig.
302
5A,a). The furanone C30 formed hydrogen bonds with Leu147, hydrophobic interactions with
303
Ile154 and Leu149, and halogen bonds (blue dashed lines) with Ala143 (Fig. 5B, b). For LuxI
304
protein, methyl anthranilate interacted via the H-bonds with Ala85, Gln166, Leu162, and Gly175,
305
and formed hydrophobic interactions with Val168, Val176, and Ala87 (Fig. 5D, d).
306
To further investigate the conformational changes in receptor protein in the presence of
307
C4-HSL and methyl anthranilate, 50 ns of molecular dynamics simulation was performed, and the
308
stability of two complexes was assessed by the values of root mean square deviation (RMSD) and
309
root mean square fluctuation (RMSF). Their lowest values indicated that the complex was more
14
310
stable. RMSD is a measure of the average distance between the backbone atoms of superimposed
311
proteins. As shown in the Fig. 6A, the RMSD values of LuxR-methyl anthranilate complex
312
reached a stable state at 0.62 nm after 20,000 ps, which suggested that its conformation was
313
relatively stable over the time period of 50 ns. However, the RMSD values of LuxR-C4-HSL
314
complex displayed wide fluctuations during the entire simulation process. RMSF represents the
315
position deviation of amino acid residues with respect to the reference position. LuxR receptor
316
protein is composed of A and B chains; no significant difference was observed in the two curves
317
of RMSF profile of A chain (Fig. S3); however, the two curves of B chain were markedly different.
318
The results showed that the interaction on B chain was consistent with the results of molecular
319
docking analysis. As shown in Fig. 6B, the RMSF values of LuxR-methyl anthranilate complex
320
were relatively low, indicating that it had high stability. Notably, the RMSF values of
321
LuxR-methyl anthranilate complex decreased gradually in the region of binding site (residues
322
135-150), while the RMSF values of LuxR-C4-HSL complex increased gradually in this region.
323
4. Discussion
324
Recently, many studies have shown that the interference of plant food extracts on bacterial
325
QS system could be an effective strategy to alleviate the virulence of foodborne pathogens (Zhou
326
et al., 2019). We reported a promising QS inhibitor, methyl anthranilate, which exhibited a strong
327
inhibitory activity on the QS-regulated phenotypes of A. sobria, such as biofilm formation,
328
motility, protease activity, and AHL production. In the present study, the sub-MIC levels of methyl
329
anthranilate were selected for subsequent experiments. To evaluate the effect of sub-MICs of
330
methyl anthranilate on cell viability of A. sobria, the integrity of cell membrane was characterized
331
by flow cytometry with PI uptake as an indicator. Cell membrane integrity plays a crucial role in
15
332
maintaining stable metabolism and resistance against harsh environment in bacterial cells. There
333
was no significant difference in the fluorescence intensity and the percentage of PI-positive cells
334
between the methyl anthranilate treatment group and negative control group. This indicated that
335
the sub-MICs of methyl anthranilate exhibited no effect on the cell membrane integrity and no
336
damage to the bacterial cells.
337
C. violaceum CV026, a reporter strain, produced purple pigment violacein upon induction
338
with the exogenous addition of short-chain autoinducer. It was widely used to monitor the
339
production of acyl-homoserine lactone or QS inhibitory activity (Ding et al., 2019). In this study,
340
the anti-QS activity of methyl anthranilate was assessed by observing the formation of inhibitory
341
zone using the reporter strain, CV026. The results showed that the production of characteristic
342
violacein was significantly inhibited by methyl anthranilate, suggesting a high QS inhibitory
343
activity. To further evaluate the extent of inhibition on the violacein, the quantitative assay of
344
violacein production was performed. This result demonstrated the sub-MICs of methyl
345
anthranilate reduced the levels of violacein in a concentration-dependent manner without affecting
346
CV026 growth. Hence, it was confirmed that the decrease in violacein production was due to the
347
interference with QS system, rather than the inhibition of bacterial growth, which was consistent
348
with above result. Consistently, Zhang et al. (2014) found that Rosa rugosa tea polyphenol extract
349
inhibited QS-controlled violacein production in CV026 without affecting its growth.
350
In order to adapt to the environmental stress, the microbial communities adhere to the contact
351
surfaces and enmesh themselves into matrices, namely the biofilm. Biofilm is widely found on the
352
surface of food machinery and equipment made of various materials, such as metal, glass, and
353
plastic. Compared with planktonic bacteria, biofilm is more resistant and difficult to remove,
16
354
which easily cause serious contamination of food and medical equipment (Li et al., 2019). QS
355
system mediated by AHL is known to play an essential role in the process of biofilm formation
356
(Rudrappa et al., 2008). Therefore, interference of QS system could be used as a novel strategy to
357
regulate the biofilm formation (Zhou et al., 2018). This result demonstrated the sub-MICs of
358
methyl anthranilate reduced the biofilm content, with no inhibition on the growth of A. sobria
359
cells. Moreover, the addition of C4-HSL promoted the biofilm formation, which suggested that
360
biofilm formation was regulated by QS system. In this study, the effect of methyl anthranilate on
361
the biofilm microstructure of A. sobria was visualized by optical microscopy and SEM. In the
362
optical microscopy images, the biofilm formed in the presence of methyl anthranilate was sparse,
363
and the density was visibly reduced. In the AHL treatment group, the biofilm showed continuous
364
and dense appearance. The SEM images revealed that the biofilm matrices treated with C4-HSL
365
were clearly increased, and large amounts of microcolonies were connected and formed a dense
366
biofilm structure. However, the biofilm treated with methyl anthranilate exhibited a small number
367
of microcolonies, as well as the pores and channels. This suggested that methyl anthranilate might
368
cause the leakage of nutrients and reduction in biofilm matrices through these pores and channels,
369
thereby inhibiting the biofilm formation. The results of biofilm microscopy supported the
370
quantitative data. Similarly, Packiavathy et al. (2014) found that the sub-MICs of Cuminum
371
cyminum could cause the loosening of biofilm architecture and inhibit biofilm formation in
372
Pseudomonas aeruginosa, Proteus mirabilis, and Serratia marcescens.
373
The effect of methyl anthranilate on the expression of QS-regulated phenotypes of A. sobria,
374
such as motility and extracellular protease activity, were also evaluated in this study. The bacterial
375
flagella-mediated motility, such as swimming and swarming, plays a major role in the process of
17
376
biofilm formation regulated by AHL-mediated QS system (Du et al., 2018). Thus, the reduction of
377
bacterial migration ability might affect the ability of biofilm formation (Gutierrez-Pacheco et al.,
378
2018). In this research, the zones of swimming and swarming motility in methyl
379
anthranilate-treated group were observed to be markedly reduced, indicating that the motility of A.
380
sobria was significantly inhibited by methyl anthranilate. However, the swimming and swarming
381
motility of bacteria after treatment with C4-HSL was significantly promoted, suggesting that
382
exogenous AHL could promote bacterial migration ability. These results were associated with the
383
inhibition of methyl anthranilate on the biofilm formation of A. sobria. Consistent with our results,
384
Lou et al. (2017) found that burdock leaf components decreased the aggregation ability, thus
385
inhibiting the biofilm formation of P. aeruginosa. Furthermore, extracellular protease is one of the
386
primary virulence factors regulated by QS system in many pathogens. Extracellular proteases
387
secreted by microorganisms could decompose the proteins and free amino acids in food and
388
produce volatile and stimulating compounds containing nitrogen and sulfur, thus accelerating the
389
spoilage process and leading to deterioration of food quality (Ding et al., 2017). Thus, the
390
inhibitory effect of methyl anthranilate on the protease activity of A. sobria was evaluated. The
391
results showed that the sub-MICs of methyl anthranilate significantly reduced protease activity.
392
Besides, the addition of exogenous C4-HSL enhanced the protease activity of A. sobria. These
393
observations were similar with those reported by Husain et al. (2017), who reported that mango
394
extract significantly inhibited the production of virulence factors of P. aeruginosa, such as
395
protease, pyocyanin, and swarming motility.
396
Due to the interference ability of methyl anthranilate on QS system, biofilm formation,
397
migration ability, and protease activity was significantly reduced in A. sobria, along with
18
398
down-regulation of QS-related genes. The inhibition of methyl anthranilate on the QS system of A.
399
sobria was further characterized by determining the expression levels of AHL synthetase (luxI
400
gene) and receptor protein (luxR gene) using RT-qPCR technology. The results showed that the
401
expression levels of luxI and luxR were significantly down-regulated after treatment with methyl
402
anthranilate, indicating that the gene expression was interfered by methyl anthranilate.
403
Furthermore, due to the vital role of AHL in QS system of Gram-negative bacteria, the effect of
404
methyl anthranilate on the AHL (C4-HSL) production in A. sobria was analyzed using GC-MS.
405
Gradual reduction of the AHL production was observed in the methyl anthranilate-treated group,
406
which suggested that AHL production of A. sobria was effectively inhibited. Our study found that
407
the inhibition of methyl anthranilate on the QS system of A. sobria might involve interference
408
with AHL biosynthesis. These results were consistent with the reports of Li et al. (2019), who
409
reported that different sub-MICs of vanillin significantly down-regulated the expression levels of
410
QS-related genes halI/halR and the production of AHL in Hafnia alvei.
411
To reveal the anti-QS mechanism of methyl anthranilate, in silico analysis was performed.
412
The interaction between methyl anthranilate and receptor protein was evaluated using molecular
413
docking analysis. Hydrogen bonds can maintain the stability of complex molecules and play a key
414
role in molecular recognition. Here, we found that methyl anthranilate and furanone C30
415
interacted with the same residues, Leu147 and Leu149, via H-bond and hydrophobic bond,
416
respectively, revealing that two compounds had common binding sites. Moreover, methyl
417
anthranilate and C4-HSL formed H-bond interactions with LuxR protein at the common site
418
Leu147, and methyl anthranilate also exhibited hydrophobic interactions at this site. Compared
419
with furanone C30 and C4-HSL, methyl anthranilate formed more H-bond interactions with LuxR
19
420
protein. These results demonstrated that the binding affinity of methyl anthranilate bound to LuxR
421
receptor protein was higher than that of its cognate ligand C4-HSL. Furthermore, molecular
422
docking studies are not competent enough to reveal the conformational changes caused by the
423
interactions between receptors and ligands (Gopu et al., 2016). Therefore, molecular dynamics
424
simulation was performed to investigate the conformational changes of receptor protein in the
425
presence of signaling molecule and methyl anthranilate. RMSD profile showed that the
426
LuxR-methyl anthranilate complex was more stable than the LuxR-C4-HSL complex during the
427
whole simulation. This might be because the interactions between the receptor protein and methyl
428
anthranilate made the conformational changes of the receptor protein relatively small, and thus, its
429
complex was more stable. RMSF profile further demonstrated that the instability of LuxR-C4-HSL
430
complex might be related to the activation of protein conformation caused by the interactions of
431
C4-HSL, while this conformation in LuxR-methyl anthranilate complex was stabilized by
432
interactions with methyl anthranilate. The in silico results suggested that methyl anthranilate acted
433
as a potential competitive inhibitor of signaling molecules that bind to LuxR receptor protein.
434
Combined with RT-qPCR results, the QS inhibitory mechanism of methyl anthranilate might
435
involve two pathways, interfering with AHL biosynthesis as well as competitively binding with
436
receptor proteins.
437
To summarize, the inhibitory effect of methyl anthranilate against A. sobria was evaluated for
438
the first time in order to determine its possible use as a novel QS inhibitor. Our results have
439
demonstrated that methyl anthranilate exhibited a remarkable inhibition on the QS-regulated
440
phenotypes in A. sobria, including biofilm formation, motility, and protease activity. Besides, the
441
results of RTq-PCR and GC-MS showed that the expression levels of QS-related genes were
20
442
down-regulated and the biosynthesis level of AHL was reduced, when treated with the different
443
sub-MICs of methyl anthranilate. To expound QS inhibitory mechanism, the interaction between
444
methyl anthranilate and receptor protein and the conformational changes in receptor protein after
445
binding were shown by molecular docking and dynamics simulation. These results demonstrated
446
that methyl anthranilate might inhibit QS system in A. sobria by interfering with AHL
447
biosynthesis, as well as competitively binding with receptor protein. Our findings strongly
448
suggested that methyl anthranilate could be used as a novel QS inhibitor and anti-biofilm agent for
449
alleviation of the damage caused by foodborne pathogens.
450
Acknowledgements
451
This research was financially supported by the National Natural Science Foundation of China
452
and
453
(2018YFD0400601, 2017YFD0400106) and the Open Project Program of Beijing Key Laboratory
454
of Flavor Chemistry, Beijing Technology and Business University (SPFW2019YB08).
455
Conflict of interest
456
National
Key
Research
(No.
31471639),
Development
Programme
of
China
All authors declare no conflict of interest.
457 458 459
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A
B
C
D
Fig. 1. Quantitative assessment of cell membrane integrity, Cells were stained with PI and analyzed by flow cytometry (A). (Con fcs) control fluorescence, (Auto fcs) Auto fluorescence, (0.0625, 0.125, 0.25, and 0.5) methyl anthranilate fluorescence. Cell membrane integrity was evaluated by PI- positive cell rate (B). Inhibitory activity of the sub-MICs of methyl anthranilate on the violacein production in CV026 (C). Quantitative analysis of violacein production and CV026 biomass after treatment with the sub-MICs of methyl anthranilate (0.0625, 0.125, 0.25, and 0.5 µL/mL) (D).
26
565 566 567 568 569 570
A
C
B
D
Fig. 2. Quantitative analysis of biofilm content (A) and biofilm biomass (B) with sub-MICs of methyl anthranilate or C4-HSL treatment. Optical microscopic images (C) and SEM images (D) of the biofilm of A. sobria with sub-MICs of methyl anthranilate or C4-HSL treatment. (a) 20 µg/mL C4-HSL, (b) control, (c–f) 0.0625, 0.125, 0.25, and 0.5 µL/mL methyl anthranilate. A
B
C
571 572 573 574
Fig. 3. Inhibitory effect of the sub-MICs of methyl anthranilate on swimming (A) and swarming (B) motility, as well as proteinase activity (C) of A. sobria. (a) 20 µg/mL C4-HSL, (b) control group, (c–f) 0.0625, 0.125, 0.25, and 0.50 µL/mL methyl anthranilate.
27
A
B
575 576 577 578 579
580 581 582 583 584 585 586
Fig. 4. Effect of the sub-MICs of methyl anthranilate on the relative expression of luxI and luxR genes (A) was assessed by RTq-PCR, and the AHL production (B) of A. sobria was evaluated by GC-MS. A
a
B
b
C
c
D
d
Fig. 5. Molecular docking of the native ligands C4-HSL, halogenated furanone C30, and methyl anthranilate with the LuxR and LuxI protein models are shown as a 3D diagram (A–D) and 2D diagram (a–d). (A,a) native ligands C4-HSL, (B,b) halogenated furanone C30, (C,c) methyl anthranilate, (D,d) methyl anthranilate combined with LuxI.
28
A
587 588 589 590 591 592
B
Fig. 6. RMSD profile (A) and RMSF profile (B) of LuxR-C4-HSL and LuxR-methyl anthranilate complex during 50 ns of molecular dynamics simulation. Black line represents the complex of LuxR protein and signaling molecule while red line indicates the complex of LuxR protein and methyl anthranilate.
29
Highlights
Methyl anthranilate significantly inhibited the QS-regulated phenotypes in A. sobria, such as biofilm formation, motility and protease activity. Methyl anthranilate down-regulated the expression levels of QS-related genes and reduced the biosynthesis level of AHL in A. sobria. The QS inhibitory mechanism of methyl anthranilate might involve two pathways, interfering with AHL biosynthesis as well as competitively binding with receptor protein. Methyl anthranilate could be as a promising QS inhibitor and anti-biofilm agent for improving food safety.