Screening of genes involved in interactions with intestinal epithelial cells in Cronobacter sakazakii
Du et al. AMB Expr
Screening of genes involved in interactions with intestinal epithelial cells in Cronobacter sakazakii
Xin‑jun Du 0
Xia Zhang 0 1
Ping Li 0
Rui Xue 0
Shuo Wang 0
0 Key Laboratory of Food Nutrition and Safety, Ministry of Education, Tianjin University of Science and Technology , Tianjin 300457 , China
1 Tianjin Entry‐Exit Inspection and Quarantine Bureau , Tianjin 300461 , China
Cronobacter sakazakii possesses a significant ability to adhere to and invade epithelial cells in its host. However, the molecular mechanisms underlying this process are poorly understood. In the current study, the adhesive and invasive capabilities of 56 C. sakazakii strains against human epithelial cells were evaluated, and one of them was selected for construction of a mutant library using the Tn5 transposon. In a systematic analysis of the adhesive and invasive capabilities of 1084 mutants, 10 mutants that showed more than a 50 % reduction in adhesion or invasion were obtained. Tail‑ PCR was used to sequence the flanking regions of the inserted transposon and 8 different genes (in 10 different mutants) were identified that encoded an exonuclease subunit, a sugar transporter, a transcriptional regulator, two flagellar biosynthesis proteins, and three hypothetical proteins. Raman spectroscopy was used to analyze variations in the biochemical components of the mutants, and the results showed that there were fewer amide III proteins, protein ‑ CH deformations, nucleic acids and tyrosines and more phenylalanine, carotenes, and fatty acids in the mutants than in the wild type strain. Real‑ time PCR was used to further confirm the involvement of the genes in the adhesive and invasive abilities of C. sakazakii, and the results indicated that the expression levels of the 8 identified genes were upregulated 1.2‑ to 11.2‑ fold. The results of this study provide us with insight into the mechanism by which C. sakazakii infects host cells at molecular level.
Cronobacter sakazakii; Epithelial cell; Adhesion; Invasion; Genes
Cronobacter is a genus of Gram-negative, rod-shaped,
non-spore-forming bacteria that belong to the family
Enterobacteriaceae. It is widely accepted that this genus
contains seven species, including Cronobacter sakazakii,
Cronobacter malonaticus, Cronobacter turicensis,
Cronobacter muytjensii, Cronobacter dublinensis, Cronobacter
condiment, and Cronobacter universalis
(Iversen et al.
2008; Joseph et al. 2012)
. Three new species
(Enterobacter pulveris, Enterobacter helveticus and Enterobacter
turicensis) that were suggested by Brady et al. (2013) to
be new species based on multi-locus sequence typing
(MLST) analysis were subsequently found to belong to
two new genera by
Stephan et al. (2014
) based on new
genome-scale analyses. Among the 7 Cronobacter
species, C. sakazakii showed a much higher isolation
frequency than the other species, accounting for 72.1 % of
all Cronobacter isolates in the Cronobacter PubMLST
(Almajed and Forsythe 2016)
Members of the genus Cronobacter exhibit greater
stress tolerance than other common pathogens, such as
Escherichia coli and Salmonella, especially to osmotic
and desiccation conditions
(Breeuwer et al. 2003;
Burgess et al. 2016)
. It was reported that C. sakazakii can
survive on dehydrated powdered infant formula (PIF)
for over than 2.5 years, which is the longest survival
time out of the Cronobacter species and other common
pathogens (Barron and Forsythe 2007). Therefore, C.
sakazakii is extensively present in the environment and a
frequent contaminator of food. The ability of C. sakazakii
to survive in PIF is a substantial threat to neonates and
infants because they are often highly dependent on PIF.
Surviving C. sakazakii is capable of rapidly multiplying
to dangerous levels during reconstitution before
feeding, and it can cause serious clinical symptoms, include
necrotizing enterocolitis (NEC), bacteremia and
meningitis, resulting in fatality rates ranging from 40 to 80 %
(Bowen and Braden 2006; Friedemann 2009)
. At least 27
deaths are known to have occurred out of approximately
120 cases that have been documented around the world
up to July 2008
Adhering to epithelial cells is a crucial step that C.
sakazakii must complete to cause pathogenic disease.
To increase our understanding of the pathogenesis of
this bacterium, several studies have attempted to explore
the adhesive and invasive capabilities of the pathogen in
different human-derived cell lines in vitro. Mange et al.
(2006) first assessed the adhesive properties of
different C. sakazakii strains to two epithelial cell lines and
a brain microvascular endothelial cell line. They found
that the adhesive capabilities varied among the strains
and that adhesion was non-fimbrial-based. Using a rat
Townsend et al. (2007
) were the first to compare
the invasive capabilities of different C. sakazakii strains
in capillary endothelial cells. In 2008, they studied
adhesion and invasion in epithelial cells and endothelial cells
in isolates obtained during a French outbreak in 1994
(Townsend et al. 2008)
. A study by Giri et al. revealed
that certain Cronobacter isolates can invade and
translocate across cultured human intestinal epithelial cells and
brain microvascular endothelial cells
(Giri et al. 2012)
Almajed and Forsythe (2016)
demonstrated that C. sakazakii clinical isolates possess a
strong capacity to invade and translocate through human
colonic epithelial cells (Caco-2) and brain
microvascular endothelial cells. All of these studies macroscopically
demonstrate the adhesive and invasive capabilities of C.
sakazakii strains. Nevertheless, we currently know very
little about how these processes occur at a molecular
level. Outer membrane protein A (OmpA) is a
well-characterized factor that plays an important role in adhesion
to and invasion of endothelial cells
(Nair et al. 2009)
Another factor, outer membrane protein X (OmpX),
was also shown by
Kim et al. (2010
) to be crucial to
invasion into human enterocyte-like epithelial and
intestinal epithelial cells. Although some progress has been
made in furthering our understanding of the adhesive
and invasive properties of C. sakazakii, the mechanisms
that underlie these pathogenic processes largely remain
In the current study, to further our understanding of
the molecular mechanisms involved in adhesion to and
invasion into host cells in C. sakazakii, we constructed a
Tn5 transposon mutant library and screened it to
identify mutants that showed defects in adhesion or invasion.
We used these mutants to identify functional genes that
are potentially responsible for adhesion or invasion. We
then performed Raman spectroscopy to analyze variation
in the biochemical components of the mutants. Our data
shed light on a variety of molecular characteristics of C.
sakazakii at the biochemical component level. Finally, we
performed real-time PCR to further explore the relative
expression levels of genes shown to contribute to
adhesion or invasion.
Materials and methods
Bacteria and culture conditions
A total of 56 C. sakazakii strains were used in the
current study, including 4 strains that were purchased from
American Type Culture Collection (ATCC) and 52
strains that were isolated from food samples that were
produced in 13 countries (Table 1). All of the food-origin
strains were collected by different Entry–Exit Inspection
and Quarantine Bureaus of China from 2005 to 2010. The
isolates were classified as Cronobacter species using the
API 20E, VITEK or Biolog system and further identified
as C. sakazakii using 16S rDNA sequencing and 10
All of the strains were cultured in Luria-Bertani (LB)
broth at 37 °C overnight while shaking (180 rpm) and
then transferred into fresh LB broth at ratio of 1:100 for
further culturing until the OD600 value reached 0.6–0.8.
Epithelial cell cultures
HCT-8 cells (ATCC CCL-244; American Type Culture
Collection, Manassas, Virginia) were grown in 24-well
tissue culture plates containing RPMI-1640 medium
(Invitrogen, Carlsbad, CA, USA) supplemented with
10 % heat-inactivated fetal bovine serum (Gibco,
Carlsbad, CA, USA), 50 IU/mL penicillin, and 50 μg/mL
streptomycin. The plates were incubated at 37 °C in 5 % CO2
until approximately 90 % of each well was covered by the
Adherence or invasion assays
The adherence and invasion abilities of C. sakazakii
against HCT-8 cells were analyzed according to methods
described in previous reports
(Mange et al. 2006;
Quintero et al. 2011)
, with some modifications. HCT-8
monolayer cells were incubated in 24-well plates and then
washed with prewarmed phosphate-buffered saline (PBS)
three times. Different C. sakazakii strains were collected
in log phase using centrifugation at 3000×g for 5 min and
then washed once with PBS buffer. The bacterial cells
were resuspended in RPMI-1640 medium to achieve
an OD600 value of 0.1. A 0.5 mL volume of bacterial
suspension was added to each well of the plates, and the
bacteria were allowed to interact with the epithelial cells
for 3 h at 37 °C in 5 % CO2. After the incubation period,
the plates were gently washed with prewarmed PBS 3
times to remove non-adhered bacterial cells. Adhered or
invaded C. sakazakii cells were released from the plates
by adding 0.5 mL/well 0.1 % Tritonx-100 and counted
using the plating method described by the manufacturer
for chromogenic cronobacter isolation agar (Oxoid,
Basingstoke, UK). Empty wells that did not contain HCT-8
cells were used as the controls to calculate the number
of bacterial cells that specifically adhered to the
epithelial cells. Adherence assays were performed in triplicate
(n = 3) using each C. sakazakii strain, and each assay was
tested in duplicate (n = 2).
Mutant generation and adhesion and invasion analyses
The isolate SAKA10119 showed the best ability to adhere
to HCT-8 cells, and it was therefore selected to
generate transposon mutants using an EZ-Tn5™ Transposome
Kit (Epicenter, Madison, USA). Briefly, a single colony of
the SAKA10119 strain was inoculated into LB broth and
cultured overnight at 37 °C while shaking. The overnight
cultures were diluted 1:100 into 30 mL fresh LB broth
and incubated until OD600 reached 0.4-0.6. Lysozyme
was added to the bacterial cells at a final concentration of
10 μg/mL and the cells were then incubated at 37 °C for
30 min to improve transformation efficiency. The treated
cells were cooled on ice for 30 min and then collected via
centrifugation at 1500×g for 10 min at 4 °C. The cell
pellets were washed with ice-cold sterile water and glycerol
(10 %, V/V), and then resuspended in 1 mL of glycerol
(10 %, V/V). To construct a mutant library, the Tn5
transposon was electrically transformed into SAKA10119 cells
according to the manufacturer’s instruction. The mutants
colonies that appeared on the LB plates containing 50 μg/
mL kanamycin were subsequently cultured in LB broth
and used in HCT-8 cell interaction assays according to
the method described above. Each mutant was analyzed
in duplicate in each assay, and each experiment was
performed in triplicate.
Identification of transposon insertion sites
In the mutants that showed a significant decrease in the
ability to adhere to HCT-8 cells, the Tail PCR method
was used to identify the transposon insertion sites
(Du et al. 2012)
. Briefly, DNA was extracted from each
mutant using QIAamp DNAmini Kit (Qiagen, Hilden,
Germany) according to the manufacturer’s instructions,
and the purified DNA was then used as a template for a
two rounds of nested PCR amplification. The obtained
fragments were cloned into the pMD18-T vector (Takara,
Dalian, China) and prepared for DNA sequencing using
vector-specific primers. The DNA sequences that flanked
the Tn5 transposon were identified using Blast analysis of
the data available in the NCBI (The National Center for
Biotechnology Information) database.
Raman spectroscopy analysis
Raman spectroscopy analyses were performed
according to methods described in a previous report
et al. 2015)
. Briefly, monoclonal colonies of the parent
strain and the 8 mutant strains were inoculated into 5 mL
of LB broth and cultured at 37 °C overnight while
shaking (180 rpm). The cultures were transferred into 5 mL
of fresh LB broth and culture under the same conditions
until the OD600 value reached 0.6–0.8. One milliliter of
each logarithmic phase bacterial culture was collected
via centrifugation at 6000×g for 5 min. The cell pellets
were rinsed 3 times using PBS (pH = 7.4), and the cells
were finally suspended in 1 mL of sterile deionized water.
Five microliters of each bacterial suspension were
transferred to a gold-coated glass slide (Thermo Scientific Inc.,
Waltham, MA, USA), and the slide was allowed to dry
at room temperature. The samples were analyzed using
a Renishaw inVia Raman system (Renishaw,
Gloucestershire, UK) equipped with a 785-nm near-infrared diode
laser and a Leica microscope (Leica Biosystems, Wetzlar,
Germany). Raman scattered light was collected using a
CCD array detector (576- by 384-pixel) during exposure
to an incident laser (300 mW). Raman spectra were
collected using a 50× objective over a 10-s exposure time
with a wave number shift range of 400–1800 cm−1. Ten
spectra were collected for each strain, and experiments
were performed in triplicate. Matlab (The MathWorks,
Inc., Natick, MA, USA) was used to construct
chemometric models based on the wavenumbers that were obtained
between 400 and 1800 cm−1. Unsupervised principal
component analysis (PCA) was performed to investigate
chemical variations in the bacterial samples by
constructing a cluster-based segregation model
(Huang et al. 2004)
Real‑time PCR analysis
The 8 putative adherence- or invasion-related genes
that were screened in the current study were submitted
to real-time PCR analysis to determine their mRNA
expression levels both before and after the bacteria were
allowed to adhere to epithelial cells. HCT-8 cells were
cultured in 75-cm2 tissue flasks until about 90 % of the
bottom of the flask was covered by monolayer cells. The
SAKA10119 strain was cultured to logarithmic phase and
washed 3 times with PBS buffer. The bacterial cells were
then resuspended in RPMI-1640 medium, and 15 mL of
the suspension was added to the epithelial cells. After
the cells were incubated at 37 °C for 3 h in 5 % CO2, the
adhered bacteria and the epithelial cells were lysed using
0.1 % (w/v) sodium dodecyl sulfate, 0.1 % (V/V) acidic
phenol, and 19 % (V/V) ethanol in water for 30 min on
(Lucchini et al. 2005)
. Total RNA was extracted from
the mixture using a Bacterial RNA Kit (Omega Bio-Tek,
Norcross, GA, USA). The total RNA that was extracted
from the SAKA10119 strain that did not interact with
epithelial cells was used as the control. cDNA was
reverse-transcribed using an ImProm-II™ Reverse
Transcription System (Promega, Madison, WI, USA). Eight
pairs of primers specific for the 8 genes and a pair of
primers specific for the 16S rDNA gene, which was used
as an internal reference, were synthesized (Table 2) and
used to perform qRT-PCR in a RealPlex 4 Master Cycler
(Eppendorf, Hamburg, Germany).
The strain SAKA10119 has been deposited in China
Center for Industrial Culture Collection (CICC) under
accession number CICC24112. The 16S rRNA gene
sequence of the strain SAKA10119 has been deposited in
GenBank database under accession number KX237756.
Adherence or invasion analyses of different strains
A total 56 strains, including food isolates and standard
strains, were analyzed to determine their ability to adhere
to and invade intestine epithelial cells. The adhesive or
invasive abilities of each strain were described using an
interaction index that indicates the difference between
the number of bacteria that adhered to or invaded the
epithelial cells and the number of bacteria that adhered
to the empty wells of the tissue culture plate. As is shown
in Fig. 1, 13 strains exhibited stronger adherence
capacities than the rest of the strains. The interaction indexes
of the 13 strains were all above 1.0 × 107 CFU. Among
these strains, the strain SAKA10119 showed the
strongest capacity to adhere to epithelial cells, with an
adhesion index that reached 2.3 × 107 CFU. A total of 24
strains showed adhesion indexes between 1 × 106 and
1 × 107 CFU. The adhesion indexes of the other 19 strains
were lower than 1 × 106 CFU, demonstrating their
comparatively weaker ability to adhere or invade the cells.
Among these strains, 4 showed a similar or even weaker
ability to adhere to epithelial cells than to the empty plate.
a hp-1, hp-2 and hp-3 indicate the three hypothetical proteins coding genes
Construction and analysis of the mutants
Based on the results of our analyses of adhesion and
invasion, the strain SAKA10119 was selected to construct a
mutant library using the EZ-Tn5™ < KAN-2 >
Transposon. A total of 1084 mutants were separated and cultured
to further analyze their adhesive and invasive
capabilities. PCR amplifications were performed using primers
specific for the kanamycin resistance gene on the Tn5
transposon to evaluate the quality of the mutant library,
and the results showed that a specific band was amplified
from all 24 of the randomly selected colonies (Additional
file 1: Figure S1). These data indicate that the mutation
library was successfully constructed. The capacity of the
1084 mutant cell lines to either adhere to or invade
epithelial cells was analyzed, and the results were compared
to the results for the parent strain. Most of the mutants
showed an adherent ability that was similar to that of
the wild type strain. However, 10 of the mutants
(designated AM 1–10, meaning Adhesion or invasion
defective Mutants) presented a higher than 50 % reduction in
adherence in comparison to the adherence observed in
the parent strain (Fig. 2). These 10 mutants were
therefore selected to identify the interrupted genes.
Identification of Tn5 transposon locations
The flanking regions of the Tn5 transposon were targeted
using Tail-PCR. After two rounds of amplification,
fragments ranging from approximately 500–1500 bp were
obtained from the 10 adhesion- or invasion-defective
mutants. These 10 fragments were sequenced, and eight
different genes showing a high similarity to sequences in
the C. sakazakii genome were obtained (the same 190 bp
sequence was obtained from 3 mutants). These genes
encoded an exonuclease subunit, a phosphotransferase
system (PTS) sugar transporter, a transcriptional
regulator, two flagellar biosynthesis proteins, and three
hypothetical proteins (Table 3). Seven out of the eight genes
were located on the chromosome, and 1 was located on
a plasmid (AM 4, which had a mutation in a PTS sugar
transporter encoding gene). Seven of the genes were
disrupted in the coding region, and one was disrupted in the
upstream non-coding region (AM 10, which was mutated
in the upstream region of a hypothetical protein
The mutants AM 1-3 had the same insertion in the
coding region of the gene recB, which encodes a subunit of
exonuclease V. These three mutants showed 89.8–92.8 %
less adhesion or invasion than the parent strain (Fig. 2).
In AM 4 and AM 5, the Tn5 transposon had inserted into
the coding regions of a PTS sugar transporter gene and
a transcriptional regulator gene, respectively. The PTS
sugar transporter gene was the only gene that was located
on a plasmid. Disruptions in two of the genes resulted
in an 89.5 and 82.9 % reduction in adhesion or invasion
(Fig. 2). The mutants AM 6 and AM 7 had disruptions
in different flagellar biosynthesis genes, both of which
contained the Tn5 transposon in their coding regions.
These two mutants showed 89.3 and 97.9 % less adhesion
or invasion than the wild type strain (Fig. 2). The mutant
AM 7 contained a disrupted fliR gene and exhibited the
weakest adherence to epithelial cells among all of the
mutants. The remaining three mutants (AM 8–10) were
disrupted in genes encoding hypothetical proteins. The
mutant AM 10 was different from the others because
the insertion took place in the upstream coding region
of the gene. These three mutants (AM 8, 9 and 10)
displayed 18.8, 30.4 and 38.3 %, respectively, of the adhesion
or invasion capacity that was observed in the wild type
strain (Fig. 2).
a hp-1, hp-2 and hp-3 indicate the three hypothetical proteins coding genes
Raman spectroscopy analysis
Raman spectroscopy was used to analyze variations
in the biochemical components of the eight C.
sakazakii mutants (Only AM 2 was selected from the three
mutants with an identical insertion site) in comparison
to the wild type strain. A PCA model was established
to differentiate the wild type and mutant strains, and
the results indicated that there was a clear segregation
between the wild type strain and the mutants (Fig. 3a).
The AM 9 strain, the AM 10 strain, and the rest of the
mutants formed three distinct groups, each of which
demonstrated a different set of variations in their
A comparison of the Raman spectra showed that five
peaks were lower (1250, 1322, 1338, 1578 and 1607 cm−1)
and five peaks were higher (1003, 1155, 1450, 1520
and 1660 cm−1) in the mutants than in the wild type
strain (Fig. 3b). The assignments of the peaks were
determined according to methods described in
(De Gelder et al. 2007; Xie et al. 2005)
results indicated that the defects in the adhesive
capacity of the mutants were related to decreases in protein
amide III (1250 cm−1), protein -CH deformations (1322
and 1338 cm−1), nucleic acids (1578 cm−1) and tyrosine
(1607 cm−1) and increases in phenylalanine (1003 cm−1),
carotenes (1155 and 1520 cm−1), and fatty acids (1450
and 1660 cm−1). The mutants AM 9 and AM 10
exhibited the most significant differences from the wild type
strain, demonstrating that the two genes (ESA_04202
and ESA_00132) that were interrupted in these strains
are strongly associated with the biosynthesis of the above
components. The mutants AM 4, AM 5, AM 7 and AM
8 showed comparatively smaller changes in these
biochemical components when compared to the results for
mutants AM 9 and AM 10. The remaining two mutants
(AM 2 and AM 6) showed the weakest variations in
comparison to the wild type strain. The decrease in the
biosynthesis of tyrosine that was observed in these two
mutants was similar to that observed in other mutants.
Real‑time PCR analysis
Real-time PCR was performed to analyze variations in
the mRNA expression levels of the genes that were
identified in the current study. We examined their expression
before and after the bacteria were allowed to adhere to
epithelial cells. The results indicated that the
transcription of all eight of the genes was upregulated 1.2 ±
0.5to 11.2 ± 0.7-fold (Fig. 4), demonstrating the relevance
of these genes to the adhesiveness or invasiveness of C.
sakazakii. Among these 8 genes, the transcriptional
regulator gene fnR was upregulated the most, and the flagellar
biosynthesis protein gene fliR was upregulated the least.
The adhesion and invasion of C. sakazakii in epithelial
cells is the first critical step in establishing a successful
(Amalaradjou et al. 2014)
many studies have been performed to analyze the
adhesiveness and invasiveness of C. sakazakii in epithelial cells
in vitro, the molecular mechanisms underlying these
processes are poorly understood. Our recent report about
variations in the transcriptome of C. sakazakii after
adhesion to or invasion into human epithelial cells shed light
on these molecular mechanism
(Jing et al. 2016)
However, too many candidate genes were identified in that
study, and much more experimental evidence was needed
to better verify the roles of the screened genes. The Tn5
transposon is an efficient tool that can be used to
identify the genes that are responsible for specific phenotypic
variations, and it has been widely used to screen genetic
systems that are involved in biofilm formation
et al. 2010; Du et al. 2012)
, responses to osmotic stress
(Alvarez-Ordóñez et al. 2014a)
and acid stress
(AlvarezOrdóñez et al. 2014b), tolerance to serum
(Schwizer et al.
, and yellow pigmentation
(Johler et al. 2010)
sakazakii. In the current study, the Tn5 transposon was
used to construct a mutant library to screen for genes
that might potentially be responsible for the adhesion and
invasion capabilities of C. sakazakii in epithelial cells.
The ability to adhere and invade are widely thought to
be critical pathogenic properties of C. sakazakii, but
little data is currently available on this subject, except for
studies on a limited number of factors, such as OmpA
and OmpX. In the current study, a total of 8 genes were
experimentally shown to be responsible for adhesion or
invasion in C. sakazakii. These included an exonuclease
V subunit beta gene (recB), a PTS sugar transporter gene
(bglF), a transcriptional regulator gene (fnr), two flagellar
biosynthesis genes (flhA and fliR) and 3 hypothetical
protein encoding genes.
recB encodes a beta subunit of the bacterial RecBCD
enzyme, which possesses DNA helicase and
nuclease activities and is involved in a major pathway during
homologous recombination that particularly contributes
to repairing double stranded (ds) DNA breaks induced
(Wu et al. 2012)
. In the trimeric RecBCD
enzyme, the RecB subunit functions with 3′ to 5′ helicase
and translocase activities
(Taylor et al. 2014)
. This gene
therefore appears to be unrelated to bacterial adhesion or
invasion capacities. However, a recB-like gene that was
identified in Helicobacter pylori was shown to play a role
in DNA repair and host colonization
(Wang et al. 2009)
Mutations in the gene resulted in the fragmentation of
genomic DNA and cell death within a short period of
time and reduced colonization in host stomachs. Their
work seemed to show that the gene has putative
functions in adhesion or invasion when the bacterium invades
host cells. However, it seems more reasonable to attribute
the reduction in colonization to one of the phenotypes
that was caused by an impaired ability to repair DNA. It
is reasonable that the putative correlation between the
recB gene and the adhesion or invasion capabilities of C.
sakazakii might be explained in the same way.
PTS is carbohydrate uptake system that is responsible
for selectively transporting sugar molecules across the
inner bacterial membrane while simultaneously
catalyzing sugar phosphorylation
(McCoy et al. 2015)
studies have shown that this gene family is involved in
virulence between bacterial pathogens and their hosts.
Rouquet et al. (2009
) identified an operon that encoded
three subunits of PTS transporters in E. coli and showed
that it was involved in adherence to host cells and the
internalization of the bacterium in different human and
chicken cells. E. coli strains with mutations in sugar
transporter genes in the PTS showed attenuated colonization
in the guts of mice and attenuated virulence in mouse
models of sepsis and pyelonephritis
et al. 2012)
. A similar function of the gene was identified
in the current study.
Fumarate nitrate reduction (FNR) regulator is
important to the virulence of pathogens when they encounter
variations in the availability of O2
(Green et al. 2014)
FNR can sense a reduction in or the absence of O2 and
activate functional genes that are required for anaerobic
respiration in addition to virulence genes during host
colonization and infection. In hypoxic niches within
a host, FNR can activate the expression of cytolysin in
many pathogens, such as E. coli and Salmonella
(Lithgow et al. 2007; Fuentes et al. 2008)
. In Shigella, FNR
mediates the type three secretion system (T3SS), which
is essential for cell invasion and virulence, when the
pathogen encounters variation in oxygen concentrations
in the gastrointestinal tract (Marteyn et al. 2010). The fnr
mutant of E. coli results in severe defects in adherence
to and the invasion of bladder and kidney epithelial cells
and was highly attenuated in a mouse model of urinary
(Barbieri et al. 2014)
. Based on these
studies, it is more likely that interrupting the fnr gene
in C. sakazakii substantially affected the survival of the
bacterium rather than affecting the cells solely by
weakening their adhesive or invasive capabilities. However,
further experimental evidence is required to answer this
It is widely recognized that flagellar
biosynthesisrelated proteins play major roles in the colonization of
the intestinal tract or invasion into host intestinal cells by
many bacterial pathogens
Hendrixson 2014; Stevenson et al. 2015)
. FlhA is a component
of the export apparatus required for flagellum assembly
and has been reported to play roles in adhesion to and
invasion into epithelial cells in many bacteria, such as
Bacillus cereus, Bacillus thuringiensis and Pseudomonas
(Fleiszig et al. 2001; Ramarao and Lereclus
. FliR is a component of the type III flagellar export
apparatus, which is mainly responsible for exporting
most of the structural components of the flagellum. It
has been reported to be associated with adhesion to and
pathogenicity against host cells in Leptospira interrogans
(Ruan et al. 2008)
. However, the functions of these two
genes have not been studied in C. sakazakii. This is first
study to explore their involvement in the adhesive or
invasive properties of C. sakazakii.
In addition to the genes mentioned above, three genes
encoding 3 small hypothetical proteins (67, 111, 113
amino acids long, respectively) were also identified in
the current study. These genes have putative functions in
adhesion to or the invasion of epithelial cells in C.
sakazakii. The identification of the unknown functions of
these genes would be helpful for determining the basis of
the pathogenicity of this opportunistic pathogen.
Raman spectroscopy is a powerful technique that is
used to characterize and image biochemical
components in living cells
(Schie and Huser 2013)
. In the
current study, Raman spectroscopy analysis of mutants
with defective adhesiveness or invasiveness against
epithelial cells showed that they contained less protein
amide III, protein -CH deformations, nucleic acids and
tyrosines. These results indicate that the mutants
exhibited depressed protein and nucleic acid biosynthesis,
reflecting the inhibited life activities of the mutants.
Conversely, the levels of phenylalanine, carotenes and
fatty acids were found to be negatively associated with
adhesion and invasion in C. sakazakii. These findings do
not clarify the mechanisms that underlie the
adhesiveness and invasiveness of C. sakazakii against host cells,
but these results do shed light on the mechanisms that
are involved at a biochemical component level in the
In summary, in the current study, eight genes were
identified and found to be associated with the adhesive
or invasive characteristics of C. sakazakii in human
epithelial cells. Raman spectroscopy was used to investigate
variations in the biochemical components in the mutants
that were caused by the interruption of the eight genes.
Real-time PCR was performed to further confirm the
relatedness of these genes to interactions between C.
sakazakii and host cells. The data presented in this study
will be useful in determining the mechanisms underlying
the pathogenesis of C. sakazakii.
Additional file 1: Figure S1. Quality evaluation of the mutant library
using PCR specific for the kanamycin resistance gene in the Tn5 transpo‑
son. M, 100 bp DNA ladder; 1‑24, kanamycin resistant gene fragments that
were amplified from randomly selected clones in the mutant library.
MLST: multi‑locus sequence typing; PIF: powdered infant formula; NEC:
necrotizing enterocolitis; OmpA: outer membrane protein A; OmpX: outer
membrane protein X; ATCC: American Type Culture Collection; LB: Luria–Ber‑
tani; PBS: phosphate‑buffered saline; PCA: principal component analysis;
CICC: China Center for Industrial Culture Collection; AM: adhesion or invasion
defective mutants; PTS: phosphotransferase system; FNR: fumarate nitrate
SW and XJD contributed to the design of the research; XZ and RX prepared
materials and performed the experiments. XJD, XZ, PL and RX analyzed the
data; XJD and SW participated in the coordination and completion of the
manuscript. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
This article does not contain any studies with human participants or animals
performed by any of the authors.
This study was funded by National Key Technology Research and Develop‑
ment Program of China
(Grant number 2014BAD04B03)
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