Transcriptional Priming of Salmonella Pathogenicity
Island-2 Precedes Cellular Invasion
Suzanne E. Osborne, Brian K. Coombes*
Department of Biochemistry and Biomedical Sciences, Michael G. DeGroote Institute for Infectious Disease Research, McMaster University, Hamilton, Ontario, Canada
Invasive salmonellosis caused by Salmonella enterica involves an enteric stage of infection where the bacteria colonize
mucosal epithelial cells, followed by systemic infection with intracellular replication in immune cells. The type III secretion
system encoded in Salmonella Pathogenicity Island (SPI)-2 is essential for intracellular replication and the regulators
governing high-level expression of SPI-2 genes within the macrophage phagosome and in inducing media thought to
mimic this environment have been well characterized. However, low-level expression of SPI-2 genes is detectable in media
thought to mimic the extracellular environment suggesting that additional regulatory pathways are involved in SPI-2 gene
expression prior to cellular invasion. The regulators involved in this activity are not known and the extracellular
transcriptional activity of the entire SPI-2 island in vivo has not been studied. We show that low-level, SsrB-independent
promoter activity for the ssrA-ssrB two-component regulatory system and the ssaG structural operon encoded in SPI-2 is
dependent on transcriptional input by OmpR and Fis under non-inducing conditions. Monitoring the activity of all SPI-2
promoters in real-time following oral infection of mice revealed invasion-independent transcriptional activity of the SPI2
T3SS in the lumen of the gut, which we suggest is a priming activity with functional relevance for the subsequent
intracellular host-pathogen interaction.
Citation: Osborne SE, Coombes BK (2011) Transcriptional Priming of Salmonella Pathogenicity Island-2 Precedes Cellular Invasion. PLoS ONE 6(6): e21648.
Editor: Olivier Neyrolles, Institut de Pharmacologie et de Biologie Structurale, France
Received April 15, 2011; Accepted June 4, 2011; Published June 28, 2011
Copyright: ? 2011 Osborne, Coombes. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This research was funded through an operating grant from the Canadian Institutes of Health Research (CIHR; MOP-82704) and an infrastructure grant
from Canada Foundation for Innovation Leaders Opportunity Fund. The funders had no role in study design, data collection and analysis, decision to publish, or
preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: firstname.lastname@example.org
Salmonella enterica causes a range of foodborne diseases from self-
limiting gastroenteritis to fatal systemic infections. The virulence
capabilities of Salmonella is mediated by two type III secretion
systems (T3SS) which function to deliver bacterial proteins, called
effectors, into host cells that can reprogram various aspects of host
biology [1,2]. The two T3SS in Salmonella are encoded by separate
horizontally acquired pathogenicity islands termed Salmonella
Pathogenicity Island (SPI)-1 and SPI-2. The T3SS-1 allows
Salmonella to invade into host epithelial cells and is needed to
establish infection in the gastrointestinal tract . Following
passage across the host epithelial barrier the bacteria are engulfed
by resident immune cells, chiefly macrophages [4,5], and induce
the expression of the T3SS-2 [6,7]. Effectors translocated by the
T3SS-2 play a critical role in protection against an arsenal of host
defences including recruitment of reactive oxygen (ROS) and
reactive nitrogen (RNS) species to the Salmonella containing
vacuole (SCV) [8,9].
The T3SS in SPI-2 is organized into four major operons; a
regulatory operon, a structural-1 operon, an effector/chaperone
operon and a structural-2 operon. Genes in these operons are
controlled by promoters in front of ssrA, ssaB, sseA and ssaG
respectively [10,11]. We recently identified two additional
promoters (ssaM and ssaR) in the structural-2 operon . The
major regulator of SPI-2 gene expression is a two-component
regulatory system encoded by the genes ssrA and ssrB in the linked
regulatory operon. In response to an unidentified environmental
cue, the SsrA sensor kinase autophosphoryates and activates the
SsrB response regulator that can bind to an evolved palindrome
sequence to induce gene expression from the SPI-2 promoters and
at several promoters outside of SPI-2 [11,12]. Expression of ssrA
and ssrB is autoregulated and also dependent on several
transcription factors including the two-component systems PhoP-
PhoQ, OmpR-EnvZ, as well as SlyA and Fis. SPI-2 is negatively
regulated by H-NS, Hha and YdgT [13,14,15,16,17,18].
It is well established that transcriptional activity in SPI-2 is
induced following intracellular invasion as well as in in vitro
conditions thought to mimic the intracellular environment
[19,20,21]. However, we and others have reported low-level
SPI-2 gene expression in non-inducing media that does not
simulate the intracellular environment [21,22,23]. Of particular
importance is that the expression under non-inducing conditions is
independent of SsrB, suggesting another transcriptional input
pathway for SPI-2 gene expression that may precede cellular
invasion. In addition to its role in systemic dissemination of
bacteria, accumulating evidence indicates that the SPI-2 T3SS
facilitates bacterial colonization of the gut and induces intestinal
inflammation [24,25,26]. It was also shown using recombinase-
based in vivo expression that three promoters in SPI-2 (sseA, ssaG
and spiC/ssaB) are activated within 15 min after entering mouse
ileal loops . These data suggest that a transcriptional
regulatory circuit operates to induce low-level gene expression in
SPI-2 prior to Salmonella’s invasion into host cells.
PLoS ONE | www.plosone.org1 June 2011 | Volume 6 | Issue 6 | e21648
We analyzed the activity of all six promoters in SPI-2 in both
inducing and non-inducing media in a variety of Salmonella
mutants lacking the regulators involved in SPI-2 gene expression.
Inducing media resulted in high simultaneous activity of each SPI-
2 promoter that was dependent on SsrB. In contrast, SPI-2
promoters had low-level activity in non-inducing media that was
independent of SsrB but instead dependent on OmpR or Fis. We
further analyzed SPI-2 promoter activity during animal infection
in real time and found that SPI-2 promoters were activated
immediately following entry into the small intestine that was
independent of invasion. Using cultured epithelial cells we
demonstrate that SPI-2 has two distinct activation steps; an initial
activation that precedes cellular invasion, followed by the classical
intracellular activation pathway for high-level induction.
Regulation of SPI-2 under non-inducing conditions
To compare the activity of SPI-2 promoters in both inducing
and non-inducing conditions in vitro we constructed bacterial
luciferase transcriptional reporters for each of the six promoters in
SPI-2 (ssrA, ssaB, sseA, ssaG, ssaM and ssaR) [10,11]. To simulate
inducing conditions we used an acidic minimal medium low in
phosphate and magnesium (LPM pH 5.8) that is well established
to activate robust SPI-2 gene expression . M9-CAA medium
containing millimolar concentrations of divalent cations and a
neutral pH was used as a non-inducing media . Wild type S.
Typhimurium containing transcriptional reporters were grown in
M9-CAA until mid-log phase at which point they were sub-
cultured into either inducing or non-inducing media followed by
continuous luminescence measurements. Following transfer to
LPM each SPI-2 promoter was induced with the same kinetics but
the magnitude of this activity varied with each promoter
(Figure 1A). Promoter activity peaked at early to mid-exponential
phase and then declined and remained constant at ,20–30% of
maximum activity (Table S1 for complete dataset). We consistently
observed an early, low-level promoter activity primarily from the
regulatory and structural-1 promoters (ssrA and ssaG) under non-
inducing conditions followed by delayed activity from the
remaining structural-2 and effector promoters (Figure 1B). These
results suggested that SPI-2 promoters had unique transcriptional
inputs under inducing and non-inducing conditions that gave rise
to differential timing and magnitude of gene expression.
SPI-2 expression in non-inducing conditions has distinct
In order to understand the regulatory input contributing to the
activity of SPI-2 promoters in non-inducing conditions, we
measured promoter activity in eight different mutants each lacking
a major regulator known to be involved in virulence gene
expression in Salmonella including ssrB, ompR, slyA, phoP, fis, ydgT,
hha and hns [11,17,18,28,29,30]. Loss of SsrB, OmpR, SlyA, PhoP
or Fis caused a marked decrease in the promoter activity observed
in LPM for each SPI-2 promoter (Table 1; Figures S1, S2, Table
S1 for full dataset). Interestingly, loss of PhoP altered the temporal
dynamics of all promoters with the exception of the ssrA promoter
(Figure S1). Deletion of the SPI-2 repressors YdgT, Hha or
expression of dominant-negative H-NS (HNSQ92am) 
increased SPI-2 promoter activity in most cases although loss of
YdgT and Hha caused a decrease in ssaG and ssaR promoter
activity (Figure S2). The ssrA and ssaG promoter activity in M9-
CAA was independent of SsrB. Instead, the ssrA promoter activity
in M9-CAA was dependent on OmpR and partially dependent on
Fis, whereas ssaG promoter activity was dependent only on Fis.
These results confirmed that the low-level SPI-2 promoter activity
under non-inducing conditions had regulatory inputs distinct from
that needed for high-level expression under inducing conditions
thought to mimic the intracellular environment.
SPI-2 promoters are induced in the lumen of the gut
following oral infection
The observation that SPI-2 promoters are modestly active
under non-inducing conditions suggested that extracellular
priming of SPI-2 gene expression may occur. Previous work using
recombinase-based in vivo expression had established that three
promoters, (sseA, ssaG, and ssaB/spiC), were active in the lumen of
the murine gut following direct injection of bacteria into ileal loops
. However, the in vivo activity of the entire complement of SPI-
2 promoters following oral infection has not been tested. Mice
infected by oral gavage with individual Salmonella strains that
report the activity of each SPI-2 promoter were subjected to in vivo
luminescence imaging immediately following infection (Figure 2).
Each SPI-2 promoter was simultaneously and immediately
activated with luciferase signal being localized exclusively to the
small intestine in the first 35 min following infection, as
determined by ex vivo imaging of individual organs at the terminal
time point (Figure 3). When we compared the normalized light
flux from each promoter, we found no significant difference in
relative promoter activity, nor differences in the number of
bacteria of each reporter strain recovered from each organ (data
not shown). To assess promoter activity in animals over a longer
time period, mice were imaged every day for three days following
oral infection. These data showed that sseA promoter activity
remained active over three days in bacteria localized in the gut
(Figure S3). Using ex vivo imaging at necropsy we also detected
luminescence signal originating from systemic tissues since S.
Typhimurium gives rise to an invasive infection in mice (Figure
The rapid increase in SPI-2 promoter activity observed
following bacterial entry into the small intestine suggested that
transcription was originating in the gut lumen prior to bacterial
invasion. To investigate this, we constructed the PsseA-lux reporter
in an invA mutant that is defective for cellular invasion  and
quantified luminescence following oral infection. Promoter activity
from the invasion-deficient strain showed a rapid increase after
infection, similar in tempo and magnitude to that from wild type
cells (Figure 4). As expected, luminescence was localized
exclusively to the small intestine, suggesting that immune cell
sampling of luminal bacteria was not responsible for this activity.
These data are consistent with results using direct injection into
murine ileal loops of recombinase-based reporter strains .
These results demonstrate that following entry of S. Typhimurium
in to the intestinal lumen, all SPI-2 promoters undergo a rapid
increase in activity that precedes cellular invasion.
SPI-2 transcriptional priming does not require host cell
Our finding that SPI-2 promoters are rapidly induced following
entry into the lumen but prior to invasion prompted us to question
whether this activity was dependent on host cell contact. PsseA-lux
reporter bacteria were pre-grown in either M9-CAA or LB then
sub-cultured into DMEM/10%FBS and luminescence activity was
recorded in 96-well plates in the presence or absence of HeLa
cells. Plates were centrifuged to synchronize host cell contact.
Regardless of the pre-growth media, PsseA-lux activity was found to
have immediate transcriptional activity within the first 15 minutes
that was independent of both invasion and the presence of HeLa
Extracellular Priming of Salmonella Virulence
PLoS ONE | www.plosone.org2 June 2011 | Volume 6 | Issue 6 | e21648
cells (Figure 5). A second peak in transcriptional activity was
observed at 1 hour post-infection which reflects activity in the
intracellular niche. This data supports the model that SPI-2
undergoes two distinct transcriptional activation events; a pre-
invasion priming activity and a transcriptional up-regulation
specific to the intracellular niche.
Since the discovery of the T3SS-2 [6,7], extensive work has
elucidated its essential role for intracellular survival of S.
Typhimurium. The regulation of this system has been well
characterized for conditions that mimic the intracellular environ-
ment encountered by the bacteria following invasion. However
little is known about the regulation of the SPI-2 T3SS preceding
cellular invasion, although we think such a regulatory input would
have relevance. Salmonella survival in macrophages and other cell
types requires deployment of bacterial effectors by the SPI-2 T3SS
that are known to block phagosome maturation and to counteract
host defensive mechanisms such as reactive oxygen and nitrogen
species [8,9,33,34]. These processes are invoked immediately
following phagocytosis, which would require a coincident
functional response from the T3SS.
Although all SPI-2 promoters had simultaneous and high
activity upon transfer to a synthetic inducing media, most –
particularly the ssaG and ssrA promoters – had significant albeit
lesser activity in non-inducing media. Surprisingly, the SPI-2
response regulator SsrB accounted for less than 5% of this activity
and instead the transcriptional input was dominated by OmpR
and Fis. Indeed, Fis binding sites have been identified upstream of
ssaG  and OmpR and SsrB binding sites overlap at the ssrA
promoter , which is entirely consistent with the transcriptional
inputs we measured. Expression of SPI-2 immediately following
entry of the bacteria into the small intestine is also consistent with
a growing body of evidence indicating that the SPI-2 T3SS
contributes to intestinal colonization. Using a recombinase-based
reporter system and mouse ileal loops it was shown that the sseA
promoter was activated within 15 min of entry into the ileum 
Figure 1. SPI-2 expression in inducing and non-inducing conditions in vitro. S. Typhimurium with luciferase transcriptional reporters for
each SPI-2 promoter were sub-cultured from actively growing cultures in M9-CAA into either (A) inducing (LPM pH 5.8) or (B) non-inducing (M9-CAA)
media. Luminescence was quantified continuously and normalized to OD600 nmat each time point (n=12). Heat maps represent the percent activity
relative to each individual promoter’s maximal expression level.
Table 1. Transcriptional reporter activity in various mutants relative to wild type.
LPM pH 5.8
PssrA 6766.4 0.160.0 5964.9 72612 75610 250667390614
PssaB 0.060.0 0.160.1 0.960.2 2065.84.664.9263652 270620
PsseA 0.260.0 0.860.11.260.33769.41268.3 296637 247625
PssaG1561.51262 1161.2 3664.8 1064.3 26610 173614
PssaM2.960.2 3.160.5 3.460.22465.89.963.0 1050641 413672
PssaR 3.660.53.660.7 4.260.5 3366.41261.92661.29663.3
PssaG10066.7 11463.69661.39666.41860.7240650 11768.8
Extracellular Priming of Salmonella Virulence
PLoS ONE | www.plosone.org3June 2011 | Volume 6 | Issue 6 | e21648
when bacterial cells are associated with the apical surface of the
host epithelium. This activity was also dependent on OmpR,
suggesting that this regulatory input may be a key source of
transcriptional priming in vivo prior to cellular invasion. Bovine
and mouse infections have shown that the SPI-2 T3SS is necessary
for enteric infection and triggers colitis in a MyD88-dependent
manner [24,25,26,36]. Our data in conjunction with these findings
provides strong evidence for the expression of the SPI-2 T3SS in
the intestine. It also implies that alternative extracellular signals
are involved in SPI-2 regulation within the intestinal lumen, with
Figure 2. SPI-2 promoter activity increases immediately following entry into the small intestine. Mice were infected by oral gavage with
S. Typhimurium strains carrying the luciferase transcriptional reporters. Animals were anesthetised and luminescence was measured as described in
Methods. Colour bars for each reporter time course are indicated. Data is representative of four biological replicates each showing similar results.
Extracellular Priming of Salmonella Virulence
PLoS ONE | www.plosone.org4 June 2011 | Volume 6 | Issue 6 | e21648
Extracellular Priming of Salmonella Virulence
PLoS ONE | www.plosone.org5 June 2011 | Volume 6 | Issue 6 | e21648
possible candidates being mammalian body temperature , the
acidity encountered during transit through the stomach, and other
signals that are presently unknown.
The exact role of SPI-2 promoter activity in the intestinal lumen
is presently unclear. Although SPI-2 is needed for enteric infection,
this phenotype does not manifest until several days after infection,
suggesting that the early transcriptional activity we measured is
unrelated to this functionality. Instead, we propose that rapid
activation of SPI-2 following entry into the lumen of the host gut
reflects transcriptional priming needed for intracellular survival.
Consistent with the notion of transcriptional priming, in mice in
which disease is dominated by a systemic infection of the
reticuloendothelial system, we found each promoter in SPI-2 to
be active within five minutes following oral infection. This activity
was sustained even in bacteria with a genetic lesion in the invasion
machinery, indicating that SPI-2 transcriptional priming precedes
cellular invasion. The T3SS-2 is needed for Salmonella to evade
host antibacterial mechanisms such as reactive oxygen and
nitrogen delivery to the nascent phagosome [8,9] and SPI-2
mutant bacteria have a marked defect in preventing NADPH
oxidase recruitment to the phagosome . However, reactive
oxygen generation inside nascent phagosomes by the host
NADPH oxidase complex is detectable within 1-min following
phagocytosis of reactive oxygen-sensitive beads  or yeast cells
, which argues strongly for transcription priming of this
bacterial defence system before the invasion event. Further
research will be needed to quantify the intracellular fitness benefit
immediately following invasion that is conferred by this early SPI-
2 gene expression.
Materials and Methods
All animal work was approved by the Animal Review Ethics
Board at McMaster University under Animal Use Protocol #09-
07-26, and conducted according to guidelines set by the Canadian
Council on Animal Care.
Bacterial strains and growth conditions
Salmonella enterica serovar Typhimurium strain SL1344 was used
for all experiments and all mutants are derivatives thereof.
Bacteria were grown at 37uC with aeration in the presence of
selective antibiotics where appropriate as follows: ampicillin
(100 mg/mL), kanamycin (50 mg/mL), chloramphenicol (34 mg/
mL), tetracycline (12 mg/mL) and streptomycin (50 mg/mL). For
transcriptional reporter experiments, bacteria were cultured
Figure 4. SPI-2 promoter activity in the small intestine does not require T3SS-1-mediated invasion. Mice were infected with an invasion-
deficient mutant (invA::Kan) carrying an sseA promoter fusion to luxCDABE. Immediately after infection, anesthetised mice were imaged as described.
(A) Whole-body luminescence from infected mice. Images are representative of three individual animals. (B) Quantification of luminescence (total
flux) is shown as the mean with standard deviation for each time point (n=3). (C) Individual organs (spleen and liver, left panel; small intestine, colon
and cecum, right panel) were imaged ex vivo at necropsy.
Figure 3. Quantification and ex vivo imaging of tissue luminescence. (A) Quantification of luminescence (total flux) is shown as the mean
with standard deviation for each time point (n=4). (B) Individual organs (S, spleen and L, liver, left panels; SI, small intestine; LI, large intestine and C,
cecum, right panels) were imaged ex vivo at necropsy. (C) Light flux from individual organs was normalized to bacterial load. Data are the means with
standard deviation from four organs for each reporter strain at the termination of the 35 min imaging session.
Extracellular Priming of Salmonella Virulence
PLoS ONE | www.plosone.org6 June 2011 | Volume 6 | Issue 6 | e21648
overnight in M9-CAA minimal media (5 mM Na2HPO4?7H2O,
22 mM KH2PO4, 8.6 mM NaCl, 18.6 mM NH4Cl, 11.1 mM
glucose, 2 mM MgSO4, 100 mM CaCl2, 0.1% casamino acids).
Low phosphate, low magnesium medium (LPM)  pH 5.8 was
used as a highly-inducing medium for SPI-2 gene expression
(5 mM KCl, 7.5 mM (NH4)2SO4, 80 mM MES, 38 mM glycerol,
0.1% casamino acids, 24 mM MgCl2, 337 mM PO432).
Cloning and mutant construction
Unmarked, in-frame deletions of slyA and ompR as well as a
marked in-frame deletion of fis (fis::Kan) were constructed using
Lambda red recombination . Transcriptional reporters with
luxCDABE were constructed in pGEN-luxCDABE  for all six of
the promoters identified in SPI-2  including ssrA, ssaB, sseA,
ssaG, ssaM, and ssaR. All primers used for mutant construction and
cloning of transcriptional reporters are listed in Table S2.
Transcriptional reporter assays
Bacteria were grown overnight in M9 CAA at 37uC with
shaking and then sub-cultured 1:100 into M9 CAA in 96-well
plates (Costar). Bacteria were grown at 37uC (150 rpm) and optical
density at 600 nm (OD600) and luminescence were measured every
15 min using an Envison 2104 plate reader (PerkinElmer).
Luminescence data was normalized to OD600 nmfor each time
point and adjusted to the luminescence at time zero.
In vivo bioluminescence imaging
Three days prior to infection, abdominal fur was removed from
the mice using a depilatory cream. Salmonella with luciferase
reporters were grown overnight in M9 CAA with selective
antibiotics at 37uC. Bacteria were washed twice and resuspended
in 0.1 M HEPES (pH 8.0), 0.9% NaCl. Female C57BL/6 mice
(Jackson Laboratories) were infected by oral gavage with ,108live
bacteria. Animals were immediately anaesthetized with 2%
isofluorane carried in 2% oxygen and imaged dorsally in an IVIS
Spectrum (Caliper Life Sciences). Greyscale and luminescence
images were captured at 5 min intervals for 35 min and processed
using Living Image Software. After the imaging session, mice were
sacrificed and individual organs were imaged ex vivo and then
processed for bacterial load determination by homogenization in a
Mixer Mill (Retsch; Haan, Germany) and selective plating on solid
media. Total flux was normalized to the initial flux recorded at
HeLa cell culture
HeLa cells were seeded in black 96-well plates with clear
bottoms at 26105cells/mL 24 h prior to infection. Overnight
cultures of wild type or an invasion-deficient DinvA strain, both
carrying (pPsseA-lux) were pre-grown in LB or M9-CAA for
3 hours then sub-cultured 1:100 into DMEM (Gibco) with 10%
fetal bovine serum (FBS). 50 mL was added to each well and
centrifuged at 500 x g for 5 min. Bioluminescence was recorded as
described above. Cells were grown at 37uC in 5% CO2.
inducing conditions has distinct regulatory inputs.
Graphs represent the entire dataset collected for the experiments
involving transcriptional activators summarized in Table 1. Wild
type S. Typhimurium carrying luciferase transcriptional reporters
for each SPI-2 promoter were sub-cultured from actively growing
cultures in M9-CAA into either inducing (LPM pH 5.8) or non-
inducing (M9-CAA) media. Luminescence was measured contin-
uously and normalized to OD600 nmat each time point (n=12).
Data are the means with standard deviation.
SPI-2 expression in inducing versus non-
inducing conditions for transcriptional repressor mu-
tants. Graphs represent the entire dataset collected for the
experiments involving transcriptional repressors summarized in
Table 1. Wild type S. Typhimurium carrying luciferase transcrip-
tional reporters for each SPI-2 promoter were sub-cultured from
actively growing cultures in M9-CAA into either inducing (LPM
pH 5.8) or non-inducing (M9-CAA) media. Luminescence was
measured continuously and normalized to OD600 nmat each time
point (n=12). Data are the means with standard deviation.
SPI-2 expression in inducing versus non-
days post infection. Mice were infected with wild type
The sseA promoter remains active from 1 to 3
Figure 5. SPI-2 undergoes two stages of transcriptional up-regulation. Wild type or invasion deficient (DinvA) Salmonella carrying sseA
bioluminescence promoter fusions were pre-grown in M9-CAA or LB then sub-cultured into DMEM/10%FBS. Bioluminescence activity was monitored
in the presence or absence of HeLa cells.
Extracellular Priming of Salmonella Virulence
PLoS ONE | www.plosone.org7 June 2011 | Volume 6 | Issue 6 | e21648
Salmonella containing the sseA transcriptional reporter. (A) Download full-text
Luminescence images were acquired every 24 h and are
representative of three individuals animals. (B) Total flux from
whole-animal imaging was quantified and is shown as the mean
with standard deviation (n=3). (C) At 3 days post-infection organs
from infected mice from (A) were imaged ex vivo (S, spleen; L, liver;
C, cecum; SI, small intestine; LI, large intestine).
promoters in wild type Salmonella and seven regulator
mutants. Experiments were conducted as described in Materials
and Methods and data is shown as the mean with standard
deviation from three separate experiments.
Transcriptional reporter data for all SPI-2
construction of mutants and transcriptional reporters.
List of primers and their sequences used for
We thank members of the Coombes lab for helpful discussions related to
Conceived and designed the experiments: SEO BKC. Performed the
experiments: SEO. Analyzed the data: SEO BKC. Wrote the paper: SEO
1. Galan JE, Wolf-Watz H (2006) Protein delivery into eukaryotic cells by type III
secretion machines. Nature 444: 567–573.
2. Cornelis GR (2006) The type III secretion injectisome. Nat Rev Microbiol 4:
3. Jones BD, Ghori N, Falkow S (1994) Salmonella typhimurium initiates murine
infection by penetrating and destroying the specialized epithelial M cells of the
Peyer’s patches. J Exp Med 180: 15–23.
4. Richter-Dahlfors A, Buchan AMJ, Finlay BB (1997) Murine Salmonellosis studied
by confocal microscopy: Salmonella typhimurium resides intracellulalr inside macro-
5. Salcedo SP, Noursadeghi M, Cohen J, Holden DW (2001) Intracellular
replication of Salmonella typhimurium strains in specific subsets of splenic
macrophages in vivo. Cell Microbiol 3: 587–597.
6. Shea JE, Hensel M, Gleeson C, Holden DW (1996) Identification of a virulence
locus encoding a second type III secretion system in Salmonella typhimurium. Proc
Natl Acad Sci U S A 93: 2593–2597.
7. Ochman H, Soncini FC, Solomon F, Groisman EA (1996) Identification of a
pathogenicity island required for Salmonella survival in host cells. Proc Natl Acad
Sci USA 93: 7800–7804.
8. Vazquez-Torres A, Xu Y, Jones-Carson J, Holden DW, Lucia SM, et al. (2000)
Salmonella pathogenicity island 2-dependent evasion of the phagocyte NADPH
oxidase. Science 287: 1655–1658.
9. Chakravortty D, Hansen-Wester I, Hensel M (2002) Salmonella pathogenicity
island 2 mediates protection of intracellular Salmonella from reactive nitrogen
intermediates. J Exp Med 195: 1155–1166.
10. Walthers D, Carroll RK, Navarre WW, Libby SJ, Fang FC, et al. (2007) The
response regulator SsrB activates expression of diverse Salmonella pathogenicity
island 2 promoters and counters silencing by the nucleoid-associated protein H-
NS. Mol Microbiol 65: 477–493.
11. Tomljenovic-Berube AM, Mulder DT, Whiteside MD, Brinkman FS,
Coombes BK (2010) Identification of the regulatory logic controlling Salmonella
pathoadaptation by the SsrA-SsrB two-component system. PLoS Genet 6:
12. Xu X, Hensel M (2010) Systematic analysis of the SsrAB virulon of Salmonella
enterica. Infect Immun 78: 49–58.
13. Bijlsma JJ, Groisman EA (2005) The PhoP/PhoQ system controls the intrama-
crophage type three secretion system of Salmonella enterica. Mol Microbiol 57: 85–96.
14. Feng X, Walthers D, Oropeza R, Kenney LJ (2004) The response regulator SsrB
activates transcription and binds to a region overlapping OmpR binding sites at
Salmonella pathogenicity island 2. Mol Microbiol 54: 823–835.
15. Stapleton MR, Norte VA, Read RC, Green J (2002) Interaction of the Salmonella
typhimurium transcription and virulence factor SlyA with target DNA and
identification of members of the SlyA regulon. J Biol Chem 277: 17630–17637.
16. Navarre WW, Porwollik S, Wang Y, McClelland M, Rosen H, et al. (2006)
Selective silencing of foreign DNA with low GC content by the H-NS protein in
Salmonella. Science 313: 236–238.
17. Coombes BK, Wickham ME, Lowden MJ, Brown NF, Finlay BB (2005)
Negative regulation of Salmonella pathogenicity island 2 is required for contextual
control of virulence during typhoid. Proc Natl Acad Sci U S A 102:
18. Silphaduang U, Mascarenhas M, Karmali M, Coombes BK (2007) Repression
of intracellular virulence factors in Salmonella by the Hha and YdgT nucleoid-
associated proteins. J Bacteriol 189: 3669–3673.
19. Cirillo DM, Valdivia RH, Monack DM, Falkow S (1998) Macrophage-
dependent induction of the Salmonella pathogenicity island 2 type III secretion
system and its role in intracellular survival. Mol Microbiol 30: 175–188.
20. Deiwick J, Nikolaus T, Erdogan S, Hensel M (1999) Environmental regulation of
Salmonella pathogenicity island 2 gene expression. Mol Microbiol 31: 1759–1773.
21. Coombes BK, Brown NF, Valdez Y, Brumell JH, Finlay BB (2004) Expression
and secretion of Salmonella pathogenicity island-2 virulence genes in response to
acidification exhibit differential requirements of a functional type III secretion
apparatus and SsaL. J Biol Chem 279: 49804–49815.
22. Bustamante VH, Martinez LC, Santana FJ, Knodler LA, Steele-Mortimer O,
et al. (2008) HilD-mediated transcriptional cross-talk between SPI-1 and SPI-2.
Proc Natl Acad Sci U S A 105: 14591–14596.
23. Miao EA, Freeman JA, Miller SI (2002) Transcription of the SsrAB regulon is
repressed by alkaline pH and is independent of PhoPQ and magnesium
concentration. J Bacteriol 184: 1493–1497.
24. Coombes BK, Coburn BA, Potter AA, Gomis S, Mirakhur K, et al. (2005)
Analysis of the contribution of Salmonella pathogenicity islands 1 and 2 to enteric
disease progression using a novel bovine ileal loop model and a murine model of
infectious enterocolitis. Infect Immun 73: 7161–7169.
25. Coburn B, Li Y, Owen D, Vallance BA, Finlay BB (2005) Salmonella enterica
serovar Typhimurium pathogenicity island 2 is necessary for complete virulence
in a mouse model of infectious enterocolitis. Infect Immun 73: 3219–3227.
26. Bispham J, Tripathi BN, Watson PR, Wallis TS (2001) Salmonella pathogenicity
island 2 influences both systemic salmonellosis and Salmonella-induced enteritis in
calves. Infect Immun 69: 367–377.
27. Brown NF, Vallance BA, Coombes BK, Valdez Y, Coburn BA, et al. (2005)
Salmonella Pathogenicity Island 2 Is Expressed Prior to Penetrating the Intestine.
PLoS Pathog 1: e32.
28. Yoon H, McDermott JE, Porwollik S, McClelland M, Heffron F (2009)
Coordinated regulation of virulence during systemic infection of Salmonella enterica
serovar Typhimurium. PLoS Pathog 5: e1000306.
29. Vivero A, Banos RC, Mariscotti JF, Oliveros JC, Garcia-del Portillo F, et al.
(2008) Modulation of horizontally acquired genes by the Hha-YdgT proteins in
Salmonella enterica serovar Typhimurium. J Bacteriol 190: 1152–1156.
30. Navarre WW, Halsey TA, Walthers D, Frye J, McClelland M, et al. (2005) Co-
regulation of Salmonella enterica genes required for virulence and resistance to
antimicrobial peptides by SlyA and PhoP/PhoQ. Mol Microbiol 56: 492–508.
31. Duong N, Osborne S, Bustamante VH, Tomljenovic AM, Puente JL, et al.
(2007) Thermosensing coordinates a cis-regulatory module for transcriptional
activation of the intracellular virulence system in Salmonella enterica serovar
Typhimurium. J Biol Chem 282: 34077–34084.
32. Galan JE, Curtiss R 3rd (1991) Distribution of the invA, -B, -C, and -D genes of
Salmonella typhimurium among other Salmonella serovars: invA mutants of Salmonella
typhi are deficient for entry into mammalian cells. Infect Immun 59: 2901–2908.
33. Waterman SR, Holden DW (2003) Functions and effectors of the Salmonella
pathogenicity island 2 type III secretion system. Cell Microbiol 5: 501–511.
34. Abrahams GL, Hensel M (2006) Manipulating cellular transport and immune
responses: dynamic interactions between intracellular Salmonella enterica and its
host cells. Cell Microbiol 8: 728–737.
35. Lim S, Kim B, Choi HS, Lee Y, Ryu S (2006) Fis is required for proper
regulation of ssaG expression in Salmonella enterica serovar Typhimurium. Microb
Pathog 41: 33–42.
36. Hapfelmeier S, Stecher B, Barthel M, Kremer M, Muller AJ, et al. (2005) The
Salmonella pathogenicity island (SPI)-2 and SPI-1 type III secretion systems allow
Salmonella serovar typhimurium to trigger colitis via MyD88-dependent and
MyD88-independent mechanisms. J Immunol 174: 1675–1685.
37. Gallois A, Klein JR, Allen LA, Jones BD, Nauseef WM (2001) Salmonella
pathogenicity island 2-encoded type III secretion system mediates exclusion of
NADPH oxidase assembly from the phagosomal membrane. J Immunol 166:
38. VanderVen BC, Yates RM, Russell DG (2009) Intraphagosomal measurement
of the magnitude and duration of the oxidative burst. Traffic 10: 372–378.
39. Tlili A, Dupre-Crochet S, Erard M, Nusse O (2011) Kinetic analysis of
phagosomal production of reactive oxygen species. Free Radical Biology and
Medicine 50: 438–447.
40. 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:
41. Lane MC, Alteri CJ, Smith SN, Mobley HL (2007) Expression of flagella is
coincident with uropathogenic Escherichia coli ascension to the upper urinary
tract. Proc Natl Acad Sci U S A 104: 16669–16674.
Extracellular Priming of Salmonella Virulence
PLoS ONE | www.plosone.org8 June 2011 | Volume 6 | Issue 6 | e21648