- Email: [email protected]
Acetylation of lysine 7 of AhyI affects the biological function in Aeromonas hydrophila
Journal Pre-proof Acetylation of lysine 7 of AhyI affects the biological function in Aeromonas hydrophila Dong Li, Srinivasan Ramanathan, Guibin Wang, Yao Wu, Qi Tang, Guohui Li PII:
S0882-4010(19)31861-3
DOI:
https://doi.org/10.1016/j.micpath.2019.103952
Reference:
YMPAT 103952
To appear in:
Microbial Pathogenesis
Received Date: 26 October 2019 Revised Date:
4 December 2019
Accepted Date: 26 December 2019
Please cite this article as: Li D, Ramanathan S, Wang G, Wu Y, Tang Q, Li G, Acetylation of lysine 7 of AhyI affects the biological function in Aeromonas hydrophila, Microbial Pathogenesis (2020), doi: https:// doi.org/10.1016/j.micpath.2019.103952. 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 Published by Elsevier Ltd.
CRediT author statement： Guohui Li: Conceptualization, Methodology, Software. Dong Li: Data curation, Writing- Original draft preparation. Yao Wu: Visualization, Investigation. Guohui Li: Supervision. Qi Tang: Software. Validation: Srinivasan Ramanathan: WritingReviewing and Editing.
1
Acetylation of lysine 7 of AhyI affects the biological function in
2
Aeromonas hydrophila
3
Dong Lia#, Srinivasan Ramanathanb#, Guibin Wangb, Yao Wub, Qi Tanga, Guohui Lia
*
a
Institute of Life Sciences, Jiangsu University, Zhenjiang 212 013, PR China.
4
b
Fujian Provincial Key Laboratory of Agroecological Processing and Safety Monitoring (School of Life
5
Sciences, Fujian Agriculture and Forestry University), Fuzhou 350 002, PR China.
6
7
8
9
10
11
12
13
14
15
16
17
#
18
*Corresponding
19
University, 301# Xuefu Road, Zhenjiang 212 013, PR China.
These authors contributed equally to this work.
author: Guohui Li, E-mail: [email protected], Institute of Life Sciences, Jiangsu
20 1
21
Abstract
22
Acyl-homoserine-lactone synthase (AhyI) of Aeromonas hydrophila can produce quorum sensing
23
(QS) auto-inducer 1 (AI-1) type signal molecule, which plays important roles in various biological
24
phenomenons such as biofilm formation, hemolysin production and motility. Previous research revealed
25
that the AhyI of A. hydrophila has acetylation modification on lysine 7 site, but its intrinsic biological
26
function is still largely unknown. To study the effect of AhyI protein and its acetylation modification on
27
the physiological traits of A. hydrophila, the site-directed mutagenesis strains including ∆ahyI::ahyI-K7Q
28
and ∆ahyI::ahyI-K7R were made in this study. The mutation at K7 site of lysine acetylation in AhyI
29
protein decreased the protease productions, but the lysine acetylations do not affect the biofilm formation
30
and hemolysin production. To further study the effect of lysine acetylation on AI-1 signal molecule
31
production, the acyl-homoserine lactones (AHLs) extraction and bioluminescence quantification were
32
performed. Compared to the rescue strain, the acetylation on K7 of AhyI resulted in a decreased level of
33
AHLs and bioluminescence productions. It indicated that the lysine acetylation modification on the AhyI
34
protein can regulate the production of signalling molecules. Overall, the obtained data in this study
35
provide a theoretical basis for further understanding the role of lysine acetylation of AhyI protein and lay
36
a foundation to systematically study the regulatory mechanism of QS.
37
Keywords: Aeromonas hydrophila; Biofilm formation; Lysine acetylation; Protein post-translational
38
modification; Quorum sensing
39
40 41
2
42
1. Introduction
43
Nowadays, the characterization of protein posttranslational modifications (PTMs) in bacterial
44
pathogens, particularly the adding of small chemical groups on the side chains of amino acids, has
45
become a vital research topic [1]. Furthermore, this type of PTMs is often dynamic, reversible and
46
conserved during the evolution process. The various types of protein PTMs such as phosphorylation,
47
acylation and methylation reversibly modify some specific amino acids residues and play a significant
48
role in various biological processes in bacterial pathogens [2]. Among the various PTMs, protein lysine
49
acetylation is one of the extensively studied PTMs due to their important roles, which transfer the acetyl
50
group from a donor metabolite to a lysine residue of specific proteins. More, the lysine acetylation is a
51
reversible process, in which the lysine deacetyltransferases remove the acetyl group from the acetylated
52
lysine residue of proteins [3]. In recent years, several researchers are continuously identifying thousands
53
of lysine acetylation sites in several bacterial pathogens including Pseudomonas aeruginosa, Bacillus
54
subtilis, Mycobacterium tuberculosis, Escherichia coli and Aeromonas hydrophila and reveal their
55
important role in bacterial virulence [2-6].
56 57
A. hydrophila is a Gram-negative pathogenic bacterium found in aquatic environments [7]. It
58
secretes a variety of virulence factors and toxins that can cause lesions and death in fish. However, the
59
disease caused by A. hydrophila can make the fish unsuitable for human ingestion. It is deliberated as an
60
emerging bacterial pathogen accountable for various systemic conditions such as skin infections,
61
peritonitis, gastroenteritis, hemolytic uremic syndrome, necrotizing fasciitis, meningitis, bacteremia and
62
cholera-like illness [8, 9]. Further, A. hydrophila secrets various virulence factors such as protease,
63
elastase, lipase, hemolysin, motility, biofilm formation and exopolysaccharide productions under the
64
control of cell density-dependent gene expression system called quorum sensing (QS) mechanism. More,
65
A. hydrophila secrets their virulence factors by two different types of the N-acyl homoserine lactone
3
66
(AHL) mediated QS systems such as LuxIR based auto-inducer 1 (AI-1) system (AhyIR) and LuxS based
67
auto-inducer-2 (AI-2) system [10-12].
68 69
In recent study, [2] identified various lysine-acetylated proteins in A. hydrophila ATCC 7966 by
70
the specific antibody enrichment combined with high-resolution mass spectrometry analysis. Further, the
71
obtained data in that study revealed the QS signal molecule protein AhyI was acetylated at the K7 site.
72
However, the specific biological function of this acetylated AhyI protein in A. hydrophila remains
73
unknown. Therefore, we have investigated the impact of lysine acetylation of AhyI protein on the
74
biological functions of A. hydrophila in the present study,
75 76
2. Materials and methods
77
2.1. Generation of ahyI knock-out strain
78
The ahyI gene deletion mutant was generated by suicide vector pRE112. Around 500 bp of
79
upstream and downstream flanking sequences of ahyI (gene name AHA_0556) was amplified from the A.
80
hydrophila ATCC7966 chromosomal DNA and then the overlap extension PCR was used for fusion into
81
the pRE112 vector for producing the recombinant constructs. The restriction sites such as BamHI and
82
SacI were used for the construction of recombinant plasmids. Further, the recombinant plasmids were
83
transformed into E. coli MC1061 (λpir) cells first to upraise the transformation efficacy and then
84
transformed into E. coli S17-1 (λpir) cells for fast propagation. After that, the plasmids were transferred
85
into the wild type A. hydrophila ATCC 7966 strain by bacterial conjugation. To select the ampicillin
86
resistance (100 µg/m AmpR) and chloramphenicol resistance (30 µg/ml CmR) single-crossover mutant
87
colonies, the first homologous recombination was done. Then, the second homologous recombination was
88
done to obtain the sacB gene depleted double-crossover mutant colonies. Moreover, PCR analysis was
89
used to confirm the single and double-crossover mutants with their corresponding primers (Table 1) [13].
90
4
91
2.2. Construction of site-directed mutagenesis strains
92
The pBBR1-MCS1 broad host-vector was used to construct the ahyI complementary strain and
93
the His-tagged pBBR1-ahyI plasmid was generated by HindIII and BamHI restriction sites as previously
94
described [14]. Addition to this, to confirm the normal expression, the corresponding promoter was also
95
inserted into the upstream of ahyI. Then, the Fast Mutagenesis System Kit was used to perform the site-
96
directed mutagenesis of ahyI (K7Q and K7R) with the corresponding primers (Table 2) according to the
97
manufacturer’s instructions (Transgen Biotech Co., Beijing, China). Then, all the resulting target
98
mutations were confirmed by DNA sequencing.
99 100
2.3. Bacterial strains used in the study and their growth conditions
101
The bacterial strains such as Vibrio harveyi BB170, Chromobacterium violaceum CV026, A.
102
hydrophila ATCC 7966 wild type strain, ∆ahyI A. hydrophila mutant strain, ∆ahyI::ahyI complementary
103
strain, ∆ahyI with original vector strain (∆ahyI::Vector) and site-directed mutagenesis (K7 point mutant
104
strains) strains such as ∆ahyI::ahyI-K7Q (acetylation strain) and ∆ahyI::ahyI-K7R (deacetylation strain)
105
were used in this study. Further, the reporter strains such as V. harveyi BB170 and C. violaceum CV026
106
strains were cultivated in Autoinducer Bioassay (AB) and Luria Bertani (LB) medium, respectively at
107
30°C, whereas the A. hydrophila ATCC 7966 wild and ∆ahyI mutant strains were cultivated in LB
108
medium at 30°C. Moreover, ∆ahyI::ahyI complementary strain, ∆ahyI with original vector strain
109
(∆ahyI::Vector) and site-directed mutagenesis strains such as ∆ahyI::ahyI-K7Q and ∆ahyI::ahyI-K7R was
110
cultivated in LB medium at 30℃along with chloramphenicol antibiotic (30 µg/ml). For experimental
111
purpose, all the strains were routinely grown overnight in their respective growth medium at their
112
optimum temperatures. Then, the obtained cultures were diluted into 1:100 ratios in their fresh respective
113
growth medium and incubated at their optimum temperatures at 200 rpm until the OD value reached 0.4
114
at 600 nm.
115 5
116
2.4. Western blotting analysis
117
For Western blotting analysis, the protein samples from strains such as ∆ahyI::ahyI, ∆ahyI::ahyI-
118
K7Q and ∆ahyI::ahyI-K7R were run on a 12% SDS-PAGE gel. Then, the electrophoresed proteins were
119
transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore, Burlington, MA, USA) in 10×
120
Transfer Buffer (Bio-Rad, Hercules, CA, USA) by a Trans-Blot Turbo Transfer System (Bio-Rad) for 15
121
mins at 25 V. After that, the PVDF membrane was blocked with 5% skim milk in PBST buffer (1×PBS
122
buffer containing 0.05% Tween-20). Then, the PVDF membrane was probed with the primary antibody
123
and incubated for 1h at room temperature with anti-acetyl lysine (PTM Biolabs, Inc. Hangzhou, China).
124
Further, the membrane was washed three times with PBST for 10 min each time and incubated with
125
horseradish peroxidase (HRP) goat anti-mouse IgG or anti-rabbit IgG secondary antibody. After that, the
126
membrane was rinsed five times with PBST for 5 mins each time and then the protein band was
127
visualized by Clarity Western ECL substrate (Bio-Rad) and imaged with a ChemiDoc XRS+ system (Bio-
128
Rad) [15, 16].
129 130
2.5. Determination of growth curve
131
The bacterial strains were cultivated in 5 ml LB medium and incubated at 30℃ for overnight at
132
200 rpm. Then, the overnight grown bacterial cultures were diluted at 1:100 ratios with LB medium and
133
incubated at 30℃ until the bacterial OD reached 0.4 at 600 nm. Then, the bacterial cultures were
134
inoculated in a HONEYCOMB® sterile 100 well plate and the OD values for growth were measured at
135
600 nm for 20 h using a Bioscreen C system (Lab Systems, Helsinki, Finland) [17].
136 137
2.6. Quantification of biofilm biomass
138
To assess the level of biofilm biomass in test bacterial pathogens, the biofilm biomass
139
quantification assay was performed as previously described method with slight modification [18]. Briefly,
140
the 1% of bacterial inoculum (cell density equivalent to 0.4 OD at 600 nm) was inoculated into 96 well 6
141
microtitre plates (MTPs) and incubated at 30℃ for 18 h. After incubation, the non-adherence planktonic
142
cells were discarded and washed twice with distilled water. Then, the plate was kept in room temperature
143
for desiccating and then the biofilm cells attached on the wells were stained by 1% crystal violet. After
144
that, the unwanted stain was washed out by distilled water and kept in room temperature for drying. Then,
145
the biofilm cells in stains were dissolved by 90% ethanol and the biofilm biomass was quantified at 595
146
nm.
147 148
2.7. Determination of extracellular protease and hemolytic activities
149
The effect of lysine acetylation on the extracellular protease and hemolysin productions were
150
measured by a previously described method with slight modifications [19]. Briefly, the 1% of skim milk
151
agar plates and hemolysin agar plates containing 5% sheep blood were prepared and the wells were made
152
by a sterile micropipette tip. Then, 5 µl of overnight bacterial cultures were loaded on their respective
153
wells of skim milk and hemolysin agar plates and incubated at 30°C for 12 h. After incubation, both
154
protease and hemolysin activities were determined based on the size of the zone of clearance on each
155
plate and the diameter of the zone of clearance were measured.
156 157
2.8. Extraction of AI-1 QS signal molecules
158
The previously described method has used for extracting the AI-1 QS signal molecules from the
159
bacterial cultures with some modifications [20]. Briefly, the bacterial strains such as ∆ahyI::ahyI,
160
∆ahyI::ahyI-K7Q and ∆ahyI::ahyI-K7R were cultured in LB medium at 30℃ for 24 h in 200 rpm. Then, 5
161
ml of bacterial culture was centrifuged at 13000 g for 5 min at 4℃ and the obtained culture supernatant
162
was filtered using 0.22 µm membrane filter. After that, the filtered supernatant was extracted with an
163
equal volume of acidified ethyl acetate (1% acetic acid) and sonicated by ultra-sonication for 20 min.
164
Then, the upper organic phase was collected and freeze-dried by vacuum freeze dryer for 2 h.
165
7
166
2.9. Detection of AI-1 QS signal molecules by thin-layer chromatography (TLC)
167
The extracted AI-1 QS signal molecules were determined by thin layer chromatography (TLC)
168
analysis by a previously described method with slight modifications [20]. Briefly, 2 µl of extracted AI-1
169
QS signal molecules were spotted on 10×10 cm size TLC plate (TLC aluminium sheets, Merck KGaA,
170
Darmstadt, Germany) and 3:2 ratio methanol-water solvent system was used as the mobile phase. Further,
171
it was allowed to the solvent system until reaching the front and then the TLC plate was dried for 10 min.
172
Then, the agar cover layer was prepared by mixing the 1% of an overnight culture of C. violaceum CV026
173
with 50 ml LB medium containing 0.7% agar and then immediately spread on the TLC plate. Then, the
174
TLC plates were transferred into a closed sterile container and incubated at 30℃ for 48 h and observed for
175
the level of AI-1 QS signal molecule productions.
176 177
2.10. Bioluminescence quantification
178
The level of AI-1 signal molecules in rescue and site-directed mutagenesis strains were further
179
determined by the bioluminescence quantification assay using V. harveyi BB170 reporter strain as
180
previously described method [21]. Briefly, the cell-free culture supernatants (CFCS) were collected from
181
all the test bacterial strains by centrifugation at 12000 g for 5 min and the collected CFCS were filtered
182
using 0.22 µm membrane filter. Then, the V. harveyi BB170 reporter strain was inoculated in AB medium
183
and incubated at 30℃ overnight. Further, overnight culture was diluted at 1:5000 ratios into fresh AB
184
medium. After that, 20 µl of CFCS of all the test bacterial strains were mixed with 180 µl of diluted V.
185
harveyi BB170 culture and incubated at 30℃ in a black flat-bottom 96-well MTP. Then, the
186
bioluminescence intensities were measured at an absorbance of 490 nm from 0-12 h using a SpectraMax
187
i3× (Molecular Devices, Sunnyvale, CA, USA) at 30℃. Then, the AI-1 activity was measured as a fold-
188
change in relative light units.
189 190
3. Results 8
191
3.1. Determining the level of acetylation by Western blotting analysis
192
In order to assess the level of acetylation in rescue strain ∆ahyI::ahyI and site-directed
193
mutagenesis strains such as ∆ahyI::ahyI-K7Q and ∆ahyI::ahyI-K7R, the Western blotting analysis was
194
performed using anti-His and anti-Kace antibodies. The obtained result showed that the level of histidine
195
expression is similar in all the strains. Further, the rescue strain ∆ahyI::ahyI showed the increasing level
196
of acetylation expression, whereas the site-directed mutagenesis strains such as ∆ahyI::ahyI-K7Q and
197
∆ahyI::ahyI-K7R showed a reduce level of acetylation signals, compared to the rescue strain (Fig. 1).
198
Therefore, it strongly suggested that the lysine residue site was modified by acetylation in rescue strain.
199
Further, this indicated that the point mutation at K7 site affects the acetylation modification of AhyI
200
protein.
201 202
3.2. Growth curve measurement
203
In order to determine the effect of a point mutation at K7 site, the growth curve analysis was
204
made along with the A. hydrophila ATCC 7966 wild type, ∆ahyI mutant, ∆ahyI::ahyI rescue, ∆ahyI with
205
original vector strains and site-directed mutagenesis strains such as ∆ahyI::ahyI-K7Q and ∆ahyI::ahyI-
206
K7R. The obtained results revealed that all the five strains including ∆ahyI mutant, ∆ahyI::ahyI rescue,
207
∆ahyI with original vector, site-directed mutagenesis strains such as ∆ahyI::ahyI-K7Q and ∆ahyI::ahyI-
208
K7R did not show any significant variation in cell densities, compared to that of the wild type strain (Fig.
209
2). Therefore, these results indicated that the mutation of the ahyI gene in A. hydrophila and the point
210
mutation at K7 acetylation site did not affect the A. hydrophila growth. Overall, the growth curve
211
measurement revealed that the lysine acetylation did not play any significant role in the growth of A.
212
hydrophila.
213 214
3.3. Effect of lysine acetylation on the biofilm formation and hemolysin productions
9
215
In order to determine the effect of lysine acetylation on the A. hydrophila biofilm formation and
216
hemolysin production, the biofilm biomass quantification and hemolytic assays were performed,
217
respectively. The obtained results in biofilm and hemolysin assays revealed that the site-directed
218
mutagenesis strains such as ∆ahyI::ahyI-K7Q and ∆ahyI::ahyI-K7R showed a similar level of biofilm
219
formation and hemolysin productions when compared to the rescue strain. Compared to the wild type
220
strain, the ahyI mutant strain showed a similar level of biofilm formation in a biofilm quantification assay.
221
More, the ahyI strain showed an increasing level of hemolysin production when compared to the wild
222
type strain in the hemolytic assay (Fig. 3).
223 224
3.4. Effect of lysine acetylation on the protease productions
225
To reveal the role of lysine acetylation on the protease productions of A. hydrophila, the
226
proteolytic assay was made. The mutation at K7 site of lysine acetylation strain ∆ahyI::ahyI-K7Q showed
227
the reduce level of the zone of clearance, compared to the rescue strain on the skim milk agar plate due to
228
decreased level of protease productions. In contrast, the mutation at K7 site of lysine deacetylation strain
229
∆ahyI::ahyI-K7R did not show any significant variation in protease inhibition (Fig. 4) .
230 231
3.5. Extraction and quantification of AI-1 QS signal molecule production
232
To assess the impact of lysine acetylation on the AI-1 QS signal molecule production, the AHLs
233
were separated by TLC plates and their intensity levels were quantified. The obtained results revealed that
234
compared to the rescue strain, the mutation at K7 site of lysine acetylation strain ∆ahyI::ahyI-K7Q
235
showed the reduce level of AHLs productions. Further, the intensity levels of AHLs production were
236
quantified and the obtained data also supported the outcome of TLC data. In contrast, the mutation at K7
237
site of lysine deacetylation strain ∆ahyI::ahyI-K7R did not show any significant variation in signal
238
molecule inhibition (Fig. 5).
239
10
240
3.6. Impact of lysine acetylation on the bioluminescence productions
241
The role of lysine acetylation on the bioluminescence production in V. harveyi BB170 upon
242
treatment with CFCS of rescue and site-directed mutagenesis strains was assessed by relative light unit
243
measurement. The mutation at K7 site of lysine acetylation strain ∆ahyI::ahyI-K7Q showed the reduce
244
level of the relative light unit due to the decreased level of bioluminescence productions, compared to the
245
rescue strain. In contrast, the mutation at K7 site of lysine deacetylation strain ∆ahyI::ahyI-K7R did not
246
show any significant variation in bioluminescence inhibition (Fig. 6).
247 248
4. Discussion
249
A. hydrophila is vital Gram-negative bacterial pathogens and commonly found in various aquatic
250
environments [8, 22]. Several researchers are continuously reporting that QS system regulates numerous
251
virulence genes expression responsible for biofilm, hemolysin, protease, elastase and endotoxin in A.
252
hydrophila for their pathogenicity [12, 23]. Therefore, it is needed to be unveiling the mechanism of QS
253
on the virulence factors production in A. hydrophila to control their infection by targeting the QS system.
254
The lysine acetylation is important protein-PTMs that alter the conformation, charge, protein-protein
255
interactions, substrate binding, enzyme activity, protein localization and stability of proteins.
256
Furthermore, the various studies are continuously reporting that the number of genome-wide
257
characterizations of lysine-acetylated proteins in bacterial pathogens regulates various biological
258
functions including transcription, quorum sensing, cell size regulation, chemotaxis and central
259
metabolism [3, 24].
260 261
The obtained mass spectrometry data in previous study revealed that the various lysine
262
acetylation modification site on A. hydrophila [2]. In which, the acetylation modification at the K7 site of
263
the AhyI protein was identified. In order to better understand the effect of lysine acetylation modification
264
on the function of the AhyI protein, we have revealed their biological functions by mutant strains 11
265
constructed by site-directed mutagenesis in this study. Initially, we have used a pull-down method to
266
purify the three proteins such as ∆ahyI::ahyI, ∆ahyI::ahyI-K7Q and ∆ahyI::ahyI-K7R. Then, the amounts
267
of protein in the samples were verified by Western blotting for revealing their consistency. After the
268
verification of the acetylated antibody by Western blotting, we found that protein acetylation level was
269
reduced after K7 locus mutation, whereas it was more prominent in the K7Q locus mutation, compared to
270
the rescue strain. Therefore, the AhyI has a certain influence on protein acetylation modification at K7
271
site.
272
After generated the site-directed mutagenesis of AhyI protein at K7 site, the effect of rescue and
273
site-directed mutagenesis strains on the biological functions of A. hydrophila were determined to reveal
274
the role of lysine acetylation modification. The biofilm is the aggregation of microorganisms in a self-
275
protected exopolysaccharide material composed of proteins, polysaccharide and extracellular DNA,
276
which protect the bacterial pathogens form various antibiotics and disinfectants. Further, it has the
277
capability to fight the invading host immune response systems [25-28]. More, the several researchers are
278
continuously reporting that the biofilm mediated bacterial infections are a major threat to both the
279
aquaculture industry and human health [29-34]. Recently [35] revealed that histone deacetylases
280
(HDACs) regulated the life cycle, morphogenic plasticity and most importantly biofilm formation in
281
Candida spp. Further, they have stated that the different types of HDACs inhibitors reduced the biofilm
282
formation of Candida spp by inhibiting the HDACs functions. Due to the interesting fact of lysine
283
acetylation on the biofilm formation in previous studies, we have performed the biofilm biomass
284
quantification assay to reveal their role on A. hydrophila biofilm. But, in our study, the mutation at K7
285
site of acetylation and deacetylation of AhyI protein showed a similar level of biofilm formation, when
286
compared to the rescue strain. Further, the previous study revealed that the AhyIR QS system regulated
287
the biofilm formation in A. hydrophila [36]. But the ahyI mutant strain showed a similar level of biofilm
288
formation in the study, compared to the wild type strain. [11] reported that the AI-2 dependent luxS gene
289
regulated the biofilm formation in A. hydrophila, in addition to the AI-1 type system. Therefore, the A. 12
290
hydrophila may utilize the AI-2 system for their biofilm regulation instead of using the AI-1 system.
291
Nevertheless, further analyses are needed to reveal the specific mechanism behind this.
292
The two different types of hemolytic toxins such as β-hemolysin and aerolysin were reported
293
previously in A. hydrophila, which are important virulence factor and regulated by QS system. Therefore,
294
the effect of mutation at the K7 site of lysine acetylation on the hemolysin production was tested by β-
295
hemolytic assay. The obtained data revealed that the mutation at K7 site of acetylation and deacetylation
296
of AhyI protein showed no significant variation on the hemolysin productions, compared to the rescue
297
strain. Whereas, the ahyI mutation strain showed an increased level of hemolysin productions, compared
298
to the wild type strain. Further, this substantiated the outcome of [37], who have reported that the ahyI
299
mutation showed an increasing level of hemolysin productions.
300 301
The A. hydrophila produces two different types of extracellular protease enzymes such as
302
metalloprotease and serine protease. More, the protease enzymes produced by A. hydrophila degrade the
303
insoluble protein elastin. Apart from these, it plays a significant role in the establishment of infections
304
through overwhelming host defences and permits A. hydrophila to persist in diverse habitats [38]. More,
305
[37] revealed the important role of AhyIR QS system on the virulence factor productions in A.
306
hydrophila. Further, they have stated that the ahyI mutant strain showed reduce level of both exoprotease
307
activities such as serine protease and metalloprotease. Therefore, in order to determine the effect of lysine
308
acetylation on the protease production in A. hydrophila, the qualitative proteolytic assay was done. In
309
which, the mutation at K7 site of lysine acetylation strain ∆ahyI::ahyI-K7Q showed a reduce level of
310
protease productions, compared to the rescue strain. More, the ahyI mutation showed a decreased level of
311
protease productions, compared to the wild type strain. So, this result also supported the outcome of [37]
312
and further revealed the importance of the ahyI gene on the A. hydrophila virulence factors productions.
313
To understand whether the sites of acetylated modification affect the signal molecule productions,
314
AI-1 QS signal molecule extraction by TLC technique was done. The obtained results revealed that 13
315
compared to the rescue strain, the mutation at K7 site of lysine acetylation strain ∆ahyI::ahyI-K7Q
316
showed a reduce level of AHLs productions. Therefore, this result indicated that the acetylation at K7 site
317
in AhyI protein may affect the signal molecule productions. The AhyI protein synthesizes the AI-1 signal
318
molecule for their QS regulation. After synthesizing of AI-1 signal molecules, it goes and binds with
319
AhyR receptor protein and triggers the virulence genes expression in A. hydrophila. Therefore, we have
320
further investigated the impact of lysine acetylation on the AI-1 signal molecule production by
321
bioluminescence bioassay in V. harveyi BB170. The exogenous AI-1 level in CFCS of test bacterial
322
strains is proficient to persuade the bioluminescence production in reporter strain V. harveyi BB170 and
323
then the AI-1 activity level was measured by relative light units. The obtained results revealed that the
324
mutation at K7 site of lysine acetylation strain ∆ahyI::ahyI-K7Q showed decrease level of
325
bioluminescence productions when compared to the rescue strain. These results also validated the
326
outcome AI-1 QS signal molecule extraction level. Therefore, these both experiments revealed that the
327
lysine acetylation modification may play a significant role in the AHLs productions in A. hydrophila.
328
Conversely, the deacetylated strain ∆ahyI::ahyI-K7R showed no significant changes in protease and
329
signal molecule productions, compared to the rescue strain due to the deacetylation. Further, it prompts
330
that targeting the K7 site of AhyI protein may reduce the A. hydrophila QS system and affects the activity
331
of the bacterial extracellular protein and signal molecule productions.
332 333
5. Conclusion
334
In this study, we have assessed the effect of lysine acetylation of the AhyI protein at K7 site for
335
revealing their biological functions in A. hydrophila. The obtained data revealed that the site-directed
336
mutagenesis strains did not affect the growth of A. hydrophila. Further, the mutation at K7 site of lysine
337
acetylation showed reduce level of protease, bioluminescence and signal molecule productions when
338
compared to the rescue strain. On the other hand, it did not show any significant effect on the biofilm
339
formation and hemolysin production. Overall, the obtained results in this study indicated that the lysine 14
340
acetylation of AhyI at the K7 site may affect the biological functions in A. hydrophila. Further, it may pay
341
the way for controlling the A. hydrophila infections by targeting the lysine acetylation. However, this
342
lysine acetylation mechanism is still unclear and requires further research and exploration.
343
Conflict of interest
344
Authors of this manuscript have declared no conflicts of interest with this work.
345
15
346
References
347
[1] Gaviard C, Cosette P, Jouenne T, Hardouin J. LasB and CbpD Virulence Factors of Pseudomonas
348
aeruginosa Carry Multiple Post-Translational Modifications on Their Lysine Residues. J
349
Proteome Res. 2019;18:923-33.
350
[2] Sun L, Yao Z, Guo Z, Zhang L, Wang Y, Mao R, et al. Comprehensive analysis of the lysine
351
acetylome in Aeromonas hydrophila reveals cross-talk between lysine acetylation and
352
succinylation in LuxS. Emerg Microbes Infect. 2019;8:1229-39.
353 354 355 356 357 358 359 360
[3] Reverdy A, Chen Y, Hunter E, Gozzi K, Chai Y. Protein lysine acetylation plays a regulatory role in Bacillus subtilis multicellularity. PLoS One. 2018;13:e0204687. [4] Zhang K, Zheng S, Yang JS, Chen Y, Cheng Z. Comprehensive profiling of protein lysine acetylation in Escherichia coli. J Proteome Res. 2013;12:844-51. [5] Gaviard C, Broutin I, Cosette P, De E, Jouenne T, Hardouin J. Lysine Succinylation and Acetylation in Pseudomonas aeruginosa. J Proteome Res. 2018;17:2449-59. [6] Xie L, Wang X, Zeng J, Zhou M, Duan X, Li Q, et al. Proteome-wide lysine acetylation profiling of the human pathogen Mycobacterium tuberculosis. Int J Biochem Cell Biol. 2015;59:193-202.
361
[7] Li Z, Wang Y, Li X, Lin Z, Lin Y, Srinivasan R, et al. The characteristics of antibiotic resistance and
362
phenotypes in 29 outer-membrane protein mutant strains in Aeromonas hydrophila. Environ
363
Microbiol. 2019.
364
[8] Citterio B, Francesca B. Aeromonas hydrophila virulence. Virulence. 2015;6:417-8.
365
[9] Rama Devi K, Srinivasan R, Kannappan A, Santhakumari S, Bhuvaneswari M, Rajasekar P, et al. In
366
vitro and in vivo efficacy of rosmarinic acid on quorum sensing mediated biofilm formation and
367
virulence factor production in Aeromonas hydrophila. Biofouling. 2016;32:1171-83.
368
[10] Kirke DF, Swift S, Lynch MJ, Williams P. The Aeromonas hydrophila LuxR homologue AhyR
369
regulates the N-acyl homoserine lactone synthase, AhyI positively and negatively in a growth
370
phase-dependent manner. FEMS Microbiol Lett. 2004;241:109-17. 16
371
[11] Kozlova EV, Popov VL, Sha J, Foltz SM, Erova TE, Agar SL, et al. Mutation in the S-
372
ribosylhomocysteinase (luxS) gene involved in quorum sensing affects biofilm formation and
373
virulence in a clinical isolate of Aeromonas hydrophila. Microb Pathog. 2008;45:343-54.
374
[12] Swift S, Karlyshev AV, Fish L, Durant EL, Winson MK, Chhabra SR, et al. Quorum sensing in
375
Aeromonas hydrophila and Aeromonas salmonicida: identification of the LuxRI homologs
376
AhyRI and AsaRI and their cognate N-acylhomoserine lactone signal molecules. J Bacteriol.
377
1997;179:5271-81.
378
[13] Yao Z, Guo Z, Wang Y, Li W, Fu Y, Lin Y, et al. Integrated Succinylome and Metabolome Profiling
379
Reveals Crucial Role of S-Ribosylhomocysteine Lyase in Quorum Sensing and Metabolism of
380
Aeromonas hydrophila. Mol Cell Proteomics. 2019;18:200-15.
381
[14] Hu F, Cao Y, Xiao F, Zhang J, Li H. Site-directed mutagenesis of Aeromonas hydrophila enoyl
382
coenzyme A hydratase enhancing 3-hydroxyhexanoate fractions of poly(3-hydroxybutyrate-co-3-
383
hydroxyhexanoate). Curr Microbiol. 2007;55:20-4.
384
[15] Su YB, Peng B, Li H, Cheng ZX, Zhang TT, Zhu JX, et al. Pyruvate cycle increases aminoglycoside
385
efficacy and provides respiratory energy in bacteria. Proc Natl Acad Sci U S A. 2018;115:E1578-
386
E87.
387
[16] Wang Y, Wang X, Ali F, Li Z, Fu Y, Yang X, et al. Comparative Extracellular Proteomics of
388
Aeromonas hydrophila Reveals Iron-Regulated Secreted Proteins as Potential Vaccine
389
Candidates. Front Immunol. 2019;10:256.
390 391 392 393
[17] Ali F, Yao Z, Li W, Sun L, Lin W, Lin X. In-Silico Prediction and Modeling of the Quorum Sensing LuxS Protein and Inhibition of AI-2 Biosynthesis in Aeromonas hydrophila. Molecules. 2018;23. [18] Sun L, Chen H, Lin W, Lin X. Quantitative proteomic analysis of Edwardsiella tarda in response to oxytetracycline stress in biofilm. J Proteomics. 2017;150:141-8.
17
394
[19] Cai Q, Wang G, Li Z, Zhang L, Fu Y, Yang X, et al. SWATH based quantitative proteomics analysis
395
reveals Hfq2 play an important role on pleiotropic physiological functions in Aeromonas
396
hydrophila. J Proteomics. 2019;195:1-10.
397 398 399 400
[20] Gui M, Liu L, Wu R, Hu J, Wang S, Li P. Detection of New Quorum Sensing N-Acyl Homoserine Lactones From Aeromonas veronii. Front Microbiol. 2018;9:1712. [21] Ju X, Li J, Zhu M, Lu Z, Lv F, Zhu X, et al. Effect of the luxS gene on biofilm formation and antibiotic resistance by Salmonella serovar Dublin. Food Res Int. 2018;107:385-93.
401
[22] Guo Z, Lin Y, Wang X, Fu Y, Lin W, Lin X. The protective efficacy of four iron-related
402
recombinant proteins and their single-walled carbon nanotube encapsulated counterparts against
403
Aeromonas hydrophila infection in zebrafish. Fish Shellfish Immunol. 2018;82:50-9.
404 405
[23] Takahashi E, Ozaki H, Fujii Y, Kobayashi H, Yamanaka H, Arimoto S, et al. Properties of hemolysin and protease produced by Aeromonas trota. PLoS One. 2014;9:e91149.
406
[24] Post DMB, Schilling B, Reinders LM, D'Souza AK, Ketterer MR, Kiel SJ, et al. Identification and
407
characterization of AckA-dependent protein acetylation in Neisseria gonorrhoeae. PLoS One.
408
2017;12:e0179621.
409
[25] Sivaranjani M, Srinivasan R, Aravindraja C, Karutha Pandian S, Veera Ravi A. Inhibitory effect of
410
alpha-mangostin on Acinetobacter baumannii biofilms - an in vitro study. Biofouling.
411
2018;34:579-93.
412
[26] Kannappan A, Sivaranjani M, Srinivasan R, Rathna J, Pandian SK, Ravi AV. Inhibitory efficacy of
413
geraniol on biofilm formation and development of adaptive resistance in Staphylococcus
414
epidermidis RP62A. J Med Microbiol. 2017;66:1506-15.
415
[27] Srinivasan R, Devi KR, Kannappan A, Pandian SK, Ravi AV. Piper betle and its bioactive
416
metabolite phytol mitigates quorum sensing mediated virulence factors and biofilm of nosocomial
417
pathogen Serratia marcescens in vitro. J Ethnopharmacol. 2016;193:592-603.
18
418
[28] Ramanathan S, Arunachalam K, Chandran S, Selvaraj R, Shunmugiah KP, Arumugam VR. Biofilm
419
inhibitory efficiency of phytol in combination with cefotaxime against nosocomial pathogen
420
Acinetobacter baumannii. J Appl Microbiol. 2018;125:56-71.
421 422
[29] Balcazar JL, Subirats J, Borrego CM. The role of biofilms as environmental reservoirs of antibiotic resistance. Front Microbiol. 2015;6:1216.
423
[30] Li W, Yao Z, Sun L, Hu W, Cao J, Lin W, et al. Proteomics Analysis Reveals a Potential Antibiotic
424
Cocktail Therapy Strategy for Aeromonas hydrophila Infection in Biofilm. J Proteome Res.
425
2016;15:1810-20.
426
[31] Srinivasan R, Santhakumari S, Ravi AV. In vitro antibiofilm efficacy of Piper betle against quorum
427
sensing mediated biofilm formation of luminescent Vibrio harveyi. Microb Pathog.
428
2017;110:232-9.
429
[32] Srinivasan R, Vigneshwari L, Rajavel T, Durgadevi R, Kannappan A, Balamurugan K, et al.
430
Biogenic synthesis of silver nanoparticles using Piper betle aqueous extract and evaluation of its
431
anti-quorum sensing and antibiofilm potential against uropathogens with cytotoxic effects: an in
432
vitro and in vivo approach. Environ Sci Pollut Res Int. 2018;25:10538-54.
433
[33] Ravindran D, Ramanathan S, Arunachalam K, Jeyaraj GP, Shunmugiah KP, Arumugam VR.
434
Phytosynthesized silver nanoparticles as antiquorum sensing and antibiofilm agent against the
435
nosocomial pathogen Serratia marcescens: an in vitro study. J Appl Microbiol. 2018;124:1425-
436
40.
437
[34] Alexpandi R, Prasanth MI, Ravi AV, Balamurugan K, Durgadevi R, Srinivasan R, et al. Protective
438
effect of neglected plant Diplocyclos palmatus on quorum sensing mediated infection of Serratia
439
marcescens and UV-A induced photoaging in model Caenorhabditis elegans. J Photochem
440
Photobiol B. 2019;201:111637.
441 442
[35] Garnaud C, Champleboux M, Maubon D, Cornet M, Govin J. Histone Deacetylases and Their Inhibition in Candida Species. Front Microbiol. 2016;7:1238. 19
443 444
[36] Talagrand-Reboul E, Jumas-Bilak E, Lamy B. The Social Life of Aeromonas through Biofilm and Quorum Sensing Systems. Front Microbiol. 2017;8:37.
445
[37] Swift S, Lynch MJ, Fish L, Kirke DF, Tomas JM, Stewart GS, et al. Quorum sensing-dependent
446
regulation and blockade of exoprotease production in Aeromonas hydrophila. Infect Immun.
447
1999;67:5192-9.
448 449
[38] Zacaria J, Delamare AP, Costa SO, Echeverrigaray S. Diversity of extracellular proteases among Aeromonas determined by zymogram analysis. J Appl Microbiol. 2010;109:212-9.
450 451
Figure legends
452 453
Figure 1. Level of protein lysine acetylation. The Western blotting analysis combined with a pull-down
454
assay showed the acetylation levels in the rescue strain and site-directed mutagenesis strains, which was
455
determined by anti-acetyl lysine antibody. Kace - lysine acetylation.
456
20
457 458
Figure 2. Effect of lysine acetylation on the growth of A. hydrophila. The site-directed mutagenesis
459
strains did not show any variation on the growth of A. hydrophila even at 20 h, compared to the wild type
460
strain. The values are expressed as mean ± standard deviation of three biological independent
461
experiments.
462
21
463
464 465
Figure 3. Impact of lysine acetylation on the biofilm formation and hemolysin production of A.
466
hydrophila. Compared to the rescue strain, the site-directed mutagenesis strains showed a similar level of
467
biofilm formation and hemolysin production. The level of biofilm formation was expressed as OD value
468
and the hemolysin production was expressed as diameter (mm) of the zone of clearance. The values are
469
expressed as mean ± standard deviation of three biological independent experiments. The statistical
470
differences were analyzed by one-way ANOVA test. ns represents non-significant.
471
22
472
473 474
Figure 4. Effect of lysine acetylation on the protease production in A. hydrophila. The skim milk agar
475
plate showed a decreased level of protease production in mutation at K7 site of lysine acetylation strain
476
∆ahyI::ahyI-K7Q, compared to the rescue strain (A). The graph represents diameter (cm) of the zone of
477
clearance (B). The values are expressed as mean ± standard deviation of three biological independent
478
experiments. The statistical differences were analyzed by one-way ANOVA test. *** represents p-value ≤
479
0.002, ns represents non-significant.
480
23
481 482
Figure 5. Impact of lysine acetylation on the AHL production in A. hydrophila. The TLC plate
483
showed a reduced level of AHL production in mutation at K7 site of lysine acetylation strain ∆ahyI::ahyI-
484
K7Q, compared to the rescue strain (A). The graph represents the intensity level of AHL production (B)..
485
The values are expressed as mean ± standard deviation of three biological independent experiments. The
486
statistical differences were analyzed by one-way ANOVA test. ** represents p-value ≤ 0.0050.
487
24
488 489
Figure 6. Effect of lysine acetylation on the bioluminescence production. The graph represents the
490
level of bioluminescence productions in a relative light unit (RLU). Compared to the rescue strain, the
491
mutation at K7 site of lysine acetylation strain ∆ahyI::ahyI-K7Q showed a decreased level of
492
bioluminescence productions. The values are expressed as mean ± standard deviation of three biological
493
independent experiments. The statistical differences were analyzed by one-way ANOVA test. * represents
494
p-value ≤ 0.0112, *** represents p-value ≤ 0.0001.
495
25
496
Table 1 The Primers used for ahyI mutant construction
497
498
Name
Primer sequence 5' to 3'
F1
CATGAATTCCCGGGAGAGCTCGGCCGGCAGAAACCAGGT
F2
AGACATCTGAAGGCGACACAAAAAAATCCCG
F3
TGTGTCGCATTCAGATGTCTCCATTTCAGTGTTCG
F4
CGATCCCAAGCTTCTTCTAGAGGTCATCTTCCCGCAGACTG
F5
TTATTCGGTGACCAGTTCGC
F6
ATGCTTGTTTTCAAAGGAAAATTAAAAGA
F7
GCTCTCGTCATCCACCAG
F8
TTTTCCCTTCACTGGCCC
Note: The underlined are restriction enzyme cutting sites.
499 500 501 502 503 504 505 506 507 508 509 510 511 26
512
Table 2 The primers used for ahyI gene rescued and site-directed mutagenesis strains constructions Stranins name
Primer sequence 5' to 3'
∆ ahyI:: ahyI-F1
GTCGACGGTATTAAGCTTGGGCACTGGTTGAACAGCAC
∆ ahyI:: ahyI-F2
GTGGTGGTGGTGGTGGGATCCTTCGGTGACCAGTTCGCGCGCCT
∆ ahyI:: ahyI-K7Q-F1
CAATTAAAAGAACACCCCAGATGGGAGGTAGA
∆ ahyI:: ahyI-K7Q-F2
GGGTGTTCTTTTAATTGTCCTTTGAAAACAAGCATTCAGAT
∆ ahyI:: ahyI-K7R-F1
CGATTAAAAGAACACCCCAGATGGGAGGTAGA
∆ ahyI:: ahyI-K7Q-F2
GGGTGTTCTTTTAATCGTCCTTTGAAAACAAGCATTCAGAT
513 514 515 516
27
Highlights The AhyI protein regulates various quorum sensing regulated virulence factors productions in Aeromonas hydrophila. The AhyI protein showed lysine acetylation at their K7 site. The lysine acetylation at K7 site of AhyI protein affects various biological functions in Aeromonas hydrophila.
S0882-4010(19)31861-3
DOI:
https://doi.org/10.1016/j.micpath.2019.103952
Reference:
YMPAT 103952
To appear in:
Microbial Pathogenesis
Received Date: 26 October 2019 Revised Date:
4 December 2019
Accepted Date: 26 December 2019
Please cite this article as: Li D, Ramanathan S, Wang G, Wu Y, Tang Q, Li G, Acetylation of lysine 7 of AhyI affects the biological function in Aeromonas hydrophila, Microbial Pathogenesis (2020), doi: https:// doi.org/10.1016/j.micpath.2019.103952. 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 Published by Elsevier Ltd.
CRediT author statement： Guohui Li: Conceptualization, Methodology, Software. Dong Li: Data curation, Writing- Original draft preparation. Yao Wu: Visualization, Investigation. Guohui Li: Supervision. Qi Tang: Software. Validation: Srinivasan Ramanathan: WritingReviewing and Editing.
1
Acetylation of lysine 7 of AhyI affects the biological function in
2
Aeromonas hydrophila
3
Dong Lia#, Srinivasan Ramanathanb#, Guibin Wangb, Yao Wub, Qi Tanga, Guohui Lia
*
a
Institute of Life Sciences, Jiangsu University, Zhenjiang 212 013, PR China.
4
b
Fujian Provincial Key Laboratory of Agroecological Processing and Safety Monitoring (School of Life
5
Sciences, Fujian Agriculture and Forestry University), Fuzhou 350 002, PR China.
6
7
8
9
10
11
12
13
14
15
16
17
#
18
*Corresponding
19
University, 301# Xuefu Road, Zhenjiang 212 013, PR China.
These authors contributed equally to this work.
author: Guohui Li, E-mail: [email protected], Institute of Life Sciences, Jiangsu
20 1
21
Abstract
22
Acyl-homoserine-lactone synthase (AhyI) of Aeromonas hydrophila can produce quorum sensing
23
(QS) auto-inducer 1 (AI-1) type signal molecule, which plays important roles in various biological
24
phenomenons such as biofilm formation, hemolysin production and motility. Previous research revealed
25
that the AhyI of A. hydrophila has acetylation modification on lysine 7 site, but its intrinsic biological
26
function is still largely unknown. To study the effect of AhyI protein and its acetylation modification on
27
the physiological traits of A. hydrophila, the site-directed mutagenesis strains including ∆ahyI::ahyI-K7Q
28
and ∆ahyI::ahyI-K7R were made in this study. The mutation at K7 site of lysine acetylation in AhyI
29
protein decreased the protease productions, but the lysine acetylations do not affect the biofilm formation
30
and hemolysin production. To further study the effect of lysine acetylation on AI-1 signal molecule
31
production, the acyl-homoserine lactones (AHLs) extraction and bioluminescence quantification were
32
performed. Compared to the rescue strain, the acetylation on K7 of AhyI resulted in a decreased level of
33
AHLs and bioluminescence productions. It indicated that the lysine acetylation modification on the AhyI
34
protein can regulate the production of signalling molecules. Overall, the obtained data in this study
35
provide a theoretical basis for further understanding the role of lysine acetylation of AhyI protein and lay
36
a foundation to systematically study the regulatory mechanism of QS.
37
Keywords: Aeromonas hydrophila; Biofilm formation; Lysine acetylation; Protein post-translational
38
modification; Quorum sensing
39
40 41
2
42
1. Introduction
43
Nowadays, the characterization of protein posttranslational modifications (PTMs) in bacterial
44
pathogens, particularly the adding of small chemical groups on the side chains of amino acids, has
45
become a vital research topic [1]. Furthermore, this type of PTMs is often dynamic, reversible and
46
conserved during the evolution process. The various types of protein PTMs such as phosphorylation,
47
acylation and methylation reversibly modify some specific amino acids residues and play a significant
48
role in various biological processes in bacterial pathogens [2]. Among the various PTMs, protein lysine
49
acetylation is one of the extensively studied PTMs due to their important roles, which transfer the acetyl
50
group from a donor metabolite to a lysine residue of specific proteins. More, the lysine acetylation is a
51
reversible process, in which the lysine deacetyltransferases remove the acetyl group from the acetylated
52
lysine residue of proteins [3]. In recent years, several researchers are continuously identifying thousands
53
of lysine acetylation sites in several bacterial pathogens including Pseudomonas aeruginosa, Bacillus
54
subtilis, Mycobacterium tuberculosis, Escherichia coli and Aeromonas hydrophila and reveal their
55
important role in bacterial virulence [2-6].
56 57
A. hydrophila is a Gram-negative pathogenic bacterium found in aquatic environments [7]. It
58
secretes a variety of virulence factors and toxins that can cause lesions and death in fish. However, the
59
disease caused by A. hydrophila can make the fish unsuitable for human ingestion. It is deliberated as an
60
emerging bacterial pathogen accountable for various systemic conditions such as skin infections,
61
peritonitis, gastroenteritis, hemolytic uremic syndrome, necrotizing fasciitis, meningitis, bacteremia and
62
cholera-like illness [8, 9]. Further, A. hydrophila secrets various virulence factors such as protease,
63
elastase, lipase, hemolysin, motility, biofilm formation and exopolysaccharide productions under the
64
control of cell density-dependent gene expression system called quorum sensing (QS) mechanism. More,
65
A. hydrophila secrets their virulence factors by two different types of the N-acyl homoserine lactone
3
66
(AHL) mediated QS systems such as LuxIR based auto-inducer 1 (AI-1) system (AhyIR) and LuxS based
67
auto-inducer-2 (AI-2) system [10-12].
68 69
In recent study, [2] identified various lysine-acetylated proteins in A. hydrophila ATCC 7966 by
70
the specific antibody enrichment combined with high-resolution mass spectrometry analysis. Further, the
71
obtained data in that study revealed the QS signal molecule protein AhyI was acetylated at the K7 site.
72
However, the specific biological function of this acetylated AhyI protein in A. hydrophila remains
73
unknown. Therefore, we have investigated the impact of lysine acetylation of AhyI protein on the
74
biological functions of A. hydrophila in the present study,
75 76
2. Materials and methods
77
2.1. Generation of ahyI knock-out strain
78
The ahyI gene deletion mutant was generated by suicide vector pRE112. Around 500 bp of
79
upstream and downstream flanking sequences of ahyI (gene name AHA_0556) was amplified from the A.
80
hydrophila ATCC7966 chromosomal DNA and then the overlap extension PCR was used for fusion into
81
the pRE112 vector for producing the recombinant constructs. The restriction sites such as BamHI and
82
SacI were used for the construction of recombinant plasmids. Further, the recombinant plasmids were
83
transformed into E. coli MC1061 (λpir) cells first to upraise the transformation efficacy and then
84
transformed into E. coli S17-1 (λpir) cells for fast propagation. After that, the plasmids were transferred
85
into the wild type A. hydrophila ATCC 7966 strain by bacterial conjugation. To select the ampicillin
86
resistance (100 µg/m AmpR) and chloramphenicol resistance (30 µg/ml CmR) single-crossover mutant
87
colonies, the first homologous recombination was done. Then, the second homologous recombination was
88
done to obtain the sacB gene depleted double-crossover mutant colonies. Moreover, PCR analysis was
89
used to confirm the single and double-crossover mutants with their corresponding primers (Table 1) [13].
90
4
91
2.2. Construction of site-directed mutagenesis strains
92
The pBBR1-MCS1 broad host-vector was used to construct the ahyI complementary strain and
93
the His-tagged pBBR1-ahyI plasmid was generated by HindIII and BamHI restriction sites as previously
94
described [14]. Addition to this, to confirm the normal expression, the corresponding promoter was also
95
inserted into the upstream of ahyI. Then, the Fast Mutagenesis System Kit was used to perform the site-
96
directed mutagenesis of ahyI (K7Q and K7R) with the corresponding primers (Table 2) according to the
97
manufacturer’s instructions (Transgen Biotech Co., Beijing, China). Then, all the resulting target
98
mutations were confirmed by DNA sequencing.
99 100
2.3. Bacterial strains used in the study and their growth conditions
101
The bacterial strains such as Vibrio harveyi BB170, Chromobacterium violaceum CV026, A.
102
hydrophila ATCC 7966 wild type strain, ∆ahyI A. hydrophila mutant strain, ∆ahyI::ahyI complementary
103
strain, ∆ahyI with original vector strain (∆ahyI::Vector) and site-directed mutagenesis (K7 point mutant
104
strains) strains such as ∆ahyI::ahyI-K7Q (acetylation strain) and ∆ahyI::ahyI-K7R (deacetylation strain)
105
were used in this study. Further, the reporter strains such as V. harveyi BB170 and C. violaceum CV026
106
strains were cultivated in Autoinducer Bioassay (AB) and Luria Bertani (LB) medium, respectively at
107
30°C, whereas the A. hydrophila ATCC 7966 wild and ∆ahyI mutant strains were cultivated in LB
108
medium at 30°C. Moreover, ∆ahyI::ahyI complementary strain, ∆ahyI with original vector strain
109
(∆ahyI::Vector) and site-directed mutagenesis strains such as ∆ahyI::ahyI-K7Q and ∆ahyI::ahyI-K7R was
110
cultivated in LB medium at 30℃along with chloramphenicol antibiotic (30 µg/ml). For experimental
111
purpose, all the strains were routinely grown overnight in their respective growth medium at their
112
optimum temperatures. Then, the obtained cultures were diluted into 1:100 ratios in their fresh respective
113
growth medium and incubated at their optimum temperatures at 200 rpm until the OD value reached 0.4
114
at 600 nm.
115 5
116
2.4. Western blotting analysis
117
For Western blotting analysis, the protein samples from strains such as ∆ahyI::ahyI, ∆ahyI::ahyI-
118
K7Q and ∆ahyI::ahyI-K7R were run on a 12% SDS-PAGE gel. Then, the electrophoresed proteins were
119
transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore, Burlington, MA, USA) in 10×
120
Transfer Buffer (Bio-Rad, Hercules, CA, USA) by a Trans-Blot Turbo Transfer System (Bio-Rad) for 15
121
mins at 25 V. After that, the PVDF membrane was blocked with 5% skim milk in PBST buffer (1×PBS
122
buffer containing 0.05% Tween-20). Then, the PVDF membrane was probed with the primary antibody
123
and incubated for 1h at room temperature with anti-acetyl lysine (PTM Biolabs, Inc. Hangzhou, China).
124
Further, the membrane was washed three times with PBST for 10 min each time and incubated with
125
horseradish peroxidase (HRP) goat anti-mouse IgG or anti-rabbit IgG secondary antibody. After that, the
126
membrane was rinsed five times with PBST for 5 mins each time and then the protein band was
127
visualized by Clarity Western ECL substrate (Bio-Rad) and imaged with a ChemiDoc XRS+ system (Bio-
128
Rad) [15, 16].
129 130
2.5. Determination of growth curve
131
The bacterial strains were cultivated in 5 ml LB medium and incubated at 30℃ for overnight at
132
200 rpm. Then, the overnight grown bacterial cultures were diluted at 1:100 ratios with LB medium and
133
incubated at 30℃ until the bacterial OD reached 0.4 at 600 nm. Then, the bacterial cultures were
134
inoculated in a HONEYCOMB® sterile 100 well plate and the OD values for growth were measured at
135
600 nm for 20 h using a Bioscreen C system (Lab Systems, Helsinki, Finland) [17].
136 137
2.6. Quantification of biofilm biomass
138
To assess the level of biofilm biomass in test bacterial pathogens, the biofilm biomass
139
quantification assay was performed as previously described method with slight modification [18]. Briefly,
140
the 1% of bacterial inoculum (cell density equivalent to 0.4 OD at 600 nm) was inoculated into 96 well 6
141
microtitre plates (MTPs) and incubated at 30℃ for 18 h. After incubation, the non-adherence planktonic
142
cells were discarded and washed twice with distilled water. Then, the plate was kept in room temperature
143
for desiccating and then the biofilm cells attached on the wells were stained by 1% crystal violet. After
144
that, the unwanted stain was washed out by distilled water and kept in room temperature for drying. Then,
145
the biofilm cells in stains were dissolved by 90% ethanol and the biofilm biomass was quantified at 595
146
nm.
147 148
2.7. Determination of extracellular protease and hemolytic activities
149
The effect of lysine acetylation on the extracellular protease and hemolysin productions were
150
measured by a previously described method with slight modifications [19]. Briefly, the 1% of skim milk
151
agar plates and hemolysin agar plates containing 5% sheep blood were prepared and the wells were made
152
by a sterile micropipette tip. Then, 5 µl of overnight bacterial cultures were loaded on their respective
153
wells of skim milk and hemolysin agar plates and incubated at 30°C for 12 h. After incubation, both
154
protease and hemolysin activities were determined based on the size of the zone of clearance on each
155
plate and the diameter of the zone of clearance were measured.
156 157
2.8. Extraction of AI-1 QS signal molecules
158
The previously described method has used for extracting the AI-1 QS signal molecules from the
159
bacterial cultures with some modifications [20]. Briefly, the bacterial strains such as ∆ahyI::ahyI,
160
∆ahyI::ahyI-K7Q and ∆ahyI::ahyI-K7R were cultured in LB medium at 30℃ for 24 h in 200 rpm. Then, 5
161
ml of bacterial culture was centrifuged at 13000 g for 5 min at 4℃ and the obtained culture supernatant
162
was filtered using 0.22 µm membrane filter. After that, the filtered supernatant was extracted with an
163
equal volume of acidified ethyl acetate (1% acetic acid) and sonicated by ultra-sonication for 20 min.
164
Then, the upper organic phase was collected and freeze-dried by vacuum freeze dryer for 2 h.
165
7
166
2.9. Detection of AI-1 QS signal molecules by thin-layer chromatography (TLC)
167
The extracted AI-1 QS signal molecules were determined by thin layer chromatography (TLC)
168
analysis by a previously described method with slight modifications [20]. Briefly, 2 µl of extracted AI-1
169
QS signal molecules were spotted on 10×10 cm size TLC plate (TLC aluminium sheets, Merck KGaA,
170
Darmstadt, Germany) and 3:2 ratio methanol-water solvent system was used as the mobile phase. Further,
171
it was allowed to the solvent system until reaching the front and then the TLC plate was dried for 10 min.
172
Then, the agar cover layer was prepared by mixing the 1% of an overnight culture of C. violaceum CV026
173
with 50 ml LB medium containing 0.7% agar and then immediately spread on the TLC plate. Then, the
174
TLC plates were transferred into a closed sterile container and incubated at 30℃ for 48 h and observed for
175
the level of AI-1 QS signal molecule productions.
176 177
2.10. Bioluminescence quantification
178
The level of AI-1 signal molecules in rescue and site-directed mutagenesis strains were further
179
determined by the bioluminescence quantification assay using V. harveyi BB170 reporter strain as
180
previously described method [21]. Briefly, the cell-free culture supernatants (CFCS) were collected from
181
all the test bacterial strains by centrifugation at 12000 g for 5 min and the collected CFCS were filtered
182
using 0.22 µm membrane filter. Then, the V. harveyi BB170 reporter strain was inoculated in AB medium
183
and incubated at 30℃ overnight. Further, overnight culture was diluted at 1:5000 ratios into fresh AB
184
medium. After that, 20 µl of CFCS of all the test bacterial strains were mixed with 180 µl of diluted V.
185
harveyi BB170 culture and incubated at 30℃ in a black flat-bottom 96-well MTP. Then, the
186
bioluminescence intensities were measured at an absorbance of 490 nm from 0-12 h using a SpectraMax
187
i3× (Molecular Devices, Sunnyvale, CA, USA) at 30℃. Then, the AI-1 activity was measured as a fold-
188
change in relative light units.
189 190
3. Results 8
191
3.1. Determining the level of acetylation by Western blotting analysis
192
In order to assess the level of acetylation in rescue strain ∆ahyI::ahyI and site-directed
193
mutagenesis strains such as ∆ahyI::ahyI-K7Q and ∆ahyI::ahyI-K7R, the Western blotting analysis was
194
performed using anti-His and anti-Kace antibodies. The obtained result showed that the level of histidine
195
expression is similar in all the strains. Further, the rescue strain ∆ahyI::ahyI showed the increasing level
196
of acetylation expression, whereas the site-directed mutagenesis strains such as ∆ahyI::ahyI-K7Q and
197
∆ahyI::ahyI-K7R showed a reduce level of acetylation signals, compared to the rescue strain (Fig. 1).
198
Therefore, it strongly suggested that the lysine residue site was modified by acetylation in rescue strain.
199
Further, this indicated that the point mutation at K7 site affects the acetylation modification of AhyI
200
protein.
201 202
3.2. Growth curve measurement
203
In order to determine the effect of a point mutation at K7 site, the growth curve analysis was
204
made along with the A. hydrophila ATCC 7966 wild type, ∆ahyI mutant, ∆ahyI::ahyI rescue, ∆ahyI with
205
original vector strains and site-directed mutagenesis strains such as ∆ahyI::ahyI-K7Q and ∆ahyI::ahyI-
206
K7R. The obtained results revealed that all the five strains including ∆ahyI mutant, ∆ahyI::ahyI rescue,
207
∆ahyI with original vector, site-directed mutagenesis strains such as ∆ahyI::ahyI-K7Q and ∆ahyI::ahyI-
208
K7R did not show any significant variation in cell densities, compared to that of the wild type strain (Fig.
209
2). Therefore, these results indicated that the mutation of the ahyI gene in A. hydrophila and the point
210
mutation at K7 acetylation site did not affect the A. hydrophila growth. Overall, the growth curve
211
measurement revealed that the lysine acetylation did not play any significant role in the growth of A.
212
hydrophila.
213 214
3.3. Effect of lysine acetylation on the biofilm formation and hemolysin productions
9
215
In order to determine the effect of lysine acetylation on the A. hydrophila biofilm formation and
216
hemolysin production, the biofilm biomass quantification and hemolytic assays were performed,
217
respectively. The obtained results in biofilm and hemolysin assays revealed that the site-directed
218
mutagenesis strains such as ∆ahyI::ahyI-K7Q and ∆ahyI::ahyI-K7R showed a similar level of biofilm
219
formation and hemolysin productions when compared to the rescue strain. Compared to the wild type
220
strain, the ahyI mutant strain showed a similar level of biofilm formation in a biofilm quantification assay.
221
More, the ahyI strain showed an increasing level of hemolysin production when compared to the wild
222
type strain in the hemolytic assay (Fig. 3).
223 224
3.4. Effect of lysine acetylation on the protease productions
225
To reveal the role of lysine acetylation on the protease productions of A. hydrophila, the
226
proteolytic assay was made. The mutation at K7 site of lysine acetylation strain ∆ahyI::ahyI-K7Q showed
227
the reduce level of the zone of clearance, compared to the rescue strain on the skim milk agar plate due to
228
decreased level of protease productions. In contrast, the mutation at K7 site of lysine deacetylation strain
229
∆ahyI::ahyI-K7R did not show any significant variation in protease inhibition (Fig. 4) .
230 231
3.5. Extraction and quantification of AI-1 QS signal molecule production
232
To assess the impact of lysine acetylation on the AI-1 QS signal molecule production, the AHLs
233
were separated by TLC plates and their intensity levels were quantified. The obtained results revealed that
234
compared to the rescue strain, the mutation at K7 site of lysine acetylation strain ∆ahyI::ahyI-K7Q
235
showed the reduce level of AHLs productions. Further, the intensity levels of AHLs production were
236
quantified and the obtained data also supported the outcome of TLC data. In contrast, the mutation at K7
237
site of lysine deacetylation strain ∆ahyI::ahyI-K7R did not show any significant variation in signal
238
molecule inhibition (Fig. 5).
239
10
240
3.6. Impact of lysine acetylation on the bioluminescence productions
241
The role of lysine acetylation on the bioluminescence production in V. harveyi BB170 upon
242
treatment with CFCS of rescue and site-directed mutagenesis strains was assessed by relative light unit
243
measurement. The mutation at K7 site of lysine acetylation strain ∆ahyI::ahyI-K7Q showed the reduce
244
level of the relative light unit due to the decreased level of bioluminescence productions, compared to the
245
rescue strain. In contrast, the mutation at K7 site of lysine deacetylation strain ∆ahyI::ahyI-K7R did not
246
show any significant variation in bioluminescence inhibition (Fig. 6).
247 248
4. Discussion
249
A. hydrophila is vital Gram-negative bacterial pathogens and commonly found in various aquatic
250
environments [8, 22]. Several researchers are continuously reporting that QS system regulates numerous
251
virulence genes expression responsible for biofilm, hemolysin, protease, elastase and endotoxin in A.
252
hydrophila for their pathogenicity [12, 23]. Therefore, it is needed to be unveiling the mechanism of QS
253
on the virulence factors production in A. hydrophila to control their infection by targeting the QS system.
254
The lysine acetylation is important protein-PTMs that alter the conformation, charge, protein-protein
255
interactions, substrate binding, enzyme activity, protein localization and stability of proteins.
256
Furthermore, the various studies are continuously reporting that the number of genome-wide
257
characterizations of lysine-acetylated proteins in bacterial pathogens regulates various biological
258
functions including transcription, quorum sensing, cell size regulation, chemotaxis and central
259
metabolism [3, 24].
260 261
The obtained mass spectrometry data in previous study revealed that the various lysine
262
acetylation modification site on A. hydrophila [2]. In which, the acetylation modification at the K7 site of
263
the AhyI protein was identified. In order to better understand the effect of lysine acetylation modification
264
on the function of the AhyI protein, we have revealed their biological functions by mutant strains 11
265
constructed by site-directed mutagenesis in this study. Initially, we have used a pull-down method to
266
purify the three proteins such as ∆ahyI::ahyI, ∆ahyI::ahyI-K7Q and ∆ahyI::ahyI-K7R. Then, the amounts
267
of protein in the samples were verified by Western blotting for revealing their consistency. After the
268
verification of the acetylated antibody by Western blotting, we found that protein acetylation level was
269
reduced after K7 locus mutation, whereas it was more prominent in the K7Q locus mutation, compared to
270
the rescue strain. Therefore, the AhyI has a certain influence on protein acetylation modification at K7
271
site.
272
After generated the site-directed mutagenesis of AhyI protein at K7 site, the effect of rescue and
273
site-directed mutagenesis strains on the biological functions of A. hydrophila were determined to reveal
274
the role of lysine acetylation modification. The biofilm is the aggregation of microorganisms in a self-
275
protected exopolysaccharide material composed of proteins, polysaccharide and extracellular DNA,
276
which protect the bacterial pathogens form various antibiotics and disinfectants. Further, it has the
277
capability to fight the invading host immune response systems [25-28]. More, the several researchers are
278
continuously reporting that the biofilm mediated bacterial infections are a major threat to both the
279
aquaculture industry and human health [29-34]. Recently [35] revealed that histone deacetylases
280
(HDACs) regulated the life cycle, morphogenic plasticity and most importantly biofilm formation in
281
Candida spp. Further, they have stated that the different types of HDACs inhibitors reduced the biofilm
282
formation of Candida spp by inhibiting the HDACs functions. Due to the interesting fact of lysine
283
acetylation on the biofilm formation in previous studies, we have performed the biofilm biomass
284
quantification assay to reveal their role on A. hydrophila biofilm. But, in our study, the mutation at K7
285
site of acetylation and deacetylation of AhyI protein showed a similar level of biofilm formation, when
286
compared to the rescue strain. Further, the previous study revealed that the AhyIR QS system regulated
287
the biofilm formation in A. hydrophila [36]. But the ahyI mutant strain showed a similar level of biofilm
288
formation in the study, compared to the wild type strain. [11] reported that the AI-2 dependent luxS gene
289
regulated the biofilm formation in A. hydrophila, in addition to the AI-1 type system. Therefore, the A. 12
290
hydrophila may utilize the AI-2 system for their biofilm regulation instead of using the AI-1 system.
291
Nevertheless, further analyses are needed to reveal the specific mechanism behind this.
292
The two different types of hemolytic toxins such as β-hemolysin and aerolysin were reported
293
previously in A. hydrophila, which are important virulence factor and regulated by QS system. Therefore,
294
the effect of mutation at the K7 site of lysine acetylation on the hemolysin production was tested by β-
295
hemolytic assay. The obtained data revealed that the mutation at K7 site of acetylation and deacetylation
296
of AhyI protein showed no significant variation on the hemolysin productions, compared to the rescue
297
strain. Whereas, the ahyI mutation strain showed an increased level of hemolysin productions, compared
298
to the wild type strain. Further, this substantiated the outcome of [37], who have reported that the ahyI
299
mutation showed an increasing level of hemolysin productions.
300 301
The A. hydrophila produces two different types of extracellular protease enzymes such as
302
metalloprotease and serine protease. More, the protease enzymes produced by A. hydrophila degrade the
303
insoluble protein elastin. Apart from these, it plays a significant role in the establishment of infections
304
through overwhelming host defences and permits A. hydrophila to persist in diverse habitats [38]. More,
305
[37] revealed the important role of AhyIR QS system on the virulence factor productions in A.
306
hydrophila. Further, they have stated that the ahyI mutant strain showed reduce level of both exoprotease
307
activities such as serine protease and metalloprotease. Therefore, in order to determine the effect of lysine
308
acetylation on the protease production in A. hydrophila, the qualitative proteolytic assay was done. In
309
which, the mutation at K7 site of lysine acetylation strain ∆ahyI::ahyI-K7Q showed a reduce level of
310
protease productions, compared to the rescue strain. More, the ahyI mutation showed a decreased level of
311
protease productions, compared to the wild type strain. So, this result also supported the outcome of [37]
312
and further revealed the importance of the ahyI gene on the A. hydrophila virulence factors productions.
313
To understand whether the sites of acetylated modification affect the signal molecule productions,
314
AI-1 QS signal molecule extraction by TLC technique was done. The obtained results revealed that 13
315
compared to the rescue strain, the mutation at K7 site of lysine acetylation strain ∆ahyI::ahyI-K7Q
316
showed a reduce level of AHLs productions. Therefore, this result indicated that the acetylation at K7 site
317
in AhyI protein may affect the signal molecule productions. The AhyI protein synthesizes the AI-1 signal
318
molecule for their QS regulation. After synthesizing of AI-1 signal molecules, it goes and binds with
319
AhyR receptor protein and triggers the virulence genes expression in A. hydrophila. Therefore, we have
320
further investigated the impact of lysine acetylation on the AI-1 signal molecule production by
321
bioluminescence bioassay in V. harveyi BB170. The exogenous AI-1 level in CFCS of test bacterial
322
strains is proficient to persuade the bioluminescence production in reporter strain V. harveyi BB170 and
323
then the AI-1 activity level was measured by relative light units. The obtained results revealed that the
324
mutation at K7 site of lysine acetylation strain ∆ahyI::ahyI-K7Q showed decrease level of
325
bioluminescence productions when compared to the rescue strain. These results also validated the
326
outcome AI-1 QS signal molecule extraction level. Therefore, these both experiments revealed that the
327
lysine acetylation modification may play a significant role in the AHLs productions in A. hydrophila.
328
Conversely, the deacetylated strain ∆ahyI::ahyI-K7R showed no significant changes in protease and
329
signal molecule productions, compared to the rescue strain due to the deacetylation. Further, it prompts
330
that targeting the K7 site of AhyI protein may reduce the A. hydrophila QS system and affects the activity
331
of the bacterial extracellular protein and signal molecule productions.
332 333
5. Conclusion
334
In this study, we have assessed the effect of lysine acetylation of the AhyI protein at K7 site for
335
revealing their biological functions in A. hydrophila. The obtained data revealed that the site-directed
336
mutagenesis strains did not affect the growth of A. hydrophila. Further, the mutation at K7 site of lysine
337
acetylation showed reduce level of protease, bioluminescence and signal molecule productions when
338
compared to the rescue strain. On the other hand, it did not show any significant effect on the biofilm
339
formation and hemolysin production. Overall, the obtained results in this study indicated that the lysine 14
340
acetylation of AhyI at the K7 site may affect the biological functions in A. hydrophila. Further, it may pay
341
the way for controlling the A. hydrophila infections by targeting the lysine acetylation. However, this
342
lysine acetylation mechanism is still unclear and requires further research and exploration.
343
Conflict of interest
344
Authors of this manuscript have declared no conflicts of interest with this work.
345
15
346
References
347
[1] Gaviard C, Cosette P, Jouenne T, Hardouin J. LasB and CbpD Virulence Factors of Pseudomonas
348
aeruginosa Carry Multiple Post-Translational Modifications on Their Lysine Residues. J
349
Proteome Res. 2019;18:923-33.
350
[2] Sun L, Yao Z, Guo Z, Zhang L, Wang Y, Mao R, et al. Comprehensive analysis of the lysine
351
acetylome in Aeromonas hydrophila reveals cross-talk between lysine acetylation and
352
succinylation in LuxS. Emerg Microbes Infect. 2019;8:1229-39.
353 354 355 356 357 358 359 360
[3] Reverdy A, Chen Y, Hunter E, Gozzi K, Chai Y. Protein lysine acetylation plays a regulatory role in Bacillus subtilis multicellularity. PLoS One. 2018;13:e0204687. [4] Zhang K, Zheng S, Yang JS, Chen Y, Cheng Z. Comprehensive profiling of protein lysine acetylation in Escherichia coli. J Proteome Res. 2013;12:844-51. [5] Gaviard C, Broutin I, Cosette P, De E, Jouenne T, Hardouin J. Lysine Succinylation and Acetylation in Pseudomonas aeruginosa. J Proteome Res. 2018;17:2449-59. [6] Xie L, Wang X, Zeng J, Zhou M, Duan X, Li Q, et al. Proteome-wide lysine acetylation profiling of the human pathogen Mycobacterium tuberculosis. Int J Biochem Cell Biol. 2015;59:193-202.
361
[7] Li Z, Wang Y, Li X, Lin Z, Lin Y, Srinivasan R, et al. The characteristics of antibiotic resistance and
362
phenotypes in 29 outer-membrane protein mutant strains in Aeromonas hydrophila. Environ
363
Microbiol. 2019.
364
[8] Citterio B, Francesca B. Aeromonas hydrophila virulence. Virulence. 2015;6:417-8.
365
[9] Rama Devi K, Srinivasan R, Kannappan A, Santhakumari S, Bhuvaneswari M, Rajasekar P, et al. In
366
vitro and in vivo efficacy of rosmarinic acid on quorum sensing mediated biofilm formation and
367
virulence factor production in Aeromonas hydrophila. Biofouling. 2016;32:1171-83.
368
[10] Kirke DF, Swift S, Lynch MJ, Williams P. The Aeromonas hydrophila LuxR homologue AhyR
369
regulates the N-acyl homoserine lactone synthase, AhyI positively and negatively in a growth
370
phase-dependent manner. FEMS Microbiol Lett. 2004;241:109-17. 16
371
[11] Kozlova EV, Popov VL, Sha J, Foltz SM, Erova TE, Agar SL, et al. Mutation in the S-
372
ribosylhomocysteinase (luxS) gene involved in quorum sensing affects biofilm formation and
373
virulence in a clinical isolate of Aeromonas hydrophila. Microb Pathog. 2008;45:343-54.
374
[12] Swift S, Karlyshev AV, Fish L, Durant EL, Winson MK, Chhabra SR, et al. Quorum sensing in
375
Aeromonas hydrophila and Aeromonas salmonicida: identification of the LuxRI homologs
376
AhyRI and AsaRI and their cognate N-acylhomoserine lactone signal molecules. J Bacteriol.
377
1997;179:5271-81.
378
[13] Yao Z, Guo Z, Wang Y, Li W, Fu Y, Lin Y, et al. Integrated Succinylome and Metabolome Profiling
379
Reveals Crucial Role of S-Ribosylhomocysteine Lyase in Quorum Sensing and Metabolism of
380
Aeromonas hydrophila. Mol Cell Proteomics. 2019;18:200-15.
381
[14] Hu F, Cao Y, Xiao F, Zhang J, Li H. Site-directed mutagenesis of Aeromonas hydrophila enoyl
382
coenzyme A hydratase enhancing 3-hydroxyhexanoate fractions of poly(3-hydroxybutyrate-co-3-
383
hydroxyhexanoate). Curr Microbiol. 2007;55:20-4.
384
[15] Su YB, Peng B, Li H, Cheng ZX, Zhang TT, Zhu JX, et al. Pyruvate cycle increases aminoglycoside
385
efficacy and provides respiratory energy in bacteria. Proc Natl Acad Sci U S A. 2018;115:E1578-
386
E87.
387
[16] Wang Y, Wang X, Ali F, Li Z, Fu Y, Yang X, et al. Comparative Extracellular Proteomics of
388
Aeromonas hydrophila Reveals Iron-Regulated Secreted Proteins as Potential Vaccine
389
Candidates. Front Immunol. 2019;10:256.
390 391 392 393
[17] Ali F, Yao Z, Li W, Sun L, Lin W, Lin X. In-Silico Prediction and Modeling of the Quorum Sensing LuxS Protein and Inhibition of AI-2 Biosynthesis in Aeromonas hydrophila. Molecules. 2018;23. [18] Sun L, Chen H, Lin W, Lin X. Quantitative proteomic analysis of Edwardsiella tarda in response to oxytetracycline stress in biofilm. J Proteomics. 2017;150:141-8.
17
394
[19] Cai Q, Wang G, Li Z, Zhang L, Fu Y, Yang X, et al. SWATH based quantitative proteomics analysis
395
reveals Hfq2 play an important role on pleiotropic physiological functions in Aeromonas
396
hydrophila. J Proteomics. 2019;195:1-10.
397 398 399 400
[20] Gui M, Liu L, Wu R, Hu J, Wang S, Li P. Detection of New Quorum Sensing N-Acyl Homoserine Lactones From Aeromonas veronii. Front Microbiol. 2018;9:1712. [21] Ju X, Li J, Zhu M, Lu Z, Lv F, Zhu X, et al. Effect of the luxS gene on biofilm formation and antibiotic resistance by Salmonella serovar Dublin. Food Res Int. 2018;107:385-93.
401
[22] Guo Z, Lin Y, Wang X, Fu Y, Lin W, Lin X. The protective efficacy of four iron-related
402
recombinant proteins and their single-walled carbon nanotube encapsulated counterparts against
403
Aeromonas hydrophila infection in zebrafish. Fish Shellfish Immunol. 2018;82:50-9.
404 405
[23] Takahashi E, Ozaki H, Fujii Y, Kobayashi H, Yamanaka H, Arimoto S, et al. Properties of hemolysin and protease produced by Aeromonas trota. PLoS One. 2014;9:e91149.
406
[24] Post DMB, Schilling B, Reinders LM, D'Souza AK, Ketterer MR, Kiel SJ, et al. Identification and
407
characterization of AckA-dependent protein acetylation in Neisseria gonorrhoeae. PLoS One.
408
2017;12:e0179621.
409
[25] Sivaranjani M, Srinivasan R, Aravindraja C, Karutha Pandian S, Veera Ravi A. Inhibitory effect of
410
alpha-mangostin on Acinetobacter baumannii biofilms - an in vitro study. Biofouling.
411
2018;34:579-93.
412
[26] Kannappan A, Sivaranjani M, Srinivasan R, Rathna J, Pandian SK, Ravi AV. Inhibitory efficacy of
413
geraniol on biofilm formation and development of adaptive resistance in Staphylococcus
414
epidermidis RP62A. J Med Microbiol. 2017;66:1506-15.
415
[27] Srinivasan R, Devi KR, Kannappan A, Pandian SK, Ravi AV. Piper betle and its bioactive
416
metabolite phytol mitigates quorum sensing mediated virulence factors and biofilm of nosocomial
417
pathogen Serratia marcescens in vitro. J Ethnopharmacol. 2016;193:592-603.
18
418
[28] Ramanathan S, Arunachalam K, Chandran S, Selvaraj R, Shunmugiah KP, Arumugam VR. Biofilm
419
inhibitory efficiency of phytol in combination with cefotaxime against nosocomial pathogen
420
Acinetobacter baumannii. J Appl Microbiol. 2018;125:56-71.
421 422
[29] Balcazar JL, Subirats J, Borrego CM. The role of biofilms as environmental reservoirs of antibiotic resistance. Front Microbiol. 2015;6:1216.
423
[30] Li W, Yao Z, Sun L, Hu W, Cao J, Lin W, et al. Proteomics Analysis Reveals a Potential Antibiotic
424
Cocktail Therapy Strategy for Aeromonas hydrophila Infection in Biofilm. J Proteome Res.
425
2016;15:1810-20.
426
[31] Srinivasan R, Santhakumari S, Ravi AV. In vitro antibiofilm efficacy of Piper betle against quorum
427
sensing mediated biofilm formation of luminescent Vibrio harveyi. Microb Pathog.
428
2017;110:232-9.
429
[32] Srinivasan R, Vigneshwari L, Rajavel T, Durgadevi R, Kannappan A, Balamurugan K, et al.
430
Biogenic synthesis of silver nanoparticles using Piper betle aqueous extract and evaluation of its
431
anti-quorum sensing and antibiofilm potential against uropathogens with cytotoxic effects: an in
432
vitro and in vivo approach. Environ Sci Pollut Res Int. 2018;25:10538-54.
433
[33] Ravindran D, Ramanathan S, Arunachalam K, Jeyaraj GP, Shunmugiah KP, Arumugam VR.
434
Phytosynthesized silver nanoparticles as antiquorum sensing and antibiofilm agent against the
435
nosocomial pathogen Serratia marcescens: an in vitro study. J Appl Microbiol. 2018;124:1425-
436
40.
437
[34] Alexpandi R, Prasanth MI, Ravi AV, Balamurugan K, Durgadevi R, Srinivasan R, et al. Protective
438
effect of neglected plant Diplocyclos palmatus on quorum sensing mediated infection of Serratia
439
marcescens and UV-A induced photoaging in model Caenorhabditis elegans. J Photochem
440
Photobiol B. 2019;201:111637.
441 442
[35] Garnaud C, Champleboux M, Maubon D, Cornet M, Govin J. Histone Deacetylases and Their Inhibition in Candida Species. Front Microbiol. 2016;7:1238. 19
443 444
[36] Talagrand-Reboul E, Jumas-Bilak E, Lamy B. The Social Life of Aeromonas through Biofilm and Quorum Sensing Systems. Front Microbiol. 2017;8:37.
445
[37] Swift S, Lynch MJ, Fish L, Kirke DF, Tomas JM, Stewart GS, et al. Quorum sensing-dependent
446
regulation and blockade of exoprotease production in Aeromonas hydrophila. Infect Immun.
447
1999;67:5192-9.
448 449
[38] Zacaria J, Delamare AP, Costa SO, Echeverrigaray S. Diversity of extracellular proteases among Aeromonas determined by zymogram analysis. J Appl Microbiol. 2010;109:212-9.
450 451
Figure legends
452 453
Figure 1. Level of protein lysine acetylation. The Western blotting analysis combined with a pull-down
454
assay showed the acetylation levels in the rescue strain and site-directed mutagenesis strains, which was
455
determined by anti-acetyl lysine antibody. Kace - lysine acetylation.
456
20
457 458
Figure 2. Effect of lysine acetylation on the growth of A. hydrophila. The site-directed mutagenesis
459
strains did not show any variation on the growth of A. hydrophila even at 20 h, compared to the wild type
460
strain. The values are expressed as mean ± standard deviation of three biological independent
461
experiments.
462
21
463
464 465
Figure 3. Impact of lysine acetylation on the biofilm formation and hemolysin production of A.
466
hydrophila. Compared to the rescue strain, the site-directed mutagenesis strains showed a similar level of
467
biofilm formation and hemolysin production. The level of biofilm formation was expressed as OD value
468
and the hemolysin production was expressed as diameter (mm) of the zone of clearance. The values are
469
expressed as mean ± standard deviation of three biological independent experiments. The statistical
470
differences were analyzed by one-way ANOVA test. ns represents non-significant.
471
22
472
473 474
Figure 4. Effect of lysine acetylation on the protease production in A. hydrophila. The skim milk agar
475
plate showed a decreased level of protease production in mutation at K7 site of lysine acetylation strain
476
∆ahyI::ahyI-K7Q, compared to the rescue strain (A). The graph represents diameter (cm) of the zone of
477
clearance (B). The values are expressed as mean ± standard deviation of three biological independent
478
experiments. The statistical differences were analyzed by one-way ANOVA test. *** represents p-value ≤
479
0.002, ns represents non-significant.
480
23
481 482
Figure 5. Impact of lysine acetylation on the AHL production in A. hydrophila. The TLC plate
483
showed a reduced level of AHL production in mutation at K7 site of lysine acetylation strain ∆ahyI::ahyI-
484
K7Q, compared to the rescue strain (A). The graph represents the intensity level of AHL production (B)..
485
The values are expressed as mean ± standard deviation of three biological independent experiments. The
486
statistical differences were analyzed by one-way ANOVA test. ** represents p-value ≤ 0.0050.
487
24
488 489
Figure 6. Effect of lysine acetylation on the bioluminescence production. The graph represents the
490
level of bioluminescence productions in a relative light unit (RLU). Compared to the rescue strain, the
491
mutation at K7 site of lysine acetylation strain ∆ahyI::ahyI-K7Q showed a decreased level of
492
bioluminescence productions. The values are expressed as mean ± standard deviation of three biological
493
independent experiments. The statistical differences were analyzed by one-way ANOVA test. * represents
494
p-value ≤ 0.0112, *** represents p-value ≤ 0.0001.
495
25
496
Table 1 The Primers used for ahyI mutant construction
497
498
Name
Primer sequence 5' to 3'
F1
CATGAATTCCCGGGAGAGCTCGGCCGGCAGAAACCAGGT
F2
AGACATCTGAAGGCGACACAAAAAAATCCCG
F3
TGTGTCGCATTCAGATGTCTCCATTTCAGTGTTCG
F4
CGATCCCAAGCTTCTTCTAGAGGTCATCTTCCCGCAGACTG
F5
TTATTCGGTGACCAGTTCGC
F6
ATGCTTGTTTTCAAAGGAAAATTAAAAGA
F7
GCTCTCGTCATCCACCAG
F8
TTTTCCCTTCACTGGCCC
Note: The underlined are restriction enzyme cutting sites.
499 500 501 502 503 504 505 506 507 508 509 510 511 26
512
Table 2 The primers used for ahyI gene rescued and site-directed mutagenesis strains constructions Stranins name
Primer sequence 5' to 3'
∆ ahyI:: ahyI-F1
GTCGACGGTATTAAGCTTGGGCACTGGTTGAACAGCAC
∆ ahyI:: ahyI-F2
GTGGTGGTGGTGGTGGGATCCTTCGGTGACCAGTTCGCGCGCCT
∆ ahyI:: ahyI-K7Q-F1
CAATTAAAAGAACACCCCAGATGGGAGGTAGA
∆ ahyI:: ahyI-K7Q-F2
GGGTGTTCTTTTAATTGTCCTTTGAAAACAAGCATTCAGAT
∆ ahyI:: ahyI-K7R-F1
CGATTAAAAGAACACCCCAGATGGGAGGTAGA
∆ ahyI:: ahyI-K7Q-F2
GGGTGTTCTTTTAATCGTCCTTTGAAAACAAGCATTCAGAT
513 514 515 516
27
Highlights The AhyI protein regulates various quorum sensing regulated virulence factors productions in Aeromonas hydrophila. The AhyI protein showed lysine acetylation at their K7 site. The lysine acetylation at K7 site of AhyI protein affects various biological functions in Aeromonas hydrophila.