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Genetic Mechanisms Underlying the Pathogenicity of Cold-Stressed
Salmonella enterica Serovar Typhimurium in Cultured Intestinal
Epithelial Cells
Jigna Shah,
a,b
* Prerak T. Desai,
a,b
* Bart C. Weimer
b
Department of Dietetics, Nutrition and Food Sciences, Utah State University, Logan, Utah, USA
a
; School of Veterinary Medicine, Department of Population Health and
Reproduction, University of California, Davis, California, USA
b
Salmonella encounters various stresses in the environment and in the host during infection. The effects of cold (5°C, 48 h), per-
oxide (5 mM H
2
O
2
, 5 h) and acid stress (pH 4.0, 90 min) were tested on pathogenicity of Salmonella. Prior exposure of Salmo-
nella to cold stress significantly (P < 0.05) increased adhesion and invasion of cultured intestinal epithelial (Caco-2) cells. This
increased Salmonella-host cell association was also correlated with significant induction of several virulence-associated genes,
implying an increased potential of cold-stressed Salmonella to cause an infection. In Caco-2 cells infected with cold-stressed Sal-
monella, genes involved in the electron transfer chain were significantly induced, but no simultaneous significant increase in
expression of antioxidant genes that neutralize the effect of superoxide radicals or reactive oxygen species was observed. In-
creased production of caspase 9 and caspase 3/7 was confirmed during host cell infection with cold-stressed Salmonella. Further,
a prophage gene, STM2699, induced in cold-stressed Salmonella and a spectrin gene, SPTAN1, induced in Salmonella-infected
intestinal epithelial cells were found to have a significant contribution in increased adhesion and invasion of cold-stressed Sal-
monella in epithelial cells.
S
almonella is an important food-borne pathogen throughout
the world. It is transmitted by the fecal-oral route, and most
infections occur due to ingestion of contaminated food. Salmo-
nella encounters and survives various stresses, such as cold, acid,
and oxidative stress, during its journey from the environment to
food to infection in the animal host. The most common stressors
include (i) cold stress, because refrigeration (5°C) is commonly
used for long-term storage of food; (ii) oxidative stress, because
peroxide is commonly used as a food sanitizer during food pro-
cessing and it is also produced by macrophages and neutrophils as
a result of inflammation and oxidative burst during infection; and
(iii) acid stress, because acids are commonly used in food process-
ing and, more importantly, gastric acidity is the first line of defense
against pathogens within the host gut. Salmonella modulates its
gene expression for survival upon exposure to the above stresses,
and this can also simultaneously alter the expression of virulence
factors and the surface structures of bacteria.
One of the major steps in the successful infection is the ability
of Salmonella to adhere to host surfaces. Salmonella expresses two
major groups of bacterial adhesins, namely, pilus (fimbrial) and
nonpilus (afimbrial) adhesins. The role of many fimbrial operons,
such as csg, bcf, fim, lpf, pef, saf, stb, stc, std, stf, sth, sti, and stj,in
adhesion of Salmonella to host cells has been investigated (
1).
Salmonella can either bind directly to host cell surfaces or bind to
components of the extracellular matrix (ECM) (
2), such as fi-
bronectin, laminin, and plasminogen (
3, 4). However, only a
small number of afimbrial adhesin factors, such as misL, ratB,
shdA, sinH (
1), and siiE (5), have been functionally characterized,
and in most cases, their binding partners on host cells are not
known. Additionally, alterations of the host cell surface due to
inflammation can provide alternate adhesin receptors for patho-
gen binding. Thus, investigating interactions of afimbrial adhesin
factors with non-ECM components is equally important. Upon
adhesion, Salmonella induces a vast array of cytoplasmic and nu-
clear responses in epithelial cells, leading to cytoskeletal rear-
rangement, membrane ruffling and macropinocytosis, induction
of transmembrane fluids and electrolyte fluxes, and synthesis of
cytokines and mediators of inflammation (
6). These functions are
shown to be carried out by type 3 secretion system (T3SS) proteins
which are encoded on two Salmonella pathogenicity islands
(SPIs), SPI1 and SPI2. Expression of SPI1 is required for epithelial
cell invasion, and SPI2 is required for intracellular survival and
replication within phagocytic cells (
6, 7).
Our hypothesis in this study was that prior exposure of Salmo-
nella enterica serovar Typhimurium to abiotic stresses will result
in modulation of gene expression and increased host cell adher-
ence and invasion. The specific objective of this study was to in-
vestigate the effect of individual stresses on S. Typhimurium. This
study tested the effect of cold stress (5°C, 48 h), peroxide stress (5
mM H
2
O
2
, 5 h), and acid stress (pH 4.0, 90 min) on the ability of
S. Typhimurium to (i) adhere to and invade intestinal epithelial
(Caco-2) cells, (ii) differentially regulate gene expression, and (iii)
maintain differential gene regulation during infection. We found
that prior exposure to cold stress significantly increased the asso-
ciation of S. Typhimurium with intestinal epithelial cells and in-
duced the expression of many virulence-associated genes. Fur-
Received 16 June 2014 Accepted 24 August 2014
Published ahead of print 5 September 2014
Editor: C. A. Elkins
Address correspondence to Bart C. Weimer bcweimer@ucdavis.edu.
* Present address: Jigna Shah, Veterinary Biomedical Sciences, University of
Minnesota, Saint Paul, Minnesota, USA; Prerak T. Desai, Department of Pathology
and Laboratory, University of California, Irvine, California, USA.
Copyright © 2014, American Society for Microbiology. All Rights Reserved.
doi:10.1128/AEM.01994-14
November 2014 Volume 80 Number 22 Applied and Environmental Microbiology p. 6943– 6953 aem.asm.org 6943
thermore, we identified a novel receptor-ligand pair involved in
the association of cold-stressed S. Typhimurium with intestinal
epithelial cells.
MATERIALS AND METHODS
Cell culture, bacterial strain, and growth conditions. Intestinal epithe-
lial cells (Caco-2; ATCC HTB-37) were obtained from the American Type
Culture Collection (Manassas, VA). Cells were seeded to a density of 10
5
cells/cm
2
using Dulbecco’s modified Eagle medium (high-glucose modi
-
fied medium) (DMEM-HMM; catalog no. SH30285; Thermo Scientific,
Rockford, IL), nonessential amino acids (Thermo Scientific), 10 mM
MOPS (morpholinepropanesulfonic acid) (Sigma, St. Louis, MO), 10
mM TES [N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid]
(Sigma), 15 mM HEPES (Sigma) and 2 mM NaH
2
PO
4
(Sigma). Addition
-
ally, 16.6% fetal bovine serum (FBS; HyClone Laboratories, Logan, UT)
was added for feeding and maintaining cells. Cells were incubated at 37°C
with 5% CO
2
for 14 days postconfluence to allow differentiation. The cells
were serum starved for 24 h prior to use by feeding the cells with cell
culture medium described above but without FBS.
S. Typhimurium LT2 ATCC 700720 was used as a wild-type strain in
this study. Cell culture medium was used to grow the bacteria at 37°C with
shaking at 220 rpm. Bacterial gene knockout procedures for STM2699 and
invA were performed as described previously (
8).
Stress treatments for bacteria. A log-phase culture of S. Typhimu-
rium was centrifuged at 7,200 ⫻ g for 5 min. The pellets were resuspended
at equal density in DMEM-HMM (i) maintained at 5°C for inducing cold
stress, (ii) with 5 mM H
2
O
2
added and maintained at 37°C for peroxide
stress, and (iii) with preadjusted pH 4.0 and maintained at 37°C for
acid stress. These stress treatments were given for 48 h, 5 h, and 90 min,
respectively, in two biological replicates. The control for these treatments
was a stationary-phase (⬃15 h) culture resuspended in DMEM-HMM
and maintained at 37°C.
Bacterium-host cell association assay. Caco-2 cells were infected
with bacteria at a multiplicity of infection (MOI) of 1:1,000, in a 96-well
plate, in three biological replicates. The infected cells were incubated for
60 min, 90 min, and 120 min at 37°C with 5% CO
2
. Upon incubation,
medium was aspirated, cells were washed three times with 200 l standard
Tyrode’s buffer and then lysed using 50 l lysis buffer (AEX Chemunex,
France) as described previously (
9), and the cell lysate was used to quantify
the number of Caco-2 cells and associated bacteria. Quantitative bacterial
analysis was done using quantitative PCR (qPCR) with a CFX 96 real-time
system (Bio-Rad, Hercules, CA). Reactions were performed with iQ SYBR
green Supermix (Bio-Rad) as per the manufacturer’s instruction. Briefly,
a 25-l reaction mixture contained 1 l of cell lysate and 100 nM forward
(F) and reverse (R) PCR primers for the 16S rRNA gene (F, 5=-TGT TGT
GGT TAA TAA CCG CA-3=;R,5=-CAC AAA TCC ATC TCT GGA-3=)
(
10) to quantify S. Typhimurium or the G3PDH gene (F, 5=-ACC ACA
GTC CAT GCC ATC AC-3=;R,5=-TCC ACC ACC CTG TTG CTG TA-3=)
to quantify Caco-2 cells (Integrated DNA Technologies, Coralville, IA).
The thermocycling parameters for both primer pairs consisted of dena-
turation at 95°C for 5 min followed by 40 cycles of denaturation, anneal-
ing, and extension at 95°C for 15 s, 56°C for 30 s, and 72°C for 30 s and a
final extension at 72°C for 1 min. The amplified product was verified using
melting curve analysis from 50°C to 95°C with a transition rate of 0.2°C/s.
At each time point, one-way analysis of variance (ANOVA) with Tukey’s
post hoc test was used to find significant differences across treatment and
control group means. Differences with P values of ⬍0.05 were considered
significant.
Gentamicin protection assay. Caco-2 cells were infected with bacteria
at an MOI of 1:1,000 in a 96-well plate, in three biological replicates. The
infected cells were incubated for 60 min at 37°C with 5% CO
2
. Medium
was aspirated, and cells were washed three times with 200 l standard
Tyrode’s buffer. To enumerate invading bacteria, cells were incubated
with 200 lof100g/ml gentamicin for2hat37°C with 5% CO
2
.To
enumerate total cell-associated bacteria, cells were incubated with
DMEM-HMM (without gentamicin). Cells were washed three times with
200 l Tyrode’s buffer and lysed with 100 l of 0.01% Triton. The total
cell-associated or invading bacteria were enumerated by the pour plate
method using nutrient agar (Difco, Detroit, MI). To calculate the number
of adherent bacteria, the mean number of invading bacteria was sub-
tracted from the mean of the total number of cell-associated bacteria. The
error for adherent bacteria was calculated using the equation (⌬Z)
2
⫽
(⌬A)
2
⫹ (⌬B)
2
, where ⌬Z is the standard error of mean (SEM) for adher
-
ent bacteria, ⌬A is the SEM for total host-associated bacteria, and ⌬B is the
SEM for invading bacteria. An independent two-sample t test was used to
find significant differences across treatment and control group means (see
Fig. 1B). When appropriate, one-way ANOVA with Tukey’s post hoc test
was done to find significant differences across treatment and control
group means (see
Fig. 6 and 7). Differences with P values of ⬍0.05 were
considered significant.
Epithelial cell infection assay for determination of gene expression.
Caco-2 cells were cultured in T-75 flasks and were serum starved 24 h
before infection. Bacteria (S. Typhimurium that had been cold stressed for
48 h and nonstressed S. Typhimurium), at an MOI of 1:1,000, were used
to infect epithelial cells. The controls included cold-stressed and non-
stressed S. Typhimurium in DMEM-HMM only (no epithelial cells) and
noninfected epithelial cells. The experiment was done in two biological
replicates. The infected cells along with the controls were incubated at
37°C with 5% CO
2
for 60 min. For infected cells, medium with extracel
-
lular bacteria was aspirated after 60 min of infection, and 10 ml of TRIzol
LS reagent (Invitrogen, Carlsbad, CA) was added to lyse the cells. Subse-
quently, the lysate was centrifuged at 7,200 ⫻ g for 5 min to pellet the
cell-associated bacteria. TRIzol LS supernatant was stored in a clean tube
and further processed for extraction of total RNA released from infected
Caco-2 cells. The cell-associated bacterial pellet was resuspended in 2
ml of fresh TRIzol LS, gently mixed, and processed for extraction of
total RNA. For controls, medium with bacteria was collected and cen-
trifuged at 7,200 ⫻ g for 5 min, and 10 ml of TRIzol LS reagent was
added, gently mixed, and processed for extraction of total RNA. For
the noninfected Caco-2 cells, medium was aspirated, and 10 ml of
TRIzol LS reagent was added to the cells, was gently mixed, and pro-
cessed for extraction of total RNA.
Bacterial RNA extraction, hybridization, and normalization. Bacte-
rial RNA was prepared from TRIzol LS reagent, and cDNA was generated
following procedures described previously (
11). Prior to hybridization,
the cDNA samples were denatured at 98°C for 10 min followed by snap
cooling at 4°C for 5 min. Labeled cDNA was hybridized onto a custom-
designed Affymetrix GeneChip covering all the annotated coding se-
quences of S. Typhimurium LT2 ATCC 700720 (
11). Labeled cDNA ob-
tained from pure culture of S. Typhimurium (500 ng) and coculture of S.
Typhimurium and Caco-2 cells (2,000 ng) was hybridized on the chips
and scanned at the Center for Integrated Biosystems (Utah State Univer-
sity, Logan, UT) following the manufacturer’s protocols. Raw data (.cel
files) were background corrected, quantile normalized, and summarized
using MS-RMA (
12).The resultant normalized log
2
-transformed inten
-
sity matrix was used for further statistical analysis.
Caco-2 RNA extraction, hybridization, and normalization. The
TRIzol LS samples (750 l) containing infected or noninfected Caco-2
cells were frozen (liquid N
2
) and thawed (70°C) twice followed by addi
-
tion of 250 l of water. RNA was extracted using TRIzol LS following
manufacturer’s instructions. RNA concentration, A260/280 and A260/
230 were measured on NanoDrop (Thermo scientific, Waltham, MA) and
analyzed for integrity on a 2100 Bioanalyzer (Agilent Technologies, Santa
Clara, CA). Synthesis of cDNA, biotin labeling of cRNA, and fragmenta-
tion and purification of cRNA were carried out using one-cycle cDNA
synthesis kit (Affymetrix, Santa Clara, CA). Labeled and fragmented
cRNA (10 g) was hybridized onto the Affymetrix U133Plus2 GeneChip
as per the manufacturer’s recommendations at the Center for Integrated
Biosystems (Utah State University, Logan, UT). Raw data (.cel files) were
background corrected, quantile normalized, and summarized using RMA
Shah et al.
6944
aem.asm.org Applied and Environmental Microbiology
(13). RMA-normalized data were then filtered through the PANP algo-
rithm (P. Warren, D. Taylor, P. G. V. Martini, J. Jackson, and J. Bien-
kowska, presented at the 7th IEEE International Conference on Bioinfor-
matics and Bioengineering, 2007) to make presence-absence calls for each
probe set. Probe sets that were called present in at least one of the samples
were included for further statistical analysis.
Antibody blocking assay. The aim of the antibody blocking assay was
to determine the role of alpha spectrin in bacterial association. Anti-
SPTAN1 antibodies (1:2,000; Novus Biologicals, Littleton, CO) were
added to the Caco-2 cells to block exposed alpha spectrin on cell surface.
Cells were incubated for 60 min at 37°C with 5% CO
2
before bacterial
treatments at an MOI of 1:100 were added. The experiment was per-
formed in two biological replicates. Invasion assays were performed as
described earlier. One-way ANOVA with Tukey’s post hoc test was used to
find significant differences across treatment and control group means.
The differences with P values of ⬍0.05 were considered significant.
Cell-based caspase assay. The aim of the cell-based caspase assay was
to measure caspase activation upon bacterial infection. Caco-2 cells were
washed with phosphate-buffered saline (PBS) before use. Upon addition
of bacteria at an MOI of 1:1,000, in three biological replicates, cells were
incubated at 37°C with 5% CO
2
for 4 h, 6 h, and 8 h. At different time
points, caspase activity was measured using Caspase-Glo 8, 9, and 3/7
assay kits (Promega, Madison, WI) following the manufacturer’s instruc-
tions. Bioluminescence was measured in a DTX 880 multimode-detector
plate reader (Beckman Coulter, Brea, CA). Two-way ANOVA with Bon-
ferroni post hoc tests was used to find significant differences between treat-
ment and control group means at different time points. The differences
with P values of ⬍0.05 were considered significant.
Statistical analysis for gene expression. Gene expression profiles for
cold-stressed (5°C, 48 h) and nonstressed S. Typhimurium alone and in
the presence of epithelial cells were obtained 60 min postinfection. The
data were analyzed as two-class unpaired data (cold stress versus no
stress), with the T statistic, using significance analysis of microarrays
(SAM) (
14). All the genes were ranked based on the score (d) from SAM
output. This preordered ranked gene list was then used in Gene Set En-
richment Analysis software (GSEA) (
15) to detect the coordinate changes
in the expression of groups of functionally related genes upon respective
treatments. The gene sets with q values of ⬍0.05 were considered signifi-
cant. The gene sets were defined based on the annotations from the Com-
prehensive Microbial Resource (CMR) (
16), clusters of orthologous
groups of proteins (COGs) (
17) and the Virulence Factors of Pathogenic
Bacteria database (VFDB) (
1).
Gene expression profiles were obtained for epithelial cells alone and
upon infection with cold-stressed (5°C, 48 h) and nonstressed S. Typhi-
murium, 60 min postinfection. The data were analyzed as two-class un-
paired data (infection with cold-stressed S. Typhimurium versus infection
with nonstressed S. Typhimurium), with the T statistic, using SAM (
14).
All the genes were ranked based on the score (d) from SAM output. This
preordered ranked gene list was then used in GSEA (
15) to detect the
coordinate changes in the expression of groups of functionally related
genes upon infection with cold-stressed S. Typhimurium. The gene sets
with q values of ⬍0.20 were considered significant. The gene sets were
defined based on the annotations in GO gene sets from the Molecular
Signatures database (MSigDB) (
15).
RESULTS AND DISCUSSION
Effect of stress treatments on bacterial association to epithelial
cells. Among the stresses tested, cold stress of S. Typhimurium
significantly (P ⬍ 0.05) increased its association with Caco-2 cells
from 60 min postinfection (p.i.) until the end of the experiment
(i.e., 120 min) compared with the nonstressed control and the
peroxide-stressed S. Typhimurium (
Fig. 1A). Because the num-
bers of cold-stressed S. Typhimurium associated with cells re-
mained significantly higher and stable through 120 min, the inva-
sion was measured only at 60 min p.i. Interestingly, the numbers
of cold-stressed S. Typhimurium bacteria that invaded Caco-2
cells were significantly (P ⬍ 0.05) higher than the numbers of
nonstressed S. Typhimurium bacteria (
Fig. 1B), suggesting that
prior exposure to cold stress significantly increased both adhesion
and invasion of S. Typhimurium in epithelial cells. Consequently,
for all other follow-up experiments, we focused on cold-stressed
Salmonella.
Gene expression profile of cold-stressed S. Typhimurium.
Cold stress of S. Typhimurium modulated expression of several
virulence-associated genes and genes associated with protein se-
cretion and trafficking, DNA metabolism and repair, and degra-
dation of RNA (
Table 1). Overall, the alteration in global gene
expression in cold-stressed S. Typhimurium suggested that both
virulence and bacterial metabolism may play a role in pathogen-
esis. However, in this study our primary focus was on the viru-
lence-associated genes and their potential link to the observed
phenotype of increased association of cold-stressed S. Typhimu-
rium to intestinal epithelial cells. To better understand the gene
expression profiles and for visualization, the differentially ex-
pressed genes from virulence-associated gene categories were
further divided into three major functional groups, namely, T3SS-
associated genes, plasmid-associated genes, and prophage-associ-
ated genes (
Fig. 2).
(i) T3SS-associated genes. Cold stress induced expression of
several T3SS-associated genes on various SPIs. The role of these
genes is to allow bacteria to remain docked at the epithelial cell
membrane in order to deliver effectors to the host cell cytosol (
18).
FIG 1 Association of S. Typhimurium with Caco-2 cells. (A) Total association
of cold-, peroxide- and acid-stressed S. Typhimurium with Caco-2 cells mea-
sured at 60 min, 90 min, and 120 min postinfection. (B) Adhesion to and
invasion of Caco-2 cells by cold-stressed S. Typhimurium measured at 60 min
postinfection. Results are means ⫾ SEM (standard errors of the means) for
three biological replicates. Asterisks and dots indicate significant differences
(P ⬍ 0.05) compared to the no-stress control and peroxide-stressed bacteria,
respectively.
Cold Stress of Salmonella Increases Its Pathogenicity
November 2014 Volume 80 Number 22
aem.asm.org 6945
These genes are also required for later stages of infection and in-
tracellular replication (
19). The two-component system ssrAB
controls the expression of genes encoding the components of the
T3SS and its effectors located within and outside the SPI2 region
(
20), including ssrA, whose expression was induced 1.8-fold. The
genes encoding effectors mainly from the ssa and sse operons,
along with sscA, sscB, sifA, sifB, pipA, pipB, and pipB2 from SPI2
and SPI5, were induced. The SPI1 genes invJ, which is responsible
for translocation of the effectors (
19), and sipC, which is involved
in actin bundling (
21), were also induced (Fig. 2B).
(ii) Plasmid-associated genes. Genes of the tra and trb clusters
forming a subset of type 4 secretion system (T4SS) (
22) were in-
duced (
Fig. 2C). These plasmid-borne genes encode the elements
of pili required for mating and conjugal DNA transfer. Transcrip-
tion of the tra operon is activated by TraJ, which is positively
regulated by FinO (
23), and induction of both traJ and finO was
observed (
Fig. 2C).
(iii) Prophage-associated genes. The genes on prophages
Fels-1 (22/35), Gifsy-2 (12/52), Gifsy-1 (24/53), and Fels-2 (9/47)
were induced (
Fig. 2A). The prophages are often identified adja-
cent to virulence genes and tRNA genes (
24) and are known to
contribute to pathogenicity, insertion of transferable elements,
and lateral spreading of the pathogenicity determinants. The roles
of Gifsy-2 and Gifsy-1 prophages have been studied (
25), but there
is no direct evidence of a direct contribution of Fels-1 and Fels-2 to
virulence other than the roles of sodC, nanH, and grvA from Fels-1
(
26) and a suspected role of abiU from Fels-2 (26, 27). Other
phage genes, such as pspA and pspD, were also induced in response
to cold stress (
Fig. 2A).
Gene expression profile of cold-stressed S. Typhimurium
during infection of epithelial cells. Infection of epithelial cells
with cold-stressed S. Typhimurium resulted in induction of pro-
phage genes, plasmid-associated genes (including the spv operon),
stress response and DNA transformation genes, and genes associ-
ated with energy metabolism, protein synthesis, polysaccharide
metabolism, and chemotaxis (
Table 2). In order to determine the
link between the gene expression and the observed phenotype of
increased epithelial cell adhesion and invasion, we again focused
on the virulence-associated gene categories, and the genes from
these categories were divided into four major functional groups
for visualization, namely, plasmid-associated genes, prophage-as-
sociated genes, DNA transposition-associated genes, and spv and
other stress response-associated genes.
(i) Plasmid-associated genes. Genes within the tra and trb
clusters, forming a subset of T4SS, were induced in response to the
cold stress alone and remained induced during the infection. Ad-
ditionally, traL, which is required for pilus tip formation on cell
surfaces (
28), and traS, which is involved in entry exclusion during
mating pair stabilization (
22), were also induced during infection
(
Fig. 3D).
(ii) Prophage-associated genes. Several genes located on the
prophages Fels-1 (23/35), Gifsy-2 (12/52), Gifsy-1 (15/53), and
Fels-2 (11/47) also remained induced during infection (
Fig. 3A).
Additionally, other phage-associated genes, such as pspABCDE,
were induced in cold-stressed S. Typhimurium during infection
(
Fig. 3A). It is important to note that the psp genes are highly
conserved in some pathogens and are involved in infection pro-
cesses.
(iii) DNA transposition-associated genes. Genes encoding re-
solvase, transposase, and integrase, on the chromosome as well as
on the plasmid, and involved in DNA transposition were induced
during infection (
Fig. 3C).
(iv) Stress response and spv operon genes. Other genes that
were induced during infection included the plasmid-associated tran-
scriptional regulator spvR and spvABC, which play a role in intracel-
lular proliferation of Salmonella (
29), and stress response-associ-
ated genes, such as cspABE, uspA, relA, htpX, and osmB (
Fig. 3B).
Additionally, the genes associated with flagellar functions from
the fli, flh, and flg operons were found to be repressed in cold-
stressed S. Typhimurium during infection. During the infection,
flagellar proteins are recognized by Toll-like receptor 5 (TLR5),
NLR, and Ipaf on the host cell, leading to induction of the proin-
flammatory cascade, caspase 1-dependent cell death, and the T-
cell-mediated immune response (
30). However, downregulation
of these genes is considered a means by which Salmonella evades
its detection (
31). Lipopolysaccharide (LPS), a component of the
outer membrane of Salmonella, has been shown to interact with
TLR4 (
32) and initiate a proinflammatory cascade. The genes en-
coding surface polysaccharides, including LPS, were also re-
pressed.
Gene expression of epithelial cells in response to infection
with cold-stressed S. Typhimurium. Infection of epithelial cells
TABLE 1 Gene categories in S. Typhimurium that were significantly (q ⬍ 0.05) regulated due to cold stress (5°C, 48 h)
Enriched gene category
a
No. of genes
regulated
No. of genes
in category Regulation FDR (q)
b
SP2-TTSS 26 27 Induced 0
TTSS 27 58 Induced 0
TTSS effectors 10 22 Induced 0
TTSS2 effectors 9 13 Induced 0
Cellular processes—pathogenesis 8 32 Induced 0.03
Prophage functions 70 143 Induced 0.04
Plasmid functions 26 28 Induced 0
Protein and peptide secretion and trafficking 21 54 Induced 0
Cellular processes—DNA transformation 28 30 Induced 0
Purine ribonucleotide biosynthesis 9 18 Induced 0.01
CogL—replication recombination and repair 79 157 Induced 0.01
Transcription—degradation of RNA 9 15 Induced 0.05
a
TTSS, type III secretion system.
b
FDR, false discovery rate.
Shah et al.
6946
aem.asm.org Applied and Environmental Microbiology
FIG 2 Heatmap representation of differentially regulated (q ⬍ 0.05) genes in S. Typhimurium due to exposure to cold stress (5°C, 48 h). (A) T3SS-associated
genes. (B) Plasmid (pSLT)-associated genes. (C) Prophage-associated genes. The blue-gray-red color scale represents low expression intensity (2.4) to high
expression intensity (13.5).
Cold Stress of Salmonella Increases Its Pathogenicity
November 2014 Volume 80 Number 22
aem.asm.org 6947
with cold-stressed S. Typhimurium regulated 18 gene categories
related to mitochondrial function along with other gene catego-
ries related to ribosomal genes, ribonucleoprotein complex, kine-
sin complex, RNA splicing, and coenzyme metabolism (
Table 3).
All the genes regulated in the categories functionally associated
with mitochondria were overlaid on the electron transport chain
(ETC) for visualization using ingenuity pathway analysis software
(IPA; Ingenuity Systems, Redwood City, CA). The genes encoding
the subunits of the four complexes of ETC, complex I (NADH
dehydrogenase/reductase), complex II (succinate dehydrogenase),
complex III (cytochrome bc
1
complex), and complex IV (cyto
-
chrome c oxidase) were induced. Additionally genes encoding
glycerol-3-phosphate dehydrogenase 2 (GPD2) and monoamine
oxidase A (MAOA), were also induced. Although ETC is a very
efficient system, it is a major site to produce superoxide radicals
and reactive oxygen species because there is a high probability of
electrons being passed to oxygen directly, instead of the next elec-
tron carrier in the chain. An appropriate balance of oxidants and
antioxidants is required for cell survival; even a slight perturbation
can lead to damage of biological macromolecules and hence cell
death. In functionally intact mitochondria, a large number of an-
tioxidant gene products are needed to neutralize the effects of
superoxide radicals and reactive oxygen species (
33). Conse-
quently, the expression intensities of the components of the anti-
oxidant defense system (
33) were further examined. No signifi-
cant changes in gene expression were observed for any of the genes
except SOD2 (superoxide dismutase 2), CAT (catalase), GPX7
(glutathione peroxidase 7), and PRX3 (peroxiredoxin 3), and
these genes were induced 1.2-, 1.2-, 1.3-, and 1.5-fold, respec-
tively.
The other genes induced in epithelial cells during infection
with cold-stressed S. Typhimurium were related to microtubule
activity and the kinesin complex. The genes located on SPI2 of S.
Typhimurium cause accumulation of microtubules around the
Salmonella-containing vacuole (SCV) (
34), recruitment of kinesin
and dynein to regulate vacuolar membrane dynamics (
35), and
interference with the activity of ubiquitination pathway (
36).
This, together with the increased adhesion and invasion of cold-
stressed Salmonella, explains the induction of genes related to mi-
crotubule activity and the kinesin complex.
These observations suggest induction of oxidative stress in ep-
ithelial cells infected with cold-stressed S. Typhimurium com-
pared with the infection with nonstressed S. Typhimurium. Oxi-
dative stress has been shown to cause damage leading to apoptosis
via caspase activation (
37, 38), and hence, activation of caspases 8,
9, and 3/7 was measured.
Activation of caspases. Caspases are intracellular cysteine-
containing, aspartic acid-specific proteases implicated in pro-
grammed cell death. Caspases are divided into the classifications
“initiators” and “executioners” (
39, 40). The activation of the ex-
ecutioner caspase, i.e., caspase 3/7, is a committed step in apopto-
sis and can occur via the extrinsic and/or intrinsic pathway involv-
ing the initiator caspase 8 and/or caspase 9, respectively (
39, 40).
The activation of caspase 3/7 was significantly increased at 8 h p.i.
with cold-stressed S. Typhimurium compared to infection with
nonstressed S. Typhimurium (
Fig. 4C). The activation of caspase 8
did not significantly change upon infection with cold-stressed S.
Typhimurium compared to nonstressed S. Typhimurium, at any
time points tested during infection (
Fig. 4A). On the other hand,
the activation of caspase 9 was significantly (P ⬍ 0.05) higher at 8
h p.i. with cold-stressed S. Typhimurium compared to infection
with nonstressed S. Typhimurium (
Fig. 4B). Moreover, activity of
caspase 9 increased with time, implying the contribution of
caspase 9 in activation of caspase 3/7 via the intrinsic pathway. To
summarize, significant activation of caspase 9 and caspase 3/7 was
concomitant with the gene regulation observations, suggesting
induced apoptosis by the intrinsic (mitochondrial) route, in epi-
thelial cells infected with cold-stressed S. Typhimurium.
Receptors involved in association of S. Typhimurium with
epithelial cells. Pathogen-host cell association is a function of the
regulation of multiple receptor-ligand interactions. A study con-
ducted using a whole-cell cross-linking method revealed a prod-
uct of STM2699 (a Fels-2 prophage gene) from S. Typhimurium
cross-linked to a receptor, SPTAN1 (spectrin), expressed on
Caco-2 cells (
41). Interestingly, in this study, the expression of
STM2699 was induced in response to cold stress alone, and it
remained induced during the infection of Caco-2 cells (
Fig. 5). As
expected, the expression of SPTAN1 in the Caco-2 cells was also
induced upon infection with cold-stressed S. Typhimurium (
Fig.
5
). Consequently, the contribution of STM2699 and SPTAN1 in-
TABLE 2 Gene categories in cold-stressed (5°C, 48 h) S. Typhimurium that were significantly (q ⬍ 0.05) regulated during infection of epithelial
cells
Enriched gene category
a
No. of genes
regulated
No. of genes
in category Regulation FDR (q)
b
Prophage functions 63 143 Induced 0
Plasmid functions 26 28 Induced 0
Transposon functions 27 44 Induced 0
SPV locus 4 4 Induced 0.04
Adaptations to atypical conditions 10 30 Induced 0.04
Cellular processes—DNA transformation 30 30 Induced 0
Energy metabolism—anaerobic 32 66 Induced 0.04
Synthesis and modification ribosomal proteins 37 61 Repressed 0
CogJ—translation 67 170 Repressed 0
Cellular processes—chemotaxis and motility 20 40 Repressed 0
Energy metabolism—pyruvate dehydrogenase 3 8 Repressed 0.02
Metabolism of surface polysaccharides and LPS 39 82 Repressed 0.04
Energy metabolism—aerobic 12 24 Repressed 0.04
a
SPV, Salmonella plasmid virulence locus.
b
FDR, false discovery rate.
Shah et al.
6948 aem.asm.org Applied and Environmental Microbiology
FIG 3 Heatmap representation of genes differentially regulated (q ⬍ 0.05) in cold-stressed (5°C, 48 h) S. Typhimurium during infection of epithelial cells. (A)
Plasmid (pSLT)-associated genes. (B) Prophage-associated genes. (C) DNA transposition-associated genes. (D) spv and other stress response-associated genes.
The blue-gray-red color scale represents low expression intensity (2.1) to high expression intensity (11.2).
Cold Stress of Salmonella Increases Its Pathogenicity
November 2014 Volume 80 Number 22
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teraction in adhesion and invasion during infection of Caco-2
cells with nonstressed as well as cold-stressed S. Typhimurium was
investigated. The gene knockout for STM2699 was created in the
wild-type parent as well as the invasion-deficient S. Typhimurium
(⌬invA) strain (
42).
Adherence of nonstressed S. Typhimurium ⌬STM2699 to the
Caco-2 cells was similar to that of the wild-type strain; however,
significantly low numbers of this mutant strain invaded cells (
Fig.
6A
). Nonstressed S. Typhimurium ⌬STM2699-⌬invA did not
show any further reduction in adhesion or invasion compared
with S. Typhimurium ⌬invA (
Fig. 6A). These results revealed con-
tribution of STM2699 specifically in cell invasion by nonstressed
S. Typhimurium during infection of Caco-2 cells. Pretreatment of
Caco-2 cells with anti-SPTANI antibodies showed that the non-
stressed wild-type S. Typhimurium adhered in significantly low
numbers to the Caco-2 cells, but no alterations in invasion were
noted (
Fig. 6B). In contrast, S. Typhimurium ⌬STM2699 infec-
tion of Caco-2 cells pretreated with anti-SPTANI antibodies re-
sulted in significant reduction in the invasion. Prior treatment of
Caco-2 cells with anti-SPTANI antibodies did not show any fur-
ther reduction in invasiveness of S. Typhimurium ⌬STM2699
⌬invA compared with the S. Typhimurium ⌬invA strain (
Fig. 6B).
These results revealed that STM2699-SPTAN1 interaction plays
an important role in the invasion of nonstressed S. Typhimurium
during infection of Caco-2 cells.
We then tested effect of prior exposure of S. Typhimurium to
cold stress on this interaction. Infection of Caco-2 cells with cold-
stressed S. Typhimurium ⌬STM2699 reduced adhesion as well as
invasion (
Fig. 7A). Infection with cold-stressed S. Typhimurium
⌬STM2699 ⌬invA additionally reduced adhesion compared with
cold-stressed S. Typhimurium ⌬invA (Fig. 7A). These results re-
vealed the contribution of STM2699 to adhesion and invasion of
FIG 4 Measurement of caspase 8 (A), caspase 9 (B), and caspase 3/7 (C) activity at 4 h, 6 h, and 8 h postinfection of epithelial cells with nonstressed and
cold-stressed (5°C, 48 h) S. Typhimurium. Asterisks indicate significant differences (P ⬍ 0.05) between infection with cold-stressed S. Typhimurium and
infection with nonstressed S. Typhimurium.
TABLE 3 Gene categories in epithelial cells that were significantly (q ⬍ 0.20) regulated during infection with cold-stressed (5°C, 48 h) S.
Typhimurium
Enriched gene category
No. of genes
regulated
No. of genes
in category Regulation FDR (q)
a
Ribosome 18 37 Induced 0.01
Ribosomal subunit 11 20 Induced 0
Organellar ribosome 12 22 Induced 0
Mitochondrial ribosome 12 22 Induced 0
Mitochondrial small ribosomal subunit 9 11 Induced 0
Organellar small ribosomal subunit 9 11 Induced 0.01
Small ribosomal subunit 9 11 Induced 0.01
Mitochondrial part 47 128 Induced 0.01
Mitochondrial envelope 28 85 Induced 0.03
Mitochondrial membrane 26 77 Induced 0.03
Mitochondrial inner membrane 24 60 Induced 0.01
Mitochondrial membrane part 20 50 Induced 0
Mitochondrial lumen 18 44 Induced 0.03
Mitochondrial matrix 18 44 Induced 0.03
Cellular respiration 9 18 Induced 0.03
NADH dehydrogenase complex 7 14 Induced 0.03
Mitochondrial respiratory chain complex I 7 14 Induced 0.03
Respiratory chain complex I 7 14 Induced 0.03
Structural constituent of ribosome 43 73 Induced 0.00
Ribosome biogenesis and assembly 6 12 Induced 0.04
Ribonucleoprotein complex 41 111 Induced 0.01
Microtubule motor activity 9 14 Induced 0.01
Kinesin complex 8 11 Induced 0.03
RNA splicing via transesterification reactions 10 30 Induced 0.03
Coenzyme metabolic process 12 31 Induced 0.04
a
FDR, false discovery rate.
Shah et al.
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aem.asm.org Applied and Environmental Microbiology
cold-stressed S. Typhimurium during infection of Caco-2 cells.
Pretreatment of Caco-2 cells with anti-SPTANI antibodies did not
alter adherence and invasion of cold-stressed wild-type S. Typhi-
murium; however, infection with cold-stressed S. Typhimurium
⌬STM2699 showed significantly reduced adhesion and invasion
(
Fig. 7B). Infection with cold-stressed S. Typhimurium ⌬STM2699
⌬invA of Caco-2 cells pretreated with anti-SPTANI antibodies ad-
ditionally reduced adhesion compared to infection with cold-
stressed S. Typhimurium ⌬invA (
Fig. 7B). These results revealed
that STM2699-SPTAN1 interaction plays an important role in
both adhesion of and invasion by cold-stressed S. Typhimurium
during infection of Caco-2 cells.
Concluding remarks. In this study, we found that prior expo-
sure of S. Typhimurium to cold stress (5°C, 48 h) significantly
increased adhesion and invasion in Caco-2 cells compared to non-
stressed S. Typhimurium. The gene expression data further re-
vealed significant induction of virulence-associated genes which
FIG 5 (Left) Gene expression intensities of the S. Typhimurium gene STM2699 in response to no stress and cold stress (5°C, 48 h) alone and during infection
of epithelial cells with nonstressed and cold-stressed (5°C, 48 h) S. Typhimurium. (Right) Gene expression intensities of the epithelial cell gene SPTAN1 in
epithelial cells alone and during infection with nonstressed and cold-stressed (5°C, 48 h) S. Typhimurium. NS, no stress; CS, cold stress. The blue-gray-red color
scale represents low expression intensity (3.7) to high expression intensity (7.5).
FIG 6 Role of STM2699 and SPTAN1 in adhesion and invasion of epithelial
cells with nonstressed S. Typhimurium. (A) Role of STM2699 in S. Typhimu-
rium adhesion to and invasion of epithelial cells. (B) Role of STM2699 and
SPTAN1 interaction in S. Typhimurium adhesion to and invasion of epithelial
cells pretreated with anti-SPTAN1 antibodies. Asterisks indicate significant
differences (P ⬍ 0.05) compared to the first bar, which represents adhesion
and invasion of wild-type S. Typhimurium.
FIG 7 Role of STM2699 and SPTAN1 in adhesion and invasion of epithelial
cells with cold-stressed (5°C, 48 h) S. Typhimurium. (A) Role of STM2699 in
adhesion and invasion of cold-stressed S. Typhimurium in epithelial cells. (B)
Role of STM2699 and SPTAN1 interaction in adhesion and invasion of cold-
stressed S. Typhimurium in epithelial cells pretreated with anti-SPTAN1 an-
tibodies. Asterisks indicate significant differences (P ⬍ 0.05) compared to the
first bar, representing adhesion and invasion of wild type S. Typhimurium.
The dot indicates a significant difference (P ⬍ 0.05) compared to the bar
representing adhesion and invasion of S. Typhimurium ⌬invA.
Cold Stress of Salmonella Increases Its Pathogenicity
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remained induced during infection of the host cells, indicating
increased pathogenicity of cold-stressed S. Typhimurium. Simul-
taneous gene expression profiling of Caco-2-cells indicated induc-
tion of mitochondrial dysfunction, induction of oxidative stress
response, and imbalance of oxidants and antioxidants upon infec-
tion with cold-stressed S. Typhimurium. Induced damage of epi-
thelial cells through intrinsic (mitochondrial) pathway was con-
firmed by induction of caspase 9 and caspase 3/7 activity during
infection with cold-stressed S. Typhimurium. Furthermore, we
found that the STM2699-SPTAN1 (protein-protein) interaction
increased the adhesion and invasion during the infection of
Caco-2 cells with cold-stressed S. Typhimurium. These observa-
tions together indicate that exposure to cold stress (5°C, 48 h) may
potentially increase the pathogenicity of S. Typhimurium. We
showed previously that preadaptation to cold stress increases the
survival during subsequent acid stress exposure, which mimics the
conditions of the gastric transit (
11). Follow-up studies to mea-
sure the transcriptional response and host-cell interaction of S.
Typhimurium upon exposure to cold stress followed by acid stress
will provide indispensable targets needed at each step for success-
ful infection.
ACKNOWLEDGMENT
Funding for this project was provided by USDA CSREES grant 2006-
34526-433 17001 to B.C.W.
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