Abstract
The application of high hydrostatic pressure (HHP) technology in the
food industry has generated potential safety hazards due to
sub-lethally injured (SI) pathogenic bacteria in food products. To
address these problems, this study explored the repair mechanisms of
HHP-induced SI Escherichia coli O157:H7. First, the repair state of SI
E. coli O157:H7 (400 MPa for 5 min) was identified, which was cultured
for 2 h (37 °C) in a tryptose soya broth culture medium. We found that
the intracellular protein content, adenosine triphosphate (ATP)
content, and enzyme activities (superoxide dismutase, catalase, and
ATPase) increased, and the morphology was repaired. The transcriptome
was analyzed to investigate the molecular mechanisms of SI repair.
Using cluster analysis, we identified 437 genes enriched in profile 1
(first down-regulated and then tending to be stable) and 731 genes in
profile 2 (up-regulated after an initial down-regulation). KEGG
analysis revealed that genes involved in cell membrane biosynthesis,
oxidative phosphorylation, ribosome, and aminoacyl-tRNA biosynthesis
pathways were enriched in profile 2, whereas cell-wall biosynthesis was
enriched in profile 1. These findings provide insights into the repair
process of SI E. coli O157:H7 induced by HHP.
Keywords: Escherichia coli O157:H7, high hydrostatic pressure,
sub-lethally injury, repair mechanism, transcriptome analysis
1. Introduction
High hydrostatic pressure (HHP) is a promising non-thermal processing
technique used for the inactivation of micro-organisms (bacteria,
yeast, and mold) in food systems, because of the advantages of
preserving nutritional and sensory characteristics [[36]1,[37]2]. HHP
technology has been successfully commercialized in the past decade
[[38]3] in fruit and vegetable juices, meat, and dairy products. The
global market for HHP foods reached approximately USD 9.8 billion in
2015 and is expected to attain a market value of USD 54.77 billion in
2025 [[39]4].
Escherichia coli O157:H7 has been implicated in many food-borne
outbreaks and posed a worldwide threat to public health [[40]5,[41]6].
According to the Centers for Disease Control and Prevention, beef and
leafy vegetables were the sources of >25% of all reported E. coli
outbreaks and >40% of related illnesses from 2003 to 2012 [[42]7]. In
addition, E. coli O157:H7 is highly virulent and causes inflammatory
reactions, hemolytic uremic syndrome, and thrombocytopenic purpura
[[43]8], establishing this organism as a high-risk pollution challenge
that must be addressed.
More dangerous still, sub-lethally injured (SI) cells (including SI E.
coli) could be induced by HHP in various food matrixes, thereby
limiting the promotion of HHP [[44]9,[45]10,[46]11]. SI cells are
induced by stresses that are not severe enough to destroy cells
[[47]12]. Studies found that typical food-borne pathogens (such as
Listeria innocua, E. coli, and Bacillus subtilis) could be sub-lethally
injured by HHP. The proposed mechanism could involve many targets,
including reversible membrane injury, nucleic acid and proteins, and
metabolic disorders. [[48]13,[49]14]. Injured cells could recover
physiological function and virulence when they are repaired in
favorable conditions, and then proliferate in food during storage,
leading to potential health threats [[50]15,[51]16,[52]17]. Though
detection methods have been developed to detect SI cells, the results
have proven inaccurate, leading to false negative results due to
differences among the culture media [[53]18].
Several studies focused on SI cells’ quantity, interfering factors, and
mechanisms. Sokolowska et al. [[54]19] found that 2.7 log of SI E. coli
cells could be detected in phosphate-buffered saline (PBS) after 400
MPa treatment for 10 min with the increase in initial bacterial
concentration, the content of SI cells increased. The SI processing
could result in changes in morphology, protein denaturation, membrane
damage, and oxidation stress [[55]20,[56]21]. Other studies focused on
the repair conditions of SI cells. Yamamoto et al. [[57]21] found that
the repair of SI E. coli, Listeria monocytogenes, and B. subtilis
induced by HHP (300–600 MPa) were related to the storage temperature
and nutrient level of the repair medium, 25 °C and nutrient-rich medium
were more suitable for the repair process. Shi et al. [[58]12] found
that the repair rate of SI E. coli O157:H7 induced by lactic acid could
be increased by sodium pyruvate, Tween 80, or certain cations (Mn, Fe,
or Zn) but not influenced by Mg or Ca. These findings suggest that the
repair of SI cells is a common phenomenon with complex mechanisms.
Unfortunately, few studies have focused on the mechanism of the repair
process, especially from the molecular level.
To fill this gap, we employed comparative transcriptome analysis for SI
(induced by HHP) and repair of E. coli O157:H7 models to determine the
differences in the transcriptional responses during the repair process.
Physiological analysis was used to augment this analysis. Our research
provided new insights into the underlying molecular and cellular
mechanisms of the repair process, which would help to explore new ways
for improving the HHP antibacterial effect and avoiding the repair of
SI cells.
2. Materials and Methods
2.1. Strain and Culture Condition
We used E. coli O157:H7 NCTC 12900, preserved in Beijing Key Laboratory
of Forest Food Processing and Safety. A single colony was inoculated
into 50 mL of tryptose soya broth culture medium (TSB, Aoboxing Biotech
Co., Ltd., Beijing, China) and then incubated at 37 °C and 180 rpm for
5 h (optical density at 600 nm = 0.86) in a shaker (TS-100B, Shanghai
Tiancheng Experimental Instrument Manufacturing Co., Ltd., Shanghai,
China) until the culture reached the medium segment of the logarithmic
phase [[59]22]. Bacterial pellets were harvested by centrifugation at
6000 rpm and 4 °C for 10 min and then washed three times with PBS (pH
7.2–7.4, Biotopped Co., Ltd., Beijing, China). The final bacterial
suspension concentration was approximately 10^8–10^9 colony-forming
units/mL.
2.2. Determination of Sub-Lethal Treatment Conditions
The bacterial suspension was divided into sterile bags equally (10 mL
per bag) and sealed tightly. The packed samples were soaked in
deionized water in an airtight cabin, and then pressurized at 100, 200,
300, 400, and 500 MPa for 1, 3, or 5 min at 25 °C using HHP equipment
(Shanghai Litu Ultra-High Voltage Equipment Co., Ltd., Shanghai,
China). The constant pressure time did not include the pressure
increase (2–3 min) or pressure-release (1–2 s) times. The treated
bacterial suspension in identical conditions was coated on a
non-selective medium (NS, tryptose soya agar medium, TSA) and a
selective medium (SC, TSA with 3% NaCl) plates, respectively [[60]22].
The plates were placed upside-down in an incubator for 24 h at 37 °C,
and the sub-lethal effects were determined using the colony counting
method. The SI rate, SI cell content, intact cell content, and dead
cell content were calculated using the following formulas: A[control],
A[ns], and A[sc] present colony-forming units of untreated control,
cells survived on the NS culture media, and cells survived on the SC
media, respectively.
[MATH:
SI rate%=(Ans<
mrow> − Asc)Ans × 100 :MATH]
(1)
[MATH:
SI cell content%=
(Ans − Asc)Acontrol<
/mfrac> × 100 :MATH]
(2)
[MATH:
Intact cell <
/mtext>content%=Asc
Acontrol<
/mfrac> × 100 :MATH]
(3)
[MATH:
Dead cell content%=100 − Ans
Acontrol<
/mfrac> × 100 :MATH]
(4)
2.3. Determination of Repair Conditions
The SI E. coli O157:H7 was repaired in TSB, buffered peptone water
(BPW), PBS, and minimal medium A (minA) medium, respectively; minA was
prepared according to Hui et al. [[61]17]. The repair method referred
to Bi et al. [[62]22]. Briefly, the SI E. coli O157:H7 induced by HHP
treatment were centrifuged at 6000 rpm and 4 °C for 10 min to remove
PBS. The bacterial pellets were re-suspended in equivalent media and
the bacterial suspensions were incubated at 37 °C and 120 rpm in a
shaker for 6 h. Then, 100 μL of bacterial solution diluted to the
appropriate concentration was coated on NS and SC every hour,
respectively. The plates were then placed in the incubator for 24 h at
37 °C, and the number of surviving colonies was recorded. The results
were displayed in the form of the logarithm of the surviving colony
number (lgS).
2.4. Determination of the Intracellular Protein Content and Adenosine
Triphosphate (ATP) Contents
The intracellular protein content was determined using a total protein
assay kit (BCA method, Beijing BioDee Biotechnology Co. Ltd., Beijing,
China). Briefly, the bacterial suspensions subjected to various
treatments were centrifuged at 6000 rpm and 4 °C for 10 min to obtain
pellets. Each pellet was re-suspended in PBS and crushed using an
ultrasonic processor (HY92-IIDN, Ningbo Scienta Biotechnology Co.,
Ltd., Ningbo, China) in an ice bath to obtain the intracellular
solution. Then, the 20 μL intracellular solution was added to the 200
μL BCA solution and incubated at 37 °C for 1 h. The absorbance was
determined at 562 nm to calculate the protein content using a standard
curve (y = 0.0009x + 0.0068, R^2 = 0.9993). The ATP content was
determined using an assay kit (Jian Cheng Bioengineering Institute,
Nanjing, China). To avoid the influence of phosphorus in PBS, the
bacterial pellet was suspended in boiled sterile water for the
subsequent ultrasonication, as described above. The intracellular
solution was then boiled in water for 15 min and extracted for two
minutes to perform the subsequent experiment according to the
manufacturer’s instructions.
2.5. Observation of the Morphological Changes
Atomic force microscopy (AFM) was performed to observe the
morphological changes of E. coli O157:H7 after various treatments.
Bacterial suspensions of the untreated (UT), HHP, and repair groups
(repaired for 4 and 8 h) were prepared, as described in [63]Section 2.2
and [64]Section 2.3, and fixed with glutaraldehyde (BioDee
Biotechnology Co., Ltd., Beijing, China) overnight at 4 °C. Each
mixture was washed three times with sterilized water and re-suspended
in sterilized water. Each sample was dropped onto the mica sheet and
naturally dried before observing using a Bruker Multimode 8 AFM (Bruker
Corporation, Karlsruhe, Germany) in auto scan mode. Then, 20 areas of
509.6 × 509.6 nm^2 were randomly selected in each treatment to count
the root mean square roughness using NanoScope Analysis (version 1.40,
Bruker Corporation, Karlsruhe, Germany).
2.6. Measurement of Enzyme Activities
Using a kit, a UV-vis spectrophotometer determined the catalase (CAT)
activity (Jian Cheng Bioengineering Institute, Nanjing, China).
Briefly, the equivalent intracellular solution was added into the
testing tube and contrast tube before and after the addition of the
stop solution, respectively. After standing at room temperature for 2
min, the absorbance was measured at 405 nm. The superoxide dismutase
(SOD) activity was determined using a WST-1 method kit and a microplate
reader (Multiskan FC, Thermo Fisher Scientific, Waltham, MA, USA). The
intracellular solution was added to the testing well, and equivalent
PBS was added to the contrast well instead of the intracellular
solution. The reaction system was incubated at 37 °C for 30 min and
then measured at 450 nm. The ATPase activity was measured using a minim
ATP enzyme test kit (Jian Cheng Bioengineering Institute, Nanjing,
China). To avoid the influence of phosphorus in PBS, the bacterial
pellet was suspended in cold sterile water, and then ultrasonicated to
obtain the enzyme solution. The results were measured at 636 nm using
the microplate reader.
2.7. RNA Isolation and Library Construction
The E. coli O157:H7 UT group, the SI (HHP group, treated at 400 MPa for
5 min), and those repaired for 2 h in TSB (repair group) were quickly
frozen in liquid nitrogen for the subsequent transcriptomic analysis.
All groups were cultured in triplicate. Total RNA was extracted using a
TTIzol reagent kit (Invitrogen, Carlsbad, CA, USA). RNA quality was
tested on an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto,
CA, USA) and checked using RNase free agarose gel electrophoresis.
After the extraction, mRNA was enriched by removing rRNA by
Ribo-Zero^TM magnetic kit (Epicentre, Madison, WI, USA). Finally, the
ligation products were size selected by agarose gel electrophoresis,
PCR amplified and sequenced using Illumina HiSeq2500 by Gene Denovo
Biotechnology Co. (Guangzhou, China).
2.8. Transcriptomic Analysis
For each transcription region, a fragment per kilobase of transcript
per million mapped reads (FPKM) value was calculated. The false
discovery rate (FDR) was calculated to correct for the multiple testing
to adjust the p-value threshold. RNAs differential expression analysis
was performed using DESeq2 software between different groups (and by
edgeR between two samples). The genes with the parameter of FDR below
0.05 and absolute fold change (FC) ≥ 2 (or |log[2]FC| ≥ 1) were
considered differential expression genes (DEGs). Gene ontology (GO) was
performed for the DEGs for each treatment stage by mapping all DEGs to
GO terms in the GO database. GO terms where p-value ≤ 0.05 were defined
as significantly enriched GO terms in DEGs. Kyoto Encyclopedia of Genes
and Genomes (KEGG, a public pathway-related database) was used to
identify significantly enriched metabolic pathways and signal
transduction pathways in DEGs. Pathways meeting the condition that
p-value ≤ 0.05 were defined as significantly enriched pathways in DEGs.
The transcriptome sequence of E. coli O157:H7 is deposited at the
National Center of Biotechnology Information (NCBI) under the
Bioproject number PRJNA769820.
2.9. Real Time Quantitative PCR (qRT-PCR) Validation
The qRT-PCR assay was conducted on E. coli O157:H7 treated with the
same method used in the transcriptome analysis. Six DEGs (fatty acid
biosynthesis, accB; ribosome, rpsS; oxidative phosphorylation, frdA and
atpG; amino acid metabolism, argG and ansB) from different pathways
were selected to confirm the RNA-seq results using the primers listed
in [65]Table S1. The annealing temperature was 56 °C. Relative gene
expression was calculated using the 2^−ΔΔCT method with 16S rRNA as an
internal control.
2.10. Statistical Analysis
All experiments were performed in triplicate. Data were expressed as
the mean ± standard deviation. The differences between groups were
analyzed using one-way analysis of variance, followed by Duncan’s
tests, using SPSS software (version 23; SPSS, Inc., Chicago, IL, USA);
p ≤ 0.05 was considered statistically significant in the physiological
experiments.
3. Results
3.1. Determination of Sublethal Injury and Repair Conditions
Following previous studies, the SI E. coli O157:H7 survived on the NS
medium but not on the SC medium, while the average E. coli O157: H7
survived on both NS and SC (when the content of NaCl in SC was ≤3%)
[[66]22,[67]23]. To obtain the SI model with an SI rate of more than
99.99% [[68]24], E. coli O157:H7 cells were treated by HHP at various
pressures (100, 200, 300, 400, and 500 MPa) and times (1, 3, and 5
min). As shown in [69]Figure 1A ([70]Table S2), the SI rate increased
with increasing pressure. When the pressures reached 400 MPa (5 min)
and 500 MPa (1, 3, and 5 min), the SI rate exceeded 99.99%, and the
intact cell content was 0.00%. Similar results were reported by
Somolonos et al. [[71]25], in which the sub-lethal injury rate reached
99.99% when treated with 400 MPa for 5 min. The SI cell content
negatively correlated with pressure ([72]Table S2). When the pressure
reached 500 MPa, the amounts of sub-lethal cells were lower than 0.05%,
which was too low to carry out the subsequent transcriptome analysis.
Consequently, samples treated with 400 MPa and 5 min (HHP group) were
used in the subsequent analysis.
Figure 1.
[73]Figure 1
[74]Open in a new tab
The sub-lethal injury rate (A) and the repair of sub-lethal E. coli
O157: H7 in TSB, BPW, PBS, and minA mediums (B). Letter a–d in (A)
indicate statistically significant within the groups treated with
different pressures and same time (p ≤ 0.05, Duncan test).
The repair of SI E. coli O157:H7 cells in TSB, BPW, PBS, and minA is
shown in [75]Figure 1B. As demonstrated by the consistent growth trend
of survival counts in NS and SC, the SI cells could be recovered over
time. When repaired in TSB and BPW, the remaining SI cells were
completely repaired after 2 h of incubation. In PBS and minA, the
repair of SI cells needed 5 h and >5 h, respectively, suggesting a
positive correlation between the repair efficiency and nutrient
abundance. Similarly, Bi et al. [[76]22] found that the trend of SI E.
coli repair efficiency induced by high-pressure carbon dioxide was TSB
> carrot juice > peptone water > PBS, which is positively related to
nutrient abundance. Thus, the SI E. coli O157: H7 repaired in TSB for 2
h was selected for further investigation of the recovery mechanism of
sub-lethal injured E. coli O157:H7 using transcriptome analysis.
3.2. Physiological Analysis Reveals the Repair Mechanism
3.2.1. Determination of the Intracellular Protein and ATP Contents
The intracellular protein contents of UT, HHP, and repair groups are
shown in [77]Figure 2A. Compared with the UT group, the intracellular
protein content decreased to 0.34 mg/mL after HHP treatment, which
could be due to the leakage of intracellular protein [[78]26]. During
the repair process, the protein content increased slightly from 4 h;
however, the content remained significantly lower than the HHP group.
The intracellular ATP contents of treatments are exhibited in
[79]Figure 2B, which showed no significant change when treated with a
single HHP. However, it started to increase rapidly after four hours of
repair. When repaired for 8 h, the ATP content was about seven times
higher than that of the UT group. In summary, the contents of protein
and ATP in cells showed an upward trend after the repair.
Figure 2.
[80]Figure 2
[81]Open in a new tab
Effects of different repair times on intracellular protein content (A),
intracellular ATP content (B), AFM images (C), surface roughness (D),
CAT and SOD activities (E), and ATPase activities (F). (C) contained
the 2-dimensional and 3-dimensional AFM images of UT, HHP, and repair
groups (repaired for 4 h and 8 h). The red arrow represented the cell
fragments and the green arrows holes. The average bacterial surface
roughness in nm was obtained on 509.6 × 509.6 nm^2 area of UT, HHP and
repair groups. For each group, 20 cells were plotted randomly.
Different letters (a to e, A to F, and a’ to e’) indicate statistically
significance between different groups (p ≤ 0.05, Duncan test).
3.2.2. Observation of Morphological Changes
The morphological changes were observed using an AFM, which has been
used to investigate biosystems, such as bacteria and eukaryotic,
because of its ability to reveal structural details with unprecedented
resolution. [82]Figure 2C shows that the UT cells demonstrated short
rod shapes with continuous and smooth surfaces. When treated with HHP,
the uniform short rod shape was broken into fragments (red arrow).
There were hollows on the cell surface (green arrows), suggesting the
destruction of cell morphology. In the 4 h repair group, the unrepaired
hollows on the cell surface remained (green arrows). After 8 h of
repair, the cells returned to full rod shape and began proliferating.
In total, 20 locations (509.6 nm × 509.6 nm) were randomly selected to
calculate the roughness ([83]Figure 2D). The HHP group showed the
highest roughness, while the repair groups were lower than the HHP
group but significantly higher than the UT group. Following a previous
study, the morphology of SI E. coli O157:H7 returned to normal levels
during repair, as seen under a scanning electron microscope (SEM)
[[84]27], similar to our results. These findings suggest that the
morphology recovers during the repair process, which requires more
time.
3.2.3. Determination of the Enzyme Activities
[85]Figure 2E shows the enzyme activities of two crucial members in the
peroxisome (SOD and CAT). The SOD activity decreased significantly
after sub-lethal treatment induced by HHP. After an 8 h repair process,
even though the SOD activity showed an upward trend, its activity
(881.35 U/mgprot) remained significantly lower than the UT group
(3641.61 U/mgprot). The CAT activity increased more than twice that of
the UT group (3.17 U/mgprot) after HHP treatment. During repair, the
CAT activity showed an increasing trend. When repaired for 6 h, the CAT
activity recovered to the same level as the UT group. When repair
continued for another two hours, the CAT activity reached 4.04
U/mgprot. Inaoka et al. [[86]28] found that the disruption of the gene
encoding SOD reduced the viability of HHP-treated B. subtilis; however,
the disruption of the gene encoding CAT had no detectable effect on the
viability of HHP-treated cells. The SOD and CAT activities showed
opposite trends in our study, possibly due to the gene encoding SOD
being more sensitive than CAT in resisting the oxidative stress caused
by HHP. HHP treatment might result in a state of metabolic imbalance,
which, in its turn, causes a burst of reactive oxygen species (ROS)
[[87]29]. However, oxidative defense mechanisms could be induced during
the cell repair process to avoid ROS accumulation [[88]30], which could
be responsible for increasing SOD and CAT activities.
The tendencies of Na^+K^+-, Ca^2+Mg^2+-, and total ATPase activities
are shown in [89]Figure 2F. These ATPases were significantly inhibited
after sub-lethal injury induced by HHP. During the repair process,
these ATPase activities continued to decrease until 6 h and increased
after 8 h of repair. Similarly, Ma et al. [[90]27] found that the
Na^+K^+-ATPase and Ca^2+Mg^2+-ATPase activities did not increase until
12 h of repair. This discrepancy could be because SI cells are repaired
in PBS rather than TSB, which is nutrient-deficient. These findings
suggest that increased SOD, CAT, and ATPases activities reflect
physiological injuries, and the repair effect might be positively
correlated with nutrient abundance.
3.3. Changes in Transcript Levels of E. coli O157:H7 during HHP and Repair
3.3.1. Transcriptional Response to the HHP and Repair
A total of 1,067,811,900, 1,093,588,200, and 922,627,500 raw reads were
collected from UT, HHP, and repair groups using the Illumina Hiseq
platform ([91]Table S3). After filtration, 1,003,731,547, 986,057,925,
and 861,150,652 clean reads were collected. Of these clean reads,
6,367,292 (UT), 6,063,864 (HHP) and 5,784,618 (Repair) reads were
mapped to the reference genome, and the mapping ratios were all higher
than 84% ([92]Table S4); i.e., these data were of high quality for
further analysis. The points representing the same treatment were
aggregative based on principal component analysis (PCA) ([93]Figure
3A). Those of UT, HHP, and repair treatments were distributed in
different quadrants, suggesting that the results of the same treatments
were repeatable, and there were significant differences among different
treatments. A total of 1304, 1071, and 1154 DEGs were detected in the
UT vs. HHP, HHP vs. repair, and UT vs. repair groups, respectively
([94]Figure 3B). In the HHP vs. repair group, there were more
upregulated DEGs (767) than downregulated DEGs (304). By contrast, the
downregulated DEGs in the UT vs. HHP and UT vs. repair groups (1081 and
818) were higher than that of upregulated DEGs (223 and 336). It could
be speculated that the HHP process inhibited the expression of DEGs
while the repair process advanced. Venn analysis was applied to
identify the similarities and differences among DEGs for the UT vs.
HHP, HHP vs. repair, and UT vs. repair groups ([95]Figure 3C,D). There
were 51 DEGs in common among the three groups: 16 upregulated and 35
downregulated ([96]Tables S5 and S6). These DEGs were related to
responses to environmental changes, such as acid (hycBCDEFG and
citCDEFXG) [[97]31,[98]32,[99]33] and nitrogen starvation (yeaGH and
astABCDE) [[100]34,[101]35]. These results suggest that the changes in
the internal and external environment induced by the HHP and repair
process could lead to responses at the transcriptional level.
Figure 3.
[102]Figure 3
[103]Open in a new tab
Analysis of the E. coli O157: H7 transcriptome in HHP (sub-lethal
cells) and repair (repaired in liquid medium for 2 h) groups. (A) the
principal component analysis of UT, HHP, and repair groups. (B) The
upregulated and downregulated differentially expressed genes (DEGs)
among UT vs. HHP, HHP vs. repair, and UT vs. repair. (C,D) The Venn
diagram analysis of upregulated and downregulated DEGs enriched in both
the HHP and repair groups, respectively. (E) The qRT-PCR results of the
6 DEGs, which were the mean values of 2^−ΔΔCT obtained from three
biological replicates with error bars representing standard deviations.
Results were normalized using 16S rRNA and expressed as fold change.
To validate the RNA-Seq data, a qRT-PCR assay was used to determine the
expression of DEGs with the same RNA samples. For all six DEGs tested
(accB, rpsS, frdA, atpG, argG, and ansB), the qRT-PCR results followed
RNA-Seq results ([104]Figure 3E), suggesting the validity of the
RNA-Seq results.
3.3.2. Cluster Analysis, GO, and KEGG Analysis
Considering that genes with similar expression patterns are likely to
possess similar functions or participate in the same regulatory
pathways, all 1942 DEGs in the UT vs. HHP and HHP vs. repair were
clustered using the short time-series expression miner (STEM,
[105]Figure 4A). Based on the cluster analysis, the DEGs could be
clustered into eight profiles, in which profiles 1 and 2 were
significantly enriched (p-value ≤ 0.05, [106]Figure 4B). Profiles 1 and
2 presented the unresponsive and upregulated DEGs, respectively, which
were downregulated following HHP. A total of 1168 DEGs were contained
in these two profiles, in which 437 genes were enriched in profile 1,
and 731 genes were enriched in profile 2. Then, the DEGs contained in
profiles 1 and 2 were subjected to GO term analysis and KEGG pathway
enrichment analysis ([107]Figure S1 and [108]Figure 4C). In GO
analysis, the DEGs were classified into three categories, molecular
function (MF), biological process (BP), and cellular component (CC). As
shown in [109]Figure 4C ([110]Table S7), under the CC category, the
most abundant sub-categories of profile 1 were: membrane (GO: 0016020),
membrane part (GO: 0044425), and intrinsic component of membrane (GO:
0031224). For profile 2, the sub-categories binding (MF), metabolic
process (BP), and ribosome (CC) were significantly enriched. For KEGG
pathway analysis, the most abundant pathways are shown in [111]Figure
S1 (Table S8). The ‘C5-branched dibasic acid metabolism (ko00660)’,
‘Cell cycle-Caulobacter (ko04112)’ and ‘Valine, leucine, and isoleucine
biosynthesis (ko00290)’ were the top three pathways enriched in profile
1. For profile 2, ‘Ribosome (ko03010)’, ‘Alanine, aspartate, and
glutamate metabolism (ko00250)’, and ‘Aminoacyl-tRNA biosynthesis
(ko00970)’ were the top enriched groups. These findings suggest that
DEGs responding to the HHP and repair processes are associated with the
membrane, genetic information transmission, and energy biosynthesis.
Figure 4.
[112]Figure 4
[113]Open in a new tab
Cluster analysis of the E. coli O157: H7 transcriptome in HHP
(sub-lethal cells) and repair (repaired in liquid medium for 2 h)
groups. (A) Patterns of gene expressions across UT, HHP, and repair
groups inferred by STEM analysis. In each profile, the light grey lines
represented the expression pattern of each gene enriched, while the
black line represented the expression tendency of all the genes in this
profile. (B) The amounts of DEGs and significances in each profile, in
which red columns defined significant difference. (C) KEGG enrichment
analysis of profile 1 and profile 2. The significance of the most
enriched pathway in these two clusters was indicated by the p-value.
The red regions represented the significant p-values, whereas the grey
regions represented the non-significant values.
3.4. Response of DEGs Related to Membrane after HHP and Repair Processes
DEGs responding to HHP and repair treatments identified in the
membrane-related pathways included ‘Peptidoglycan biosynthesis’ and
‘Fatty acid biosynthesis,’ which were associated with membrane
biosynthesis ([114]Figure 5A,B, [115]Table S9). Peptidoglycan is a
component of the cell wall (also called outer membrane) in bacteria
[[116]36]. In total, 12 DEGs were involved in the ‘Peptidoglycan
biosynthesis’ pathway, in which 5 DEGs were promoted, and 7 showed no
significant change during the repair process ([117]Table S9). Deleting
the genes murA and murB directly decreased peptidoglycan synthesis in
Corynebacterium glutamicum [[118]37]. It could be inferred that the
upregulation of these genes might lead to repair the outer membrane.
Figure 5.
[119]Figure 5
[120]Open in a new tab
Transcriptional changes of genes involved in the significantly enriched
pathway. (A) The peptidoglycan biosynthesis pathway. (B) The fatty acid
biosynthesis pathway.
Conversely, gene dacC (encoding the low-molecular-weight
penicillin-binding protein necessary for synthesis and stabilization)
showed no significant change. Gao et al. [[121]38] found that the
downregulation of gene murD in Staphylococcus aureus resulted in a
thinner cell wall. The unresponsiveness of these DEGs might reduce the
repair efficiency of the outer membrane.
Fatty acids in E. coli are produced for the biosynthesis of lipids and
the inner membrane. Most DEGs involved in the ‘Fatty acid biosynthesis’
pathway were significantly induced after repair. The fabB gene
(encoding beta-ketoacyl-ACP synthase I, which is required for
unsaturated fatty acid biosynthesis [[122]39]) increased by 1.43
log[2]FC compared to the HHP group. The fadG gene (an essential gene in
this pathway) increased by 0.91 log[2]FC. It was worth noting that the
fadD gene showed an opposite trend, downregulated 0.62 log[2]FC after
the repair process. The product of fadD participates in the initial
stages of long-chain fatty acid β-oxidation, which oxidizes fatty acid
into acetyl-CoA [[123]40]. It could be inferred that promoting fatty
acid synthesis-related genes and inhibiting its oxidation-related gene
should be a method of how the inner membrane was repaired at the
transcriptional level.
3.5. Response of DEG Related to Energy Biosynthesis after HHP and Repair
‘Oxidative phosphorylation’ is the pathway that produces ATP, the
energy currency in bacteria. There are five complexes in this pathway
([124]Figure 6A, [125]Table S10), including NADH dehydrogenase (complex
I), succinate dehydrogenase, cytochrome bc1 complex, cytochrome c
oxidase, and ATP synthase (complex V). The nuo genes encode the proton
pump, which catalyzes the first step of electron transport [[126]41].
Six of the nine nuo genes upregulated 1.04 to 2.25 log[2]FC after
repair, except nuoA, nuoH, and nuoI. F[0]F[1]-ATP synthase (complex V)
is responsible for final ATP synthesis by oxidative or
photophosphorylation in membranes [[127]42]. As shown in [128]Figure 6,
the genes atpADG (encoding the F[0]F[1] ATP synthase subunits α, β, and
γ, respectively) and atpC (encoding the F[1] complex subunit ε) were
all upregulated during the repair. It could be inferred that the
upregulation of genes involved in the oxidative phosphorylation pathway
led to an increase in energy synthesis, thereby meeting the needs of
the repair process.
Figure 6.
[129]Figure 6
[130]Open in a new tab
Transcriptional changes of genes involved in the significantly enriched
pathway. (A) The oxidative phosphorylation pathway. (B) The ribosome
and aminoacyl-tRNA biosynthesis pathways.
3.6. Response of DEG Related to Genetic Information Transmission after HHP
and Repair
DEGs involved in the genetic information transmission in response to
HHP and repair treatment were identified, enriched in ’aminoacyl-tRNA
biosynthesis‘ and ’ribosome’. The DEGs involved in these pathways
showed an identical trend of change (i.e., they were promoted after
repair treatment; [131]Figure 6B, [132]Table S11). Concerning the
expressions of genes involved in ‘aminoacyl-tRNA biosynthesis’, 19 of
the 22 DEGs are amino acid ligases, which activate the amino acid and
transfer this moiety to tRNA [[133]43]. The ribosome is where the codon
translates into amino acid and is related to the polypeptide
elongation. The products of DEGs enriched in ‘ribosome’ are in EF-Tu
(rpmC, rplBCDPVW, and rpsCJS), EF-G (rpsGL), IF1 (initiation factor 1)
and IF 3 (rpsDKM and rpmI), SecY (rplFORX, rpmD, and rpsEHNQ), and FtsY
(rpsAP and rplYS), which covers the entire process from the binding of
tRNA and mRNA to the transport of proteins; all these genes were
promoted. In particular, the rpmE2-1 gene, whose product is involved in
the essential release factor RF1, increased 16.20 log[2]FC after
repair. In prokaryotes, the native function of RF1 is to recognize
codon UAA/UAG and terminate translation [[134]44]. The relationship
between HHP and RF1 is unclear; however, it might be a critical point
for responding to HHP injuries and assisting in repair. The
upregulation of these DEGs suggests enhancing the genetic information
transmission process.
4. Discussion
The SI E. coli O157:H7 model (treated with 400 MPa for 5 min) was
repaired when cultured for 2 h in TSB. Then, the repair mechanism was
studied using physiological and transcriptome analysis.
The intracellular protein content increased slightly after 4 h of
repair, similar to Pan et al. [[135]45], who found that the protein
content of sub-lethal L. monocytogenes induced by Ar/O[2] plasma only
slightly increased after the repair process. The ribosome is the site
of protein biosynthesis. Ribosome reconstruction is crucial for the
recovery of growth in HHP-injured B. subtilis [[136]46]. Similarly, the
DEGs involved in ribosome were significantly upregulated in our study
([137]Figure 6B), which could be the reason for the promotion of
protein content. However, the protein content did not increase
substantially. Leucine plays a vital role in regulating the signal
transduction of translation initiation [[138]47]. As shown in
[139]Table S12, the ‘valine, leucine, and isoleucine biosynthesis’
pathway was enriched in profile 1. The essential leuABC operon involved
in this pathway (which encodes enzymes catalyzing the conversation of
α-ketoisovalerate to leucine) showed no significant change
[[140]48,[141]49]. This phenomenon could be a reason for the
insignificant increase in protein content. These findings suggest that
the promotion of protein content could be due to the upregulation of
the ribosome pathway; however, the process of protein synthesis is
affected by complex factors leading to inefficient protein synthesis.
Conversely, the content of ATP increased significantly during repair
([142]Figure 2B). The mechanism of the ATP content increase is unclear;
however, a significantly positive correlation between ATP content and
viability was detected [[143]50], suggesting better cell viability
after repair treatment. Gao et al. [[144]51] found that the
mitochondria complex I subunit was upregulated in the radioresistant
glioma U87MG cells, and the copy numbers of mitochondria increased.
Gurgan et al. [[145]52] found that the downregulation of nuo genes and
atpCD impair energy production of Rhodobacter capsulatus by heat
stress. Similar trends of these DEGs were detected in our transcriptome
analysis ([146]Figure 6A and [147]Table S10). The ATP content, that
substantially increased during the repair process, is due to the
promotion of the ‘oxidative phosphorylation’ pathway. Repairing the SI
cells, especially the cytoplasmic membrane, is energy-dependent and
requires RNA and protein synthesis [[148]53,[149]54]. It could be
speculated that the cell repair process is accompanied by the protein
and ATP synthesis, which could be responses to physiological activity
demands.
In terms of cell morphology, it appears that cells were repaired;
however, the roughness was only partially recovered ([150]Figure 2C).
Nikparvar et al. [[151]55] observed that membrane holes in L.
monocytogenes were not repaired until 48 h, when they were treated with
400 MPa for 8 min using a quantitative model. Thus, it could be
concluded that the cell membrane was not completely repaired after 8 h
of repair. Combined with the transcriptomic results ([152]Figure 5A,B),
we speculate that the repair of the outer membrane was partially
inhibited, and that of the inner membrane was promoted. Arroyo et al.
[[153]56] found that the membrane structure of pulsed electric field
induced SI in Enterobacter sakazakii (also a Gram-negative organism)
could be repaired with different kinetics. The repair of the outer
membrane was much slower than the plasma membrane at the initial
stages, shown indirectly by our results. In other words, even though
the number of colonies was restored, the physiological function of
cells remained in the repair process after culture in TSB for 2 h. In
terms of the membrane part, the repair of the inner membrane could
occur before that of the outer membrane, which led to failure in
restoring the roughness.
5. Conclusions
The repair mechanism of HHP-induced SI E. coli O157:H7 includes the
following: repair of the inner membrane of cells by promoting fatty
acid biosynthesis and then restoring cell morphology; generation of
energy by promoting oxidative phosphorylation process; and accelerating
protein synthesis by promoting ribosome function. Our findings suggest
that the inner membrane is an essential repair site, and the repair
process requires energy and protein. These findings provide insight
into the repair process of HHP-induced SI E. coli O157:H7, which could
help avoid potential food safety hazards caused by SI cells
Supplementary Materials
The following supporting information can be downloaded at:
[154]https://www.mdpi.com/article/10.3390/foods11152377/s1, Table S1:
Information of primers for qRT-PCR; Table S2: The sub-lethally injured
(SI) cell contents (%) and SI rate (%) of different HHP conditions.
Table S3: Statistics of transcriptome analysis reads and assembly
quality. Table S4: Statistics of transcriptome analysis reads aligned
to reference genome. Table S5: The commonly up-regulated different
expressed genes (DEGs). Table S6: The commonly down-regulated (DEGs).
Table S7: GO enrichment analysis of profile 1 and profile 2. Table S8:
KO enrichment analysis of profile 1 and profile 2. Table S9: DEGs
involved in pathways related to membrane. Table S10: DEGs involved in
pathways related to energy biosynthesis. Table S11: DEGs involved in
pathways related to genetic information transmission. Table S12: DEGs
involved in pathways related to other metabolisms. Figure S1: The top
20 GO terms enriched in profile 1 and profile 2 (two significant
clusters). Each fan-shaped region represented a GO term, the red square
represented significance, while the purple square represented the
amount of DEGs. ([[155]57] is cited in the Supplementary Materials).
[156]Click here for additional data file.^ (538.1KB, zip)
Author Contributions
Conceptualization, A.-D.S.; methodology, J.-Y.H. and Y.-Q.L.; software,
J.-Y.H. and Y.-Q.L.; validation, J.-Y.H., Y.-Q.L. and J.-Y.S.; formal
analysis, X.H.; investigation, W.-B.Z.; resources, A.-D.S. and Z.-L.G.;
data curation, Y.-Q.L.; writing—original draft preparation, J.-Y.H.;
writing—review and editing, J.-Y.S.; visualization, J.-Y.H. and
Y.-Q.L.; supervision, A.-D.S.; project administration, A.-D.S.; funding
acquisition, A.-D.S. All authors have read and agreed to the published
version of the manuscript.
Institutional Review Board Statement
Not applicable.
Informed Consent Statement
Not applicable.
Data Availability Statement
Data is contained within the article and [157]supplementary materials.
Conflicts of Interest
The authors declare no conflict of interest.
Funding Statement
This research was funded by the National Natural Science Foundation of
China, grant number 31871817 and 32172222 (Ai-Dong Sun), and the
National Key Research and Development Program of China, grant number
2016YFD0400302 (Ai-Dong Sun).
Footnotes
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References