ArticlePDF Available

Genetic Mechanisms Underlying the Pathogenicity of Cold-Stressed Salmonella enterica Serovar Typhimurium in Cultured Intestinal Epithelial Cells

Authors:

Abstract and Figures

Salmonella encounters various stresses in the environment and in the host during infection. The effects of cold (5°C, 48 h), peroxide (5 mM H2O2, 5 h) and acid stress (pH 4.0, 90 min) were tested on pathogenicity of Salmonella. Prior exposure of Salmonella 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 Salmonella, 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. Increased 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 Salmonella in epithelial cells.
Content may be subject to copyright.
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
aem.asm.org 6949
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.
6950
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
November 2014 Volume 80 Number 22
aem.asm.org 6951
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.
REFERENCES
1. Chen L, Yang J, Yu J, Yao Z, Sun L, Shen Y, Jin Q. 2005. VFDB: a
reference database for bacterial virulence factors. Nucleic Acids Res. 33:
D325–D328. http://dx.doi.org/10.1093/nar/gki008.
2. Patti JM, Allen BL, McGavin MJ, Hook M. 1994. MSCRAMM-mediated
adherence of microorganisms to host tissues. Annu. Rev. Microbiol. 48:
585–617. http://dx.doi.org/10.1146/annurev.mi.48.100194.003101.
3. Olsen A, Jonsson A, Normark S. 1989. Fibronectin binding mediated by
a novel class of surface organelles on Escherichia coli. Nature 338:652–
655. http://dx.doi.org/10.1038/338652a0.
4. Sjobring U, Pohl G, Olsen A. 1994. Plasminogen, absorbed by Esche-
richia coli expressing curli or by Salmonella enteritidis expressing thin
aggregative fimbriae, can be activated by simultaneously captured tissue-
type plasminogen activator (t-PA). Mol. Microbiol. 14:443– 452. http://dx
.doi.org/10.1111/j.1365-2958.1994.tb02179.x.
5. Gerlach RG, Jäckel D, Stecher B, Wagner C, Lupas A, Hardt WD,
Hensel M. 2007. Salmonella pathogenicity island 4 encodes a giant non-
fimbrial adhesin and the cognate type 1 secretion system. Cell. Microbiol.
9:1834–1850. http://dx.doi.org/10.1111/j.1462-5822.2007.00919.x.
6. Galan JE. 1996. Molecular genetic bases of Salmonella entry into host
cells. Mol. Microbiol. 20:263–271. http://dx.doi.org/10.1111/j.1365-2958
.1996.tb02615.x.
7. Marcus SL, Brumell JH, Pfeifer CG, Finlay BB. 2000. Salmonella patho-
genicity islands: big virulence in small packages. Microbes Infect. 2:145–
156. http://dx.doi.org/10.1016/S1286-4579(00)00273-2.
8. Datsenko KA, Wanner BL. 2000. One-step inactivation of chromosomal
genes in Escherichia coli K-12 using PCR products. Proc. Natl. Acad. Sci.
U. S. A. 97:6640 6645. http://dx.doi.org/10.1073/pnas.120163297.
9. Desai PT, Walsh MK, Weimer BC. 2008. Solid-phase capture of patho-
genic bacteria by using gangliosides and detection with real-time PCR.
Appl. Environ. Microbiol. 74:2254 –2258. http://dx.doi.org/10.1128/AEM
.02601-07.
10. Ziemer C, Steadham S. 2003. Evaluation of the specificity of Salmonella
PCR primers using various intestinal bacterial species. Lett. Appl. Micro-
biol. 37:463–469. http://dx.doi.org/10.1046/j.1472-765X.2003.01430.x.
11. Shah J, Desai PT, Chen D, Stevens JR, Weimer BC. 2013. Preadaptation
to cold stress in Salmonella enterica serovar Typhimurium increases sur-
vival during subsequent acid stress exposure. Appl. Environ. Microbiol.
79:7281–7289. http://dx.doi.org/10.1128/AEM.02621-13.
12. Stevens JR, Ganesan B, Desai P, Rajan S, Weimer BC. 2008. Statistical
issues in the normalization of multi-species microarray data, p 47– 62. In
Proceedings of Conference on Applied Statistics in Agriculture.
13. Irizarry RA, Hobbs B, Collin F, Beazer-Barclay YD, Antonellis KJ,
Scherf U, Speed TP. 2003. Exploration, normalization, and summaries of
high density oligonucleotide array probe level data. Biostatistics 4:249
264.
http://dx.doi.org/10.1093/biostatistics/4.2.249.
14. Tusher VG, Tibshirani R, Chu G. 2001. Significance analysis of microar-
rays applied to the ionizing radiation response. Proc. Natl. Acad. Sci.
U. S. A. 98:5116–5121.
http://dx.doi.org/10.1073/pnas.091062498.
15. Subramanian A, Tamayo P, Mootha VK, Mukherjee S, Ebert BL,
Gillette MA, Paulovich A, Pomeroy SL, Golub TR, Lander ES, Mesirov
JP. 2005. Gene set enrichment analysis: A knowledge-based approach for
interpreting genome-wide expression profiles. Proc. Natl. Acad. Sci.
U. S. A. 102:15545–15550.
http://dx.doi.org/10.1073/pnas.0506580102.
16. Peterson JD, Umayam LA, Dickinson T, Hickey EK, White O. 2001. The
Comprehensive Microbial Resource. Nucleic Acids Res. 29:123–125.
http:
//dx.doi.org/10.1093/nar/29.1.123
.
17. Tatusov RL, Koonin EV, Lipman DJ. 1997. A genomic perspective on
protein families. Science 278:631–637.
http://dx.doi.org/10.1126/science
.278.5338.631
.
18. Cornelis GR. 2006. The type III secretion injectisome. Nat. Rev. Micro-
biol. 4:811–825.
http://dx.doi.org/10.1038/nrmicro1526.
19. Hansen-Wester I, Hensel M. 2001. Salmonella pathogenicity islands en-
coding type III secretion systems. Microbes Infect. 3:549 –559.
http://dx
.doi.org/10.1016/S1286-4579(01)01411-3
.
20. Garmendia J, Beuzon CR, Ruiz-Albert J, Holden DW. 2003. The roles of
SsrA-SsrB and OmpR-EnvZ in the regulation of genes encoding the Sal-
monella typhimurium SPI-2 type III secretion system. Microbiology 149:
2385–2396.
http://dx.doi.org/10.1099/mic.0.26397-0.
21. Hayward RD, Koronakis V. 1999. Direct nucleation and bundling of
actin by the SipC protein of invasive Salmonella. EMBO J. 18:49264934.
http://dx.doi.org/10.1093/emboj/18.18.4926.
22. Lawley TD, Klimke WA, Gubbins MJ, Frost LS. 2003. F factor conjuga-
tion is a true type IV secretion system. FEMS Microbiol. Lett. 224:1–15.
http://dx.doi.org/10.1016/S0378-1097(03)00430-0.
23. Camacho EM, Casadesus J. 2002. Conjugal transfer of the virulence
plasmid of Salmonella enterica is regulated by the leucine-responsive reg-
ulatory protein and DNA adenine methylation. Mol. Microbiol. 44:1589
1598.
http://dx.doi.org/10.1046/j.1365-2958.2002.02981.x.
24. McClelland M, Sanderson KE, Spieth J, Clifton SW, Latreille P, Court-
ney L, Porwollik S, Ali J, Dante M, Du F, Hou S, Layman D, Leonard
S, Nguyen C, Scott K, Holmes A, Grewal N, Mulvaney E, Ryan E, Sun
H, Florea L, Miller W, Stoneking T, Nhan M, Waterston R, Wilson RK.
2001. Complete genome sequence of Salmonella enterica serovar Typhi-
murium LT2. Nature 413:852–856.
http://dx.doi.org/10.1038/35101614.
25. Figueroa-Bossi N, Bossi L. 1999. Inducible prophages contribute to Sal-
monella virulence in mice. Mol. Microbiol. 33:167–176.
http://dx.doi.org
/10.1046/j.1365-2958.1999.01461.x
.
26. Brussow H, Canchaya C, Hardt WD. 2004. Phages and the evolution of
bacterial pathogens: from genomic rearrangements to lysogenic conver-
sion. Microbiol. Mol. Biol. Rev. 68:560 602.
http://dx.doi.org/10.1128
/MMBR.68.3.560-602.2004
.
27. Boyd EF, Brussow H. 2002. Common themes among bacteriophage-
encoded virulence factors and diversity among the bacteriophages in-
volved. Trends Microbiol. 10:521–529.
http://dx.doi.org/10.1016/S0966
-842X(02)02459-9
.
28. Anthony KG, Klimke WA, Manchak J, Frost LS. 1999. Comparison of
proteins involved in pilus synthesis and mating pair stabilization from the
related plasmids F and R100-1: insights into the mechanism of conjuga-
tion. J. Bacteriol. 181:5149–5159.
29. Gulig PA, Danbara H, Guiney DG, Lax AJ, Norel F, Rhen M. 1993.
Molecular analysis of spv virulence genes of the salmonella virulence plas-
mids. Mol. Microbiol. 7:825– 830.
http://dx.doi.org/10.1111/j.1365-2958
.1993.tb01172.x
.
30. Miao EA, Andersen-Nissen E, Warren SE, Aderem A. 2007. TLR5 and
Ipaf: dual sensors of bacterial flagellin in the innate immune system. Se-
min. Immunopathol. 29:275–288.
http://dx.doi.org/10.1007/s00281-007
-0078-z
.
31. Cummings LA, Wilkerson WD, Bergsbaken T, Cookson BT. 2006. In
vivo, fliC expression by Salmonella enterica serovar Typhimurium is het-
erogeneous, regulated by ClpX, and anatomically restricted. Mol. Micro-
biol. 61:795–809.
http://dx.doi.org/10.1111/j.1365-2958.2006.05271.x.
32. Du X, Poltorak A, Silva M, Beutler B. 1999. Analysis of Tlr4-mediated
Shah et al.
6952
aem.asm.org Applied and Environmental Microbiology
LPS signal transduction in macrophages by mutational modification of
the receptor. Blood Cells Mol. Dis. 25:328 –338. http://dx.doi.org/10.1006
/bcmd.1999.0262.
33. Lin MT, Beal MF. 2006. Mitochondrial dysfunction and oxidative stress
in neurodegenerative diseases. Nature 443:787–795. http://dx.doi.org/10
.1038/nature05292.
34. Kuhle V, Jackel D, Hensel M. 2004. Effector proteins encoded by Salmo-
nella pathogenicity island 2 interfere with the microtubule cytoskeleton
after translocation into host cells. Traffic 5:356–370.
http://dx.doi.org/10
.1111/j.1398-9219.2004.00179.x.
35. Guignot J, Caron E, Beuzon C, Bucci C, Kagan J, Roy C, Holden DW.
2004. Microtubule motors control membrane dynamics of Salmonella-
containing vacuoles. J. Cell Sci. 117:1033–1045. http://dx.doi.org/10.1242
/jcs.00949.
36. Rytkonen A, Poh J, Garmendia J, Boyle C, Thompson A, Liu M,
Freemont P, Hinton JC, Holden DW. 2007. SseL, a Salmonella deubiq-
uitinase required for macrophage killing and virulence. Proc. Natl. Acad.
Sci. U. S. A. 104:3502–3507. http://dx.doi.org/10.1073/pnas.0610095104.
37. Hampton MB, Orrenius S. 1997. Dual regulation of caspase activity by
hydrogen peroxide: implications for apoptosis. FEBS Lett. 414:552–556.
http://dx.doi.org/10.1016/S0014-5793(97)01068-5.
38. Zamzami N, Marchetti P, Castedo M, Decaudin D, Macho A, Hirsch T,
Susin SA, Petit PX, Mignotte B, Kroemer G. 1995. Sequential reduction
of mitochondrial transmembrane potential and generation of reactive ox-
ygen species in early programmed cell death. J. Exp. Med. 182:367–377.
http://dx.doi.org/10.1084/jem.182.2.367.
39. Chandra J, Samali A, Orrenius S. 2000. Triggering and modulation of
apoptosis by oxidative stress. Free Radic. Biol. Med. 29:323–333. http://dx
.doi.org/10.1016/S0891-5849(00)00302-6.
40. Pop C, Salvesen GS. 2009. Human caspases: activation, specificity, and
regulation. J. Biol. Chem. 284:21777–21781. http://dx.doi.org/10.1074
/jbc.R800084200.
41. Desai PT. 2011. Molecular Interactions of Salmonella with the host epi-
thelium in presence of commensals. Utah State University, Logan, UT.
42. Galan JE, Curtiss R, III. 1989. Cloning and molecular characterization of
genes whose products allow Salmonella typhimurium to penetrate tissue
culture cells. Proc. Natl. Acad. Sci. U. S. A. 86:6383– 6387. http://dx.doi
.org/10.1073/pnas.86.16.6383.
Cold Stress of Salmonella Increases Its Pathogenicity
November 2014 Volume 80 Number 22
aem.asm.org 6953
... For example, the comparison of avian pathogenic E. coli (APEC) DE205B with and without prophage phiv205-1-infecting chicken embryo fibroblasts cells DF-1 indicated the adhesion abilities of the bacteria without the prophage significantly decreased from 3.3 × 10 6 CFU/mL to 2.1 × 10 6 CFU/mL compared to the bacteria with the prophage [60]. A later discovery by Wahl et al. demonstrated that the gpE gene on prophage SopEϕ could increase the adhesion of S. enterica to epithelial cells by encoding a putative tail-spike protein at cold temperatures [4,61]. In addition, prophage CJIE1 has been demonstrated to increase the adherence of host C. jejuni to human intestine cells INT-407; the C. jejuni isolates containing prophages had approximately 6-to-7-fold greater adherence than the isolates without prophages [62]. ...
... Previous studies proposed three potential mechanisms that explain how prophages can increase biofilm formation: induction of the prophage causes extracellular DNA (eDNA) release and enhances biofilm production, the prophage encodes proteins that promote the formation of biofilms, or prophage excision activates motility operators (discussed in the next section) [60,83]. Further, Shah et al. found evidence of a temperate phage encoding biofilm-associated proteins in Salmonella Typhimurium strain [61]. The authors found that the Salmonella strain expressed the protein STM2699, encoded by the Fels-2 prophage of S. Typhimurium, to alter the bacterial cell surface for better biofilm formation on the surface of Caco-2 cells during long-term refrigeration storage at 5 • C. Another study found that deletion of prophage CTXϕ from the V. cholerae strain decreased the bacterial biofilm formation by more than two times compared to the wild-type strain; the hypothesis for the decreased biofilm was that the expression of the ctxAB operon within prophage CTXϕ also regulated the expression of primary biofilm-related genes, such as vpsT and vpsR (Vibrio polysaccharide) [84]. ...
Article
Full-text available
Bacteriophages are viruses that infect bacteria and archaea and are classified as virulent or temperate phages based on their life cycles. A temperate phage, also known as a lysogenic phage, integrates its genomes into host bacterial chromosomes as a prophage. Previous studies have indicated that temperate phages are beneficial to their susceptible bacterial hosts by introducing additional genes to bacterial chromosomes, creating a mutually beneficial relationship. This article reviewed three primary ways temperate phages contribute to the bacterial pathogenicity of foodborne pathogens, including phage-mediated virulence gene transfer, antibiotic resistance gene mobilization, and biofilm formation. This study provides insights into mechanisms of phage-bacterium interactions in the context of foodborne pathogens and provokes new considerations for further research to avoid the potential of phage-mediated harmful gene transfer in agricultural environments.
... On the other hand, whereas Kröger et al. (2013) did not observe any significant change in the expression of SPI-1 and SPI-2 genes after a 10-minute shock at 10 °C [115], Shah et al. (2014) observed that exposure of S. Typhimurium cells to a temperature of 5 °C for 48 h resulted in induction of several groups of virulence genes, including T3SSassociated genes located on SPI-1 and SPI-2 [190]. The latter authors also studied the effect of this cold shock on the ability of Salmonella to adhere to and invade Caco-2 cells, and observed an increase in both phenomena. ...
... On the other hand, whereas Kröger et al. (2013) did not observe any significant change in the expression of SPI-1 and SPI-2 genes after a 10-minute shock at 10 °C [115], Shah et al. (2014) observed that exposure of S. Typhimurium cells to a temperature of 5 °C for 48 h resulted in induction of several groups of virulence genes, including T3SSassociated genes located on SPI-1 and SPI-2 [190]. The latter authors also studied the effect of this cold shock on the ability of Salmonella to adhere to and invade Caco-2 cells, and observed an increase in both phenomena. ...
Article
Full-text available
The success of Salmonella as a foodborne pathogen can probably be attributed to two major features: its remarkable genetic diversity and its extraordinary ability to adapt. Salmonella cells can survive in harsh environments, successfully compete for nutrients, and cause disease once inside the host. Furthermore, they are capable of rapidly reprogramming their metabolism, evolving in a short time from a stress-resistance mode to a growth or virulent mode, or even to express stress resistance and virulence factors at the same time if needed, thanks to a complex and fine-tuned regulatory network. It is nevertheless generally acknowledged that the development of stress resistance usually has a fitness cost for bacterial cells and that induction of stress resistance responses to certain agents can trigger changes in Salmonella virulence. In this review, we summarize and discuss current knowledge concerning the effects that the development of resistance responses to stress conditions encountered in food and food processing environments (including acid, osmotic and oxidative stress, starvation, modified atmospheres, detergents and disinfectants, chilling, heat, and non-thermal technologies) exerts on different aspects of the physiology of non-typhoidal Salmonellae, with special emphasis on virulence and growth fitness.
... Though the impact of prophage genes on symbiosis initiation in marine invertebrates has not been explored, studies of the bacterial colonization of humans and chicks have provided a basis for our assumptions. Functional genes encoded by prophages play a role in adhesion or biofilm formation, including ssa, speC, and spd1 in prophage HKU.vir of Streptococcus pyogenes (Brouwer et al. 2020), pblA and pblB in prophages of Streptococcus mitis (Bensing et al. 2001), the prophage gene for STM2699 protein in Salmonella (Shah et al. 2014 (Müller et al. 2013), and a gene encoding endo-alpha-sialidase in the prophage of Escherichia coli DE205B (Liu et al. 2020). Therefore, similar functional genes in prophages may induce bacterial colonization in marine invertebrates. ...
Article
Full-text available
Marine invertebrates are ecologically and economically important and have formed holobionts by evolving symbiotic relationships with cellular and acellular microorganisms that reside in and on their tissues. In recent decades, significant focus on symbiotic cellular microorganisms has led to the discovery of various functions and a considerable expansion of our knowledge of holobiont functions. Despite this progress, our understanding of symbiotic acellular microorganisms remains insufficient, impeding our ability to achieve a comprehensive understanding of marine holobionts. In this review, we highlight the abundant viruses, with a particular emphasis on bacteriophages; provide an overview of their diversity, especially in extensively studied sponges and corals; and examine their potential life cycles. In addition, we discuss potential phage–holobiont interactions of various invertebrates, including participating in initial bacterial colonization, maintaining symbiotic relationships, and causing or exacerbating the diseases of marine invertebrates. Despite the importance of this subject, knowledge of how viruses contribute to marine invertebrate organisms remains limited. Advancements in technology and greater attention to viruses will enhance our understanding of marine invertebrate holobionts. Expected final online publication date for the Annual Review of Marine Science, Volume 16 is January 2024. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.
... ASCs were cultured in 5% CO 2 / 37°C, and used at passage six. Colonic epithelial cells (Caco-2; ATCC HTB-37) were obtained from American Type Culture Collection (Manassas, VA) and grown according the method defined by Shah et al. (2014). ...
Article
Full-text available
The potential of mesenchymal stem cells (MSCs) for tissue repair and regeneration has garnered great attention. While MSCs are likely to interact with microbes at sites of tissue damage and inflammation, like in the gastrointestinal system, the consequences of pathogenic association on MSC activities have yet to be elucidated. This study investigated the effects of pathogenic interaction on MSC trilineage differentiation paths and mechanisms using model intracellular pathogen Salmonella enterica ssp enterica serotype Typhimurium. The examination of key markers of differentiation, apoptosis, and immunomodulation demonstrated that Salmonella altered osteogenic and chondrogenic differentiation pathways in human and goat adipose-derived MSCs. Anti-apoptotic and pro-proliferative responses were also significantly upregulated ( p < 0.05) in MSCs during Salmonella challenge. These results together indicate that Salmonella , and potentially other pathogenic bacteria, can induce pathways that influence both apoptotic response and functional differentiation trajectories in MSCs, highlighting that microbes have a potentially significant role as influencers of MSC physiology and immune activity.
... Yet another example is the STM2699 protein, encoded by prophage Fels-2 in S. enterica serovar Typhimurium. STM2699 crosslinks the Salmonella cells to the eukaryotic spectrin receptor, but expression is only induced after a cold shock (Shah, Desai and Weimer 2014). On the other hand, some bacteriophages of V. cholera and S. aureus encode colonization factors that are displayed on the bacterial surface. ...
Article
Bacteria-infecting viruses (phages) and their hosts maintain an ancient and complex relationship. Bacterial predation by lytic phages drives an ongoing phage-host arms race, whereas temperate phages initiate mutualistic relationships with their hosts upon lysogenisation as prophages. In human pathogens, these prophages impact bacterial virulence in distinct ways: by secretion of phage-encoded toxins, modulation of the bacterial envelope, mediation of bacterial infectivity and the control of bacterial cell regulation. This review builds the argument that virulence-influencing prophages hold extensive, unexplored potential for biotechnology. More specifically, it highlights the development potential of novel therapies against infectious diseases, to address the current antibiotic resistance crisis. First, designer bacteriophages may serve to deliver genes encoding cargo proteins which repress bacterial virulence. Secondly, one may develop small molecules mimicking phage-derived proteins targeting central regulators of bacterial virulence. Thirdly, bacteria equipped with phage-derived synthetic circuits which modulate key virulence factors could serve as vaccine candidates to prevent bacterial infections. The development and exploitation of such antibacterial strategies will depend on the discovery of other prophage-derived, virulence control mechanisms and, more generally, on the dissection of the mutualistic relationship between temperate phages and bacteria, as well as on continuing developments in the synthetic biology field.
Article
Salmonella enterica is capable of entering the interior of leafy greens and establishing in the apoplastic area, a phenomenon known as internalization. The ability of internalized bacteria to evade common disinfection practices poses a well-established risk. Our aim was to study the effect of: i) inoculum size and ii) prior adaptation of Salmonella to sublethal stresses, on the internalization of the pathogen in four leafy vegetables. Spinach, lettuce, arugula and chicory were inoculated, by immersion for 2 min at room temperature with: i) Salmonella Enteritidis at 3.0, 4.0, 5.0, 6.0, 7.0 log CFU/mL and ii) non-adapted or adapted S. Enteritidis to acid (in TSB with 1% glucose, incubated for 24 h at 37 °C) cold (in TSB for 7 days at 4 °C), starvation (0.85% NaCl of pH 6.6, 48 h at 37 °C) or desiccation (1.5 h at 42 °C, 4 days at 21 °C) stress at appx 3.5 log CFU/mL). Inoculated leafy greens were subsequently stored at 5 °C and 20 °C for 2 h and 48 h (n = 2 × 2). Population of internalized Salmonella, after surface decontamination with 1% w/v AgNO3, was assessed on selective media. Even the lowest initial bacterial inoculum was adequate for internalization of Salmonella to occur in leafy vegetables. Non-adapted Salmonella inoculum of 7.0 (maximum) and 3.0 log CFU/mL (lowest inoculation level tested) after short storage (2 h) resulted in 3.7–4.3 and 1.3–1.5 log CFU/g internalized bacterial population, respectively. Colonization (including both attachment and internalization processes), as well as internalization process, were positively correlated to initial inoculum level. These processes reached a different plateau beyond which, no further increase in internalization was observed. Adaptation of the pathogen to mild stresses enhanced internalization (P < 0.05), with desiccation- and acid-adapted Salmonella demonstrating the highest internalization capacity, regardless of the vegetable and storage temperature. These findings could contribute to further elucidation of colonization capacity of Salmonella in leafy vegetables and assist in selecting the proper conditions that contribute to the prevention of fresh produce Salmonella contamination.
Chapter
Low temperature is often used in food processing. However, a series of physical and biochemical modifications can be produced by foodborne pathogens in the process of transient stress and low temperature adaptation, these changes make foodborne pathogenic bacteria continue to grow at low temperature, and even increase its pathogenicity and drug resistance, causing huge damage to food safety and human health. This chapter mainly introduces the application of low temperature in food processing, the changes of physiological and biochemical characteristics of bacteria exposed to a cold environment, and the factors affecting the development of cold resistance of foodborne pathogens, with emphasis on various cold resistance mechanisms of foodborne pathogens.
Article
Avian colibacillosis caused by avian pathogenic Escherichia coli (APEC) causes significant economic losses to the poultry industry worldwide and is also a leading potential threat to human health. Bacteriophages integrate into the host bacterial chromosome, and are an important source of genetic variation and have a major impact on bacterial evolution. Previously, we predicted prophage phiv205-1 in APEC strain DE205B. Here, to determine the function of prophage phiv205-1, we constructed the prophage deletion mutant DE205BΔphiv205-1. Compared with the wild-type (WT) APEC strain DE205B, the adherence and invasive abilities of DE205BΔphiv205-1 were reduced by 41.88%(P < 0.05). Further, the mutant strain had 52.38% reduced biofilm formation compared with the WT strain (P < 0.001). Chick challenge showed that the median lethal dose (LD50) of the mutant strain and WT strain was 3.13 × 10⁵ colony-forming units (CFU) and 3.86 × 10⁴ CFU, respectively, indicating that the mutant strain had decreased virulence compared with the WT strain. Furthermore, in vivo studies showed that, compared with the WT strain, DE205BΔphiv205-1 bacterial loads were reduced by 1.6-fold (P < 0.05) and 4.8-fold (P < 0.001) in the lungs and brains, respectively, of the infected chicks. In conclusion, the prophage phiv205-1 contributes to the virulence of APEC strain DE205B by facilitating the adherence, biofilm formation, and colonization abilities of its host strain.
Article
Adaptation is an important phenomenon for the survival of any organism in various in-vitro and in-vivo conditions leading to their better survival. We studied Salmonella, for the evolutionary adaptation under different conditions. When Salmonella was passaged for 12 rounds in Luria Bertani (P12-STM) media, F media (F12-STM) and Caenorhabditis elegans (Ce12-STM), we found certain phenotypic variations with respect to unpassaged strain. The passaged strains showed a significant increase in motility, body size and number of flagella they possessed, increased tolerance to ROS and RNS under in-vitro condition. They were more invasive in the human intestinal epithelial cells (Int-407) and showed a higher proliferation rate in the phagocytic cells (U-937 and RAW-264.7) suggesting their efficient intracellular proliferation. The evolved strains were hyper-infectious and reduced host viability significantly in C. elegans. The upregulation of various virulent and regulatory genes observed could be associated with the evolved adaptive virulent phenotype by Salmonella in a given condition. Whole genome sequencing also indicates the genetic alteration including insertion and deletions, single nucleotide polymorphisms which are common in all the strains as well as exposure specific, giving varied phenotype among the passaged strains. This study indicates that Salmonella is showing adaptation, genetic alteration and have become hypervirulent hence is capable of causing disease severity.
Article
Salmonella is a foodborne bacterial pathogen associated with red meat production worldwide. Survival of, and cross contamination with, this pathogen at any stage from the farm to the processing facility may result in human disease. Cattle, goat and sheep are common animal reservoirs of Salmonella. Various interventions to control the spread of this pathogen during red meat processing have been applied. Physical interventions, such as chilling, thermal treatments, ionizing radiation, ultraviolet radiation, as well as novel interventions such as high pressure treatment and ultrasound, have been used with varying success to reduce Salmonella during processing. This review examines the use of physical interventions to control Salmonella during processing and the stress response of Salmonella towards these interventions. Most existing studies do not provide a very strong insight into the molecular mechanisms of survival of Salmonella after exposure to these physical interventions. Attachment of Salmonella onto red meat species may be strain specific and the genetic profile of these strains may contribute to the survival of Salmonella during red meat processing. Bacterial sigma factors which may act as DNA repair proteins may induce the expression of virulence genes. Salmonella may develop a tolerance mechanism to these physical interventions by enhancing the expression of genes which may provide protection to cells during processing. Developing effective approaches for reduction and elimination of this pathogen on red meat still remains a challenge due to a lack of understanding of the stress response of different serovars of Salmonella. Genome wide stress responses and validation of the gene expression of Salmonella transcriptomes and proteomes need to be intensified to determine phenotypic and genotypic profiles of Salmonella on different red meat species under these different stressors.
Article
Full-text available
Comparative genomics demonstrated that the chromosomes from bacteria and their viruses (bacteriophages) are coevolving. This process is most evident for bacterial pathogens where the majority contain prophages or phage remnants integrated into the bacterial DNA. Many prophages from bacterial pathogens encode virulence factors. Two situations can be distinguished: Vibrio cholerae, Shiga toxin-producing Escherichia coli, Corynebacterium diphtheriae, and Clostridium botulinum depend on a specific prophage-encoded toxin for causing a specific disease, whereas Staphylococcus aureus, Streptococcus pyogenes, and Salmonella enterica serovar Typhimurium harbor a multitude of prophages and each phage-encoded virulence or fitness factor makes an incremental contribution to the fitness of the lysogen. These prophages behave like "swarms" of related prophages. Prophage diversification seems to be fueled by the frequent transfer of phage material by recombination with superinfecting phages, resident prophages, or occasional acquisition of other mobile DNA elements or bacterial chromosomal genes. Prophages also contribute to the diversification of the bacterial genome architecture. In many cases, they actually represent a large fraction of the strain-specific DNA sequences. In addition, they can serve as anchoring points for genome inversions. The current review presents the available genomics and biological data on prophages from bacterial pathogens in an evolutionary framework.
Article
Full-text available
Salmonella is an important cause of bacterial food-borne gastroenteritis. Salmonella encounters multiple abiotic stresses during pathogen elimination methods used in food processing, and these stresses may influence its subsequent survivability within the host or in the environment. Upon ingestion, Salmonella is exposed to gastrointestinal acidity, a first line of the host innate defense system. This study tested the hypothesis that abiotic stresses encountered during food processing alter the metabolic mechanisms in Salmonella that enable survival and persistence during subsequent exposure to the host gastrointestinal acidic environment. Out of the four different abiotic stresses tested, viz., cold, peroxide, osmotic, and acid, preadaptation of the log-phase culture to cold stress (5°C for 5 h) significantly enhanced survival during subsequent acid stress (pH 4.0 for 90 min). The gene expression profile of Salmonella preadapted to cold stress revealed induction of multiple genes associated with amino acid metabolism, oxidative stress, and DNA repair, while only a few of the genes in the above-mentioned stress response and repair pathways were induced upon exposure to acid stress alone. Preadaptation to cold stress decreased the NAD+/NADH ratio and hydroxyl (OH·) radical formation compared with those achieved with the exposure to acid stress alone, indicating alteration of aerobic respiration and the oxidative state of the bacteria. The results from this study suggest that preadaptation to cold stress rescues Salmonella from the deleterious effect of subsequent acid stress exposure by induction of genes involved in stress response and repair pathways, by modification of aerobic respiration, and by redox modulation.
Conference Paper
Full-text available
Several species of bacteria are involved in the production of cheese, including Lactobacillus brevis and Lactococcus lactis. A custom-designed Affymetrix microarray was recently developed to study gene expression in three organisms on a single chip. This array contains only perfect match features for the coding and non-coding regions in the genomes of all three sequences. The multi-species nature of this array version raises interesting questions regarding the preprocessing or normalization strategies for the analysis of gene expression data. We present and evaluate several possible strategies using both cDNA dilution data and experimental expression data from a repeated measures design. The statistical protocols highlighted in this work are applicable to other multi-species microarrays.
Article
The type III secretion system (TTSS) encoded by Salmonella typhimurium pathogenicity island 2 (SPI-2) is expressed after bacterial entry into host cells. The SPI-2 TTSS secretes the translocon components SseBCD, which translocate across the vacuolar membrane a number of effector proteins whose action is required for intracellular bacterial replication. Several of these effectors, including SifA and SifB, are encoded outside SPI-2. The two-component regulatory system SsrA-SsrB, encoded within SPI-2, controls the expression of components of the SPI-2 TTSS apparatus as well as its translocated effectors. The expression of SsrA-B is in turn regulated by the OmpR-EnvZ two-component system, by direct binding of OmpR to the ssrAB promoter. Several environmental signals have been shown to induce in vitro expression of genes regulated by the SsrA-B or OmpR-EnvZ systems. In this work, immunoblotting and flow cytometry were used to analyse the roles of SsrA-B and OmpR-EnvZ in coupling different environmental signals to changes in expression of a SPI-2 TTSS translocon component (SseB) and two effector genes (sifA and sifB). Using single and double mutant strains the relative contribution of each regulatory system to the response generated by low osmolarity, acidic pH or the absence of Ca 2 + was determined. SsrA-B was found to be essential for the induction of SPI-2 gene expression in response to each of these individual signals. OmpR-EnvZ was found to play a minor role in sensing these signals and to require a functional SsrA-B system to mediate their effect on SPI-2 TTSS gene expression.