Virulence

ISSN: (Print) (Online) Journal homepage: https://www.tandfonline.com/loi/kvir20

A novel quorum sensing regulator LuxT

contributes to the virulence of Vibrio cholerae

Yuehua Li, Junxiang Yan, Jinghao Li, Xinke Xue, Ying Wang & Boyang Cao

To cite this article: Yuehua Li, Junxiang Yan, Jinghao Li, Xinke Xue, Ying Wang & Boyang Cao
(2023) A novel quorum sensing regulator LuxT contributes to the virulence of Vibrio cholerae,
Virulence, 14:1, 2274640, DOI: 10.1080/21505594.2023.2274640

To link to this article: https://doi.org/10.1080/21505594.2023.2274640

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VIRULENCE
2023, VOL. 14, NO. 1, 2274640
https://doi.org/10.1080/21505594.2023.2274640

RESEARCH ARTICLE

A novel quorum sensing regulator LuxT contributes to the virulence of Vibrio

cholerae
Yuehua Lia,b,c#, Junxiang Yana,b,c#, Jinghao Lia,b,c, Xinke Xuea,b,c, Ying Wanga,b,c, and Boyang Caoa,b,c
a
TEDA Institute of Biological Sciences and Biotechnology, Nankai University, Tianjin, China; bKey Laboratory of Molecular Microbiology and
Technology of the Ministry of Education, Nankai University, Tianjin, China; cTianjin Key Laboratory of Microbial Functional Genomics, TEDA
College, Nankai University, Tianjin, China

ABSTRACT ARTICLE HISTORY

Vibrio cholerae is a waterborne bacterium that primarily infects the human intestine and causes Received 25 April 2023
cholera fatality. Quorum sensing (QS) negatively regulates the expression of V. cholerae virulence Revised 12 September 2023
gene. However, the primary associated mechanisms remain undetermined. This investigation identi­ Accepted 18 October 2023
fied a new QS regulator from the TetR family, LuxT, which increases V. cholerae virulence by directly
KEYWORDS
inhibiting hapR expression. HapR is a master QS regulator that suppresses virulence cascade expres­ Vibrio cholerae; quorum
sion. The expression of luxT increased 4.8-fold in the small intestine of infant mice than in Luria-Bertani sensing; luxT; hapR;
broth. ΔluxT mutant strain revealed a substantial defect in the colonizing ability of the small intestines. virulence; ArcA
At low cell densities, the expression level of hapR was upregulated by luxT deletion, suggesting that
LuxT can suppress hapR transcription. The electrophoretic mobility shift analysis revealed that LuxT
directly binds to the hapR promoter region. Furthermore, luxT expression was upregulated by the two-
component system ArcB/ArcA, which responses to changes in oxygen levels in response to the host’s
small intestine’s anaerobic signals. In conclusion, this research reveals a novel cell density-mediated
virulence regulation pathway and contributes to understanding the complex association between
V. cholerae virulence and QS signals. This evidence furnishes new insights for future studies on
cholerae’s pathogenic mechanisms.

Introduction Quorum sensing is a cell density-dependent interac­

Vibrio cholerae is a gram-negative, facultative anaerobic
bacterium that causes cholera. Cholera is responsible for
enhanced mortality globally, causing 3–5 million new
infections and > 100,000 deaths yearly [1,2]. Upon entrance
into the small intestine, V. cholerae releases two critical
virulence factors: the ctxAB-encoded cholera toxin (CT)
on the lysogenic CTXΦ bacteriophage, directly causing
diarrhea [3] and the toxin-co-regulated pilus (TCP), essen­
tial for enterocytes attachment and intestinal colonization
[4]. CT and TCP are controlled by a tightly regulated
signaling cascade [5]. toxT transcription is a prerequisite
for virulence factor expression and is controlled by the two
membrane-localized complexes, ToxRS and TcpPH. ToxT
directly stimulates the transcription of the ctx and tcp genes
[6,7]. Several environmental stimuli can affect V. cholerae
virulence, such as, pH and temperature regulate the expres­
sion of tcpP [8,9], bile salts regulate the expression of toxT
[10], and quorum sensing (QS) signals regulate the pro­
duction of virulence factor [11].
tion among bacteria [12], used by V. cholerae to mod­
ulate virulence factors release, biofilm generation, type
VI release, metabolic modulation, and natural compe­
tence [13–17]. QS is modulated via generating, releas­
ing, and identifying signaling molecules called
autoinducers (AIs), which control population-wide
behavioral alterations. AIs signals control the activity
of the membrane receptors CqsSR, LuxPQ, and VpsS.
These membrane receptors, in turn, activate luxO tran­
scription. LuxO stimulates small RNA at low cell den­
sities, which represses hapR expression, whereas, at
high cell densities, LuxO is suppressed, activating
hapR transcription [18,19]. HapR, the master QS reg­
ulator, which represses V. cholerae virulence gene, is
not expressed at this particular stage of infection. As
a consequence, expression of virulence factors, includ­
ing TCP and CT, are derepressed and timely produced
[11]. This investigation demonstrated another new reg­
ulator, LuxT, in the QS cascade that substantially reg­
ulates V. cholerae virulence.

CONTACT Ying Wang yingwang@nankai.edu.cn; Boyang Cao boyangcao@nankai.edu.cn

#
Yuehua Li and Junxiang Yan contributed equally to this work.
Supplemental data for this article can be accessed online at https://doi.org/10.1080/21505594.2023.2274640.
© 2023 Nankai University. Published by Informa UK Limited, trading as Taylor & Francis Group.
This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial License (http://creativecommons.org/licenses/by-nc/4.0/), which
permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. The terms on which this article has been
published allow the posting of the Accepted Manuscript in a repository by the author(s) or with their consent.
2 Y. LI ET AL.
LuxT is a transcriptional repression regulator of the
TetR family that was initially discovered as a luxO start
upstream codon region binding protein in V. harveyi
[20,21]. Subsequently, Eickhoff et al. reported that
LuxT represses qrr1 (a small regulatory RNA) tran­
scription but not luxO transcription [22].
Furthermore, LuxT modulates low cell density beha­
viors in V. harveyi, including type III release and pro­
duction of siderophore and aerolysin [23]. In
V. vulnificus, LuxO stimulates luxT transcription, and
LuxT suppresses the transcription of smcR (HapR
homolog) [24]. In V. alginolyticus, LuxT is associated
with the QS cascade by positively regulating LuxO and
LuxR (HapR homologs) [25]. In V. parahaemolyticus,
LuxT homolog SwrT positively modulates swarming
motility by inhibiting SwrZ [26]. However, the LuxT
regulation in V. cholerae is undefined and poorly
understood.
The redox-modulated anoxic binary system
responds to oxygen as a signal in the environmental
[27]. ArcA, a global transcription factor, regulates hun­
dreds of genes in different bacteria [28,29]. In
V. cholerae, ArcA positively modulates toxT expression
and enhances the V. cholerae virulence [30]. ArcA
regulates vpsT expression, associated with V. cholerae
biofilm formation [31]. Furthermore, it critically regu­
lates flrA expression, thereby controlling V. cholerae
motility [32]. Further comprehensive analysis of ArcA-
regulated genes will help better understand their patho­
genic role in V. cholerae.
This investigation identified that LuxT, a previously
unrecognized QS system regulator is essential for the
virulence of V. cholerae and provided new insights on
regulating the ArcA/ArcB two-component system.
Results
ArcA binds to LuxT promoter and positively
regulates its expression
ArcA is a global modulator of various biological pro­
cesses, with a regulon comprising hundreds of target
genes in various bacteria [28,29]. However, its function
in V. cholerae has been overlooked. Much research has
been carried out on its conserved binding sites [33,34].
A putative ArcA-binding site was identified in
V. cholerae E12382 (5′-AGTTCATTGT-3′; −111 to
−120 from the VCA0917 translational start site) located
in the promoter site of VCA0917 (Figure S1). This site
was compared with the E. coli and Salmonella con­
served binding sites, and a highly similar sequence
was identified (Figure S2). To elucidate if VCA0917
was modulated directly by ArcA, ArcA with a 6× His-
tagged fusion was isolated, and an electrophoretic
mobility shift assay (EMSA) was carried out using the
VCA0917 promoter region. Slow-migrating VCA0917
promoter bands were observed with increasing phos­
phorylated ArcA (ArcA-P) (Figure 1a). However, nega­
tive control 4.5s RNA revealed no migrating bands with
increasing concentrations of ArcA-P (Figure 1a).
Therefore, it was validated that ArcA directly interacts
with the VCA0917 promoter.
To investigate whether ArcA modulates VCA0917
expression, quantitative real-time PCR (qRT-PCR)
was carried out to elucidate VCA0917 expression in
the ΔarcA mutant and wild-type (WT) strains. About
2-fold reduction was in VCA0917 expression in the
ΔarcA strain than the WT strain, which was reversed
after the complementation of ΔarcA with arcA
(Figure 1b). These findings indicate that ArcA directly
positively regulated the expression of VCA0917.
The BlastP analysis revealed the sequence homology
of VCA0917 with TetR family regulator LuxT, compris­
ing the helix-turn-helix (HTH) domain for DNA bind­
ing. The luxT gene has also been observed in other
Vibrio species [20–26]. V. cholerae LuxT protein exhib­
ited 90%, 92%, 93%, and 93% identity to those of
V. alginolyticus (E1U688), V. vulnificus (CMCP6),
V. harveyi (Q9ANS7), and V. parahaemolyticus
(Q87J33), respectively, indicating that LuxT is con­
served in Vibrio spp. We concluded that ArcA binds
to luxT promoter and positively regulates its
expression.
luxT expression is induced by a low oxygen signal
in the small intestine via ArcA
In our study, it was found that ArcA directly positively
regulated the expression of luxT. ArcA can sense changes
in oxygen availability and regulate the expression of
downstream genes [28]. Therefore, we studied whether
ArcA regulates luxT expression in response to the low
oxygen concentration in the small intestine. We per­
formed the qRT-PCR assays in vitro [35]. The expression
level of luxT in the WT strain increased approximately
5.8-fold in a low-oxygen state compared with that under
high-oxygen conditions in LB broth (Figure 2a).
However, luxT expression in the ΔarcA strain under
low- and high-oxygen states was not markedly different
(Figure 2a), indicating that low oxygen activates luxT
expression via ArcA. In addition, qRT-PCR assay showed
that luxT expression in V. cholerae infected infant mouse
intestines increased approximately 4.8-fold compared
with that in LB broth (Figure 2b), indicating that
V. cholerae induces luxT expression in the small intestine.
Furthermore, qRT-PCR assays showed that the
 VIRULENCE 3

Figure 1. ArcA positively regulates the expression of luxT by directly interacting with its promoter. (a) EMSA of ArcA-P protein and
luxT promoter DNA. The luxT promoter DNA (50 ng) and ArcA-P protein (0 to 1.25 µM) were used in each reaction; 4.5s RNA was used
as a negative control. (b) qRT-PCR elucidated luxT expression level in the WT, ΔarcA, and ΔarcA::ParcA strains in LB broth. Data were
depicted as mean ± SD (n = 3); unpaired Student’s t-test was applied to assess p-values (*p < 0.05; **p < 0.01; ***p < 0.001; NS, not
significant, p > 0.05).

expression of luxT was significantly decreased in the
ΔarcA strain compared to WT in the small intestine
(Figure 2c). Complementation of ΔarcA with arcA
restored the expression level of luxT to the WT level in
the small intestine (Figure 2c). These results indicate that
V. cholerae induces luxT expression in the small intestine
in response to the low oxygen conditions via ArcA.
LuxT promotes V. cholerae colonization in infant
mouse intestine
Once ingested by the host, V. cholerae colonizes intest­
inal epithelial cells’ surface and causes chronic diarrhea.
A mouse intestinal colonization assay was carried out
to investigate LuxT’s impact on the pathogenicity of
V. cholerae. WT and ΔluxT mutant strains were admi­
nistered intragastrically in 3-day-old CD-1 infant mice,
and the bacteria recovered from the intestinal homo­
genates was quantified. It was revealed that mouse
intestines infected with the ΔluxT mutant strain con­
tained a relatively lower number of bacteria than that
infected with the WT strain, which was reversed after
the complementation of ΔluxT with luxT (Figure 3a).
In addition, we performed an competition assay in vivo.
The competitive assay in vivo showed that the intestinal
colonization ability of the ΔluxT strain was significantly
reduced compared with that of the WT strain by CI
value at 0.23 (Figure 3b). Furthermore, the complemen­
tation of ΔluxT with luxT restored the colonization
ability back to the WT level (Figure 3b). These findings
indicate that LuxT positively influences intestinal colo­
nization in V. cholerae.
LuxT enhances V. cholerae virulence gene
expression by directly repressing hapR expression
To assess the LuxT mechanism that regulates
V. cholerae virulence, the expression of virulence gene
in the ΔluxT mutant and WT strains were compared
via qRT-PCR, revealing that the virulence gene expres­
sion was repressed in the ΔluxT mutant strain
(Figure 4a). For example, tcpPH and toxT were inhib­
ited in the ΔluxT mutant strain. TCP island genes were
suppressed 1.5- to 3.6-fold, and ctxA and ctxB to 2-fold
in the ΔluxT mutant strain (Figure 4a). In contrast, the
expression of hapR, a master QS regulator, was ~
5.2-fold higher in the ΔluxT strain than in the WT
strain (Figure 4b). The literature reported that HapR
4 Y. LI ET AL.

Figure 2. luxT expression is induced by a low oxygen signal in the small intestine via ArcA. (a) qRT-PCR expression level of luxT in the WT
and ΔarcA mutant strains in LB broth during anaerobic or aerobic conditions. (b) luxT expression in LB broth and in the small intestine. (c)
qRT-PCR expression level of luxT in the WT, ΔarcA, and ΔarcA::ParcA strains in the small intestine. Data were depicted as mean ± SD (n = 3);
unpaired Student’s t-test was applied to assess p-values (*p < 0.05; **p < 0.01; ***p < 0.001; NS, not significant, p > 0.05).

inhibits the expression of virulence gene [13,36,37],
indicating that LuxT enhances V. cholerae virulence
gene expression by repressing hapR expression.
Furthermore, it was also elucidated if LuxT directly
modulated hapR expression by EMSA, which indicated
that LuxT-maltose-binding protein (MBP) directly bound
to the promoter region of hapR, whereas LuxT-MBP pro­
tein lacked binding with the negative control 4.5s RNA
(Figure 4c). With the help of dye-primer-based DNase
I foot-printing assay, a distinct LuxT-bound sequence com­
prising 18 bp motif (5′-CTTTGTTATGTACCCACT-3′;
−98 to −115 from hapR translational start site) was identi­
fied (Figure 4d). These findings indicate that LuxT
enhances V. cholerae virulence gene expression by directly
repressing hapR expression.
LuxT inhibits hapR transcription at a low cell
density
The effects of cell density on LuxT-mediated regulation
were investigated by monitoring hapR expression in the
ΔluxT mutant and WT strains during the growth phase.
It was noticed that at low cell densities (optical density at
600 nm [OD600] ≤0.6), hapR level were reduced in the
WT strain than in the ΔluxT mutant strain (Figure 5).
However, at high cell densities (OD600 ≥1.5), its level was
comparable in both strains (Figure 5). They suggested
that LuxT primarily inhibits hapR level at low cell
densities.
Discussion
Because V. cholerae is the cause of increasing deaths
worldwide, it is essential to elucidate its virulence
mechanisms and their regulation. This research identi­
fied a novel regulatory mechanism mediated by LuxT
that is associated with the virulence of V. cholerae in
a HapR-dependent manner, and cell density was
demonstrated as a host cue for this contribution.
LuxT genetic analysis suggested that it is substan­
tially conserved in Vibrio spp., with 85–93% sequence
homology. However the roles of LuxT in regulating QS

Figure 3. LuxT promotes V. cholerae colonization in infant
mouse intestine. (a) the capability of the WT, ΔluxT, and
ΔluxT::PluxT strains to colonize the intestines of infant mice
was assessed via a mouse intestinal colonization assay.
Bacterial counts in the mouse intestine (CFU per milliliter)
were determined 22–24 h after intragastric inoculation with
V. cholerae E12382 strain. (b) competition assay comparing
the colonizing ability of WT, ΔluxT, and ΔluxT::PluxT in infant
mouse intestine. Each symbol represents the CI in an individual
mouse, horizontal bars indicate the median. Unpaired Student’s
t-test was applied to assess p-values (*p < 0.05; **p < 0.01; ***p
< 0.001; NS, not significant, p > 0.05).
and the associated phenotypes seem to be elusive in
various Vibrio spp. LuxT was first discovered in
V. harveyi. LuxT could bind directly to the promoter
region of luxO and could inhibit luxO transcription
[20,21]. This conclusion was later overturned by
Michaela J. Eickhoff et al., who found that LuxT did
not inhibit the transcription of luxO in V. harveyi but
inhibited the transcription of sRNA qrr1 [22]. In a later
study by Michaela J. Eickhoff et al., in V. harveyi, LuxT
directly inhibits the transcription of a GntR family
transcription factor SwrZ and controls of type III secre­
tion, siderophore, and aerolysin [23]. In
V. parahaemolyticus, SwrT (homolog of LuxT)
VIRULENCE 5
repressed the transcription of swrZ and SwrZ repressed
the laf genes encoding the lateral flagellar [26]. In
V. vulnificus, LuxT inhibits elastase production by inhi­
biting smcR transcription, and LuxO directly positively
regulates luxT transcription [24]. In V. alginolyticus,
LuxT positively regulates extracellular protease produc­
tion, motility, and virulence. The transcription of luxT
was cell density dependent and was positively regulated
by LuxU. In addition, LuxT positively regulated both
luxO at transcriptional level and luxR at post-
transcriptional level, which is thoroughly different
from the established QS regulation mode in
V. harveyi and V. vulnificus [25]. In our study, we
found that LuxT promote V. cholerae virulence by
inhibiting the expression of hapR at low cell density
(Figure 6). Consistent with our research, previous stu­
dies have shown QS induces virulence factor produc­
tion by inhibiting the expression of hapR at low cell
density [38]. When V. cholerae cells initially entered
inside the host small intestine, the population cell num­
ber is relatively low and the master QS regulator HapR
is repressed. So the virulence factors, including TCP
and CT, were produced to enables the pathogens to
colonize inside the host small intestine and cause the
watery diarrhea.
V. cholerae colonizes the surface of intestinal epithe­
lial cells with low oxygen concentrations. Therefore,
during infection, it must survive in an anaerobic envir­
onment and ArcB/ArcA two-component system is cri­
tical for this low-oxygen environment. The research
indicates that ArcA positively modulates V. cholerae
virulence by increasing toxT expression, and this effect
is more pronounced during anaerobic growth [30].
Here, it was revealed that in low-oxygen conditions,
V. cholerae promotes luxT expression in the small
intestine via ArcA, and LuxT then increases virulence
(Figure 6). It is highly likely that ArcA’s influence on
V. cholerae virulence partially depends on regulating
luxT expression. When the environment changes,
ArcB, membrane sensor protein, is phosphorylated
and transfers its phosphoryl group to ArcA. ArcA-P is
then stimulated as a transcription factor, upregulating
or downregulating many downstream genes [39].
Further studies are required to confirm the redox signal
in the small intestine for ArcB activation, which
increases luxT expression.
In conclusion, this investigation elucidated the viru­
lence and QS system modulatory activity of novel LuxT
in V. cholerae. It was suggested that at low cell densi­
ties, LuxT enhances V. cholerae virulence by directly
6 Y. LI ET AL.

Figure 4. LuxT enhances V. cholerae virulence gene expression by directly repressing hapR expression. (a) qRT-PCR elucidated
virulence gene expression levels in the WT, ΔluxT, and ΔluxT::PluxT strains in LB broth. (b) qRT-PCR expression level of hapR in the
WT, ΔluxT, and ΔluxT::PluxT strains in LB broth. Data were represented as the mean ± SD (n = 3); unpaired Student’s t-test was
applied to assess p-values (*p < 0.05; **p < 0.01; ***p < 0.001; NS, not significant, p > 0.05). (c) EMSA of LuxT protein and hapR
promoter DNA. The hapR promoter DNA (50 ng) and LuxT protein (0 to 2 µM) were used in each reaction; 4.5s RNA was used as
a negative control. (d) LuxT binds to a motif in the hapR promoter region. Electropherograms indicated the hapR promoter region’s
protection pattern after DNase I digestion and incubation with and without LuxT protein (2 μM). The protected region revealed
a substantially decreased peak intensities pattern (red) than the control (blue). The bottom of the figure depicts elucidated LuxT-
binding motif in a box.

Figure 5. LuxT inhibits hapR transcription at low cell densities.
qRT-PCR assessed hapR expression level in the WT, ΔluxT, and
ΔluxT::PluxT strains at the cell densities indicated. Data were
represented as the mean ± SD (n = 3); unpaired Student’s t-test
was applied to assess p-values (*p < 0.05; **p < 0.01; ***p <
0.001; NS, not significant, p > 0.05).
repressing hapR expression. Moreover, V. cholerae
induces luxT expression under low-oxygen conditions
in the small intestine via ArcB/ArcA (Figure 6).
Materials and methods
Bacterial strains, plasmids, and growth conditions
The V. cholerae O1 El Tor biotype strain El2382 was
isolated in 1994 and was acquired from the Shanghai
Municipal Center for Disease Control and Prevention.
Table S1 enlists the bacterial plasmids and strains uti­
lized. Table S2 represents the primers applied. Bacteria
were propagated in LB broth at 37 °C without shaking
(anaerobic state) or with shaking (aerobic state) as per
literature [35]. Antibiotics were used as follows: poly­
myxin B (40 μg/mL), ampicillin (100 μg/mL), kanamy­
cin (50 μg/mL), and chloramphenicol (25 μg/mL). All
the chemicals utilized were acquired from Sigma
(St. Louis, MO, USA).
Establishing mutant strains and complementation
Isogenic deletion mutant strains (ΔarcA and ΔluxT)
were established with the help of suicide vector
pRE112, as per literature [40]. For complementation,
first, PCR amplification of the gene was carried out;
Figure 6. Diagram depicting the LuxT-mediated virulence regulatory networks in V. cholerae strain E12382. The expression of luxT in
V. cholerae was induced in the small intestine in response to the low oxygen conditions via ArcA. Meanwhile, LuxT directly inhibited
hapR translation at low cell density. As a consequence, the expression of virulence gene was derepressed and timely produced.
VIRULENCE 7
then, the gene was cloned into the vector pBAD33 [41],
and lastly, using electroporation, the vector was incor­
porated into V. cholerae. Table S2 represents all the
primers utilized.
RNA isolation and quantitative real-time PCR
V. cholerae cells were propagated overnight in LB
media at 37°C and subcultured aerobically or anaero­
bically at a 1:100 inoculation ratio. When the OD600
was 0.6, the cells were acquired via centrifugation for 5
min at 5000 rpm. Whole RNA was acquired with the
help of TRIzol reagent (#15, 596–018; Invitrogen,
Waltham, MA, USA) as directed on the kit. cDNA
was prepared using the Prime Script RT Reagent Kit
(Takara, Shiga, Japan). qRT-PCR was conducted in the
Applied Biosystems 7500 sequence detection system
with SYBR green fluorescence dye. For normalization,
16S rRNA was utilized [42]. The 2–ΔΔCT method calcu­
lated fold change in the assessed gene expression [43].
Electrophoretic mobility shift assay
The LuxT-MBP and 6×His-tagged ArcA were
expressed and then purified as per the literature [42].
Then, luxT and hapR promoter region’s DNA frag­
ments (300 bp) were amplified via PCR. The luxT
promoter (50 ng) DNA fragments were pre-incubated
with an accelerating concentration of purified ArcA
protein in a binding buffer (20 µL) (10 mM Tris –
HCl [pH 8.0], 1 mM EDTA, 1 mM DTT, 1.5 mg/mL
poly IC, 50 mM KCl, 50 μg/mL BSA, and 10% glycerol)
augmented with acetyl phosphate (20 nM) for 30 min at
ambient temperature (AT) [31]. Similarly, hapR pro­
moter (50 ng) DNA fragments and various concentra­
tions of purified LuxT-MBP protein were incubated at
AT for 30 min in a binding buffer (20 µL) (40 mM
HEPES-KOH [pH 7.9], 400 mM KCl, 10 mM MgCl2,
2 mM DTT, 10% glycerol, and 1 μg of poly IC)) [24].
4.5s RNA was set as the negative control [31]. For
electrophoresis, the prepared samples were loaded on
the 6% polyacrylamide gel in 0.5 × Tris-Borate EDTA
(TBE); the gels were stained using Gel Red (1: 10000
dilutions in TBE) and visualized via a UV transillumi­
nator (Tanon).
8 Y. LI ET AL.
Dye primer-based DNase I footprinting assay
The 300 bp promoter of hapR was amplified using forward
(with a 5’ end 6-FAM modification) and reverse primers.
Different quantities of LuxT protein (0–2 μM) were added
to 6-FAM-labeled hapR promoter (50 ng) in a binding
buffer(10 mM Tris-HCl [pH 7.5], 0.2 mM DTT, 5 mM
MgCl2, 10 mM KCl, and 10% glycerol) for 30 min at AT.
With the help of 0.05 U DNase I (Thermo; EN0521), the
protein-DNA mixtures were partially digested at 25 °C for
5 min and terminated by incubation with EDTA (250 mM)
at 65 °C for 10 min. Using the QIAquick PCR Purification
Kit (Qiagen; 28104), the DNA fragments were purified,
eluted in distilled water (20 μL), and then assessed by
MAP Biotech Co., Ltd. (Shanghai, China). The data were
measured using Peak Scanner (v1.0).
Intestinal colonization assay
This assay was performed per the literature, with opti­
mizations [40]. CD-1 infant mice (age = 3 days) were
acquired from the Beijing Vital River Laboratory
Animal Technology (Beijing, China). Overnight
V. cholerae cultures were subcultured in fresh LB
broth with a 1:100 inoculation ratio. For OD600 of 1.5
V. cholerae (WT or mutant strains) (105 colony-
forming units (CFU) in 10 µL PBS) was intragastrically
administered to each mouse. After 22–24 h, their intest­
inal specimens were collected for CFU determination.
Competition assay for mouse intestinal
colonization
In this study, the V. cholerae lacZ+ strains (both mutant
and WT) and lacZ− strains (ΔlacZ) were grown overnight
in LB broth under 37°C. Later, about 105 lacZ+ strains
were blended with lacZ− cells at an equivalent amount,
and then the resultant mixture was given into mice
through intragastric administration. Small intestine was
dissected after incubation for 22–24 h, the weight was
measured, and the sample was put onto the LB agar plates
supplemented with 5-bromo-4-chloro-3-indoyl-β-D
galactopyranoside (X-gal) to enumerate those recovered
bacteria and obtain the output ratios. The competitive
index (CI) was calculated as lacZ+ to lacZ− cell output
ratio divided by lacZ+ to lacZ− cell input ratio.
Statistical analyses
Statistical analysis was performed using SPSS (version
12.0; IBM Corp., Armonk, NY, USA). Origin 8.5
(Origin Lab Corporation) was utilized for acquiring fig­
ures. The data are depicted as mean ± standard deviation
(SD) from three independent experiments. The Student’s
t-test assessed the intergroup comparison. Differences
were deemed significant at p < 0.05 (*, p < 0.05; **, p <
0.01; ***, p < 0.001; NS, not significant, p > 0.05).
Disclosure statement
No potential conflict of interest was reported by the author(s).
Funding
This work was supported by the National Key Program for
Infectious Diseases of China (Nos. 2017ZX10303405-001,
2017ZX10104002-001-006, 2018ZX10712001-017).
Author contributions
Yuehua Li: conception and design, acquisition of data, ana­
lysis and interpretation of data, and drafting the article.
Junxiang Yan: conception and design, analysis and interpre­
tation of data, and drafting the article. Jinghao Li: conception
and design, analysis and interpretation of data. Xinke Xue:
analysis and interpretation of data. Wang Ying: conception
and design, analysis and interpretation of data, and review­
ing/revising it critically for important intellectual content.
Boyang Cao: conception and design, analysis and interpreta­
tion of data, and drafting the article.
Data availability statement
The authors confirm that the data supporting the findings of
this study are available in the article and its supplementary
materials.
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