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Effect of the catabolic control protein A of Lactiplantibacillus plantarum AR113 on its colonization in vivo

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Lactiplantibacillus plantarum is selective for carbohydrate utilization, which is primarily regulated by the catabolic control protein A (ccpA). To investigate the impact of carbohydrate metabolism on the in vivo colonization of L. plantarum AR113, we constructed a ccpA knockout strain (AR113ΔccpA). In vitro assays showed that AR113ΔccpA had a 0.34 decrease in maximum biomass, and a 2.63 h increase in hysteresis time compared to AR113. In a single administration, there was no significant difference in the number of AR113 and AR113ΔccpA in the mucus layers, and the number of AR113 was approximately 34‐times higher than AR113ΔccpA at 48 h in the intestinal lumen. Notably, the knockout of the ccpA gene did not affect the colonization time of AR113 in the intestine during continuous administration. Therefore, the present work demonstrated that the ccpA did not play a crucial role in the in vivo colonization time of AR113 and provided valuable insights into the role of carbohydrate metabolism in bacterial colonization time in vivo.
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Received: 3 January 2024 Revised: 28 January 2024 Accepted: 6 February 2024
DOI: 10.1002/fft2.378
LETTER
Effect of the catabolic control protein A of Lactiplantibacillus
plantarum AR113 on its colonization in vivo
Wenfei Qin1,2Yue Zeng2Weilian Hung3Jiaqi Sun3Yongjun Xia1
Zhiqiang Xiong1Xin Song1Lianzhong Ai1Guangqiang Wang1
1School of Health Science and Engineering,
Shanghai Engineering Research Center of
Food Microbiology, University of Shanghai for
Science and Technology,Shanghai, China
2Shanghai Key Laboratory of Pancreatic
Diseases, Shanghai General Hospital, Shanghai
Jiao TongUniversity School of Medicine,
Shanghai, China
3Inner Mongolia Yili Industrial Group Co., Ltd.,
Hohhot, China
Correspondence
Guangqiang Wang, School of Health Science
and Engineering, Shanghai Engineering
Research Center of Food Microbiology,
University of Shanghai for Science and
Technology, Shanghai 200093, China. Email:
1015wanggq@163.com
Funding information
National Natural Science Foundation of China,
Grant/AwardNumber: No.31972056; USST
Medical-Engineering Cross-Project,
Grant/AwardNumber: No.10-21-308-420;
National Science Foundation for Distinguished
Young scholars, Grant/Award Number:
32025029; ClFST-Yili Foundationof Health
science; Shanghai Education Committee
Scientific Research Innovation Projects, China,
Grant/AwardNumber: 2101070007800120
Abstract
Lactiplantibacillus plantarum is selective for carbohydrate utilization, which is primarily
regulated by the catabolic control protein A (ccpA). To investigate the impact of carbo-
hydrate metabolism on the in vivo colonization of L. plantarum AR113, we constructed
a ccpA knockout strain (AR113ΔccpA). In vitro assays showed that AR113ΔccpA had
a 0.34 decrease in maximum biomass, and a 2.63 h increase in hysteresis time com-
pared to AR113. In a single administration, there was no significant difference in the
number of AR113 and AR113ΔccpA in the mucus layers, and the number of AR113
was approximately 34-times higher than AR113ΔccpA at 48 h in the intestinal lumen.
Notably, the knockout of the ccpA gene did not affect the colonization time of AR113
in the intestine during continuous administration. Therefore, the present work demon-
strated that the ccpA did not play a crucial role in the in vivo colonization time of AR113
and provided valuable insights into the role of carbohydrate metabolism in bacterial
colonization time in vivo.
KEYWORDS
carbohydrate metabolism, catabolic control protein A, gut colonization, Lactiplantibacillus plan-
tarum
1INTRODUCTION
Lactobacillus species are important probiotics with the capacity to
adapt to various environments, proliferate rapidly, and form commu-
nal relationships with other microorganisms, which are related to their
ability to utilize a variety of carbohydrates (Borges et al., 2014;Goh&
Klaenhammer, 2015; Kant et al., 2011). However, Lactobacillus species
are rarely able to utilize different carbon sources simultaneously
This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided
the original work is properly cited.
© 2024 The Authors. Food Frontiers published by John Wiley & Sons Australia, Ltd and Nanchang University, Northwest University, Jiangsu University, Zhejiang
University, FujianAgriculture and Forestry University
(Chen et al., 2018). Lactobacillus species preferentially selects pre-
ferred carbohydrate (usually glucose) when multiple carbohydrates are
present, and their metabolites repress the expression of genes related
to the metabolism of non-preferred carbohydrates, which causes a
complex physiological effect known as carbon catabolite repression
(CCR) (Zeng et al., 2017). In Gram-positive bacteria, catabolic control
protein A (ccpA) is a key regulator of the CCR effects (Chen et al.,
2015). The ccpA, a DNA-binding protein, belongs to the LacI/GalR
1796 wileyonlinelibrary.com/journal/fft2 Food Frontiers. 2024;5:1796–1805.
QIN ET AL.1797
family of transcriptional regulators (Crasnier-Mednansky, 2008; Schu-
macher et al., 2007; Swint-Kruse & Matthews, 2009). In the presence
of preferred carbon sources such as glucose, ccpA with the assis-
tance of serine-phosphorylated HPr (HPr-Ser-P) binds to the catabolic
response element sites of non-preferred carbohydrates metabolism
genes, thereby repressing their transcription and preventing bacterial
utilization of non-preferred carbon sources (Deutscher et al., 2006;Wu
et al., 2015). Nevertheless, studies have shown that ccpA gene deletion
leads to partial or complete abolition in function of CCR, which restor-
ing normal utilization of other carbohydrates (Moye et al., 2014;Zeng
et al., 2017).
As a globally critical regulatory protein in low-GC Gram-positive
bacteria, ccpA was widely involved in several physiological activities
(Zhang et al., 2021). It was believed that preferred carbohydrates
are the main determinant of microbial growth rate and a key factor
in the competition between Lactobacillus and other microorganisms.
The selective use of carbohydrates by Lactobacillus species is closely
related to their survival in the digestive tract, competition with the
gut microbiome and specific functions in an unfavorable environment
for growth (Xiao et al., 2021). To date, numerous studies on the num-
ber and colonization ability of Lactobacillus in the intestinal tract have
focused on the transcriptional regulation of genes by ccpA directly or
indirectly (Grand et al., 2020; Peng et al., 2020; Weme et al., 2015;
Zheng et al., 2012). However, it is unclear whether ccpA-regulated car-
bohydrate metabolism affects the ability of bacterial colonization in
vivo.
The purpose of the present study was to investigate the effect
of carbohydrate metabolism on the colonization ability of Lactiplan-
tibacillus plantarum AR113 in the intestine. We constructed the ccpA
gene knockout strain of L. plantarum AR113 using the clustered reg-
ularly interspaced short palindromic repeats—associated protein 9
(CRISPR/Cas9) gene-editing technique and determined the strain’s
physiological properties (e.g., acid tolerance, bile salts tolerance, and
adhesion ability with HT-29 cells). In addition, the effect of carbon
metabolism on the number and colonization time of L. plantarum AR113
was investigated in vivo using 5-(6)-carboxyfluorescein diacetate N-
succinimidyl ester (cFDA-SE) fluorescent staining and red fluorescent
protein (rfp) gene labeling.
2MATERIALS AND METHODS
2.1 Bacterial strains and growth conditions
Bacteria used in this study are listed in Table S1.Escherichia coli To p10
was grown at 30C in Lysogeny Broth (Sangon Biotech Co., Ltd). L. plan-
tarum AR113 and L. plantarum AR113 ΔccpA were cultured at 37C
in deMan, Rogosa, and Sharpe (MRS) agar or broth. Bacterial toler-
ance to acid and bile salts was assessed as described by Mishra and
Prasad (Matz et al., 1999), with appropriate modifications. Acid toler-
ance was assessed in modified MRS broth (pH adjusted to 3.0, 3.5, and
4.0 using 10 M of HCl (Sinopharm Chemical Reagent Co., Ltd)), and bile
salt tolerance was assessed in modified MRS broth (bile salt (Sangon
Biotech Co., Ltd) added 0.2%, 0.3%, and 0.4% w/v). The OD600 nm values
of AR113 and AR113ΔccpA were adjusted to 1.0, then inoculated at 3%
v/v into fresh modified MRS broth, mixed, and inoculated at 200 µLin
sterile microplate (Bioscreen C. Oy Growth Curves Ab Ltd), uninocu-
lated MRS broth was used as control, and sterile microplate was placed
in a fully automated growth curve analyzer (Bioscreen C. Oy Growth
Curves Ab Ltd) and incubated at 37C for 24 h. The OD600 nm values
was measured every 30 min. Three replicates were performed for each
sample.
2.2 Construction of ccpA knockout strain
The ccpA knockout plasmid (pLdccpA) was constructed as follows:
1000-bp fragments flanking ccpA (up and down) were amplified from
AR113 by polymerase chain reaction (PCR) using up-F/up-R and
down-F/down-R. A 103-bp guide RNA (single guide-RNA, sgRNA) was
amplified from pHSP01 by PCR using sgRNA-F/sgRNA-R. The up,
down, and sgRNA fragments were fused by overlapping extension PCR
using up-F/sgRNA-R to generate an up-down-sgRNA fragment. Then,
the fragment up-down-sgRNA was assembled into pHSP01 backbone,
which was obtained by double digestion with ApaI (Cat.1604, Takara)
and XbaI (Cat.1643, Takara) enzymes, using a single-step cloning kit
(Cat.C112-01, Vazyme), then incubated at 37C for 1 h, and coated in
plates with kanamycin (50 µg/mL, Sangon Biotech Co., Ltd). The primers
are shown in Table S2.
The ccpA knockout strain was constructed as follows (Huang et al.,
2019): First, the RecE/T-assisted plasmid pLH01 was electroporated
into AR113 competent cells. Then, AR113 (pLH01) competent cells
were prepared. After that, the verified knockout plasmid pLdccpA was
electroporated (parameters: 5 kV, 200 Ω,25µF) into AR113 (pLH01)
competent cells. Then, 900 µL of recovery medium SMRS (MRS broth
with 500 mM of sucrose, 20 mM of MgCl2) (bbi-lifesciences) was imme-
diately added, incubated for 2–3 h, coated in MRS agar supplemented
with antibiotics (erythromycin and chloramphenicol working concen-
trations were 10 µg/mL), incubated for 2–3 days at 37C. The colonies
were verified by PCR using primers (Table S2) and sequencing, the
strain was stored in glycerol (40% v/v) at 80C. The construction of
recombinant rfp strain follows the same procedure as above.
2.3 Aggregation assays
Auto- and co-aggregation assays were performed according to Col-
lado et al. (2007), with certain modifications. Bacteria grown in MRS
broth were harvested by centrifugation at 5000 ×gfor 15 min and
then washed twice and resuspended in phosphate-buffered saline
(PBS, NO. E607016, bbi-lifesciences), and the final absorbance of
the bacterial suspension was adjusted to 0.25 ±0.05 (approximately
1×108CFU/mL) at 600 nm. Bacterial suspensions (5 mL) were mixed
by vortexing for 15 s, and auto-aggregation was determined after 0 and
1798 QIN ET AL.
FIGURE 1 Construction of catabolic control protein A (ccpA) gene knockout plasmid and strain: (A) M: 2000 bp Marker, Line 1–3: ccpA-up;
Line 4–7: ccpA-down; Line 8–11: ccpA-sgRNA; (B) M: 5000 bp Marker, Line 1–3: ccpA-up-down-sgRNA; (C) M: 15,000 bp Marker, Line 1–3:
pHSP01; (D) M: 5000 bp Marker, Line 1–3: pLdccpA; (E) M: 5000 bp Marker, Line 1: positive control, Line 2: negative control, Line 3, 4:
Lactiplantibacillus plantarum AR113 (pLH01); and (F) 5000 bp Marker, Line 1: L. plantarum AR113. Line 2: negative control, Line 3, 4: L. plantarum
AR113ΔccpA.
5 h. The auto-aggregation was calculated using the following equation:
RAA =A0At
At ×100%
where Atand A0represent absorbance at t=0 or 5 h, respectively.
For co-aggregation assay, equal volumes (2 mL) of L. plantarum and E.
coli suspensions were mixed in pairs by vortexing for 15 s, and control
tubes were set up at the same time. The OD600 nm value of the suspen-
sions was measured at different times (0 and 5 h). The co-aggregation
was calculated using the following equation:
RCA =A0+A12Amix
A0+A1×100%
where A0and A1represent the OD600 nm value of the initial
absorbances of L. plantarum and E. coli suspensions, respectively, and
Amix represents the OD600 nm value of the mixed bacterial suspensions
at 5 h.
2.4 Measurement of bacterial hydrophobicity
For hydrophobicity assay, bacterial suspension was prepared in the
same way as in the aggregation assay. The OD600 nm value of bacte-
rial suspension was adjusted to 0.25 ±0.05, then 3 mL of bacterial
suspension was added to the test glass tube, an equal volume of
xylene (Sinopharm Chemical Reagent Co., Ltd.) was added, and the two-
phase system was vortexed for 3 min to mix thoroughly. The OD600 nm
value of the aqueous layer was measured, and the hydrophobicity was
calculated using the following equation:
RHC =A0At
At ×100%
where Atrepresents the absorbance at time t=1h,andA0represents
the absorbance at t=0h.
2.5 Adhesion ability of L. plantarum AR113 to
HT-29 Cells
The adhesion abilities of L. plantarum to HT-29 cells were performed
as previously described (Wang et al., 2019), with some modifications.
L. plantarum were harvested by centrifugation (3145 ×g,10min)and
washed twice with PBS (pH 7.2) and then resuspended in Roswell Park
Memorial Institute 1640 culture medium, and L. plantarum suspen-
sion concentration was adjusted to 108CFU/mL. The HT-29 cells were
cultured in 12-well plates with cover slips for adhesion experiments.
When the cells covered 80%–90% of the plates, they were rinsed twice
with PBS and add equal volumes of L. plantarum and cell suspension to
each well, and the plates were placed in a cell incubator at 37C for
2 h. Then, each well in the plates was washed three times with PBS to
remove free and nonattached bacteria, followed by methyl alcohol fix-
ation (1 mL) at room temperature for 20 min and then washed twice
with PBS, Gram staining, and microscopic observation. Twenty views
were randomly chosen for imaging. The adhesion number of L. plan-
tarum to HT-29 cells was calculated based on 100 cells. The experiment
was performed in triplicate.
QIN ET AL.1799
2.6 Detection of fluorescence
L. plantarum AR113-rfp and L. plantarum AR113ΔccpA-rfp were cul-
tured on MRS a broth for overnight cultivation. Then, L. plantarum
suspensions were adjusted to the same OD600 nm value, and the flu-
orescence intensities of L. plantarum (unlabeled and labeled) were
measured by an enzymatic standard (excitation wavelength 600 nm
and emission wavelength 630 nm). In addition, 10 µLofL. plantarum
suspension was aspirated onto a slide and covered with a cover-
slip, and L. plantarum AR113-rfp and L. plantarum AR113ΔccpA-rfp
were observed in bright-field and fluorescence modes of fluorescence
microscopy.
2.7 Flow cytometry analysis
Fluorescent labeling of L. plantarum was performed as described in pre-
vious studies (Lee et al., 2004). L. plantarum suspension was adjusted
to 109CFU/mL, and the suspension was mixed with cFDA-SE (50 µM,
Sangon Biotech Co., Ltd) in equal volumes, followed by incubation in
a37
C water bath for 20 min in the dark. Cell was collected by cen-
trifugation at 8000 ×gfor 3 min and rinsed with PBS twice to remove
unreacted cFDA-SE. Flow cytometry (FACSCalibur, BD Bioscience) was
used to detect the cell fluorescent labeling percentage. The experiment
was repeated in triplicate.
2.8 Animal experiments
Male C57 BL/6 mice (Jieshijie) were subjected to 1 week of acclima-
tization under the following conditions: 23 ±2C and humidity of
50% ±10%. In vivo colonization experiments were performed as
described previously (Lee e al., 2004). Mice were divided into control
group and experimental groups (L. plantarum AR113 and L. plantarum
AR113ΔccpA) with four mice in each group. In experimental groups,
each mouse was gavaged with bacterial suspension (the fluorescence
intensity was adjusted to 2 ×104RFU) for 6 days. The control group
was gavaged with an equal volume of sterile water. All animal pro-
cedures were performed in accordance with the Guidelines for Care
and Use of Laboratory Animals of Shanghai Jiao Tong University and
approved by the Animal Ethics Committee of Shanghai Jiao Tong
University (201902066).
2.9 Collection of feces and intestinal mucus
Feces collection was performed as described in a previous study
(Fornelos et al., 2020). Mouse feces were collected into sterilized
tubes (1.5 mL) using forceps, and forceps were sterilized before and
after use. Fecal handling was performed as described in the previous
research (Tuo et al., 2013), with some modifications. In brief, feces
(0.025 ±0.005 g) were homogenized with 1 mL of sterilized PBS in
sterile tubes (5 mm diameter steel filters) at 1000 rpm for 3 min. Then,
the supernatant was obtained by centrifugation at 400 ×gfor 3 min
at 4C and filtered through a 300-mesh filter. The duodenum, jejunum,
ileum, and colon tissues were collected from mice of approximately
2 cm, and the intestinal contents were collected. Then, the intestine
was dissected longitudinally, the mucosal layerwas scraped with a cov-
erslip into sterile tube with 1 mL of sterilized PBS, and the rest of
the procedure are consistent with fecal treatment methods. Fluores-
cence intensity was measured using a microplate reader (SpectraMax
i3x, Molecular Devices) at an excitation wavelength of 600 nm and an
emission wavelength of 635 nm.
2.10 Statistical analysis
Data are presented as means with standard deviations. Significant dif-
ferences among the data were examined using a one-way ANOVA
analysis of variance and Duncan’s test with Statistical Product and Ser-
vice Solutions software (SPSS). GraphPad Prism software (GraphPad
Software) was used to plot the graphs.
3RESULTS
3.1 Construction of the ccpA gene knockout
strain
The ccpA-up and ccpA-down were amplified using L. plantarum AR113
as a template for recovery of the up, down, and the ccpA-sgRNA was
amplified using plasmid pHSP01 as a template for recovery of the
sgRNA fragments (Figure 1A). The up, down, and sgRNA fragments
overlapped. The up-down-sgRNA fragment (Figure 1B) was cloned
seamlessly with the vector cleaved by ApaIandXbaI for pHSP01
(Figure 1C), and the ligated product was transferred to competent
cells of E. coli Top10 and coated onto kanamycin-resistant plates. Sin-
gle colonies were verified by electrophoresis and sequenced, and the
successfully sequenced plasmid was named pLdccpA (Figure 1D).
The knockout plasmid pLdccpA was electrotransferred to compe-
tent cells of L. plantarum AR113 (pLH01) (Figure 1E), and positive
clones were screened on erythromycin- and chloramphenicol-resistant
plates. Then, PCR verification and sequencing were performed. The
successfully sequenced strain was named L. plantarum AR113ΔccpA
(Figure 1F).
3.2 Effect of ccpA on the physiological properties
of L. plantarum AR113
To determine the effects of the ccpA gene knockout on the physio-
logical properties of L. plantarum AR113, L. plantarum AR113 and L.
plantarum AR113ΔccpA were cultured in the MRS broth. The growth
curves of the strains were obtained (Figure 2A), and the maximum
biomass, hysteresis time, and maximum specific growth rate were ana-
lyzed (Table 1). The ccpA knockout decreased the maximum biomass of
1800 QIN ET AL.
FIGURE 2 Growth and physiological characteristics of the strains. (A) Growth curves of Lactiplantibacillus plantarum AR113 (AR113) and
Lactiplantibacillus plantarum AR113ΔccpA (AR113ΔccpA) in deMan, Rogosa, and Sharpe (MRS) broth. (B–D) Growth of the strains in MRS broth
with bile salt concentrations of 0.2%, 0.3%, and 0.4% w/v, respectively. (E–G) Growth of the strains in MRS broth at pH 3.0, 3.5, and 4.0,
respectively. (H) Hydrophobicity, auto-aggregation, co-aggregation ability, and adhesion ability of the strains. Data are represented as
means ±standard deviations, and each experiment was performed in triplicate (n=3. Note: Means differ significantly (p<0.01).
the strain by 0.34 (OD600nm ), increased the hysteresis time by 1.63 h,
and decreased the maximum specific growth rate by 54.5%.
Next, we determined the environmental tolerance of the strains.
MRS broth was added with 0.2%, 0.3%, and 0.4% bile salts to assess the
bile salt tolerance of the strains (Figure 2B–D and Table 1). At a bile salt
concentration of 0.2%, compared with L. plantarum AR113, L. plantarum
AR113ΔccpA showed a 0.32 decrease in maximum biomass, a 6.09 h
increase in hysteresis time, and a 0.04 decrease in maximum specific
growth rate. L. plantarum AR113ΔccpA exhibited no growth at bile salt
concentrations above 0.3%. In terms of acid tolerance, the effects of
the ccpA gene knockout on acid tolerance are shown in Figure 2E–G
and Table 2.BothL. plantarum AR113 and L. plantarum AR113ΔccpA
failed to grow at pH 3.0. In addition, the effects of ccpA knockout on
hydrophobicity, auto-aggregation, co-aggregation ability, and adhesion
to HT-29 cells are shown in Figure 2H. No significant difference was
found between L. plantarum AR113 and L. plantarum AR113ΔccpA in
any of these physiological characteristics.
3.3 Effect of ccpA on the number of L. plantarum
AR113 in vivo
To investigate the effect of carbon metabolism on the number of
L. plantarum AR113 in vivo, we took advantage of the highly uni-
form and stable fluorescence of cFDA-SE-labeled cells, whereby the
fluorescence of the offspring cells was reduced by half with each divi-
sion. The labeling rate of cFDA-SE for bacteria was detected by flow
cytometry (Figure 3A,B). The labeling rate of the bacteria by the fluo-
rescent dye was greater than 98%, thus allowing for subsequent animal
experiments.
MiceweregavagedbyasingleadministrationofthecFDA-SE-
labeled L. plantarum AR113 or L. plantarum AR113ΔccpA. In the intesti-
nal mucus layer (Figure 3C–F), the numbers of L. plantarum AR113
and L. plantarum AR113ΔccpA in the duodenum, jejunum, ileum, and
colon all showed a decrease within 24 h and a rebound after 24 h.
After 72 h, L. plantarum AR113 and L. plantarum AR113ΔccpA were
QIN ET AL.1801
FIGURE 3 Changes in the number of wild-type and catabolic control protein A (ccpA) knockout strains in vivo with time. Parts (A) and (B)
represent Lactiplantibacillus plantarum AR113 (AR113) and Lactiplantibacillus plantarum AR113ΔccpA (AR113ΔccpA), respectively. Flow cytometry
of FDA-SE-labeled strains before (a) and after (b). (C–J) Changes in the number of colonies in intestinal mucus and lumen with time, where parts
(C–F) represent the duodenal mucus layer, the jejunum mucus layer, the ileum mucus layer, and the colon mucus layer, respectively, whereas parts
(G–J) represent the duodenal lumen, the jejunum lumen, the ileum lumen, and the colon lumen, respectively. Data are represented as
means ±standard deviations, and each experiment was performed in triplicate (n=3).
mainly distributed in the colon mucus layer. Similarly, in the intesti-
nal lumen (Figure 3G–J), the numbers of L. plantarum AR113 and L.
plantarum AR113ΔccpA in the duodenal, jejunal, ileal, and colonic con-
tents all showed a decrease within 24 h and a rebound after 24 h.
Notably, the difference in bacterial number between L. plantarum
AR113 and L. plantarum AR113ΔccpA wasgreatestat48h,withthe
former (1925 ±158 CFU/cm) being approximately 34 times higher
than the latter (57 ±12 CFU/cm). In short, the numbers of L. plantarum
AR113 and L. plantarum AR113ΔccpA showed the same trend in the
mucus layer and lumen, but the number of L. plantarum AR113ΔccpA
was significantly less than that of L. plantarum AR113.
3.4 Effect of ccpA on in vivo colonization time of
L. plantarum AR113
To further investigate, the effect of carbon metabolism on bacterial
colonization ability in vivo, we recombined the rfp gene into the L.
plantarum AR113 and L. plantarum AR113ΔccpA chromosomes. The
strains obtained after sequencing and plasmid loss (Figure 4A)were
named L. plantarum AR113-rfp and L. plantarum AR113ΔccpA-rfp. The
labeled and unlabeled strains were significantly different in fluores-
cence intensity at the same OD600 nm value (Figure 4B). Observations
of L. plantarum AR113-rfp and L. plantarum AR113ΔccpA-rfp in bright-
field and fluorescence modes of fluorescence microscopy (Figure 4C)
demonstrated that the strains could stably express the rfp gene.
The fluorescence intensity of L. plantarum AR113-rfp and L. plan-
tarum AR113ΔccpA-rfp was adjusted to 2 ×104RFU, followed by
continuous administration to mice. The colonization time of the strains
in the intestine is shown in Figure 4D. L.plantarumAR113-rfp and L.
plantarum AR113ΔccpA-rfp reached the highest fluorescence intensity
on day 7 (6.031 ×104RFU and 5.548 ×104RFU, respectively), after
which both showed a gradual decrease over time until reaching the
lowest fluorescence intensity of 0.390 ×104and 0.511 ×104RFU.
Remarkably, L. plantarum AR113-rfp and L. plantarum AR113ΔccpA-rfp
were able to colonize in vivo for up to 2 weeks, indicating that ccpA-
mediated carbohydrate metabolism did not alter the colonization time
of L. plantarum AR113 in vivo.
4DISCUSSION
In our work, we used CRISPR/Cas9 gene-editing technique to knockout
the ccpA gene in L. plantarum AR113, the results of in vitro physiolog-
1802 QIN ET AL.
FIGURE 4 Lactiplantibacillus plantarum AR113 in vivo colonization assay. (A) M: 5000 bp Marker, Line 1: L. plantarum AR113, Line 2: negative
control, Line 3: L. plantarum AR113- rfp (AR113-rfp), Line 4: L. plantarum AR113ΔccpA-rfp (AR113ΔccpA-rfp). The red fluorescent protein (rfp)
gene size was 863 bp. (B) Fluorescence intensity measurement of strains. (C) Fluorescence microscope observation, where (i) is a bright-field
image, (ii) is a fluorescence observation image, and (iii) is an overlay image of bright field and fluorescence, “**” represents significant difference
(p<.01). (D) Grouping of in vivo colonization experiments. (E) Fluorescence intensity of L. plantarum in feces with time. Data are represented as
means ±standard deviations, and each experiment was performed in triplicate (n=3).
ical characterization assays showed that L. plantarum AR113 growth
was inhibited after knockout of the ccpA gene. This was consistent with
a study by Zotta et al. (2012), who found that when the ccpA gene
was replaced by other gene fragments in L. plantarum WCFS1, the spe-
cific growth rate of the mutant strain was approximately 60% of the
wild-type strain, indicating that the growth of L. plantarum WCFS1 was
significantly inhibited. This was associated with the abolition of ccpA-
mediated inhibition when functional ccpA was not available, causing
the strain to lose its ability to utilize preferred carbon sources (usually
glucose) (Chen et al., 2018).
Probiotics are living microorganisms that benefit host health when
administered in sufficient amounts (García-Cayuela et al., 2014;
Guidone et al., 2014). The ability of probiotics to survive after expe-
riencing stressful conditions in the gut is critical in ensuring their
probiotic function. In our work, we found no significant difference in
the acid tolerance of L. plantarum AR113 and L. plantarum AR113ΔccpA.
However, the growth of L. plantarum AR113ΔccpA stalled when the
bile salt concentration was above 0.3%, and knockout of the ccpA
gene appeared to increase the sensitivity of the strain to bile salt. A
study showed that Listeria monocytogenes was associated with ccpA
in response to bile salt stress (Duché et al., 2002). It had also been
shown that bile salt mutant strains could utilize carbon sources more
efficiently, and alterations in carbohydrate metabolism suggested an
association between ccpA and bile salt tolerance, but the mechanism
of action needed to be further explored (Noriega et al., 2004;Sánchez
et al., 2004). In addition, we found that the knockout of the ccpA gene
did not alter the hydrophobicity, auto-aggregation, co-aggregation
ability, and adhesion to HT-29 cells of the strain.Many studies had con-
firmed that cell model experiments were simple, rapid, intuitive, and
effective in detecting the adherence capacity of probiotics (Laparra &
Sanz, 2009; Rodes et al., 2013). Adhesion ability was usually considered
a critical role for probiotics in vivo colonization, but in vitro adhesion
assays could only be used as one of the indicators to assess bacte-
rial adhesion and colonization in vivo and those indicators could not
simulate the complex environment in vivo (Calatayud et al., 2019; San-
tarmaki et al., 2017). Therefore, performing in vivo experiments was
necessary.
Carbohydrate metabolism is the main source of energy for gut com-
mensal bacteria and is vital for the survival of bacteria in the intestine
(Chen et al., 2015; Cui et al., 2021;Kantetal.,2011). The available
carbohydrates in the human enteric environment include diet-derived
components, human milk oligosaccharides, and host-secreted mucus
glycoproteins (Xiao et al., 2021). The ccpA gene, as a global regula-
tor of bacterial response to carbon sources in the environment, plays
an important role in bacterial growth and proliferation (Zhang et al.,
2021). In vivo, the numbers of L. plantarum AR113ΔccpA was smaller
than that of L. plantarum AR113. It was worth noting that L. plan-
tarum AR113 and L. plantarum AR113ΔccpA were able to colonize in
vivo for up to 2 weeks, which showed that ccpA knockout had a sig-
nificant effect on L. plantarum AR113 proliferation in the gut and did
QIN ET AL.1803
TAB L E 1 Maximum biomass, hysteresis time, and maximum specific growth rate of strains in deMan, Rogosa, and Sharpe (MRS) broth at different pH levels.
Strains
Maximum biomass (OD600 nm) Hysteresis time (h) Maximum specific growth rate (OD600nm /h)
Control pH3.0 pH3.5 pH4.0 Control pH3.0 pH3.5 pH4.0 Control pH3.0 pH3.5 pH4.0
Lactiplantibacillus plantarum AR113 1.67 0.76 1.04 2.33 8.35 5.13 0.33 0.05 0.11
L. plantarum AR113ΔccpA 1.33a0.45 0.88 3.96a21.21a9.46a0.15a0.05 0.08
aindicates significant difference in AR113ΔccpA versus AR113 (p<.01).
TAB L E 2 Maximum biomass, hysteresis time, and maximum specific growth rate of strains in deMan, Rogosa, and Sharpe (MRS) broth with different bile salt concentrations.
Strains
Maximum biomass (OD600 nm) Hysteresis time (h) Maximum specific growth rate (OD600nm /h)
Control 0.2% 0.3% 0.4% Control 0.2% 0.3% 0.4% Control 0.2% 0.3% 0.4%
Lactiplantibacillus plantarum AR113 1.67 1.08 0.88 0.50 2.33 4.68 7.80 7.01 0.33 0.10 0.07 0.04
L. plantarum AR113ΔccpA 1.33a0.76a––3.96a10.77a 0.15a0.06
aindicates significant difference in AR113ΔccpA versus AR113 (p<0.01).
1804 QIN ET AL.
not alter the colonization time of L. plantarum AR113 in vivo. Previous
studies have shown that the inactivation of genes involved in carbo-
hydrate metabolism in Lactobacillus reuteri 10023 (LJ1654, LJ1655,
LJ1656) (Sims et al., 2011; Tannock et al, 2012), Lactobacillus acidophilus
NCK1909 (glgA) (Goh & Klaenhammer, 2014), and Lactobacillus john-
sonii NCC533 (malA) (Denou et al., 2008) resulted in a significant
reduction in strain colonization activity. In addition, a study showed
that the maintenance of intestinal colonization time depended mainly
on the adhesion ability of the strains (Qin et al., 2022). This was highly
consistent with the fact that there was no difference in the numbers of
L. plantarum AR113 and L. plantarum AR113ΔccpA in the mucus layer in
our study. In addition, the complexityof the intestinal environment, the
scarcity of intestinal carbon sources, and the competition of intestinal
flora for carbon sources may also contribute to the lack of the differ-
ence in the duration of colonization between L. plantarum AR113 and
L. plantarum AR113ΔccpA in vivo. The ccpA acted as a global regulator
of carbon sources in the environment and the effect of ccpA-mediated
carbon metabolism on bacterial colonization in vivo needed further
investigation.
5CONCLUSIONS
In conclusion, the ccpA gene knockout significantly inhibited the
growth of L. plantarum AR113 in vitro. However, there were no
significant differences in the adherence ability, hydrophobicity, auto-
aggregation, and co-aggregation between L. plantarum AR113 and
L. plantarum AR113ΔccpA. In addition, the ccpA gene knockout did
not change the duration of colonization of the strain in vivo, con-
sistent with the lack of a difference in the numbers of L. plantarum
AR113 and L. plantarum AR113ΔccpA in the intestinal mucus layer.
In summary, this work provided important insights into the effects
of ccpA-mediated carbohydrate metabolism on bacterial colonization
in vivo.
AUTHOR CONTRIBUTIONS
Conducted the experiments; data curation; writing—original draft:Wen-
fei Qin. Writing—review and editing: Yue Zeng. Methodology: Weilian
Hung. Formal analysis; methodology: Jiaqi Sun. Validation; visualization;
funding acquisition: Yongjun Xia. Validation; investigation; writing—review
and editing: Zhiqiang Xiong. Supervision; writing—review and editing:Xin
Song. Supervision; project administration; funding acquisition: Lianzhong
Ai. Conceived and designed the experiments; supervision; funding acquisi-
tion; project administration: Guangqiang Wang. All authors have read
and agreed to the published version of the manuscript.
ACKNOWLEDGMENTS
The work was supported by the National Science Foundation for
Distinguished Young Scholars (No. 32025029), Shanghai Educa-
tion Committee Scientific Research Innovation Projects, China
(2101070007800120), National Natural Science Foundation of China
(No. 31972056), and CIFST-Yili Foundation of Health Science and
USST Medical-Engineering Cross-Project (No. 10-21-308-420).
CONFLICT OF INTEREST STATEMENT
The authors declare that they have no known conflicts of interests or
personal relationships that could have appeared to influence the work
reported in this paper.
ETHICS STATEMENT
In this work, all mice were housed for acclimatization for at least
one week before the following experiments, and the procedures were
performed according to institutional and governmental regulations
about the use of experimental animals. All animal procedures were
performed in accordance with the Guidelines for Care and Use of
Laboratory Animals of Shanghai Jiao Tong University and approved
by the Animal Ethics Committee of Shanghai Jiao Tong University
(201902066).
ORCID
Lianzhong Ai https://orcid.org/0000-0002-6681-9102
Guangqiang Wang https://orcid.org/0000-0002- 7762-743X
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How to cite this article: Qin, W., Zeng, Y., Hung, W., Sun, J., Xia,
Y., Xiong, Z., Song, X., Ai, L., & Wang, G. (2024). Effect of the
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AR113 on its colonization in vivo.Food Frontiers,5, 1796–1805.
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Lactobacilli are members of a large family involved in industrial food fermentation, therapeutics, and health promotion. However, the development of genetic manipulation tools for this genus lags behind its relative industrial and medical significance. The development of CRISPR‐based genome engineering for Lactobacillus is now underway. Generally, the CRISPR‐Cas9 system relies on targeted Cas9/sgRNA complex‐induced double‐strand breaks (DSBs) to stimulate recombination repair, which allows chromosomal genetic editing. However, some Lactobacillus species are sensitive to DSBs due to a deficiency in homology‐directed repair. Here, we coupled phage‐derived RecE/T with CRISPR‐Cas9 and assessed the transcriptional activity of broad‐spectrum host promoters to set up a versatile toolbox containing a recombination helper plasmid and a broad host CRISPR‐Cas9 editing plasmid, which enables efficient genome editing in Lactobacillus plantarum WCFS1 and Lactobacillus brevis ATCC367. The RecE/T‐assisted CRISPR‐Cas9 toolbox realized single gene deletions at an efficiency of 50–100% in 7 days. Furthermore, we accomplished the chromosomal gene replacement of Lp_0537 using a P23‐pdc (pyruvate decarboxylase) expression cassette with an efficiency of 35.7%. Our work established a RecE/T‐assisted CRISPR genome editing toolbox for L. plantarum WCFS1 and L. brevis ATCC367 and also demonstrated that RecE/T‐assisted CRISPR‐Cas9 is an effective genome editing system, which can be readily implemented in lactobacilli. This article is protected by copyright. All rights reserved.
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This study was to effectively screen out a Lactobacillus strain of excellent adhesion ability and ameliorative effect on disease symptom of a murine ulcerative colitis model. The auto-aggregation rate (RAA), co-aggregation rate with Escherichia coli O157 (RCA), and cell surface hydrophobicity (AHC) of 17 Lactobacillus strains were measured for primary selection. The results indicate that Lactobacillus plantarum AR326 displayed the topmost overall aggregation performance among all strains examined. A positive and linear relationship between RCA and RAA values was observed for 15 Lactobacillus strains with RAA = 16-46% and RCA = 15-27%. For adhesion ability to human adenocarcinoma HT-29 cells, it showed a good, positive dependence on RAA for five representative Lactobacillus strains, where L. plantarum AR326 and L. fermentum AR184 showed a higher adhesion ability than the others. Using 5 (and 6)-carboxyfluorescein diacetate N-succinimidyl ester (cFDA SE)-labeling technique, L. plantarum AR326 was confirmed to adhere to and colonize well in the intestinal mucosa of mice mainly in the ileum and colon.Finally, L. plantarum AR326 at the dosage applied (daily 2x10⁹ cfu/mouse) could attenuate murine dextran sulfate sodium (DSS)-induced colitis by effectively decreasing the body weight loss, disease activity index, colon length shortening, myeloperoxidase activity, and colon epithelial damage of experimental animals. The protective effects involved the restoration of the tight junction protein expression and reduction of the abnormal expression of pro-inflammatory cytokines. Conclusively, L. plantarum AR326 can be used as promising probiotics to ameliorate DSS-induced colitis.